CN101460521A

CN101460521A - Hepatocyte growth factor (HGF) binding proteins

Info

Publication number: CN101460521A
Application number: CNA2007800201545A
Authority: CN
Inventors: M·韩; S·K·赖特; W·M·小温斯特; L·布劳尔特; 林杰; B·伊特麦德-吉尔伯森; C·克尼赫; J·久里斯
Original assignee: Aveo Pharmaceuticals Inc
Current assignee: Aveo Pharmaceuticals Inc
Priority date: 2006-06-02
Filing date: 2007-06-01
Publication date: 2009-06-17
Also published as: SI2027156T1; ZA200809483B; ZA200809484B; CN101460520B; CN101460520A

Abstract

The present invention provides a family of binding proteins that bind and neutralize the activity of hepatocyte growth factor (HGF), in particular human HGF. The binding proteins can be used as diagnostic and/or therapeutic agents. With regard to their therapeutic activity, the binding proteins can be used to treat certain HGF responsive disorders, for example, certain HGF responsive tumors.

Description

The conjugated protein of pHGF (HGF)

Related application

The application requires the U.S. Provisional Application No.60/810 of submission on June 2nd, 2006, the U.S. Provisional Application No.60/860 that on November 21st, 714 and 2006 submitted to, 461 interests and right of priority, the disclosure of these applications is incorporated this paper at this into way of reference.

Technical field

Technical field of the present invention relates to molecular biology, immunology and oncology.More particularly, technical field of the present invention is and the conjugated protein of human hepatocyte growth factor (HGF) bonded based on antibody.

Background technology

PHGF (HGF), be also referred to as spreading factor (Scatter Factor, SF), be a kind of multi-functional heterodimer protein that mainly produces by mesenchymal cell, and be the effector (referring to people such as Bottaro (1991) SCIENCE 251:802-804, people such as Rubin (1993) BIOCHIM.BIOPHYS.ACTA 1155:357-371) of expressing the cell of Met tyrosine kinase receptor.People Met acceptor also is called as " c-Met ".Sophisticated HGF comprises two polypeptide chains, that is, and and α chain and β chain.Disclosed research points out that the α chain comprises the c-Met receptor binding domains of HGF.

When HGF combined with its homoreceptor, HGF had regulated the various kinds of cell activity.The HGF-Met signal transduction pathway works in liver regeneration, wound healing, neurotization, blood vessel generation and malignant tumour.For example referring to people such as Cao (2001) PROC.NATL.ACAD.SCI.USA 98:7443-7448, people such as Burgess (2006) CANCER RES.66:1721-1729, and United States Patent (USP) sequence number 5,997,868 and 5,707,624.The investigator has developed many HGF conditioning agents (comprising antibody) and has treated and relate to the active multiple disorder of HGF, for example the cancer (HGF responsive cancer) of some HGF response.For example referring to the open sequence number WO 2005/017107 of international patent application.

Fig. 1 has schematically shown the common basic structure of all antibody.Antibody is the polymer protein that contains 4 polypeptide chains.2 polypeptide chains wherein are called as heavy chain or H chain, and other 2 polypeptide chains are called as light chain or L chain.The heavy chain and the light chain that connect immunoglobulin (Ig) by the interchain disulfide linkage.The heavy chain that connects immunoglobulin (Ig) by a plurality of interchain disulfide bonds.Light chain is by 1 variable region (V among Fig. 1 _L) and 1 constant region (C among Fig. 1 _L) constitute, and heavy chain is by 1 variable region (V among Fig. 1 _H) and at least 3 constant region (CH among Fig. 1 ₁, CH ₂And CH ₃) constitute.The variable region has determined the specificity of antibody, and constant region then comprises other functions.

Amino acid and structural information show that each variable region all comprises 3 hypervariable regions (being also referred to as complementarity-determining region or CDR), and its flank is 4 conservative relatively framework region or FR.Described 3 CDR (are called CDR ₁, CDR ₂And CDR ₃) determined the binding specificity of single antibody.When with antibody during, it is desirable to usually produce and comprise for the binding specificity of target molecule topnotch and the antibody of avidity as diagnostic reagent and therapeutical agent.The specificity and the avidity that it is believed that the difference antagonist of variable region comprise far-reaching influence.

United States Patent (USP) sequence number 5,707,624 has been described the purposes of antibody in the treatment Kaposi sarcoma of anti-HGF.Similarly, United States Patent (USP) sequence number 5,997,868 has been described by using the antibody of anti-HGF to patient to be treated, thereby blocks the oncotherapy that endogenous HGF promotes the ability that the tumour medium vessels takes place.Recently, the investigator proposes, and comprises the potentiality (Burgess (2006) is the same) that become the therapeutical agent of suffering from the tumour patient that HGF relies on the antibody of the β chain combination of HGF.

Yet, still need to be used as other HGF conditioning agents of therapeutical agent and diagnostic reagent.

Summary of the invention

The present invention is based in part on specifically the discovery in conjunction with the conjugated protein family of HGF (particularly people HGF).Described conjugated protein is based on antibody, to such an extent as to it comprises based on specifically in conjunction with the antigen of the CDR of the antibody family of HGF (that is HGF) binding site.Described CDR has given the binding specificity of described conjugated protein confrontation HGF.Described conjugated protein can be used as diagnostic reagent and therapeutical agent.When with described conjugated protein when the therapeutical agent, this conjugated protein is transformed (for example humanization), thereby reduces or eliminate the risk of when being applied to recipient (for example people), inducing at the immunne response of described conjugated protein.

Therefore the neutralized activity of HGF of described conjugated protein can be used as therapeutical agent.In certain embodiments, described conjugated protein inhibition HGF combines the activity of the HGF that neutralized thus with its homoreceptor (c-Met).In other embodiments, described conjugated protein combines with HGF, and its biological activity that neutralizes, but does not suppress combining of HGF and c-Met acceptor.Because HGF relates to the growth and the propagation of cancer cells, therefore described conjugated protein can be used for the propagation of anticancer.In addition, when when being applied to Mammals, described conjugated protein can suppress or reduce growth of tumor in the Mammals.

The description of accompanying drawing, detailed Description Of The Invention and claim by hereinafter described shows these and other aspects of the present invention and advantage clearly.

Description of drawings

Can understand the present invention more up hill and dale referring to the following drawings.

Fig. 1 is the figure of the schematic representative of classical antibody.

Fig. 2 is the synoptic diagram that shows below aminoacid sequence, and described aminoacid sequence has defined the complete immunoglobulin heavy chain variable region of antibody, and wherein said antibody is represented with 1A3,1D3,1F3,2B8,2F8,3A12,3B6 and 3D11.The aminoacid sequence of each antibody is all compared with the aminoacid sequence of another antibody, and defines definition signal peptide, CDR in square frame ₁, CDR ₂And CDR ₃The zone.Do not represent the FR sequence with the sequence of square frame.

Fig. 3 is for showing the CDR of each the immunoglobulin heavy chain variable region sequence that shows among Fig. 2 ₁, CDR ₂And CDR ₃The synoptic diagram of sequence.

Fig. 4 is the synoptic diagram that shows below aminoacid sequence, and described aminoacid sequence has defined the complete immunoglobulin light chain variable region of antibody 1A3,1D3,1F3,2B8,2F8,3A12,3B6 and 3D11.The aminoacid sequence of each antibody is all compared with the aminoacid sequence of another antibody, and defines definition signal peptide, CDR in square frame ₁, CDR ₂And CDR ₃The zone.Do not represent the FR sequence with the sequence of square frame.

Fig. 5 is for showing the CDR of each the immunoglobulin light chain variable region sequence that demonstrates among Fig. 4 ₁, CDR ₂And CDR ₃The synoptic diagram of sequence.

The serve as reasons summary view of the result of gained in the test of measuring the tumors inhibition activity of antibody 1D3,1F3,1A3 and the 2B8 of anti-HGF in the U87MG heteroplastic transplantation model of Fig. 6.Rhombus is corresponding to PBS; Trilateral is corresponding to the antibody 1A3 of anti-HGF; X is corresponding to the antibody 1D3 of anti-HGF; Square antibody 1F3 corresponding to anti-HGF; And circular antibody 2B8 corresponding to anti-HGF.

The serve as reasons summary view of the result of gained in the test of measuring the tumors inhibition activity of antibody 1D3,1F3,1A3 and the 2B8 of anti-HGF in the U118 heteroplastic transplantation model of Fig. 7.Rhombus is corresponding to IgG; Square antibody 1F3 corresponding to anti-HGF; Trilateral is corresponding to the antibody 1D3 of anti-HGF; X is corresponding to the antibody 1A3 of anti-HGF; And circular antibody 2B8 corresponding to anti-HGF.

Fig. 8 is a table, and this table has been summed up interactional dynamic (dynamical) surface plasma resonance data between antigen-binding affinity and HGF and chimeric 2B8 antibody, chimeric/humanized 2B8 antibody or the humanized 2B8 antibody.This tabular has gone out tested many to Kappa light chain and IgG1 heavy chain.In 3 are independently tested, these antibody that comprise standard deviation (STDEV) of listing are analyzed.

Fig. 9 shows the epi-position that Hu2B8 combination and mouse monoclonal antibody 2B8 repel mutually for summing up the histogram of testing data.Humanized or chimeric 2B8 is captured on anti-people's the chip of Fc.Then, HGF is combined with humanized or chimeric 2B8.Measure mouse 2B8 or control antibodies (antibody of the anti-HGF of polyclonal goat) and captive HGF bonded ability.Humanized 2B8 antibody and chimeric 2B8 suppress combining of mouse 2B8 and HGF.The post of white is corresponding to chimeric 2B8 antibody; Gray post is corresponding to humanization Hu2B8 antibody (Kappa variable region Kv1-39.1 and variable region of heavy chain Hv5-51.1); The post of black is corresponding to humanization Hu2B8 antibody (Kappa variable region Kv3-15.1 and variable region of heavy chain Hv5-51.1).

Detailed Description Of The Invention

The present invention be based in part on specifically in conjunction with HGF and in and the discovery of the conjugated protein family of the activity of HGF (particularly people HGF). Described conjugated protein can be used for many diagnosis and treatment is used. Described conjugated protein based on regard to its in conjunction with HGF and in and the ability of the activity of HGF and the antigen binding site of selecteed some monoclonal antibody. Particularly, described conjugated protein comprises a plurality of immune globulin variable region CDR sequences, and it has defined the binding site that is used for HGF together.

The neutralization of considering these antibody is active, and they are specially adapted to mediate growth and/or the propagation of HGF responsive cell (for example cancer cell). When described conjugated protein is used as therapeutic agent, can transforms it, thereby minimize or eliminate the risk of when being applied to the recipient, inducing for the immune response of described conjugated protein. In addition, according to concrete application, comprise described conjugated protein and other parts (for example detectable label (such as radioactive labels) and effector molecule (such as other protein with based on micromolecular therapeutic agent)) are puted together. Hereinafter will be described in detail in these features of the present invention and the aspect each.

I-in conjunction with the conjugated protein of HGF

In one aspect, the invention provides a kind of isolating conjugated protein in conjunction with people HGF, this conjugated protein comprises: (i) comprise structure C DR _L1-CDR _L2-CDR _L3Immunoglobulin light chain variable region; The immunoglobulin heavy chain variable region that (ii) comprises 3 complementarity-determining regions (CDR), wherein said immunoglobulin light chain variable region and described immunoglobulin heavy chain variable region have defined the single binding site that is used in conjunction with people HGF together.CDR _L1Comprise aminoacid sequence X ₁X ₂Ser X ₄X ₅X ₆X ₇X ₈X ₉X ₁₀X ₁₁X ₁₂X ₁₃X ₁₄X ₁₅, amino acid X wherein ₁Be Arg, Lys or Ser, X ₂Be Ala or Thr, X ₄Is Glu, Gln or Ser, X ₅Be Asn, Asp or Ser, X ₆Be Ile or Val, X ₇Be Asp, Lys, Ser, Val or Tyr, X ₈Be peptide bond or Tyr, X ₉Be peptide bond or Asp, X ₁₀Be peptide bond or Gly, X ₁₁Be peptide bond or Asn, X ₁₂Be peptide bond, Ile or Ser, X ₁₃Be Asn or Tyr, X ₁₄Be Ile, Leu, Met or Val, X ₁₅Be Ala, Asn, His or Ser.CDR _L2Comprise aminoacid sequence X ₁₆X ₁₇X ₁₈X ₁₉X ₂₀X ₂₁X ₂₂, amino acid X wherein ₁₆Be Ala, Asp, Arg, Gly or Val, X ₁₇Be Ala, Thr or Val, X ₁₈Be Asn, Ser or Thr, X ₁₉Be Arg, Asn, Lys or His, X ₂₀Be Leu or Arg, X ₂₁Be Ala, Asn, Glu, Val or Pro, X ₂₂Be Asp, Ser or Thr.CDR _L3Comprise aminoacid sequence X ₂₃X ₂₄X ₂₅X ₂₆X ₂₇X ₂₈ProX ₃₀Thr, wherein amino acid X ₂₃Be Leu, Gly or Gln, X ₂₄Be His or Gln, X ₂₅Be Phe, Ser, Trp or Tyr, X ₂₆Be Asp, Ile, Ser, Trp or Tyr, X ₂₇Be Gly, Glu, Asn or Ser, X ₂₈Be Asp, Asn, Phe, Thr or Tyr, X ₃₀Be Leu, Phe, Pro or Tyr.

In other respects, the invention provides a kind of isolating conjugated protein in conjunction with HGF, this conjugated protein comprises: (i) comprise structure C DR _H1-CDR _H2-CDR _H3Immunoglobulin heavy chain variable region; The immunoglobulin light chain variable region that (ii) comprises 3 complementarity-determining regions (CDR), wherein said immunoglobulin heavy chain variable region and described immunoglobulin light chain variable region have defined the single binding site that is used in conjunction with HGF together.CDR _H1Comprise aminoacid sequence X ₁TyrX ₃X ₄X ₅, amino acid X wherein ₁Be Asp, Asn, Ser or Thr, X ₃Be Phe, Ser, Trp or Tyr, X ₄Be Ile, Leu or Met, X ₅Be Asn, His or Ser.CDR _H2Comprise aminoacid sequence X ₆IleX ₈X ₉X ₁₀X ₁₁GlyX ₁₃X ₁₄X ₁₅TyrX ₁₇X ₁₈X ₁₉X ₂₀X ₂₁X ₂₂, amino acid X wherein ₆Be Lys, Gln, Glu, Val or Tyr, X ₈Be Asn, Gly, Ser, Trp or Tyr, X ₉Be Ala, Pro or Ser, X ₁₀Be Gly or Thr, X ₁₁Be peptide bond, Asp, Asn, Gly or Ser, X ₁₃Be Asp, Asn, His or Ser, X ₁₄Be Ser or Thr, X ₁₅Be Asn or Tyr, X ₁₇Be Asn or Pro, X ₁₈Be Ala, Asp, Gly, Gln, Glu, Pro or Ser, X ₁₉Be Asn, Lys, Met or Ser, X ₂₀Be Leu, Phe or Val, X ₂₁Be Lys, Met or Gln, X ₂₂Be Asp, Gly or Ser.CDR _H3Comprise aminoacid sequence X ₂₃X ₂₄X ₂₅X ₂₆X ₂₇X ₂₈X ₂₉X ₃₀X ₃₁X ₃₂X ₃₃X ₃₄Tyr, wherein amino acid X ₂₃Be Arg, Asn, Gln or Glu, X ₂₄Be Gly, Leu, Arg or Tyr, X ₂₅Be peptide bond, Asp or Gly, X ₂₆Be peptide bond or Gly, X ₂₇Be peptide bond or Tyr, X ₂₈Be peptide bond, Leu or Tyr, X ₂₉Be peptide bond, Gly, Leu, Arg or Val, X ₃₀Be peptide bond, Asp, Gly or Glu, X ₃₁Be peptide bond, Asn, Arg, Ser or Tyr, X ₃₂Be peptide bond, Ala, Gly, Ile or Tyr, X ₃₃Be Met or Phe, X ₃₄Be Ala or Asp.

Should be appreciated that described conjugated protein comprises aforesaid light chain immunoglobulin and heavy chain immunoglobulin or its fragment.In addition, should be appreciated that described conjugated protein can be complete antibody or its Fab or biosynthetic antibody sites.

In certain embodiments, in the CDR of light chain immunoglobulin and heavy chain immunoglobulin sequence, insert framework region (FR).

, in other the embodiment CDR sequence of light chain immunoglobulin and heavy chain immunoglobulin is inserted between people or the humanized framework region at some.

On the other hand, the invention provides a kind of specifically in conjunction with the isolating conjugated protein of HGF.This conjugated protein comprises: (a) comprise structure C DR _L1-CDR _L2-CDR _L3Immunoglobulin light chain variable region; (b) immunoglobulin heavy chain variable region, wherein said immunoglobulin light chain variable region and described immunoglobulin heavy chain variable region have defined the single binding site that is used in conjunction with people HGF together.CDR _L1Comprise the sequence that is selected among SEQ ID NO.8 (1A3), SEQ ID NO.18 (2B8), SEQ ID NO.28 (2F8), SEQ ID NO.38 (3B6), SEQ ID NO.48 (3D11), SEQ ID NO.58 (1D3), SEQ ID NO.68 (1F3) and the SEQ ID NO.78 (3A12).CDR _L2Comprise the sequence that is selected among SEQ ID NO.9 (1A3), SEQ ID NO.19 (2B8), SEQ ID NO.29 (2F8), SEQ ID NO.39 (3B6), SEQ ID NO.49 (3D11), SEQ ID NO.59 (1D3), SEQ ID NO.69 (1F3), SEQ ID NO.79 (3A12) and the SEQ ID NO.206 (LRMR2B8LC).CDR _L3Comprise the sequence that is selected among SEQ ID NO.10 (1A3), SEQ ID NO.20 (2B8), SEQ ID NO.30 (2F8), SEQ ID NO.40 (3B6), SEQ ID NO.50 (3D11), SEQ ID NO.60 (1D3), SEQ ID NO.70 (1F3) and the SEQ ID NO.80 (3A12).In whole specification sheets and claims, by after the represented sequence of concrete SEQ ID NO. for being drawn together the antibody in bracket, this antibody is the source of concrete sequence.For example, SEQ ID NO.8 (1A3) expression, the sequence of SEQ ID NO.8 is based on the sequence among the antibody 1A3.

In one embodiment, described conjugated protein comprises immunoglobulin light chain variable region, and this immunoglobulin light chain variable region comprises CDR _L1, CDR _L2And CDR _L3, CDR wherein _L1Comprise the sequence shown in the SEQID NO.8 (1A3), CDR _L2Comprise the sequence shown in the SEQ ID NO.9 (1A3), and CDR _L3Comprise the sequence shown in the SEQ ID NO.10 (1A3).

In another embodiment, described conjugated protein comprises immunoglobulin light chain variable region, and this immunoglobulin light chain variable region comprises CDR _L1, CDR _L2And CDR _L3, CDR wherein _L1Comprise the sequence shown in the SEQID NO.18 (2B8), CDR _L2Comprise the sequence shown in SEQ ID NO.19 (2B8) or the SEQID NO.206 (LRMR2B8LC), and CDR _L3Comprise the sequence shown in the SEQ ID NO.20 (2B8).

In another embodiment, described conjugated protein comprises immunoglobulin light chain variable region, and this immunoglobulin light chain variable region comprises CDR _L1, CDR _L2And CDR _L3, CDR wherein _L1Comprise the sequence shown in the SEQID NO.28 (2F8), CDR _L2Comprise the sequence shown in the SEQ ID NO.29 (2F8), and CDR _L3Comprise the sequence shown in the SEQ ID NO.30 (2F8).

In another embodiment, described conjugated protein comprises immunoglobulin light chain variable region, and this immunoglobulin light chain variable region comprises CDR _L1, CDR _L2And CDR _L3, CDR wherein _L1Comprise the sequence shown in the SEQID NO.38 (3B6), CDR _L2Comprise the sequence shown in the SEQ ID NO.39 (3B6), and CDR _L3Comprise the sequence shown in the SEQ ID NO.40 (3B6).

In another embodiment, described conjugated protein comprises immunoglobulin light chain variable region, and this immunoglobulin light chain variable region comprises CDR _L1, CDR _L2And CDR _L3, CDR wherein _L1Comprise the sequence shown in the SEQID NO.48 (3D11), CDR _L2Comprise the sequence shown in the SEQ ID NO.49 (3D11), and CDR _L3Comprise the sequence shown in the SEQ ID NO.50 (3D11).

In another embodiment, described conjugated protein comprises immunoglobulin light chain variable region, and this immunoglobulin light chain variable region comprises CDR _L1, CDR _L2And CDR _L3, CDR wherein _L1Comprise the sequence shown in the SEQID NO.58 (1D3), CDR _L2Comprise the sequence shown in the SEQ ID NO.59 (1D3), and CDR _L3Comprise the sequence shown in the SEQ ID NO.60 (1D3).

In another embodiment, described conjugated protein comprises immunoglobulin light chain variable region, and this immunoglobulin light chain variable region comprises CDR _L1, CDR _L2And CDR _L3, CDR wherein _L1Comprise the sequence shown in the SEQID NO.68 (1F3), CDR _L2Comprise the sequence shown in the SEQ ID NO.69 (1F3), and CDR _L3Comprise the sequence shown in the SEQ ID NO.70 (1F3).

In another embodiment, described conjugated protein comprises immunoglobulin light chain variable region, and this immunoglobulin light chain variable region comprises CDR _L1, CDR _L2And CDR _L3, CDR wherein _L1Comprise the sequence shown in the SEQID NO.78 (3A12), CDR _L2Comprise the sequence shown in the SEQ ID NO.79 (3A12), and CDR _L3Comprise the sequence shown in the SEQ ID NO.80 (3A12).

In each embodiment in above-described embodiment, preferably with described CDR _L1, CDR _L2And CDR _L3Sequence is inserted between people or the humanized IgF R.Should be appreciated that described conjugated protein can be complete antibody, its Fab or biosynthetic antibody sites.

On the other hand, the invention provides a kind of isolating conjugated protein in conjunction with people HGF.This conjugated protein comprises: (a) comprise structure C DR _H1-CDR _H2-CDR _H3Immunoglobulin heavy chain variable region; (b) immunoglobulin light chain variable region, wherein said immunoglobulin heavy chain variable region and described immunoglobulin light chain variable region have defined the single binding site that is used in conjunction with people HGF together.CDR _H1Comprise the sequence that is selected among SEQ ID NO.5 (1A3), SEQ ID NO.15 (2B8), SEQ ID NO.25 (2F8), SEQ ID NO.35 (3B6), SEQ ID NO.45 (3D11), SEQ ID NO.55 (1D3), SEQ ID NO.65 (1F3) and the SEQ ID NO.75 (3A12); CDR _H2Comprise and be selected from SEQ ID NO.6 (1A3), SEQ ID NO.16 (2B8), SEQ ID NO.26 (2F8), SEQ ID NO.36 (3B6), SEQ ID NO.46 (3D11), SEQ ID NO.56 (1D3), SEQ ID NO.66 (1F3), SEQ ID NO.76 (3A12), SEQ ID NO.202 (Hu2B8 Hvlf.1), the sequence among SEQ ID NO.203 (Hu2B8 Hv5a.1 or Hu2B8 Hv5-51.1), SEQ ID NO.204 (LR2B8HC) and the SEQ ID NO.205 (LRMR2B8HC); And CDR _H3Comprise the sequence that is selected among SEQ ID NO.7 (1A3), SEQID NO.17 (2B8), SEQ ID NO.27 (2F8), SEQ ID NO.37 (3B6), SEQ IDNO.47 (3D11), SEQ ID NO.57 (1D3), SEQ ID NO.67 (1F3) and the SEQ IDNO.77 (3A12).

In one embodiment, described conjugated protein comprises immunoglobulin heavy chain variable region, and this immunoglobulin heavy chain variable region comprises CDR _H1, CDR _H2And CDR _H3, CDR wherein _H1Comprise the sequence shown in the SEQID NO.5 (1A3); CDR _H2Comprise the sequence shown in the SEQ ID NO.6 (1A3); And CDR _H3Comprise the sequence shown in the SEQ ID NO.7 (1A3).

In another embodiment, described conjugated protein comprises immunoglobulin heavy chain variable region, and this immunoglobulin heavy chain variable region comprises CDR _H1, CDR _H2And CDR _H3, CDR wherein _H1Comprise the sequence shown in the SEQID NO 15 (2B8); CDR _H2Comprise the sequence shown in SEQ ID NO.16 (2B8), SEQ IDNO.202 (Hu2B8 Hv1f.1), SEQ ID NO.203 (Hu2B8 Hv5a.1 or Hu2B8Hv5-51.1), SEQ ID NO.204 (LR2B8HC) or the SEQ ID NO.205 (LRMR2B8HC); And CDR _H3Comprise the sequence shown in the SEQ ID NO.17 (2B8).

In another embodiment, described conjugated protein comprises immunoglobulin heavy chain variable region, and this immunoglobulin heavy chain variable region comprises CDR _H1, CDR _H2And CDR _H3, CDR wherein _H1Comprise the sequence shown in the SEQID NO.25 (2F8); CDR _H2Comprise the sequence shown in the SEQ ID NO.26 (2F8); And CDR _H3Comprise the sequence shown in the SEQ ID NO.27 (2F8).

In another embodiment, described conjugated protein comprises immunoglobulin heavy chain variable region, and this immunoglobulin heavy chain variable region comprises CDR _H1, CDR _H2And CDR _H3, CDR wherein _H1Comprise the sequence shown in the SEQID NO.35 (3B6); CDR _H2Comprise the sequence shown in the SEQ ID NO.36 (3B6); And CDR _H3Comprise the sequence shown in the SEQ ID NO.37 (3B6).

In another embodiment, described conjugated protein comprises immunoglobulin heavy chain variable region, and this immunoglobulin heavy chain variable region comprises CDR _H1, CDR _H2And CDR _H3, CDR wherein _H1Comprise the sequence shown in the SEQID NO.45 (3D11); CDR _H2Comprise the sequence shown in the SEQ ID NO.46 (3D11); And CDR _H3Comprise the sequence shown in the SEQ ID NO.47 (3D11).

In another embodiment, described conjugated protein comprises immunoglobulin heavy chain variable region, and this immunoglobulin heavy chain variable region comprises CDR _H1, CDR _H2And CDR _H3, CDR wherein _H1Comprise the sequence shown in the SEQID NO.55 (1D3); CDR _H2Comprise the sequence shown in the SEQ ID NO.56 (1D3); And CDR _H3Comprise the sequence shown in the SEQ ID NO.57 (1D3).

In another embodiment, described conjugated protein comprises immunoglobulin heavy chain variable region, and this immunoglobulin heavy chain variable region comprises CDR _H1, CDR _H2And CDR _H3, CDR wherein _H1Comprise the sequence shown in the SEQID NO.65 (1F3); CDR _H2Comprise the sequence shown in the SEQ ID NO.66 (1F3); And CDR _H3Comprise the sequence shown in the SEQ ID NO.67 (1F3).

In another embodiment, described conjugated protein comprises immunoglobulin heavy chain variable region, and this immunoglobulin heavy chain variable region comprises CDR _H1, CDR _H2And CDR _H3, CDR wherein _H1Comprise the sequence shown in the SEQID NO.75 (3A12); CDR _H2Comprise the sequence shown in the SEQ ID NO.76 (3A12); And CDR _H3Comprise the sequence shown in the SEQ ID NO.77 (3A12).

In each embodiment in above-described embodiment, preferably with described CDR _H1, CDR _H2And CDR _H3Sequence is inserted between people or the humanized IgF R.Should be appreciated that described conjugated protein can be complete antibody, its Fab or biosynthetic antibody sites.

On the other hand, the invention provides a kind of conjugated protein in conjunction with people HGF.This conjugated protein comprises immunoglobulin heavy chain variable region and immunoglobulin light chain variable region, described immunoglobulin heavy chain variable region is selected from: the residue 20-141 of SEQ ID NO.2 (1A3), the residue 20-137 of SEQID NO.12 (2B8), the residue 20-137 of SEQ ID NO.22 (2F8), the residue 20-139 of SEQ ID NO.32 (3B6), the residue 20-132 of SEQ ID NO.42 (3D11), the residue 20-141 of SEQ ID NO.52 (1D3), the residue 20-141 of the residue 20-141 of SEQ ID NO.62 (1F3) and SEQ ID NO.72 (3A12), described immunoglobulin light chain variable region is selected from: the residue 21-127 of SEQ ID NO.4 (1A3), the residue 21-127 of SEQ IDNO.14 (2B8), the residue 20-131 of SEQ ID NO.24 (2F8), the residue 23-129 of SEQ ID NO.34 (3B6), the residue 23-128 of SEQ ID NO.44 (3D11), the residue 21-127 of SEQ ID NO.54 (1D3), the residue 21-127 of SEQ ID NO.64 (1F3), and the residue 21-127 of SEQ ID NO.74 (3A12).

In another embodiment, described conjugated protein comprises immunoglobulin heavy chain variable region and immunoglobulin light chain variable region, described immunoglobulin heavy chain variable region comprises the aminoacid sequence of the residue 20-141 of SEQ IDNO.2 (1A3), and described immunoglobulin light chain variable region comprises the aminoacid sequence of the residue 21-127 of SEQ ID NO.4 (1A3).

In another embodiment, described conjugated protein comprises immunoglobulin heavy chain variable region and immunoglobulin light chain variable region, described immunoglobulin heavy chain variable region comprises the aminoacid sequence of the residue 20-137 of SEQ IDNO.12 (2B8), and described immunoglobulin light chain variable region comprises the aminoacid sequence of the residue 21-127 of SEQ ID NO.14 (2B8).

In another embodiment, described conjugated protein comprises immunoglobulin heavy chain variable region and immunoglobulin light chain variable region, described immunoglobulin heavy chain variable region comprises the aminoacid sequence of the residue 20-137 of SEQ IDNO.22 (2F8), and described immunoglobulin light chain variable region comprises the aminoacid sequence of the residue 20-131 of SEQ ID NO.24 (2F8).

In another embodiment, described conjugated protein comprises immunoglobulin heavy chain variable region and immunoglobulin light chain variable region, described immunoglobulin heavy chain variable region comprises the aminoacid sequence of the residue 20-139 of SEQ IDNO.32 (3B6), and described immunoglobulin light chain variable region comprises the aminoacid sequence of the residue 23-129 of SEQ ID NO.34 (3B6).

In another embodiment, described conjugated protein comprises immunoglobulin heavy chain variable region and immunoglobulin light chain variable region, described immunoglobulin heavy chain variable region comprises the aminoacid sequence of the residue 20-132 of SEQ IDNO.42 (3D11), and described immunoglobulin light chain variable region comprises the aminoacid sequence of the residue 23-128 of SEQ ID NO.44 (3D11).

In another embodiment, described conjugated protein comprises immunoglobulin heavy chain variable region and immunoglobulin light chain variable region, described immunoglobulin heavy chain variable region comprises the aminoacid sequence of the residue 20-141 of SEQ IDNO.52 (1D3), and described immunoglobulin light chain variable region comprises the aminoacid sequence of the residue 21-127 of SEQ ID NO.54 (1D3).

In another embodiment, described conjugated protein comprises immunoglobulin heavy chain variable region and immunoglobulin light chain variable region, described immunoglobulin heavy chain variable region comprises the aminoacid sequence of the residue 20-141 of SEQ IDNO.62 (1F3), and described immunoglobulin light chain variable region comprises the aminoacid sequence of the residue 21-127 of SEQ ID NO.64 (1F3).

In another embodiment, described conjugated protein comprises immunoglobulin heavy chain variable region and immunoglobulin light chain variable region, described immunoglobulin heavy chain variable region comprises the aminoacid sequence of the residue 20-141 of SEQ IDNO.72 (3A12), and described immunoglobulin light chain variable region comprises the aminoacid sequence of the residue 21-127 of SEQ ID NO.74 (3A12).

In each embodiment in above-described embodiment, described conjugated protein is complete antibody, its Fab or biosynthetic antibody sites.

On the other hand, the invention provides a kind of isolating conjugated protein in conjunction with people HGF.This conjugated protein comprises: (i) immunoglobulin light chain variable region, and it is selected from: SEQ ID NO.173 (Hu2B8 Kv1-39.1 variable region of light chain), SEQ ID NO.179 (Hu2B8 Kv3-15.1 variable region of light chain), SEQ ID NO.193 (LR2B8LC variable region of light chain) and SEQ ID NO.199 (LRMR2B8LC variable region of light chain); And (ii) immunoglobulin heavy chain variable region, it is selected from: SEQ ID NO.159 (Hu2B8 Hvlf.1 variable region of heavy chain), SEQ ID NO.165 (Hu2B8 Hv5a.1 variable region of heavy chain), SEQ ID NO.169 (Hu2B8 Hv5-51.1 variable region of heavy chain), SEQ ID NO.183 (LR2B8HC variable region of heavy chain) and SEQ ID NO.189 (LRMR2B8LC variable region of light chain).Described conjugated protein can be complete antibody, its Fab or biosynthetic antibody sites.

On the other hand, the invention provides a kind of isolating conjugated protein in conjunction with people HGF.This conjugated protein comprises: (i) light chain immunoglobulin, and it is selected from: SEQ ID NO.177 (Hu2B8 Kv1-39.1+Kappa constant region (Km (3) allotype (allelotrope 2)), SEQ ID NO.181 (Hu2B8 Kv3-15.1+Kappa constant region (Km (3) allotype (allelotrope 2)), SEQ ID NO.197 (LR2B8LC+Kappa constant region (Km (3) allotype (allelotrope 1)) and SEQ ID NO.201 (LRMR2B8LC+Kappa constant region (Km (3) allotype (allelotrope 1)); And (ii) heavy chain immunoglobulin, it is selected from: ((Glm (17 for Hu2B8 Hv1f.1+IgG1 constant region for SEQ IDNO.163,1) allotype)), ((Glm (17 for Hu2B8 Hv5a.1+IgG1 constant region for SEQID NO.167,1) allotype)), SEQ ID NO.171 (Hu2B8 Hv5-51.1+IgG1 constant region (Glm (17,1) allotype)), SEQ ID NO.187 (LR2B8HC+IgG1 constant region (Glm (3) allotype) (allelotrope 1)) and SEQ ID NO.191 (LRMR2B8HC+IgG1 constant region (Glm (3) allotype) (allelotrope 1)).Described conjugated protein can be complete antibody, its Fab or biosynthetic antibody sites.

On the other hand, the invention provides a kind of isolating conjugated protein in conjunction with reductive people HGF.This conjugated protein comprises: (i) comprise the immunoglobulin light chain variable region of 3 CDR, and the immunoglobulin heavy chain variable region that (ii) comprises 3 CDR.CDR is inserted between the FR usually.The CDR of light chain immunoglobulin and heavy chain immunoglobulin has defined together in conjunction with the binding site of reductive people HGF (for example α chain of reductive HGF).Reductive HGF is meant to use is enough to reduce the HGF that the reductive agent (for example dithiothreitol (DTT) (DTT), 2 mercapto ethanol or gsh) of the amount of disulfide linkage is handled between α chain and the β chain.Exemplary concentration comprises, for example the DTT of 100mM and 5% 2 mercapto ethanol.

In certain embodiments, described conjugated protein comprises immunoglobulin light chain variable region, and this immunoglobulin light chain variable region comprises and is selected from CDR _L1, CDR _L2And CDR _L3In at least 1 CDR.Alternatively, described conjugated protein comprises 2 CDR, for example CDR _L1And CDR _L2, or CDR _L1And CDR _L3, or CDR _L1And CDR _L3Alternatively, described conjugated protein comprises 3 all CDR, that is, and and CDR _L1, CDR _L2And CDR _L3CDR _L1Comprise aminoacid sequence X ₁X ₂SerX ₄X ₅X ₆X ₇X ₈X ₉X ₁₀X ₁₁X ₁₂X ₁₃X ₁₄X ₁₅, amino acid X wherein ₁Be Arg or Lys, X ₂Be Ala or Thr, X ₄Be Glu or Gln, X ₅Be Asn, Ser or Asp, X ₆Be Ile or Val, X ₇Be Tyr, Asp or Lys, X ₈Be peptide bond or Tyr, X ₉Be peptide bond or Asp, X ₁₀Be peptide bond or Gly, X ₁₁Be peptide bond or Asn, X ₁₂Be peptide bond or Ser, X ₁₃Be Asn or Tyr, X ₁₄Be Ile or Leu, X ₁₅Be Ala, Asn or Ser.CDR _L2Comprise aminoacid sequence X ₁₆X ₁₇X ₁₈X ₁₉LeuX ₂₁X ₂₂, amino acid X wherein ₁₆Be Ala, Asp, Val or Arg, X ₁₇Be Ala or Val, X ₁₈Be Asn, Ser or Thr, X ₁₉Be Arg, Asn or His, X ₂₁Be Ala, Glu, Val or Pro, X ₂₂Be Asp or Ser.CDRL ₃Comprise aminoacid sequence X ₂₃X ₂₄X ₂₅X ₂₆X ₂₇X ₂₈ProX ₃₀Thr, wherein amino acid X ₂₃Be Leu or Gln, X ₂₄Be His or Gln, X ₂₅Be Phe, Ser or Tyr, X ₂₆Be Asp, Ile or Trp, X ₂₇Be Gly or Gln, X ₂₈Be Asp, Phe or Thr, X ₃₀Be Phe, Pro or Tyr.

In another embodiment, described conjugated protein comprises immunoglobulin heavy chain variable region, and this immunoglobulin heavy chain variable region comprises and is selected from CDR _H1, CDR _H2And CDR _H3In at least 1 CDR.Alternatively, described conjugated protein comprises 2 CDR, for example CDR _H1And CDR _H2, or CDR _H1And CDR _H3, or CDR _H1And CDR _H3Alternatively, described conjugated protein comprises 3 all CDR, that is, and and CDR _H1, CDR _H2And CDR _H3CDR _H1Comprise aminoacid sequence X ₁TyrX ₃X ₄X ₅, amino acid X wherein ₁Be Asp, Asn, Ser or Thr, X ₃Be Phe, Trp or Tyr, X ₄Be Ile or Met, X ₅Be Asn, His or Ser.CDR _H2Comprise aminoacid sequence X ₆IleX ₈X ₉GlyX ₁₁GlyX ₁₃X ₁₄X ₁₅TyrX ₁₇X ₁₈X ₁₉X ₂₀LysX ₂₂, amino acid X wherein ₆Be Lys, Gln or Tyr, X ₈Be Gly, Ser or Tyr, X ₉Be Pro or Ser, X ₁₁Be Asp, Gly or Ser, X ₁₃Be Asp or Ser, X ₁₄Be Ser or Thr, X ₁₅Be Asn or Tyr, X ₁₇Be Asn or Pro, X ₁₈Be Ala, Asp, Gly or Glu, X ₁₉Be Asn, Met or Ser, X ₂₀Be Phe or Val, X ₂₂Be Asp or Gly.CDR _H3Comprise aminoacid sequence X ₂₃X ₂₄X ₂₅X ₂₆X ₂₇X ₂₈X ₂₉X ₃₀X ₃₁X ₃₂X ₃₃AspTyr, wherein amino acid X ₂₃Be Arg or Gln, X ₂₄Be Gly or Leu, X ₂₅Be Asp, Gly or peptide bond, X ₂₆Be Gly or peptide bond, X ₂₇Be peptide bond or Tyr, X ₂₈Be Leu, peptide bond or Tyr, X ₂₉Be Gly, Arg or Leu, X ₃₀Be Asp, Gly or Glu, X ₃₁Be Tyr, Arg or Asn, X ₃₂Be Ala, Gly or Tyr, X ₃₃Be Met or Phe.

Should be appreciated that described conjugated protein comprises aforesaid heavy chain immunoglobulin and light chain immunoglobulin or its fragment.In addition, should be appreciated that described conjugated protein is complete antibody or its Fab or biosynthetic antibody sites.

In certain embodiments, described conjugated protein comprises immunoglobulin light chain variable region, and this immunoglobulin light chain variable region comprises: (i) CDRL ₁, it has the sequence that is selected from SEQ ID NO.8 (1A3), SEQ ID NO.28 (2F8), SEQ ID NO.38 (3B6), SEQ ID NO.58 (1D3) and SEQ ID NO.68 (1F3), (ii) CDR _L2, it has the sequence that is selected from SEQ IDNO.9 (1A3), SEQ ID NO.29 (2F8), SEQ ID NO.39 (3B6), SEQ IDNO.59 (1D3) and SEQ ID NO.69 (1F3), and (iii) CDR _L3, it has the sequence that is selected from SEQ ID NO.10 (1A3), SEQ ID NO.30 (2F8), SEQ ID NO.40 (3B6), SEQ ID NO.60 (1D3) and SEQ ID NO.70 (1F3).The CDR sequence can be inserted between people or the humanized FR.In other embodiments, described conjugated protein comprises immunoglobulin light chain variable region, and this immunoglobulin light chain variable region comprises the aminoacid sequence among the residue 21-127 of the residue 21-127 of residue 23-129, SEQ ID NO.54 (1D3) of residue 20-131, SEQ ID NO.34 (3B6) of the residue 21-127, the SEQ ID NO.24 (2F8) that are selected from SEQ ID NO.4 (1A3) and SEQ ID NO.64 (1F3).

In certain embodiments, described conjugated protein comprises immunoglobulin heavy chain variable region, and this immunoglobulin heavy chain variable region comprises: (i) CDR _H1, it has the sequence that is selected from SEQ ID NO.5 (1A3), SEQ ID NO.25 (2F8), SEQ ID NO.35 (3B6), SEQ ID NO.55 (1D3) and SEQ ID NO.65 (1F3), (ii) CDR _H2, it has the sequence that is selected from SEQ IDNO.6 (1A3), SEQ ID NO.26 (2F8), SEQ ID NO.36 (3B6), SEQ IDNO.56 (1D3) and SEQ ID NO.66 (1F3), and (iii) CDR _H3, it has the sequence that is selected from SEQ ID NO.7 (1A3), SEQ ID NO.27 (2F8), SEQ ID NO.37 (3B6), SEQ ID NO.57 (1D3) and SEQ ID NO.67 (1F3).The CDR sequence can be inserted between people or the humanized FR.In another embodiment, described immunoglobulin heavy chain variable region comprises the aminoacid sequence among the residue 20-141 of the residue 20-141 of residue 20-139, SEQ ID NO.52 (1D3) of residue 20-137, SEQ ID NO.32 (3B6) of the residue 20-141, the SEQID NO.22. (2F8) that are selected from SEQ ID NO.2 (1A3) and SEQ ID NO.62 (1F3).

On the other hand, the invention provides a kind of isolating conjugated protein, it is in conjunction with people HGF, and comprises immunoglobulin light chain variable region and immunoglobulin heavy chain variable region.Described isolating conjugated protein with at least a kind with reference to antibody competition combine HGF, wherein saidly be selected from: the antibody of immunoglobulin heavy chain variable region that (i) has the residue 20-137 of the immunoglobulin light chain variable region of residue 20-131 of SEQ ID NO.24 (2F8) and SEQ ID NO.22 (2F8) with reference to antibody, (ii) have the antibody of immunoglobulin heavy chain variable region of the residue 20-139 of the immunoglobulin light chain variable region of residue 23-129 of SEQ ID NO.34 (3B6) and SEQ ID NO.32 (3B6), and the antibody of immunoglobulin heavy chain variable region that (iii) has the residue 20-132 of the immunoglobulin light chain variable region of residue 23-128 of SEQ ID NO.44 (3D11) and SEQ ID NO.42 (3D11).Under certain conditions, described conjugated protein is in conjunction with the HGF phase epi-position identical with one of reference antibody.

Should be appreciated that every kind of conjugated protein above being discussed can be complete antibody, for example monoclonal antibody.Alternatively, described conjugated protein can be antigen-binding fragments of antibodies, perhaps can be biosynthetic antibody combining site.Antibody fragment comprises Fab, Fab ', (Fab ') ₂Or Fv fragment.The technology that is used to prepare this antibody fragment is known to those skilled in the art.Multiple biosynthetic antibody combining site is known in the art, and it comprises, for example single Fv or sFv molecule, and for example the United States Patent (USP) sequence number 5,476, and 786 is described.Other biological synthetic antibody combining site comprises the conjugated protein of dual specific or bi-functional, the antibody of dual specific or bi-functional for example, and it is in conjunction with at least two kinds of different antigenic antibody or antibody fragments.For example, the dual specific conjugated protein can be in conjunction with HGF (for example people HGF) and other purpose antigens.The method that is used to prepare bi-specific antibody is well known in the art, and it comprises, for example by merging hybridoma or by connecting Fab ' fragment.For example referring to people such as Songsivilai (1990) CLIN.EXP.IMMUNOL.79:315-325; People such as Kostelny (1992) J.IMMUNOL.148:1547-1553.

Conjugated protein of the present invention can be replaced by arginine with the halfcystine that is included in 561 positions or the glycine in 555 positions is combined by the hHGF that L-glutamic acid replaces.

In another aspect, this law provides a kind of isolating conjugated protein, itself and people HGF bonded k _dBe 4.0 x 10 ^-5s ^-1Or lower, 3.0 x 10 ^-5s ^-1Or lower or 2.0 x 10 ^-5s ^-1Or it is lower.Described isolating protein and people HGF bonded k _dBe 5.0 x 10 ^-5s ^-1To 0.5 x 10 ^-5s ^-1, 4.0 x 10 ^-5s ^-1To 1.0 x 10 ^-5s ^-1, or 3.0 x 10 ^-5s ^-1To 1.5 x 10 ^-5s ^-1In another aspect, the invention provides a kind of isolating conjugated protein, itself and people HGF bonded K _DBe 100pM or lower, 20pM or lower, 10pM or lower or 5pM or lower.Described isolating protein and people HGF bonded K _DBe 100pM to 5pM, 20pM to 5pM, 15pM to 10pM, 20pM to 10pM or 15pM to 5pM.Unless otherwise mentioned, measure K by method and condition described in the embodiment 6 _DValue.

On the other hand, this law provides a kind of isolating conjugated protein in conjunction with people HGF, and wherein said antibody is at 37 ℃ of following and people HGF bonded K _DCompare under 25 ℃ and people HGF bonded K _DLow.Described conjugated protein is randomly under 37 ℃, to be lower than the K of 5pM _DCombine with people HGF.

In other respects with embodiment in, described conjugated protein suppresses combining of hHGF and c-Met.For example, when adopting the scheme described in the embodiment 7 (a) to measure, the IC of described conjugated protein ₅₀(maximum suppress 50% o'clock concentration) is at least about 4.0,4.5,5.0,5.5,6.0,6.5 and 7.0nM.In other the embodiment, by using the described method of embodiment 7 (b), described conjugated protein neutralization is incorporated into the HGF BrdU in the 4MBr-5 cell (ATCC, Catalog No.CCL208) at some.

When using the scheme described in the embodiment 7 (b) to analyze, the IC of described conjugated protein ₅₀For 50nM or lower, be preferably 45,40,35,30,25,20,15,10,5,1,0.5nM or lower.In other the embodiment, by using the analysis described in the embodiment 9, described conjugated protein can be used for suppressing the c-Met phosphorylation that HGF stimulates in the PC-3 cell (ATCC, Manassus, VACatalog No.CRL-1435) at some.Use the analysis described in the embodiment 9, described conjugated protein is with 2nM or lower IC ₅₀(referring to table 8) suppresses the c-Met phosphorylation that HGF stimulates (1.25nM) in the PC-3 cell.

The preparation of II-conjugated protein

Can use methods known in the art to prepare conjugated protein of the present invention in many ways.For example, by using the commercially available sequence information chemical synthesis coding variable region of light chain that synthetic instrument and this paper provided and the dna molecular of variable region of heavy chain.This synthetic dna molecular can be connected to other suitable nucleotide sequences, for example comprise the constant region and the expression control sequenc of encoding sequence, thereby prepare the conventional genetic expression construct of the required conjugated protein of coding.The method for preparing defined gene construct is the ordinary skill in the art.Alternatively, can use the synthetic nucleic acid probe by routine hybridization technique or round pcr by cloning the sequence that this paper provides in the hybridoma, the sequence of wherein said nucleic acid probe is based on sequence information that this paper provided or about the sequence information of the prior art of the gene of the heavy chain of encoding murine antibody in the hybridoma and light chain.The preparation of this probe and use are the ordinary skills of this area.

The nucleic acid of conjugated protein that can coding is required is introduced (connection) in expression vector, and can this expression vector be incorporated in the host cell by the rotaring dyeing technology or the transformation technology of standard known in the art.Exemplary host cell comprises, for example Bacillus coli cells, Chinese hamster ovary (CHO) cell, HeLa cell, young hamster kidney (BHK) cell, monkey-kidney cells (COS), human liver cancer cell (for example Hep G2) and myeloma cell, these cells can not produce immunoglobulin (Ig) in addition.Can under the condition that allows host cell expression goal gene (for example expressing the gene of immunoglobulin light chain variable region or variable region of heavy chain), cultivate host cell through transfection.Use the expression product of technology results gained known in the art.

Concrete expression and purification condition change according to employed expression system.For example, if at the expression in escherichia coli gene, at first will be in expression vector with this gene clone.Realize this process by the downstream that engineered gene is placed suitable bacterium promotor (for example Trp or Tac) and signal sequence (for example sequence of the fragment B of coded protein A (FB)).The fusion rotein that the quilt of gained is expressed is accumulated in the refractile body in the tenuigenin of cell or the inclusion body usually, and can gather in the crops behind smudge cells by French press (French press) or supersonic method.Then, the dissolving refractile body, and use method refolding and the shearing expressed protein of having set up at other recombinant proteins.

If in eukaryotic host cell (for example myeloma cell or Chinese hamster ovary celI), express cydorge gene, at first this project gene be inserted in the expression vector that comprises suitable eukaryotic promoter, secretion signal, immunoglobulin (Ig) enhanser and a plurality of introns.This expression vector randomly comprises the sequence of all constant regions of coding or part constant region, thereby heavy chain or light chain whole or part are expressed.Can use the transfection method set up with described gene construct transfection in myeloma cell or Chinese hamster ovary celI.This transfectional cell can be expressed V _LOr V _HFragment, V _L-V _HHeterodimer, V _H-V _LOr V _L-V _HSingle chain polypeptide, complete heavy chain immunoglobulin or light chain or its part, wherein each can link to each other with the protein domain that comprises another function (for example cytotoxicity).

The modification of III-conjugated protein

Should be appreciated that, according to the purpose purposes of described conjugated protein, can modify, thereby optimize its performance this conjugated protein.For example, when described conjugated protein is used as therapeutical agent, can modify this conjugated protein, to reduce its immunogenicity in the purpose recipient.Alternatively or additionally, described conjugated protein and another protein or peptide (for example somatomedin, cytokine or cytotoxin) can be merged or coupling.Can use the gene manipulation techniques of routine known in the art to realize this modification.

The antigenic multiple technologies that are used to reduce antibody and antibody fragment are as known in the art.These technology can be used for reducing or eliminating the antigenicity of conjugated protein of the present invention.For example,, preferably this conjugated protein is transformed, to reduce its antigenicity in human body when described conjugated protein is applied to man-hour.This process is commonly called the humanization process.Preferably, humanized conjugated protein antigenic avidity is derived with it and come, the non-humanized conjugated protein of primary is identical or basic identical to antigenic avidity.

In a known humanization method, produce chimeric protein, wherein the constant region for immunoglobulin that is derived from second kind of different species (for example people) from the constant region for immunoglobulin of the antibody of a kind of species (for example mouse) replaces.In this embodiment, the antibody of gained is mouse-people's mosaic, and wherein the immunogenicity than the sequence of the mouse of correspondence is little in principle for the immunogenicity of human constant region sequence.This antibody engineering is learned at people (1984) PROC.NAT.ACAD.SCI.81:6851-6855 such as (for example) Morrison; People such as Neuberger (1984) NATURE 312:604-608; And describe to some extent among United States Patent (USP) sequence number 6,893,625 (Robinson), 5,500,362 (Robinson) and 4,816,567 (Cabilly).

In other method (being called CDR transplants), the variable region of light chain CDR of purpose antibody and the CDR of variable region of heavy chain are transplanted in the framework (FR) of another kind of antibody.For example, mouse CDR is transplanted in the people FR sequence.In some embodiments, the light chain of the antibody of anti-HGF and the CDR of variable region of heavy chain are transplanted among people FR or the joint owner FR.In order to form joint owner FR, comparison is from the FR of the aminoacid sequence of a plurality of people's heavy chains or light chain, to determine total aminoacid sequence.CDR is implanted in for example United States Patent (USP) sequence number 7,022,500 (Queen), 6,982,321 (Winter), 6,180,370 (Queen), 6,054,297 (Carter), 5,693,762 (Queen), 5,859,205 (Adair), 5,693,761 (Queen), 5,565,332 (Hoogenboom), 5,585, among 089 (Queen), 5,530,101 (Queen); And people (1986) NATURE 321:522-525 such as Jones; People such as Riechmann (1988) NATURE 332:323-327; People such as Verhoeyen (1988) SCIENCE 239:1534-1536; And describe to some extent among Winter (1998) the FEBS LETT 430:92-94.

In the method that is called as " super humanization ", produce the antibody that people's immunogenicity wherein is reduced or eliminates by the another kind of form of transplanting.In super humanization, based on people CDR with treat the structural similarity of the CDR of humanized mouse antibodies, from the lineup plants the gene of system, select people FR sequence.This method exists, and for example the United States Patent (USP) sequence number 6,881, describes to some extent among people (2002) the J.IMMUNOL 169:1119-1125 such as 557 (Foote) and Tan.

Other reduce immunogenic method and comprise the technology for " changing shape ", " highly chimeric (hyperchimerization) " or " frosting (veneering)/reinvent (resurfacing) " of being called that is used to produce humanized antibody.For example referring to people such as Vaswami (1998) ANNALSOF ALLERGY, ASTHMA ， ﹠amp; IMMUNOL.81:105; People such as Roguska (1996) PROT.ENGINEER 9:895-904; And United States Patent (USP) sequence number 6,072,035 (Hardman).At frosting/reinvent in the method, the amino-acid residue near the surface in the mouse antibodies is replaced by the amino-acid residue of the more frequent appearance in same position place in people's antibody.This antibody is reinvented technology and is existed, and for example the United States Patent (USP) sequence number 5,639, describes to some extent among 641 (Pedersen).

A kind ofly be used for that mouse antibodies is changed into the illustrative methods that is suitable for human medicinal use form and be called as ACTIVMAB ^TM(NY), this technology relates to the carrier based on vaccinia virus of expressing antibodies in mammalian cell to technology for Vaccinex company, Rochester.It is said, will produce the high-caliber combination diversity of heavy chain immunoglobulin and light chain.For example referring to United States Patent (USP) sequence number 6,706,477 (Zauderer), 6,800,442 (Zauderer) and 6,872,518 (Zauderer).

It is by KaloBios Pharmaceuticals company (Palo Alto, CA) technology of commercialization enforcement that another kind is used for mouse antibodies is changed into the illustrative methods that is suitable for the human form of using.This technology relates to uses special somebody " acceptor " storehouse to produce " epi-position is compiled " storehouse that is used to carry out antibody screening

Being used for mouse antibodies is modified into the another kind of illustrative methods that is suitable for human medicinal use form is HUMAN ENGINEERING ^TM(HE ^TM) technology, this technology is to be implemented by the commercialization of XOMA (US) LLC company.For example referring to open sequence number WO 93/11794 of international application and United States Patent (USP) sequence number 5,766,886,5,770,196,5,821,123 and 5,869,619.

Comprise that any suitable method of any aforesaid method may be used to reduce or eliminate people's immunogenicity of purpose conjugated protein.

In addition, it is possible producing complete people's antibody in mouse.In the method, use transgenic mice to prepare people's antibody, the substantive part that the gene that produces mouse antibodies in the described transgenic mice is produced the gene of people's antibody replaces.This mouse has produced the human normal immunoglobulin molecule and has not produced the mouse immuning ball protein molecule.For example referring to people (1997) NATURE GENETICS 15:146-156 such as WO 98/24893 (people such as Jacobovitz) and Mendez.Can use following method to produce the monoclonal antibody of the complete anti-HGF of people.Application target antigen (for example HGF) immunity contains human immunoglobulin gene's transgenic mice.Then, from described mouse, obtain the lymphocyte of this mouse, afterwards itself and marrow class clone are merged, thus preparation immortalization hybridoma cell line.Screening is also selected this hybridoma cell line, thereby identifies the hybridoma cell line that produces the HGF specific antibody.

According to the purpose purposes of conjugated protein of the present invention, this conjugated protein and other molecules are puted together.For example, if described conjugated protein as therapeutical agent, is then puted together this conjugated protein and another reagent (for example mediation or the effector molecule of promotion treatment in addition) subsequently.With regard to described effector is non-situation based on proteinic reagent (for example small-molecule drug, radioactive labels or radiotoxin), and the interior coupling chemical method of body that can use standard so is with this reagent and described conjugated protein chemical coupling.On the other hand, if described effector molecule is protein or peptide (for example enzyme, acceptor, toxin, somatomedin, cytokine or other immunomodulators), can use the interior coupling chemical method of body to make described conjugated protein and effector chemical coupling so, perhaps can make described conjugated protein and effector coupling form fused protein.Can use the technology similar to make up and expressed fusion protein matter to the method discussed among the part II.

The purposes of IV-conjugated protein

Conjugated protein described herein can be used as diagnostic reagent or therapeutical agent.

(1) treatment is used

Owing in the conjugated protein of the present invention and the activity of HGF, use so it can be used for multiple treatment.For example, some conjugated protein of the present invention can be used for prevention or treatment high proliferation disease or disorder (for example cancer of various ways).

Can use described conjugated protein to suppress or reduce the propagation of tumour cell.In the method, described tumour cell is exposed in the conjugated protein of treatment significant quantity, thus the propagation of inhibition or minimizing tumour cell.In certain embodiments, described conjugated protein inhibition tumor cell proliferation is at least 50%, 60%, 70%, 80%, 90%, 95% or 100%.

In certain embodiments, described conjugated protein is used to suppress or reduce the propagation of tumour cell, and wherein said conjugated protein has reduced the binding ability of hHGF and c-Met.In other embodiments, though at described conjugated protein in conjunction with hHGF but when substantially not suppressing the combining of hHGF and c-Met, described conjugated protein is used to suppress or reduce the propagation of tumour cell, shown in the antibody 3B6 in table 5 and 6.

In addition, described conjugated protein can be used for growth of tumor or development in the inhibition or the Mammals that slows down.In the method, to the described conjugated protein of administration significant quantity, thus growth of tumor in the inhibition or the Mammals that slows down.Therefore, described conjugated protein can be used for treatment, for example tumour in the Mammals.Described method comprises the described conjugated protein to administration treatment significant quantity.Described conjugated protein can be used separately or use with another kind of pharmaceutical activity molecular combinations, so that the treatment tumour.

Comprise that conjugated protein of the present invention can be used for treating multiple HGF and replys disorder, comprise that the HGF in for example lung cancer, mammary cancer, colorectal carcinoma, prostate cancer, ovarian cancer, head and neck cancer, ovarian cancer, multiple myeloma, liver cancer, cancer of the stomach, esophagus cancer, kidney, nasopharyngeal carcinoma, carcinoma of the pancreas, mesothelioma, melanoma and the glioblastoma replys tumour cell.

As used herein, " treatment ", " therapy " and " treatment handle " be meant to morbid state in the treatment Mammals (particularly people) treatment handle, but comprise: (a) the preventing disease state takes place in Mammals, particularly tends to take place morbid state this Mammals is not also diagnosed out when having this morbid state; (b) suppress morbid state, that is, stop the development of morbid state; And/or (c) state that palliates a disease, that is, morbid state is disappeared.

In general, the scope of the treatment significant quantity of activeconstituents is extremely about 100mg/kg of about 0.1mg/kg, randomly is extremely about 100mg/kg of about 1mg/kg, randomly is about 1mg/kg to 10mg/kg.Amount of application depends on multiple factor, existence and the kind and the route of administration of vehicle in the relative biological efficacy of the disease for example to be treated or the type of indication and degree, concrete patient's overall health, the conjugated protein of being sent, the formulation of conjugated protein, the formulation.In order to reach required blood levels fast or to organize level, initial application dosage is brought up to beyond the scope of highest level, perhaps make initial application dosage be lower than optimal dose and in therapeutic process, the every day application dosage is increased gradually according to concrete situation.For example, optimize people's dosage in the I phase dose escalation study of design by the routine of 0.5mg/kg to 20mg/kg scope.Medicine frequency is according to changing such as route of administration, dosage with in the factor of the morbid state of treatment.Exemplary medicine frequency be every day 1 time, weekly 1 time and per 2 the week 1 time.Preferred route of administration is parenteral administration, for example intravenous infusion.Preparation based on the medicine of monoclonal antibody is the ordinary skill of this area.In some embodiments of the present invention, with described conjugated protein (for example monoclonal antibody) freeze-drying, and when using, with its reconstruct in buffer saline.

Described conjugated protein can be used separately, perhaps with other medicine activity component combined administration.Described other active ingredients (for example immunomodulator) can be used with described conjugated protein, perhaps can use before or after described conjugated protein.

The preparation that comprises the conjugated protein of therepic use contains the conjugated protein with pharmaceutically useful carrier combinations usually.As used herein, " pharmaceutically useful carrier " is meant buffer reagent, carrier and vehicle, in reliable medical judgment (sound medical judgment) scope, described buffer reagent, carrier and vehicle are applicable to and contact with the humans and animals tissue and do not have too much toxicity, stimulation, transformation reactions or other problems or complication, match with rational benefit/risk ratio.From with preparation the meaning of other component compatibility say that described carrier should be " acceptable ", and be harmless to the recipient.In this regard, pharmaceutically useful carrier be intended to comprise the compatible mutually buffer reagent of any and all and medicament administration mode, solvent, dispersion medium, dressing, etc. blend absorption delay agent etc.It is known in the art being used for these media of pharmaceutically active substance and the use of reagent.

Described preparation can be easily exists with the form of dose unit, and can prepare by any suitable method (comprising known any method in the pharmaceutics field).Pharmaceutical composition of the present invention should be made compatible mutually with the route of administration of its plan.The example of route of administration comprises that parenteral administration or parenteral use outward, and for example intravenously is used, intradermal administration, suction are used, transdermal administration (part), saturating mucosal administration and rectal administration.Can be by known any method in the pharmaceutical field (for example at Remington ' s Pharmaceutical Sciences, method described in the 18th ed. (Mack Publishing Company, 1990)) preparation is used for the solution of oral administration or parenteral administration.

The preparation that is applicable to oral administration is following form: isolating unit, and for example injection, capsule, gelatine capsule, packed (sachet), tablet, tablet or lozenge all contain the conjugated protein of predetermined amount in each isolating unit; Powder or particulate composition; Solution in waterborne liquid or non-aqueous liquid or suspension; Oil-in-water emulsion or water in oil emulsion.

The preparation that is applicable to parenteral administration comprises, for example following composition: sterile diluent for example is used to water, salt brine solution, fixed oil, polyoxyethylene glycol, glycerine, propylene glycol or other synthetic solvents injected; Antiseptic-germicide, for example phenylcarbinol or para methyl paraben; Antioxidant, for example xitix or sodium bisulfite; Sequestrant, for example ethylenediamine tetraacetic acid (EDTA); Buffer reagent, for example acetate, Citrate trianion or phosphoric acid salt; And the reagent that is used for adjustment of tonicity, for example sodium-chlor or glucose.Can use acid or alkali (for example hydrochloric acid or sodium hydroxide) to regulate pH.The prepared product of parenteral administration can be encapsulated in ampoule, disposable syringe or the bottle of the multiple doses made by glass or plastics in.

Substantially, the composition that is applicable to the injection purposes comprises aqueous solution (under water-soluble situation) or dispersion liquid and the powder that is used for preparing immediately aseptic injectable solution or dispersion liquid.For intravenously is used, suitable carriers comprise physiological saline, bacteriostatic water, CremophorELTM (BASF, Parsippany, NJ) or phosphate buffered saline buffer (PBS).It should be stable under the condition of producing and storing, and should be saved to prevent the microbiological contamination such as bacterium and fungi.Described carrier can be to contain, for example the solvent or the dispersion medium of water, ethanol, polyvalent alcohol (for example glycerine, propylene glycol and liquid polyethylene glycol) and their suitable mixture.

Pharmaceutical preparation is preferably aseptic.For example sterilize by the filtration of aseptic filter membrane.Under the situation of the described composition of freeze-drying, can before or after freeze-drying and reconstruct, use the sterilization of aforesaid method.In case be mixed with pharmaceutical composition, it just can be stored in solution, suspension, gel, emulsion, solid or dehydration or freeze dried form of powder, for example in the bottle.

(2) diagnostic is used

No matter when, use the direct mark of detectable part or this conjugated protein of indirect labelling usually with the described conjugated protein external or intravital diagnosis that is used for.Detectable part is any part that can directly or indirectly produce detectable signal.For example, detectable part can be radio isotope, as ³Hydrogen ( ³H), ¹⁴Carbon (14C), ³²Phosphorus ( ³²P), ³⁵Sulphur ( ³⁵S) or ¹²⁵Iodine ( ¹²⁵I); Fluorescent chemicals or chemiluminescence compound are as fluorescein isothiocyanate, rhodamine or fluorescein; Enzyme is as alkaline phosphatase, beta galactosidase enzyme or horseradish peroxidase; Spin probe is as spin labeling; Or coloured particle, as latex or gold grain.Should be appreciated that, use several different methods as known in the art (for example, at people such as Hunter (1962) NATURE 144:945; People such as David (1974) BIOCHEMISTRY 13:1014; People such as Pain (1981) J.IMMUNOL.METH.40:219; And the method described in Nygren (1982) J.HISTOCHEM.ANDCYTOCHEM.30:407) conjugated protein and detectable part are puted together.Detect described mark by for example range estimation or by spectrophotometer or other detectors.

Use described conjugated protein in the art in the available panimmunity analytical method.Exemplary immunoassay comprises, for example sandwich immunoassays method, competitive immunization analytical method, immunohistochemical methods method.

In the sandwich immunoassays method, used two kinds and analyte or purpose antigen bonded antibody, for example, wherein a kind of antibody is fixed on the solid support, and another kind of antibody is free in the solution also with detectable part mark.In the time will containing antigenic sample and be incorporated in this system, this antigen not only with immobilized antibodies but also with the antibodies of mark, thereby on the surface of upholder, formed " sandwich " immunocomplex.Detect compound protein by unconjugated sample composition of flush away and unnecessary traget antibody, and measure the amount of the protein compound traget antibody on the support surface.Alternatively, but by using the 3rd antibody with free antibodies bonded test section mark to detect the antibody that is free in the solution.The detailed summary of the design of immune analysis, theory and scheme can find in many articles, comprises people such as Butt (1984) PRACTICAL IMMUNOLOGY, Marcel Dekker, New York; People such as Harlow, editor (1988) ANTIBODIES, A LABORATORY APPROACH, Cold Spring HarborLaboratory; And people such as Diamandis, editor (1996) IMMUNOASSAY, Academic Press, Boston.

The conjugated protein that the present invention includes mark can be used as intravital developer, and therefore, conjugated protein is with the intravital purpose of developer target recipient particular organization.But the preferred remote test section that is used for the interior video picture of body comprises reflectivity atom technetium ^-99m( ^99mTc), it is about 6 hours gamma emitter for a kind of transformation period.The on-radiation that uses in the video picture partly comprises nitroxide spin labeling and lanthanon and transition metal ion in vivo, and all these materials can both be induced the original position proton relaxation.Except immune video picture, can in the radioimmunotherapy method of standard, use the compound radioactive segment, thereby destroy target cell.The preferred Nucleotide that is used for the high dosage radioimmunotherapy comprises radioactive atom ⁹⁰Yttrium ( ⁹⁰Yt), ¹³¹Iodine ( ¹³¹I) or ¹¹¹Indium ( ¹¹¹In).Can use known coupling technology in the video picture field, use ¹³¹I, ¹¹¹In and ^99mTc comes the mark conjugated protein.Similarly, be used for preparing and the method using developer and catch and handle image is known in the video picture field, therefore no longer describe in detail in this article.Similarly, be used to carry out be well known in the art based on the method for the immunotherapy of antibody.For example referring to United States Patent (USP) sequence number 5,534,254.

In whole specification sheets, have, comprise or when containing concrete composition, comprise that composition also is grouped into by described one-tenth substantially or is grouped into by described one-tenth when composition is described as.Similarly, have, comprise or when comprising concrete treatment step, this method also is made up of described treatment step substantially or is made up of described treatment step when method is described as.Unless otherwise mentioned, be unessential otherwise the order of step or be used to carries out the order of some operation, as long as the present invention is exercisable.

In addition, unless otherwise mentioned, otherwise can implement two or more steps or operation simultaneously.

Embodiment

Following examples have been discussed multiple anti-hHGF MONOCLONAL ANTIBODIES SPECIFIC FOR and characteristic.

The MONOCLONAL ANTIBODIES SPECIFIC FOR of embodiment 1-anti-hHGF

Present embodiment has been described the MONOCLONAL ANTIBODIES SPECIFIC FOR of a certain amount of anti-hHGF.

Repeat immunity (RIMMS) method according to multidigit point, (Portland ME) carries out immunity, fusion and primary dcreening operation by MBS company.Use recombinant human HGF (R﹠amp; D Systems, Minneapolis, MN; Catalog No.294-HGN-025) 5 AJ mouse of immunity and 5 Balb/c mouse.Being chosen in enzyme linked immunological absorption detects its serum in (ELISA) and demonstrates active 2 mouse of the highest anti-HGF and be used for subsequently fusion.Collect from the spleen and the lymphoglandula that are fit to mouse.Collect the B cell then, and itself and myeloma cell line are merged.On one or more flat boards, fusion product is carried out serial dilution, until cloning property near one-tenth.By ELISA, just the supernatant liquor from the fusions of gained is screened in conjunction with hHGF.Be accredited as the supernatant liquor that comprises at the antibody of HGF, further characterize by function test in the body as described in the following Examples.Select one group of hybridoma, and to this hybridoma subclone and amplification.Then, by under standard conditions, the affinitive layer purification monoclonal antibody on the ProteinA/G resin.

The sequential analysis of the monoclonal antibody of embodiment 2-anti-hHGF

Present embodiment has been described the isotype and the sequential analysis of the monoclonal antibody of the anti-hHGF of preparation among the embodiment 1.

The mensuration of the mouse monoclonal antibody isotype of a.HGF

Use IsoStrip Mouse Monoclonal Antibody Isotyping test kit (Roche Applied Science), measure the light chain kind of every kind of monoclonal antibody and the isotype of heavy chain according to the specification sheets that manufacturers provides.

All antibody all is detected and comprises Kappa light chain immunoglobulin and IgG1 heavy chain immunoglobulin.

B. the encode survey of nucleotide sequence of immunoglobulin heavy chain variable region and variable region of light chain Fixed

Use RNeasy Miniprep test kit (Qiagen Venlo, The Netherlands), the specification sheets that provides according to manufacturers extracts total RNA from every kind of monoclonal hybridoma system.Use BD SMART ^TMRACE cDNA Amplification test kit (Clontech), the specification sheets that provides according to manufacturers, use Oligonucleolide primers BD SMART II A (5 ' aagcagtggtatcaacgcagagtacgcggg 3 ') (SEQ ID NO.85) and 5 '-RACECDS primer (5 ' tttttttttttttttttttttttttvn 3 ', wherein v=a, g or c and n=a, g, c or t) (SEQ ID NO.86) generate the first chain cDNA of total length, to carry out 5 ' RACE (the terminal rapid amplifying of cDNA).

Use Expand High-Fidelity PCR System (Roche AppliedScience), the specification sheets that provides according to manufacturers is by PCR (polymerase chain reaction) the increase variable region of immunoglobulin (Ig) Kappa chain and the variable region of heavy chain (IgG1).Use 5 ' oligonucleotide primer mixture Universal Primer Mix A, (5 ' ctaatacgactcactatagggcaagcagtggtatcaacgcagagt 3 ', (SEQ ID NO.87) and 5 ' ctaatacgactcactatagggc 3 ', the mixture of (SEQ ID NO.88)) and the specific primer of 3 ' IgG1 constant region, (5 ' tatgcaaggcttacaaccaca 3 ', (SEQID NO.89) or 5 ' gccagtggatagacagatgggggtgtcg 3 ', (SEQ ID NO.90)) the amplification variable region of heavy chain.Use the variable region of 5 ' oligonucleotide primer mixture Universal Primer MixA and 3 ' Kappa constant region specific primer (5 ' ctcattcctgttgaagctcttgacaat 3 ' (SEQ ID NO.91) or 5 ' cgactgaggcacctccagatgtt3 ' (SEQ ID NO.92)) amplification Kappa chain.

Separate independent PCR product by agarose gel electrophoresis, and use Qiaquick GelPurification test kit (Qiagen), the specification sheets that provides according to manufacturers carries out purifying.Use clone's test kit subsequently based on topoisomerase

Test kit (has

Carrier) (CA), in pCR2.1 TOPO plasmid, and the transformation technology of the standard of use is transformed into the gained plasmid in the DH5 bacterium specification sheets that provides according to manufacturers with the PCR product cloning for Invitrogen, Carlsbad.Use the dideoxy dna sequence measurement of standard by Agencourt Bioscience company, use T7 (5 ' TAATACGACTCACTATAGGG 3 ') (SEQ ID NO.93), M13 forward primer (5 ' GTAAAACGACGGCCAGT3 ') (SEQ ID NO.94) and M13 reverse primer (5 ' CAGGAAACAGCTATGACC 3 ') (SEQ ID NO.95) to checking order by isolating plasmid DNA in the bacterial clone that transforms, thus the order of evaluation variable region sequences.(Invitrogen, Carlsbad CA) analyze institute's calling sequence with the IMGT/V-Quest webserver (http://imgt.cines.fr/textes/vquest), thereby identify and verify the sequence of variable region to utilize Vector NTI software.

C. the encode Kappa of 1A3,1D3,1F3 and 2B8 and the immune globulin of IgG1 chain The mensuration of the nucleotide sequence of Bai Chonglian and constant region of light chain sequence

Use forward primer 5 ' ggggacaagtttgtacaaaaaagcaggctgccacc AtgAactttgggctcagattgattttcc 3 ' (the line part is an initiator codon) (SEQ ID NO.96) and reverse primer 5 ' ggggaccactttgtacaagaaagctgggt TcaTttaccaggagagtgggagagg 3 ' (the line part is a terminator codon) (SEQ ID NO.97), the cDNA of the total length of the IgG1 chain of pcr amplification 1A3,1D3 and 1F3 from the cDNA of above-mentioned preparation.Use forward primer 5 ' ggggacaagtttgtacaaaaaagcaggctgccacc AtgGgatggagctatatcatcctcttt3 ' (the line part is an initiator codon) (SEQ ID NO.98) and reverse primer 5 ' ggggaccactttgtacaagaaagctgggttcatt TacCaggagagtgggagag 3 ' (the line part is a terminator codon) (SEQ ID NO.99), the full-length cDNA of the IgG1 chain of amplification 2B8 from the cDNA of above-mentioned preparation.

Use forward primer 5 ' ggggacaagtttgtacaaaaaagcaggctgccacc AtgGaatcacagactctggtcttcata 3 ' (the line part is an initiator codon) (SEQ ID NO.100) and reverse primer 5 ' ggggaccactttgtacaagaaagctgggt CtaThe cDNA of the total length of the Kappa chain of acactcattcctgttgaagctc 3 ' (the line part is a terminator codon) (SEQ ID NO.101) amplification 2B8.By Gateway BP recombining reaction (Invitrogen, Carlsbad, CA), with PCR fragment subclone to pDONR221 (Invitrogen, Carlsbad, CA) in, and the dioxygen dna sequencing method of being used standard by Agencourt Bioscience company checks order to the plasmid of gained, thereby identify the sequence of constant region, the sequence of the step of going forward side by side card variable region.

D. sequential analysis

Utilize IMGT/V-QUEST web server software (http://imgt.cines.fr/textes/vquest/) to identify variable region (normal text).Initiator codon (ATG) according to the reading frame of the upstream of the variable region of confirming to be arranged in mensuration is come the prediction signal peptide sequence.The identification signal peptide sequence is also write down line in its subscript.

Last Nucleotide of each variable region is first base by variable region/Next Password that the constant region linker is produced.This Nucleotide is included in the variable region, and this is because it is the part of exon.Below the aminoacid sequence of listed constant region comprise the translation product of this linker codon.

In order to produce complete heavy chain or kappa chain antibody sequence, below Biao Zhu variable region sequences combines (signal sequence has underscore) with its constant region sequence separately.

(1) 1A3 variable region of heavy chain(SEQ ID NO.1)

1 atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgaa

61 gtgcagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaactctcc

121 tgtgcagcct ctgaattcac tttcagtaac tattacatgt cttgggttcg ccagactcca

181 gagaagaggc tgcagtgggt cgcatacatt agtcctggtg gtggtagctc ctactatcca

241 gccagtgtga agggtcgatt caccatctcc agagacaatg ccaagaacac cctgtacctg

301 caaatgagca gtctgaagtc tgaggacaca gccatgtatt actgtgcaag acaaggggat

361 ggttactacg gggactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc

421 tcag

(2) 1A3 Kappa variable region of light chain (SEQ ID NO.3)

1 atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt

61 gacatccaga tgactcagtc tccagcctcc ctatctgttt ctgtgggaga aactgtcacc

121 atcacatgtc gagcaagtga gaatatttat agtaatttag catggtatca gcagaaacag

181 ggaaaatctc ctcagctcct ggtctatgct gcaacaaact tagcagatgg tgtgccatca

241 aggttcagtg gcagtggatc aggcacacag ttttccctca agatcaacag cctgcagtct

301 gaagattttg ggacttatta ctgtcaacat ttttggggta ctccgtacac gttcggaggg

361 gggaccaagc tggaaataaa ac

(3) 2B8 variable region of heavy chain(SEQ ID NO.11)

1 atgggatgga gctatatcat cctctttttg gtagcaacag ctacagatgt ccactcccag

61 gtccaactgc agcagcctgg ggctgaactg gtgaagcctg ggacttcagt gaagctgtcc

121 tgcaaggctt ctggctacac cttcaccacc tactggatgc actgggtgaa tcagaggcct

181 ggacaaggcc ttgagtggat tggagagatt aatcctacca acggtcatac taactacaat

241 gagaagttca agagcaaggc cacactgact gtagacaaat cctccagcac agcctacatg

301 caactcagca gcctgacatc tgaggactct gcggtctatt actgtgcaag aaactatgtt

361 ggtagcatct ttgactactg gggccaaggc accactctca cagtctcctc ag

(4) 2B8 Kappa variable region of light chain(SEQ ID NO.13)

1 atggaatcac agactctggt cttcatatcc atactgctct ggttatatgg tgctgatggg

61 aacattgtaa tgacccaatc tcccaaatcc atgtccatgt cagtaggaga gagggtcacc

121 ttgagctgca aggccagtga gaatgtggtt tcttatgtat cctggtatca acagaaacca

181 gcgcagtctc ctaaactgct gatatacggg gcatccaacc ggaacactgg ggtccccgat

241 cgcttcacag gcagtggatc tgcaacagatttcactctga ccatcagcag tgtgcgggct

301 gaagaccttg cagattatca ctgtgggcag agttacaact atccgtacac gttcggaggg

361 gggaccaggc tggaaataaa ac

(5) 2F8 variable region of heavy chain(SEQ ID NO.21)

1 atggaatgga gctgggtctt tctcttcctc ctgtcagtaa ctgcaggtgt ccactgccag

61 gtccagctga agcagtctgg agctgagctg gtgaggcctg ggacttcagt gaagatgtcc

121 tgcaaggctt ctggctacac cttcactacc tactatatac actgggtgaa tcagaggcct

181 ggacagggcc ttgagtggat tggaaagatt ggtcctggaa gtggtagtac ttactaca at

241 gagatgttca aagacaaggc cacattgact gtagacacat cctccagcac agcctacatg

301 cagctcagca gcctgacatc tgacgactct gcggtctatt tctgtgcaag aaggggactg

361 ggacgtggct ttgactactg gggccaaggc accactctca cagtctcctc ag

(6) 2F8 Kappa variable region of light chain(SEQ ID NO.23)

1 atggagacag acacaatcct gctatgggtg ctgctgctct gggttccagg ctccactggt

61 gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc

121 atctcctgca aggccagcca aagtgttgat tatgatggta atagttatat caactggtac

181 caacagaaac caggacagcc acccaaagtc ctcatctatg ttgcatccaa tctagaatct

241 gggatcccag ccaggtttag tggcagtggg tctgggacag acttcaccct caacatccat

301 cctgtggagg aggaggatgc tgcaacctattactgtcagc aaagtattga ggatcctccc

361 acgttcggtg ctgggaccaa gctggagctg aaac

(7) 3B6 variable region of heavy chain(SEQ ID NO.31)

1 atggaatggc cttgtatctt tctcttcctc ctgtcagtaa ctgaaggtgt ccactcccag

61 gttcagctgc agcagtctgg ggctgaactg gtgaggcctg ggtcctcagt gaagatttcc

121 tgcaaggctt ctggctatgt attcagtagc tactggatga actgggtgaa gcagaggcct

181 ggacagggtc ttgagtggat tggacagatt tatcctggag atggtgatag taactacaat

241 ggaaacttca agggtaaagc cacactgact gcagacaaat cctccagtac agcctacatg

301 cagctcagca gcctaacatc tgaggactct gcggtctatt tctgtgcatc ccagctcggg

361 ctacgtgaga actactttga ctactggggc caaggcacca ctctcacagt ctcctcag

(8) 3B6 Kappa variable region of light chain(2 possible ATG initiator codons (capitalization is represented)) (SEQ ID NO.33)

1 ATGgacATGa ggacccctgc tcagtttctt ggaatcttgt tgctctggtt tccaggtatc

61 aaatgtgaca tcaagatgac ccagtctcca tcttccatgt atgcatctct aggagagaga

121 gtcacaatca cttgcaaggc gagtcaggac attaaaagct atttaagctg gttccagcag

181 aaaccaggga aatctcctaa gaccctgatc tatcgtgtaa acagattggt agatggggtc

241 ccatcaaggt tcagtggcag tggatctggg caagattctt ctctcaccat caccagcctg

301 gagaatgaag atatgggaat ttattattgt ctacagtatg atgagtttcc gttcacgttc

361 ggagggggga ccaagctgga aataaagc

(9) 3D11 variable region of heavy chain(SEQ ID NO.41)

1 atggctgtcc cgggtctgtt cctctgcctg gttgcatttc caagctgtgt cctgtcccag

61 gtacagctga aggagtcagg acctggcctggtggcgccct cacagagcctgtccatcact

121 tgcactgtct ctgggttttc attaaccagc tatagtttac actgggttcg ccagcctcca

181 ggaaagggtc tggaatggct gggagtaata tgggctggtg gaaacacaaa ttataattcg

241 tctctcatgt ccagactgac catcaggaaa gacaactcca agagccaagt tttcttaaaa

301 atgaacagtc tgcaaactga tgacacagcc atgactact gtgccagaga gaggtttgct

361 tactggggcc aagggactctggtcactgtc tctgcag

(10) 3D11 Kappa variable region of light chain(SEQ ID NO.43)

1 atggattttc aagtgcagat tttcagcttc ctgctaatca gtgcctcagt caaaatatcc

61 agaggacaaa ttgttctcac ccagtctcca gcaatcatgt ctgcatatcc aggggagaag

121 gtcaccatga cctgcagtgc cagctcaagt gtaagttaca tgcactggta ccagcagaag

181 tcaggcacct cccccaaaag atggatttat gacacatcca aactggcttc tggagtccct

241 gctcgcttca gtggcagtgg gtctgggacc tcttactccc tcacaatcag tagtatggag

301 gctgaagatg ctgccactta ttactgccag cagtggagta gtaacccact cacgttcggt

361 gctgggacca agctggagct gaaac

(11) 1D3 variable region of heavy chain(SEQ ID NO.51)

1 atgaactttg ggctcagatt gattttccttgt ccttgttt taaaaggtgt gaagtgtgaa

61 gtgcagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaactctcc

121 tgtgcagcct ctggattcac tttcagtgac tattacatgt cttgggttcg ccagactcca

181 gagaagaggc tggagtgggt cgcatacatt agtagtggtg gtggtagcac ctactatcca

241 gacagtgtga agggtcgatt caccatctcc cgagacaatg ccaagaacac cctgtacctg

301 caaatgagca gtctgaagtc tgaggacaca gccatatatt actgtgtgag acaaggggat

361 ggttattacg gggactatgc tatggactac tggggtcaag gaacctcagt catcgtctcc

421 tcag

(12) 1D3 Kappa variable region of light chain(SEQ ID NO.53)

1 atgagtgtg cccactcaggt cctggggttg ctgctgctgt ggcttacaga tgtcagatgt

61 gacatccaga tgactcagtc tccagcctcc ctatctgtat ctgtgggaga aactgtcacc

121 atcacatgtc gaacaagtga gaatatttac agtaatttag cgtggtatca gcagaaacag

181 ggaaaatctc ctcagctcct aatctatgct gcaacaaact tagcagatgg tgtgccatca

241 aggttcagtg gcagtggatc aggcacacag ttttccctca ggatcaacag cctgcagtct

301 gaagattttg ggaggtatta ctgtcaacat ttttggggga ctccgtacac gttcggaggg

361 gggaccaaac tggaaataaa ac

(13) 1F3 variable region of heavy chain(SEQ ID NO.61)

1 atgaacttt gggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgag

61 gtgcagctgg tggagtctgg gggaggctta gtgcagtctg gagggtccct gaaactctcc

121 tgtgcggcct ctggattcac tttcagtaac tatttcatgt cttgggttcg ccagactcca

181 gagaagaggc tggagtgggt cgcatatatt agtagtggtg gtggtagcac ctactatcca

241 gacagtgtga agggtcgatt caccatctct agagacaatg ccaagaacac cctgtacctg

301 caaatgagca gtctgaagtc tgaggacaca gccatgtatt actgtgtaag acaaggggat

361 ggttactacg gggactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc

421 tcag

(14) 1F3 Kappa variable region of light chain(SEQ ID NO.63)

1 atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt

61 gacatccaga tgactcagtc tccagcctcc ctatctgtat ctgtgggaga aactgtcacc

121 atcacatgtc gagcaagtga gaatatttac agtaatttag catggtatca gcagaaacag

181 ggaaaatctc ctcagctcct ggtctatgat gcaacacact taccagatgg tgtgccatca

241 aggttcagtg gcagtggatc aggcacacag ttttccctca agatcaacag cctgcagtct

301 gaagattttg ggagttattactgtcaacat ttttggggta ctccgtacac gtttggaggg

361 gggaccagac tggaaattaa ac

(15) 3A12 variable region of heavy chain(SEQ ID NO.71)

1 atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgaa

61 gtgcagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaaatctcc

121 tgtgcagcct ctggatttac tttcagtaac tatttcatgt cttgggttcg ccagactcca

181 gagaagaggc tggagtgggt cgcatacatt agtagtggtg gtggtagcac ctactatcca

241 gacagtgtga agggtcgatt caccatctcc agagacaatg ccaagaacac cctgtacctg

301 caaatgaaca gtctgaagtc tgaggacaca gccatgtatt actgtgtaag acaaggagat

361 ggttactatg gggactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc

421 tcag

(16) 3A12 Kappa variable region of light chain(SEQ ID NO.73)

1 atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt

61 gacatccaga tgactcagtc gccagcctcc ctatctgtat ctgtgggaga aactgtcacc

121 atcacatgtc gagcaagtga gaatatttac attaatttag catggtatca gcagaaacag

181 ggaaaatctc ctcagctcct ggtccatgct gcaacaaagt tagcagatgg tgtgccatca

241 aggttcagtg gcagtggatc aggcacacag tattccctca agatcaacag cctgcagtct

301 gaagattttg ggagttatta ctgtcaacat ttttggggta ctccgtacac gttcggaggg

361 gggaccaaac tagaaataaa ac

(17) with reference to mouse IgG1 CH (J00453)(SEQ ID NO.81)

1 ccaaaacgac acccccatct gtctatccac tggcccctgg atctgctgcc caaactaact

61 ccatggtgac cctgggatgc ctggtcaagg gctatttccc tgagccagtg acagtgacct

121 ggaactctgg atccctgtcc agcggtgtgc acaccttccc agctgtcctg gagtctgacc

181 tctacactct gagcagctca gtgactgtcc cctccagccc tcggcccagc gagaccgtca

241 cctgcaacgt tgcccacccg gccagcagca ccaaggtgga caagaaaatt gtgcccaggg

301 attgtggttg taagccttgc atatgtacag tcccagaagt atcatctgtc ttcatcttcc

361 ccccaaagcc caaggatgtg ctcaccatta ctctgactcc taaggtcacg tgtgttgtgg

421 tagacatcag caaggatgat cccgaggtcc agttcagctg gtttgtagat gatgtggagg

481 tgcacacagc tcagacgcaa ccccgggagg agcagttcaa cagcactttc cgctcagtca

541 gtgaacttcc catcatgcac caggactggc tcaatggcaa ggagttcaaa tgcagggtca

601 acagtgcagc tttccctgcc cccatcgaga aaaccatctc caaaaccaaa ggcagaccga

661 aggctccaca ggtgtacacc attccacctc ccaaggagca gatggccaag gataaagtca

721 gtctgacctg catgataaca gacttcttcc ctgaagacat tactgtggag tggcagtgga

781 atgggcagcc agcggagaac tacaagaaca ctcagcccat catgaacacg aatggctctt

841 acttcgtcta cagcaagctc aatgtgcaga agagcaactg ggaggcagga aatactttca

901 cctgctctgt gttacatgag ggcctgcaca accaccatac tgagaagagc ctctcccact

961 ctcctggtaa atga

(18) the mouse IgG1 CH of the mensuration of 1A3,1D3,1F3 and 2B8 (spreads out Being conigenous AJ is mouse)(SEQ ID NO.82)

1 ccaaaacgac acccccatct gtctatccac tggcccctgg atctgctgcc caaactaact

61 ccatggtgac cctgggatgc ctggtcaagg gctatttccc tgagccagtg acagtgacct

121 ggaactctgg atccctgtcc agcggtgtgc acaccttccc agctgtcctg cagtctgacc

181 tctacactct gagcagctca gtgactgtcc cctccagcac ctggcccagc gagaccgtca

241 cctgcaacgt tgcccacccg gccagcagca ccaaggtgga caagaaaatt gtgcccaggg

301 attgtggttg taagccttgc atatgtacag tcccagaagt atcatctgtc ttcatcttcc

361 ccccaaagcc caaggatgtg ctcaccatta ctctgactcc taaggtcacg tgtgttgtgg

421 tagacatcag caaggatgat cccgaggtcc agttcagctg gtttgtagat gatgtggagg

481 tgcacacagc tcagacgcaa ccccgggagg agcagttcaa cagcactttc cgctcagtca

541 gtgaacttcc catcatgcac caggactggc tcaatggcaa ggagttcaaa tgcagggtca

601 acagtgcagc tttccctgcc cccatcgaga aaaccatctc caaaaccaaa ggcagaccga

661 aggctccaca ggtgtacacc attccacctc ccaaggagca gatggccaag gataaagtca

721 gtctgacctg catgataaca gacttcttcc ctgaagacat tactgtggag tggcagtgga

781 atgggcagcc agcggagaac tacaagaaca ctcagcccat catggacaca gatggctctt

841 acttcgtcta cagcaagctc aatgtgcaga agagcaactg ggaggcagga aatactttca

901 cctgctctgt gttacatgag ggcctgcaca accaccatac tgagaagagc ctctcccact

961 ctcctggtaa atga

(19) with reference to mouse Kappa constant region of light chain (V00807) and 1D3,1F3 Mouse Kappa constant region of light chain (is mouse derived from AJ) with the mensuration of 2B8(SEQ ID NO.83)

1 gggctgatgc tgcaccaact gtatccatct tcccaccatc cagtgagcag ttaacatctg

61 gaggtgcctc agtcgtgtgc ttcttgaaca acttctaccc caaagacatc aatgtcaagt

121 ggaagattga tggcagtgaa cgacaaaatg gcgtcctgaa cagttggact gatcaggaca

181 gcaaagacag cacctacagc atgagcagca ccctcacgtt gaccaaggac gagtatgaac

241 gacataacag ctatacctgt gaggccactc acaagacatc aacttcaccc attgtcaaga

301 gcttcaacag gaatgagtgt tag

(20) comparing the Nucleotide that contains 1 change with the situation of 1D3,1F3 and 2B8 (uses Underscore is represented), the mouse Kappa constant region of light chain of the mensuration of 1A3(SEQ ID NO.84)

1 gggctgatgc tgcaccaact gtatccatct tcccaccatc cagtgagcag ttaacatctg

61 gaggtgcctc agtcgtgtgc ttcttgaaca acttctaccc caaagacatc aatgtcaagt

121 ggaagattga tggcagtgaa cgacaaaatg gcgtcctgaa cagttggact gatcaggaca

181 gcaaagacag cacctacagc atgagcagca ccctcatgtt gaccaaggac gagtatgaac

241 gacataacag ctatacctgt gaggccactc acaagacatc aacttcaccc attgtcaaga

301 gcttcaacag gaatgagtgt tag

Each aminoacid sequence that limits the immunoglobulin heavy chain variable region of the prepared antibody of embodiment 1 is all listed among Fig. 2.Each sequence all with other sequence alignments, and in square frame, determined qualification signal peptide, CDR ₁, CDR ₂And CDR ₃Sequence.Fig. 3 has shown CDR each antibody, independent ₁, CDR ₂And CDR ₃Comparison.

Each aminoacid sequence that defines the immunoglobulin light chain variable region of each prepared antibody of embodiment 1 is all listed among Fig. 4.Each sequence all with other sequence alignments, and in square frame, determined qualification signal peptide, CDR ₁, CDR ₂And CDR ₃Sequence.Fig. 5 reality be used for CDR each antibody, independent ₁, CDR ₂And CDR ₃Arrangement.

For convenience, table 1 provides the concordance list of the corresponding relation between those sequences of listing in the antibody sequence discussed in the present embodiment and the sequence table.

Table 1

SEQ ID NO.	Protein or nucleic acid
SEQ ID NO.	Protein or nucleic acid	1	Variable region of heavy chain 1A3-nucleic acid
2	Variable region of heavy chain 1A3-protein	1	Variable region of heavy chain 1A3-nucleic acid
2	Variable region of heavy chain 1A3-protein	3	Light (kappa) chain variable region 1A3-nucleic acid
4	Light (kappa) chain variable region 1A3-protein	3	Light (kappa) chain variable region 1A3-nucleic acid
4	Light (kappa) chain variable region 1A3-protein	5	Heavy chain CDR ₁1A3
6	Heavy chain CDR ₂1A3	5	Heavy chain CDR ₁1A3
6	Heavy chain CDR ₂1A3	7	Heavy chain CDR ₃1A3
8	Light (kappa) CDR ₁1A3	7	Heavy chain CDR ₃1A3

9	Light (kappa) CDR ₂1A3
9	Light (kappa) CDR ₂1A3	10	Light (kappa) CDR ₃1A3
11	Variable region of heavy chain 2B8-nucleic acid	10	Light (kappa) CDR ₃1A3
11	Variable region of heavy chain 2B8-nucleic acid	12	Variable region of heavy chain 2B8-protein
13	Light (kappa) chain variable region 2B8-nucleic acid	12	Variable region of heavy chain 2B8-protein
13	Light (kappa) chain variable region 2B8-nucleic acid	14	Light (kappa) chain variable region 2B8-protein
15	Heavy chain CDR ₁2B8	14	Light (kappa) chain variable region 2B8-protein
15	Heavy chain CDR ₁2B8	16	Heavy chain CDR ₂2B8
17	Heavy chain CDR ₃2B8	16	Heavy chain CDR ₂2B8
17	Heavy chain CDR ₃2B8	18	Light (kappa) CDR ₁2B8
19	Light (kappa) CDR ₂2B8	18	Light (kappa) CDR ₁2B8
19	Light (kappa) CDR ₂2B8	20	Light (kappa) CDR ₃2B8
21	Variable region of heavy chain 2F8-nucleic acid	20	Light (kappa) CDR ₃2B8
21	Variable region of heavy chain 2F8-nucleic acid	22	Variable region of heavy chain 2F8-protein
23	Light (kappa) chain variable region 2F8-nucleic acid	22	Variable region of heavy chain 2F8-protein
23	Light (kappa) chain variable region 2F8-nucleic acid	24	Light (kappa) chain variable region 2F8-protein
25	Heavy chain CDR ₁2F8	24	Light (kappa) chain variable region 2F8-protein
25	Heavy chain CDR ₁2F8	26	Heavy chain CDR ₂2F8
27	Heavy chain CDR ₃2F8	26	Heavy chain CDR ₂2F8
27	Heavy chain CDR ₃2F8	28	Light (kappa) CDR ₁2F8
29	Light (kappa) CDR ₂2F8	28	Light (kappa) CDR ₁2F8
29	Light (kappa) CDR ₂2F8	30	Light (kappa) CDR ₃2F8
31	Variable region of heavy chain 3B6-nucleic acid	30	Light (kappa) CDR ₃2F8
31	Variable region of heavy chain 3B6-nucleic acid	32	Variable region of heavy chain 3B6-protein
33	Light (kappa) chain variable region 3B6-nucleic acid	32	Variable region of heavy chain 3B6-protein
33	Light (kappa) chain variable region 3B6-nucleic acid	34	Light (kappa) chain variable region 3B6-protein
35	Heavy chain CDR ₁3B6	34	Light (kappa) chain variable region 3B6-protein
35	Heavy chain CDR ₁3B6	36	Heavy chain CDR ₂3B6
37	Heavy chain CDR ₃3B6	36	Heavy chain CDR ₂3B6

38	Light (kappa) CDR ₁3B6
38	Light (kappa) CDR ₁3B6	39	Light (kappa) CDR ₂3B6
40	Light (kappa) CDR ₃3B6	39	Light (kappa) CDR ₂3B6
40	Light (kappa) CDR ₃3B6	41	Variable region of heavy chain 3D11-nucleic acid
42	Variable region of heavy chain 3D11-protein	41	Variable region of heavy chain 3D11-nucleic acid
42	Variable region of heavy chain 3D11-protein	43	Light (kappa) chain variable region 3D11-nucleic acid
44	Light (kappa) chain variable region 3D11-protein	43	Light (kappa) chain variable region 3D11-nucleic acid
44	Light (kappa) chain variable region 3D11-protein	45	Heavy chain CDR ₁3D11
46	Heavy chain CDR ₂3D11	45	Heavy chain CDR ₁3D11
46	Heavy chain CDR ₂3D11	47	Heavy chain CDR ₃3D11
48	Light (kappa) CDR ₁3D11	47	Heavy chain CDR ₃3D11
48	Light (kappa) CDR ₁3D11	49	Light (kappa) CDR ₂3D11
50	Light (kappa) CDR ₃3D11	49	Light (kappa) CDR ₂3D11
50	Light (kappa) CDR ₃3D11	51	Variable region of heavy chain 1D3-nucleic acid
52	Variable region of heavy chain 1D3-protein	51	Variable region of heavy chain 1D3-nucleic acid
52	Variable region of heavy chain 1D3-protein	53	Light (kappa) chain variable region 1D3-nucleic acid
54	Light (kappa) chain variable region 1D3-protein	53	Light (kappa) chain variable region 1D3-nucleic acid
54	Light (kappa) chain variable region 1D3-protein	55	Heavy chain CDR ₁1D3
56	Heavy chain CDR ₂1D3	55	Heavy chain CDR ₁1D3
56	Heavy chain CDR ₂1D3	57	Heavy chain CDR ₃1D3
58	Light (kappa) CDR ₁1D3	57	Heavy chain CDR ₃1D3
58	Light (kappa) CDR ₁1D3	59	Light (kappa) CDR ₂1D3
60	Light (kappa) CDR ₃1D3	59	Light (kappa) CDR ₂1D3
60	Light (kappa) CDR ₃1D3	61	Variable region of heavy chain 1F3-nucleic acid
62	Variable region of heavy chain 1F3-protein	61	Variable region of heavy chain 1F3-nucleic acid
62	Variable region of heavy chain 1F3-protein	63	Light (kappa) chain variable region 1F3-nucleic acid
64	Light (kappa) chain variable region 1F3-protein	63	Light (kappa) chain variable region 1F3-nucleic acid
64	Light (kappa) chain variable region 1F3-protein	65	Heavy chain CDR ₁1F3
66	Heavy chain CDR ₂1F3	65	Heavy chain CDR ₁1F3

67	Heavy chain CDR ₃1F3
67	Heavy chain CDR ₃1F3	68	Light (kappa) CDR ₁1F3
69	Light (kappa) CDR ₂1F3	68	Light (kappa) CDR ₁1F3
69	Light (kappa) CDR ₂1F3	70	Light (kappa) CDR ₃1F3
71	Variable region of heavy chain 3A12-nucleic acid	70	Light (kappa) CDR ₃1F3
71	Variable region of heavy chain 3A12-nucleic acid	72	Variable region of heavy chain 3A12-protein
73	Light (kappa) chain variable region 3A12-nucleic acid	72	Variable region of heavy chain 3A12-protein
73	Light (kappa) chain variable region 3A12-nucleic acid	74	Light (kappa) chain variable region 3A12-protein
75	Heavy chain CDR ₁3A12	74	Light (kappa) chain variable region 3A12-protein
75	Heavy chain CDR ₁3A12	76	Heavy chain CDR ₂3A12
77	Heavy chain CDR ₃3A12	76	Heavy chain CDR ₂3A12
77	Heavy chain CDR ₃3A12	78	Light (kappa) CDR ₁3A12
79	Light (kappa) CDR ₂3A12	78	Light (kappa) CDR ₁3A12
79	Light (kappa) CDR ₂3A12	80	Light (kappa) CDR ₃3A12

In addition, for convenience, following sequence has been represented total length sequence of heavy chain reality or that estimate of described each antibody of present embodiment and sequence of light chain (that is, comprise variable region sequences and constant region sequence the two).Notice that the constant region of mouse antibodies 2F8,3A12,3B6 and 3D11 is order-checking not, but suppose that they comprise and 1D3,1F3 and the identical constant region sequence of 2B8 antibody (it is through order-checking), this is because they are mouse derived from AJ all.But, should be appreciated that variable region sequences as herein described can be connected with each sequence in a plurality of other constant region sequences well known by persons skilled in the art, thereby produces activated total length heavy chain immunoglobulin and light chain.

The nuclear of coding total length 1A3 sequence of heavy chain (1A3 variable region of heavy chain and IgG1 constant region) Acid sequence (underscore be signal sequence)(SEQ ID NO.122)

1 atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgaa

61 gtgcagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaactctcc

121 tgtgcagcct ctgaattcac tttcagtaac tattacatgt cttgggttcg ccagactcca

181 gagaagaggc tgcagtgggt cgcatacatt agtcctggtg gtggtagctc ctactatcca

241 gccagtgtga agggtcgatt caccatctcc agagacaatg ccaagaacac cctgtacctg

301 caaatgagca gtctgaagtc tgaggacaca gccatgtatt actgtgcaag acaaggggat

361 ggttactacg gggactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc

421 tcagccaaaa cgacaccccc atctgtctat ccactggccc ctggatctgc tgcccaaact

481 aactccatgg tgaccctggg atgcctggtc aagggctatt tccctgagcc agtgacagtg

541 acctggaact ctggatccct gtccagcggt gtgcacacct tcccagctgt cctgcagtct

601 gacctctaca ctctgagcag ctcagtgact gtcccctcca gcacctggcc cagcgagacc

661 gtcacctgca acgttgccca cccggccagc agcaccaaggtggacaagaa aattgtgccc

721 agggattgtg gttgtaagcc ttgcatatgt acagtcccag aagtatcatc tgtcttcatc

781 ttccccccaa agcccaagga tgtgctcacc attactctga ctcctaaggt cacgtgtgtt

841 gtggtagaca tcagcaagga tgatcccgag gtccagttca gctggtttgt agatgatgtg

901 gaggtgcaca cagctcagac gcaaccccgg gaggagcagt tcaacagcac tttccgctca

961 gtcagtgaac ttcccatcatgcaccaggac tggctcaatg gcaaggagtt caaatgcagg

1021 gtcaacagtg cagctttccc tgcccccatc gagaaaacca tctccaaaac caaaggcaga

1081 ccgaaggctc cacaggtgta caccattcca cctcccaagg agcagatggc caaggataaa

1141 gtcagtctga cctgcatgat aacagacttc ttccctgaag acattactgt ggagtggcag

1201 tggaatgggc agccagcgga gaactacaag aacactcagc ccatcatgga cacagatggc

1261 tcttacttcg tctacagcaa gctcaatgtg cagaagagca actgggaggc aggaaatact

1321 ttcacctgct ctgtgttaca tgagggcctg cacaaccacc atactgagaa gagcctctcc

1381 cactctcctg gtaaatga

(2) define total length 1A3 sequence of heavy chain (1A3 variable region of heavy chain and IgG1 constant region) Protein sequence (not having signal sequence)(SEQ IDNO.123)

1 evqlvesggg lvqpggslkl scaaseftfs nyymswvrqt pekrlqwvay ispgggssyy

61 pasvkgrfti srdnakntly lqmsslksed tamyycarqg dgyygdyamd ywgqgtsvtv

121 ssakttppsv yplapgsaaq tnsmvtlgcl vkgyfpepvt vtwnsgslss gvhtfpavlq

181 sdlytlsssv tvpsstwpse tvtcnvahpa sstkvdkkiv prdcgckpci ctvpevssvf

241 ifppkpkdvl titltpkvtc vvvdiskddp evqfswfvdd vevhtaqtqp reeqfnstfr

301 svselpimhq dwlngkefkc rvnsaafpap iektisktkg rpkapqvyti pppkeqmakd

361 kvsltcmitd ffpeditvew qwngqpaeny kntqpimdtd gsyfvyskln vgksnweagn

421 tftcvlheg lhnhhteksl shspgk

(3) nucleic acid of coding total length 1A3 sequence of light chain (1A3Kappa variable region and constant region) Sequence (underscore be signal sequence)(SEQ ID NO.124)

1 atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt

61 gacatccaga tgactcagtc tccagcctcc ctatctgttt ctgtgggaga aactgtcacc

121 atcacatgtc gagcaagtga gaatatttat agtaatttag catggtatca gcagaaacag

181 ggaaaatctc ctcagctcct ggtctatgct gcaacaaact tagcagatgg tgtgccatca

241 aggttcagtg gcagtggatc aggcacacag ttttccctca agatcaacag cctgcagtct

301 gaagattttg ggacttatta ctgtcaacat ttttggggta ctccgtacac gttcggaggg

361 gggaccaagc tggaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca

421 tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac

481 cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg

541 aacagttgga ctgat cagga cagcaaagac agcacctaca gcatgagcag caccct catg

601 ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca

661 tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag

Define the egg of total length 1A3 sequence of light chain (1A3Kappa variable region and constant region) White matter sequence (not having signal sequence)(SEQ ID NO.125)

1 diqmtqspas lsvsvgetvt itcraseniy snlawyqqkq gkspqllvya atnladgvps

61 rfsgsgsgtq fslkinslqs edfgtyycqh fwgtpytfgg gtkleikrad aaptvsifpp

121 sseqltsgga svvcflnnfy pkdinvkwki dgserqngvl nswtdqdskd stysmsstlm

181 ltkdeyerhn sytceathkt stspivksfn rnec

(5) nucleic acid of coding total length 2B8 sequence of heavy chain (2B8 variable region of heavy chain and IgG1 constant region) Sequence (underscore be signal sequence)(SEQ ID NO.126)

1 atgggatgga gctatatcat cctctttttg gtagcaacag ctacagatgt ccactcccag

61 gtccaactgc agcagcctgg ggctgaactg gtgaagcctg ggacttcagt gaagctgtcc

121 tgcaaggctt ctggctacac cttcaccacc tactggatgc actgggtgaa tcagaggcct

181 ggacaaggcc ttgagtggat tggagagatt aatcctacca acggtcatac taactacaat

241 gagaagttca agagcaaggc cacactgact gtagacaaat cctccagcac agcctacatg

301 caac tcagca gcctgacatc tgaggactct gcggtctatt actgtgcaag aaactatgtt

361 ggtagcatct ttgactactg gggccaaggc accactctca cagtctcctc agccaaaacg

421 acacccccat ctgtctatcc actggcccct ggatctgctg cccaaactaa ctccatggtg

481 accctgggat gcctggtcaa gggctatttc cctgagccag tgacagtgac ctggaactct

541 ggatccctgt ccagcggtgt gcacaccttc ccagctgtcc tgcagtctga cctctacact

601 ctgagcagct cagtgac tgt cccctccagc acctggccca gcgagaccgt cacctgcaac

661 gttgcccacc cggccagcag caccaaggtg gacaagaaaa ttgtgcccag ggattgtggt

721 tgtaagcctt gcatatgtac agtcccagaa gtatcatctg tcttcatctt ccccccaaag

781 cccaaggatg tgctcaccat tactctgact cctaaggtca cgtgtgttgt ggtagacatc

841 agcaaggatg atcccgaggt ccagttcagc tggtttgtag atgatgtgga ggtgcacaca

901 gctcagacgc aaccccggga ggagcagttc aacagcactt tccgctcagt cagtgaactt

961 cccatcatgc accaggactg gctcaatggc aaggagttca aatgcagggtcaacagtgca

1021 gctttccctg cccccatcga gaaaaccatc tccaaaacca aaggcagacc gaaggctcca

1081 caggtgtaca ccattccacc tcccaaggag cagatggcca aggataaagt cagtctgacc

1141 tgcatgataa cagacttctt ccctgaagac attactgtgg agtggcagtg gaatgggcag

1201 ccagcggaga actacaagaa cactcagccc atcatggaca cagatggctc ttacttcgtc

1261 tacagcaagc tcaatgtgca gaagagcaac tgggaggcag gaaatacttt cacctgctct

1321 gtgttacatg agggcctgca caaccaccat actgagaaga gcctctccca ctctcctggt

1381 aaatga

(6) define total length 2B8 sequence of heavy chain (2B8 variable region of heavy chain and IgG1 constant region) Protein sequence (not having signal sequence)(SEQ ID NO.127)

1 qvqlqapqae lvkpgtsvkl sckasgytft tywmhwvnqr pggglewige inptnghtny

61 nekfkskatl tvdkssstay mqlssltsed savyycarny vgsifdywgq gtticvssak

121 ttppsvypla pgsaaqtnsm vtlgclvkgy fpepvtvtwn sgslssgvht fpavlqsaly

181 tlsssvtvps stwpsetvtc nvahpasstk vdkkivprdc gckpcictvp evssvrifpp

241 kpkdvltitl tpkvtcvvvd iskddpevqf swfvddvevh taqtqpreeq fnsttrsvse

301 lpimhqdwln gkefkcrvns aafpapiekt isktkgrpka pqvytipppk eqmakdkvsl

361 tcmitdffpe ditvewqwng qpaenykntq pimdtdgsyf vysklnvqks nweagntrtc

421 svlheglhnh htekslshsp gk

(7) coding total length 2B8 sequence of light chain (2B8 Kappa variable region of light chain and constant region) Nucleotide sequence (underscore be signal sequence)(SEQ ID NO.128)

1 atggaatcac agactctggt cttcatatcc atactgctct ggttatatgg tgctgatggg

61 aacattgtaatgacccaatc tcccaaatcc atgtccatgt cagtaggaga gagggtcacc

121 ttgagctgca aggccagtga gaatgtggtt tcttatgtat cctggtatca acagaaacca

181 gcgcagtctc ctaaactgct gatatacggg gcatccaacc ggaacactgg ggtccccgat

241 cgcttcacag gcagtggatc tgcaacagat ttcactctga ccatcagcag tgtgcgggct

301 gaagaccttg cagattatca ctgtgggcag agttacaact atccgtacac gttcggaggg

361 gggaccaggc tggaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca

421 tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac

481 cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg

541 aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg

601 ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca

661 tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag

(8) define the albumen of total length 2B8 sequence of light chain (2B8 Kappa variable region and constant region) Matter sequence (not having signal sequence)(SEQ ID NO.129)

1 nivmtqspks msmsvgervt lsckasenvv syvswyqqkp aqspklliyg asnrntgvpd

61 rftgsgsatd ftltissvra edladyhcgq synypytfgg gtrleikrad aaptvsifpp

121 ssegltsgga svvcflnnfy pkdinvkwki dgserqngvl nswtdqdskd stysmsstlt

181 ltkdeyerhn sytceathkt stspivksfn rnec

(9) nuclear of coding total length 2F8 sequence of heavy chain (2F8 variable region of heavy chain and IgG1 constant region) Acid sequence (underscore be signal sequence)(SEQ ID NO.130)

1 atggaatgga gctgggtctt tctcttcct cctgtcagtaa ctgcaggtgt ccactgccag

61 gtccagctga agcagtctgg agctgagctg gtgaggcctg ggacttcagt gaagatgtcc

121 tgcaaggctt ctggctacac cttcactacc tactatatac actgggtgaa tcagaggcct

181 ggacagggcc ttgagtggat tggaaagatt ggtcctggaa gtggtagtac ttactacaat

241 gagatgttc aaagacaaggc cacattgact gtagacacat cctccagcac agcctacatg

301 cagctcagca gcctgacatc tgacgactctgcggtctatt tctgtgcaag aaggggactg

361 ggacgtggct ttgactactg gggccaaggc accactctca cagtctcctc agccaaaacg

421 acacccccat ctgtctatcc actggcccct ggatctgctg cccaaactaa ctccatggtg

481 accctggga t gcctggtcaa gggctatttc cctgagccag tgacagtgac ctggaactct

541 ggatccctgt ccagcggtgt gcacaccttc ccagctgtcc tgcagtctga cctctacact

601 ctgagcagct cagtgactgt cccctccagc acctggcccagcgagaccgt cacctgcaac

661 gttgcccacc cggccagcag caccaaggtg gacaagaaaa ttgtgcccag ggattgtggt

721 tgtaagcctt gcatatgtac agtcccagaagtatcatctg tcttcatctt ccccccaaag

781 cccaaggatg tgctcaccattactctgact cctaaggtca cgtgtgttgt ggtagacatc

841 agcaaggatg atcccgaggt ccagttcagc tggtttgtag atgatgtgga ggtgcacaca

901 gctcagacgc aaccccggga ggagcagttc aacagcactt tccgctcagt cagtgaactt

961 cccatcatgc accaggactg gctcaatggc aaggagttcaaatgcagggt caacagtgca

1021 gctttccctg cccccatcga gaaaaccatc tccaaaacca aaggcagacc gaaggctcca

1081 caggtgtaca ccattccacc tcccaaggag cagatggcca aggataaagt cagtctgacc

1141 tgcatgataa cagacttctt ccctgaagac attactgtgg agtggcagtg gaatgggcag

1201 ccagcggaga actacaagaa cactcagccc atcatggaca cagatggctcttacttcgtc

1261 tacagcaagc tcaatgtgca gaagagcaac tgggaggcag gaaatacttt cacctgctct

1321 gtgttacatg agggcctgca caaccaccat actgagaaga gcctctccca ctctcctggt

1381 aaatga

(10) define total length 2F8 sequence of heavy chain (2F8 variable region of heavy chain and IgG1 constant region) Protein sequence (not having signal sequence)(SEQ ID NO.131)

1 qvqlkqsgae lvrpgtsvkm sckasgytft tyyihwvnqr pgqglewigk igpgsgstyy

61 nemfkdkatl tvdtssstay mqlssltsdd savyfcarrg lgrgfdywgq gttltvssak

121 ttppsvypla pgsaaqtnsm vtlgclvkgy fpepvtvtwn sgslssgvht fpavlqsdly

181 tlsssvtvps stwpsetvtc nvahpasstk vdkkivprdc gckpcictvp evssvfifpp

241 kpkdvltitl tpkvtcvvvd iskddpevqf swfvddvevh taqtqpreeq fnstfrsvse

301 lpimhqdwln gkefkcrvns aafpapiekt isktkgrpka pqvytipppk eqmakdkvsl

361 tcmitdffpe ditvewqwng qpaenykntq pimdtdgsyf vysklnvqks nweagntftc

421 svlheglhnh htekslshsp gk

(11) nucleic acid of coding total length 2F8 sequence of light chain (2F8 Kappa variable region and constant region) Sequence (underscore be signal sequence)(SEQ ID NO.132)

1 atggagacag acacaatcct gctatgggtg ctgctgctct gggttccagg ctccactggt

61 gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc

121 atctcctgca aggccagcca aagtgttgat tatgatggta atagttatat caactggtac

181 caacagaaac caggacagcc acccaaagtc ctcatctatg ttgcatccaa tctagaatct

241 gggatcccag ccaggtttag tggcagtggg tctgggacag acttcaccct caacatccat

301 cctgtggagg aggaggatgc tgcaacctat tactgtcagc aaagtattga ggatcctccc

361 acgttcggtg ctgggaccaa gctggagctg aaacgggctg atgctgcacc aactgtatcc

421 atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg

481 aacaacttct accccaaaga catcaatgtc aagtggaaga ttgatggcag tgaacgacaa

541 aatggcgtcc tgaacagttg gactgatcag gacagcaaag acagcaccta cagcatgagc

601 agcaccctca cgttgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc

661 actcacaaga catcaacttc acccattgtc aagagcttca acaggaatga gtgttag

(12) define the egg of total length 2F8 sequence of light chain (2F8 Kappa variable region and constant region) White matter sequence (not having signal sequence)(SEQ ID NO.133)

1 divltqspas lavslgqrat isckasqsvd ydgnsyinwy qqkpgqppkv liyvasnles

61 giparfsgsg sgtdftlnih pveeedaaty ycqqsiedpp tfgagtklel kradaaptvs

121 ifppsseqlt sggasvvcfl nnfypkdinv kwkidgserq ngvlnswtdq dskdstysms

181 stltltkdey erhnsytcea thktstspiv ksfnrnec

(13) coding total length 3B6 sequence of heavy chain (3B6 variable region of heavy chain and IgG1 constant region) Nucleotide sequence (underscore be signal sequence)(SEQ ID NO.134)

1 atggaatggc cttgtatctt tctcttcctc ctgtcagtaa ctgaaggtgt ccactcccag

61 gttcagctgc agcagtctgg ggctgaactg gtgaggcctg ggtcctcagt gaagatttcc

121 tgcaaggctt ctggctatgt attcagtagc tactggatga actgggtgaa gcagaggcct

181 ggacagggtc ttgagtggat tggacagatt tatcctggag atggtgatag taactacaat

241 ggaaacttca agggtaaagc cacactgact gcagacaaat cctccagtac agcctacatg

301 cagctcagca gcctaacatc tgaggactct gcggtctatt tctgtgcatc ccagctcggg

361 ctacgtgaga actactttga ctactggggc caaggcacca ctctcacagt ctcctcagcc

421 aaaacgacac ccccatctgt ctatccactg gcccctggat ctgctgccca aactaactcc

481 atggtgaccc tgggatgcct ggtcaagggc tatttccctg agccagtgac agtgacctgg

541 aactctggat ccctgtccag cggtgtgcac accttcccag ctgtcctgca gtctgacctc

601 tacactctga gcagctcagt gactgtcccc tccagcacct ggcccagcga gaccgtcacc

661 tgcaacgttg cccacccggc cagcagcacc aaggtggaca agaaaattgt gcccagggat

721 tgtggttgta agccttgcat atgtacagtc ccagaagtat catctgtctt catcttcccc

781 ccaaagccca aggatgtgct caccattact ctgactccta aggtcacgtg tgttgtggta

841 gacatcagca aggatgatcc cgaggtccag ttcagctggt ttgtagatga tgtggaggtg

901 cacacagctc agacgcaacc ccgggaggag cagttcaaca gcactttccg ctcagtcagt

961 gaacttccca tcatgcacca ggactggctc aatggcaagg agttcaaatg cagggtcaac

1021 agtgcagctt tccctgcccc catcgagaaa accatctcca aaaccaaagg cagaccgaag

1081 gctccacagg tgtacaccat tccacctccc aaggagcaga tggccaagga taaagtcagt

1141 ctgacctgca tgataacaga cttcttccct gaagacatta ctgtggagtg gcagtggaat

1201 gggcagccag cggagaacta caagaacact cagcccatca tggacacaga tggctcttac

1261 ttcgtctaca gcaagctcaa tgtgcagaag agcaactggg aggcaggaaa tactttcacc

1321 tgctctgtgt tacatgaggg cctgcacaac caccatactg agaagagcct ctcccactct

1381 cctggtaaat ga

(14) define total length 3B6 sequence of heavy chain (3B6 variable region of heavy chain and IgG1 constant region) Protein sequence (not having signal sequence)(SEQ ID NO.135)

1 qvqlqqsgae lvrpgssvki sckasgyvfs sywmnwvkqr pgqglewigq iypgdgdsny

61 ngnfkgkatl tadkssstay mqlssltsed savyfcasql glrenyfdyw gqgttltvss

121 akttppsvyp lapgsaaqtn smvtlgclvk gyfpepvtvt wnsgslssgv htfpavlqsd

181 lytlsssvtv psstwpsetv tcnvahpass tkvdkkivpr dcgckpcict vpevssvfif

241 ppkpkdvlti tltpkvtcvv vdiskddpev qfswfvddve vhtaqtqpre eqfnstfrsv

301 selpimhqdw lngkefkcrv nsaafpapie ktisktkgrp kapqvytipp pkeqmakdkv

361 sltcmitdff peditvewqw ngqpaenykntqpimdtdgs yfvysklnvq ksnweagntf

421 tcsvlheglh nhhtekslsh spgk

(15) nucleic acid of coding total length 3B6 sequence of light chain (3B6 Kappa variable region and constant region) Sequence (underscore be signal sequence)(SEQ ID NO.136)

1 ATGgacATGa ggacccctgc tcagtttctt ggaatcttgt tgctctggtt tccaggtatc

61 aaatgtgaca tcaagatgac ccagtctcca tcttccatgt atgcatctct aggagagaga

121 gtcacaatca cttgcaaggc gagtcaggac attaaaagct atttaagctg gttccagcag

181 aaaccaggga aatctcctaa gaccctgatc tatcgtgtaa acagattggt agatggggtc

241 ccatcaaggt tcagtggcag tggatctggg caagattctt ctctcaccat caccagcctg

301 gagaatgaag atatgggaat ttattattgt ctacagtatg atgagtttcc gttcacgttc

361 ggagggggga ccaagctgga aataaagcgg gctgatgctg caccaactgt atccatcttc

421 ccaccatcca gtgagcagtt aacatctgga ggtgcctcag tcgtgtgctt cttgaacaac

481 ttctacccca aagacatcaa tgtcaagtgg aagattgatg gcagtgaacg acaaaatggc

541 gtcctgaaca gttggactga tcaggacagc aaagacagca cctacagcat gagcagcacc

601 ctcacgttga ccaaggacga gtatgaacga cataacagct atacctgtga ggccactcac

661 aagacatcaa cttcacccat tgtcaagagc ttcaacagga atgagtgtta g

(16) define the egg of total length 3B6 sequence of light chain (3B6 Kappa variable region and constant region) White matter sequence (not having signal sequence)(SEQ ID NO.137)

1 dikmtqspss myaslgervt itckasqdik sylswfqqkp gkspktliyr vnrlvdgvps

61 rfsgsgsgqd ssltitslen edmgiyyclq ydefpftfgg gtkleikrad aaptvsiipp

121 ssegltsgga svvcflnnfy pkdinvkwki dgserqngvl nswtdqdskd stysmsstlt

181 ltkdeyerhn sytceathkt stspivksfn rnec

(17) coding total length 3D11 sequence of heavy chain (3D11 variable region of heavy chain and IgG1 constant region) Nucleotide sequence (underscore be signal sequence)(SEQ ID NO.138)

1 atggctgtcc cggtgctgtt cctctgcctg gttgcatttc caagctgtgt cctgtcccag

61 gtacagctga aggagtcagg acctggcctg gtggcgccct cacagagcct gtccatcact

121 tgcactgtct ctgggttttc attaaccagc tatagtttac actgggttcg ccagcctcca

181 ggaaagggtc tggaatggct gggagtaata tgggctggtg gaaacacaaa ttataattcg

241 tctctcatgt ccagactgac catcaggaaa gacaactcca agagccaagt tttcttaaaa

301 atgaacagtc tgcaaactga tgacacagcc atgtactact gtgccagaga gaggtttgct

361 tactggggcc aagggactct ggtcactgtc tctgcagcca aaacgacacc cccatctgtc

421 tatccactgg cccctggatc tgctgcccaa actaactcca tggtgaccct gggatgcctg

481 gtcaagggct atttccctga gccagtgaca gtgacctgga actctggatc cctgtccagc

541 ggtgtgcaca ccttcccagc tgtcctgcag tctgacctct acactctgag cagctcagtg

601 actgtcccct ccagcacctg gcccagcgag accgtcacct gcaacgttgc ccacccggcc

661 agcagcacca aggtggacaa gaaaattgtg cccagggatt gtggttgtaa gccttgcata

721 tgtacagtcc cagaagtatc atctgtcttc atcttccccc caaagcccaa ggatgtgctc

781 accattactc tgactcctaa ggtcacgtgt gttgtggtag acatcagcaa ggatgatccc

841 gaggtccagt tcagctggtt tgtagatgat gtggaggtgc acacagctca gacgcaaccc

901 cgggaggagc agttcaacag cactttccgc tcagtcagtg aacttcccat catgcaccag

961 gactggctca atggcaagga gttcaaatgc agggtcaaca gtgcagcttt ccctgccccc

1021 atcgagaaaa ccatctccaa aaccaaaggc agaccgaagg ctccacaggt gtacaccatt

1081 ccacctccca aggagcagat ggccaaggat aaagtcagtc tgacctgcat gataacagac

1141 ttcttccctg aagacattac tgtggagtgg cagtggaatg ggcagccagc ggagaactac

1201 aagaacactc agcccatcat ggacacagat ggctcttact tcgtctacag caagctcaat

1261 gtgcagaaga gcaactggga ggcaggaaat actttcacct gctctgtgtt acatgagggc

1321 ctgcacaacc accatactga gaagagcctc tcccactctc ctggtaaatg a

(18) define total length 3D11 sequence of heavy chain (3D11 variable region of heavy chain and IgG1 constant region) Protein sequence (not having signal sequence)(SEQ ID NO.139)

1 qvqlkesgpg lvapsqslsi tctvsgfslt syslhwvrqp pgkglewlgv iwaggntnyn

61 sslmsrltir kdnsksqvfl kmnslqtddt amyycarerf aywgqgtlvt vsaakttpps

121 vyplapgsaa qtnsmvtlgc lvkgyfpepv tvtwnsgsls sgvhtfpavl qsdlytlsss

181 vtvpsstwps etvtcnvahp asstkvdkki vprdcgckpc ictvpevssv fifppkpkdv

241 ltitltpkvt cvvvdiskdd pevqfswfvd dvevhtaqtq preeqfnstf rsvselpimh

301 qdwlngkefk crvnsaafpa piektisktk grpkapqvyt ipppkeqmak dkvsltcmit

361 dffpeditve wqwngqpaen ykntqpimdtdgsyfvyskl nvqksnweag ntftcsvlhe

421 glhnhhteks lshspgk

(19) nuclear of coding total length 3D11 sequence of light chain (3D11 Kappa variable region and constant region) Acid sequence (underscore be signal sequence)(SEQ ID NO.140)

1 atggattttc aagtgcaga ttttcagcttc ctgctaatca gtgcctcagt caaaatatcc

61 agaggacaaa ttgttctc acccagtctcca gcaatcatgt ctgcatatcc aggggagaag

121 gtcaccatga cctgcagtgc cagctcaagt gtaagttaca tgcactggta ccagcagaag

181 tcaggcacct cccccaaaag atggatttat gacacatcca aactggcttc tggagtccct

241 gctcgcttca gtggcagtgg gtctgggacc tcttactccctcacaatcagtagtatggag

301 gctgaagatg ctgccactta ttactgccag cagtggagta gtaacccact cacgttcggt

361 gctgggacca agctggagct gaaacgggct gatgctgcac caactgtatc catcttccca

421 ccatccagtg agcagttaac atctggaggt gcctcagtcg tgtgcttctt gaacaacttc

481 taccccaaag acatcaatgt caagtggaag attgatggca gtgaacgaca aaatggcgtc

541 ctgaacagtt ggactgatca ggacagcaaa gacagcacct acagcatgag cagcaccctc

601 acgttgacca aggacgagta tgaacgacat aacagctata cctgtgaggc cactcacaag

661 acatcaactt cacccattgt caagagcttc aacaggaatg agtgttag

(20) define total length 3D11 sequence of light chain (3D11 Kappa variable region and constant region) Protein sequence (not having signal sequence)(SEQ ID NO.141)

1 givltqspai msaypgekvt mtcsasssvs ymhwyggksg tspkrwiydt sklasgvpar

61 fsgsgsgtsy sltissmeae daatyycggw ssnpltfgag tklelkrada aptvsifpps

121 seqltsggas vvcflnnfyp kdinvkwkid gserqngvln swtdqdskds tysmsstltl

181 tkdeyerhns ytceathkts tspivksfnr nec

(21) coding total length 1D3 sequence of heavy chain (1D3 variable region of heavy chain and IgG1 constant region) Nucleotide sequence (underscore be signal sequence)(SEQ ID NO.142)

1 atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgaa

61 gtgcagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaactctcc

121 tgtgcagcct ctggattcac tttcagtgac tattacatgt cttgggttcg ccagactcca

181 gagaagaggc tggagtgggt cgcatacatt agtagtggtg gtggtagcac ctactatcca

241 gacagtgtga agggtcgatt caccatctcc cgagacaatg ccaagaacac cctgtacctg

301 caaatgagca gtctgaagtc tgaggacaca gccatatatt actgtgtgag acaaggggat

361 ggttattacg gggactatgc tatggactac tggggtcaag gaacctcagt catcgtctcc

421 tcagccaaaa cgacaccccc atctgtctat ccactggccc ctggatctgc tgcccaaact

481 aactccatgg tgaccctggg atgcctggtc aagggctatt tccctgagcc agtgacagtg

541 acctggaact ctggatccct gtccagcggt gtgcacacct tcccagctgt cctgcagtct

601 gacctctaca ctctgagcag ctcagtgact gtcccctcca gcacctggcc cagcgagacc

661 gtcacctgca acgttgccca cccggccagc agcaccaagg tggacaagaa aattgtgccc

721 agggattgtg gttgtaagcc ttgcatatgt acagtcccag aagtatcatc tgtcttcatc

781 ttccccccaa agcccaagga tgtgctcacc attactctga ctcctaaggt cacgtgtgtt

841 gtggtagaca tcagcaagga tgatcccgag gtccagttca gctggtttgt agatgatgtg

901 gaggtgcaca cagctcagac gcaaccccgg gaggagcagt tcaacagcac tttccgctca

961 gtcagtgaac ttcccatcat gcaccaggac tggctcaatg gcaaggagtt caaatgcagg

1021 gtcaacagtg cagctttccc tgcccccatc gagaaaacca tctccaaaac caaaggcaga

1081 ccgaaggctc cacaggtgta caccattcca cctcccaagg agcagatggc caaggataaa

1141 gtcagtctga cctgcatgat aacagacttc ttccctgaag acattactgt ggagtggcag

1201 tggaatgggc agccagcgga gaactacaag aacactcagc ccatcatgga cacagatggc

1261 tcttacttcg tctacagcaa gctcaatgtg cagaagagca actgggaggc aggaaatact

1321 ttcacctgct ctgtgttaca tgagggcctg cacaaccacc atactgagaa gagcctctcc

1381 cactctcctg gtaaatga

(22) define total length 1D3 sequence of heavy chain (1D3 variable region of heavy chain and IgG1 constant region) Protein sequence (not having signal sequence)(SEQ ID NO.143)

1 evqlvesggg lvqpggslkl scaasgftfs dyymswvrqt pekrlewvay issgggstyy

61 pdsvkgrfti srdnakntly lqmsslksed taiyycvrqg dgyygdyamd ywgqgtsviv

121 ssakttppsv yplapgsaaq tnsmvtlgcl vkgyfpepvt vtwnsgslss gvhtfpavlq

181 sdlytlsssv tvpsstwpse tvtcnvahpa sstkvdkkiv prdcgckpcictvpevssvf

241 ifppkpkdvl titltpkvtc vvvdiskddp evqfswfvdd vevhtaqtqp reeqfnstfr

301 svselpimhq dwlngkefkc rvnsaafpap iektisktkg rpkapqvyti pppkeqmakd

361 kvsltcmitd ffpeditvew qwngqpaeny kntqpimdtd gsyfvyskln vqksnweagn

421 tftcsvlheg lhnhhteksl shspgk

(23) nucleic acid of coding total length 1D3 sequence of light chain (1D3 Kappa variable region and constant region) Sequence (underscore be signal sequence)(SEQ ID NO.144)

1 atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgtcagatgt

61 gacatccaga tgactcagtc tccagcctcc ctatctgtat ctgtgggaga aactgtcacc

121 atcacatgtc gaacaagtga gaatatttac agtaatttag cgtggtatca gcagaaacag

181 ggaaaatctc ctcagctcct aatctatgct gcaacaaact tagcagatgg tgtgccatca

241 aggttcagtg gcagtggatc aggcacacag ttttccctca ggatcaacag cctgcagtct

301 gaagattttg ggaggtatta ctgtcaacat ttttggggga ctccgtacac gttcggaggg

361 gggaccaaac tggaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca

421 tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac

481 cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg

541 aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg

601 ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca

661 tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag

(24) define total length 1D3 sequence of light chain (1D3 Kappa variable region and constant region) Protein sequence (not having signal sequence)(SEQ ID NO.145)

1 diqmtqspas lsvsvgetvt itcrtseniy snlawyqqkq gkspqlliya atnladgvps

61 rfsgsgsgtq fslrinslqs edfgryycqh fwgtpytfgg gtkleikrad aaptvsifpp

121 sseqltsgga svvcflnnfy pkdinvkwki dgserqngvl nswtdqdskd stysmsstlt

181 ltkdeyerhn sytceathkt stspivksfn rnec

(25) coding total length 1F3 sequence of heavy chain (1F3 variable region of heavy chain and IgG1 constant region) Nucleotide sequence (underscore be signal sequence)(SEQ ID NO.146)

1 atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgag

61 gtgcagctgg tggagtctgg gggaggctta gtgcagtctg gagggtccct gaaactctcc

121 tgtgcggcct ctggattcac tttcagtaac tatttcatgt cttgggttcg ccagactcca

181 gagaagaggc tggagtgggt cgcatatatt agtagtggtg gtggtagcac ctactatcca

241 gacagtgtga agggtcgatt caccatctct agagacaatg ccaagaacac cctgtacctg

301 caaatgagca gtctgaagtc tgaggacaca gccatgtatt actgtgtaag acaaggggat

361 ggttactacg gggactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc

421 tcagccaaaa cgacaccccc atctgtctat ccactggccc ctggatctgc tgcccaaact

481 aactccatgg tgaccctggg atgcctggtc aagggctatt tccctgagcc agtgacagtg

541 acctggaact ctggatccct gtccagcggt gtgcacacct tcccagctgt cctgcagtct

601 gacctctaca ctctgagcag ctcagtgact gtcccctcca gcacctggcc cagcgagacc

661 gtcacctgca acgttgccca cccggccagc agcaccaagg tggacaagaa aattgtgccc

721 agggattgtg gttgtaagcc ttgcatatgt acagtcccag aagtatcatc tgtcttcatc

781 ttccccccaa agcccaagga tgtgctcacc attactctga ctcctaaggt cacgtgtgtt

841 gtggtagaca tcagcaagga tgatcccgag gtccagttca gctggtttgt agatgatgtg

901 gaggtgcaca cagctcagac gcaaccccgg gaggagcagt tcaacagcac tttccgctca

961 gtcagtgaac ttcccatcat gcaccaggac tggctcaatg gcaaggagtt caaatgcagg

1021 gtcaacagtg cagctttccc tgcccccatc gagaaaacca tctccaaaac caaaggcaga

1081 ccgaaggctc cacaggtgta caccattcca cctcccaagg agcagatggc caaggataaa

1141 gtcagtctga cctgcatgat aacagacttc ttccctgaag acattactgt ggagtggcag

1201 tggaatgggc agccagcgga gaactacaag aacactcagc ccatcatgga cacagatggc

1261 tcttacttcg tctacagcaa gctcaatgtg cagaagagca actgggaggc aggaaatact

1321 ttcacctgct ctgtgttaca tgagggcctg cacaaccacc atactgagaa gagcctctcc

1381 cactctcctg gtaaatga

(26) define total length 1F3 sequence of heavy chain (1F3 variable region of heavy chain and IgG1 constant region) Protein sequence (not having signal sequence)(SEQ ID NO.147)

1 evqlvesggg lvqsggslkl scaasgftfs nyfmswvrqt pekrlewvay issgggstyy

61 pdsvkgrfti srdnakntly lgmsslksed tamyycvrqg dgyygdyamd ywgqgtsvtv

121 ssakttppsv yplapgsaaq tnsmvtlgcl vkgyfpepvt vtwnsgslss gvhtfpavlq

181 sdlytlsssv tvpsstwpse tvtcnvahpa sstkvdkkiv prdcgckpci ctvpevssvf

241 ifppkpkdvl titltpkvtc vvvdiskddp evqfswfvdd vevhtaqtqp reeqfnstfr

301 svselpimhq dwlngkefkc rvnsaafpap iektisktkg rpkapqvyti pppkegmakd

361 kvsltcmitd ffpeditvew qwnggpaeny kntqpimdtd gsyfvyskln vqksnweagn

421 tftcsvlheg lhnhhteksl shspgk

(27) nucleic acid of coding total length 1F3 sequence of light chain (1F3 Kappa variable region and constant region) Sequence (underscore be signal sequence)(SEQ ID NO.148)

1 atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt

61 gacatccaga tgactcagtc tccagcctcc ctatctgtat ctgtgggaga aactgtcacc

121 atcacatgtc gagcaagtga gaatatttac agtaatttag catggtatca gcagaaacag

181 ggaaaatctc ctcagctcct ggtctatgat gcaacacact taccagatggtgtgccatca

241 aggttcagtg gcagtggatc aggcacacag ttttccctca agatcaacag cctgcagtct

301 gaagattttg ggagttatta ctgtcaacat ttttggggta ctccgtacac gtttggaggg

361 gggaccagac tggaaattaa acgggctgat gctgcaccaa ctgtatccat cttcccacca

421 tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac

481 cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaatggcgtcctg

541 aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg

601 ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca

661 tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag

(28) define the egg of total length 1F3 sequence of light chain (1F3 Kappa variable region and constant region) White matter sequence (not having signal sequence)(SEQ ID NO.149)

1 diqmtqspas lsvsvgetvt itcraseniy snlawyqqkq gkspqllvyd athlpdgvps

61 rfsgsgsgtq fslkinslqs edfgsyycqh fwgtpytfgg gtrleikrad aaptvsifpp

121 sseqltsgga svvcflnnfy pkdinvkwki dgserqngvl nswtdqdskd stysmsstlt

181 ltkdeyerhn sytceathkt stspivksfn rnec

(29) coding total length 3A12 sequence of heavy chain (3A12 variable region of heavy chain and IgG1 constant region) Nucleotide sequence (underscore be signal sequence)(SEQ ID NO.150)

1 atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgaa

61 gtgcagctggtggagtctgg gggaggctta gtgcagcctg gagggtccct gaaaatctcc

121 tgtgcagcct ctggatttac tttcagtaac tatttcatgt cttgggttcg ccagactcca

181 gagaagaggc tggagtgggt cgcatacatt agtagtggtg gtggtagcac ctactatcca

241 gacagtgtga agggtcgatt caccatctcc agagacaatg ccaagaacac cctgtacctg

301 caaatgaaca gtctgaagtc tgaggacaca gccatgtatt actgtgtaag acaaggagat

361 ggttactatg gggactatgctatggactac tggggtcaag gaacctcagt caccgtctcc

421 tcagccaaaa cgacaccccc atctgtctat ccactggccc ctggatctgc tgcccaaact

481 aactccatgg tgaccctggg atgcctggtc aagggctatt tccctgagcc agtgacagtg

541 acctggaact ctggatccct gtccagcggt gtgcacacct tcccagctgt cctgcagtct

601 gacctctaca ctctgagcag ctcagtgact gtcccctcca gcacctggcc cagcgagacc

661 gtcacctgca acgttgccca cccggccagc agcaccaagg tggacaagaa aattgtgccc

721 agggattgtg gttgtaagcc ttgcatatgt acagtcccag aagtatcatc tgtcttcatc

781 ttccccccaa agcccaagga tgtgctcacc attactctga ctcctaaggt cacgtgtgtt

841 gtggtagaca tcagcaagga tgatcccgag gtccagttca gctggtttgt agatgatgtg

901 gaggtgcaca cagctcagac gcaaccccgg gaggagcagt tcaacagcac tttccgctca

961 gtcagtgaac ttcccatcat gcaccaggac tggctcaatg gcaaggagtt caaatgcagg

1021 gtcaacagtg cagctttccc tgcccccatc gagaaaacca tctccaaaac caaaggcaga

1081 ccgaaggctc cacaggtgta caccattcca cctcccaagg agcagatggc caaggataaa

1141 gtcagtctga cctgcatgat aacagacttc ttccctgaag acattactgt ggagtggcag

1201 tggaatgggc agccagcgga gaactacaag aacactcagc ccatcatgga cacagatggc

1261 tcttacttcg tctacagcaa gctcaatgtg cagaagagca actgggaggc aggaaatact

1321 ttcacctgct ctgtgttaca tgagggcctg cacaaccacc atactgagaa gagcctctcc

1381 cactctcctg gtaaatga

(30) define total length 3A12 sequence of heavy chain (3A12 variable region of heavy chain and IgG1 constant region) Protein sequence (not having signal sequence)(SEQ ID NO.151)

1 evqlvesggg lvqpggslki scaasgftfs nyfmswvrqt pekrlewvay issgggstyy

61 pdsvkgrfti srdnakntly lqmnslksed tamyycvrqg dgyygdyamd ywgqgtsvtv

121 ssakttppsv yplapgsaaq tnsmvtlgcl vkgyfpepvt vtwnsgslss gvhtfpavlq

181 sdlytlsssv tvpsstwpse tvtcnvahpa sstkvdkkiv prdcgckpci ctvpevssvf

241 ifppkpkdvl titltpkvtc vvvdiskddp evqfswfvdd vevhtaqtqp reeqfnstfr

301 svselpimhq dwlngkefkc rvnsaafpap iektisktkg rpkapqvyti pppkeqmakd

361 kvsltcmitd ffpeditvew qwngqpaeny kntqpimdtd gsyfvyskln vqksnweagn

421 tftcsvlheg lhnhhteksl shspgk

(31) nuclear of coding total length 3A12 sequence of light chain (3A12 Kappa variable region and constant region) Acid sequence (underscore be signal sequence)(SEQ ID NO.152)

1 atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt

61 gacatccaga tgactcagtc gccagcctcc ctatctgtat ctgtgggaga aactgtcacc

121 atcacatgtc gagcaagtga gaatatttac attaatttag catggtatca gcagaaacag

181 ggaaaatctc ctcagctcct ggtccatgct gcaacaaagt tagcagatgg tgtgccatca

241 aggttcagtg gcagtggatc aggcacacag tattccctca agatcaacag cctgcagtct

301 gaagattttg ggagttatta ctgtcaacat ttttggggta ctccgtacac gttcggaggg

361 gggaccaaac tagaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca

421 tccagtgagc agttaacatc tggaggtgcctcagtcgtgt gcttcttgaa caacttctac

481 cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg

541 aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg

601 ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccactcacaagaca

661 tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag

(32) define total length 3A12 sequence of light chain (3A12 Kappa variable region and constant region) Protein sequence (not having signal sequence)(SEQ ID NO.153)

1 diqmtqspas lsvsvgetvt itcraseniy inlawyqqkq gkspqllvha atkladgvps

61 rfsgsgsgtq yslkinslqs edfgsyycqh fwgtpytfgg gtkleikrad aaptvsifpp

121 sseqltsgga svvcflnnfy pkdinvkwki dgserqngvl nswtdqdskd stysmsstlt

181 ltkdeyerhn sytceathkt stspivksfn rnec

For convenience, table 2 provides the concordance list of the corresponding relation between those sequences of listing in the full length sequence of the antibody of being discussed in the present embodiment and the sequence table.

Table 2

SEQ ID NO.	Protein or nucleic acid
SEQ ID NO.	Protein or nucleic acid	122	1A3 variable region of heavy chain+IgG1 constant region-nucleic acid
123	1A3 variable region of heavy chain+IgG1 constant region-protein	122
123		124	1A3 variable region of light chain+constant region-nucleic acid
125	1A3 variable region of light chain+constant region-protein	124
125	1A3 variable region of light chain+constant region-protein	126	2B8 variable region of heavy chain+IgG1 constant region-nucleic acid
127	2B8 variable region of heavy chain+IgG1 constant region-protein	126
127		128	2B8 variable region of light chain+constant region-nucleic acid
129	2B8 variable region of light chain+constant region-protein	128
129	2B8 variable region of light chain+constant region-protein	130	2F8 variable region of heavy chain+IgG1 constant region-nucleic acid
131	2F8 variable region of heavy chain+IgG1 constant region-protein	130
131		132	2F8 variable region of light chain+constant region-nucleic acid
133	2F8 variable region of light chain+constant region-protein	132
133	2F8 variable region of light chain+constant region-protein	134	3B6 variable region of heavy chain+IgG1 constant region-nucleic acid

135	3B6 variable region of heavy chain+IgG1 constant region-protein
135		136	3B6 variable region of light chain+constant region-nucleic acid
137	3B6 variable region of light chain+constant region-protein	136
137	3B6 variable region of light chain+constant region-protein	138	3D11 variable region of heavy chain+IgG1 constant region-nucleic acid
139	3D11 variable region of heavy chain+IgG1 constant region-protein	138
139		140	3D11 variable region of light chain+constant region-nucleic acid
141	3D11 variable region of light chain+constant region-protein	140
141	3D11 variable region of light chain+constant region-protein	142	1D3 variable region of heavy chain+IgG1 constant region-nucleic acid
143	1D3 variable region of heavy chain+IgG1 constant region-protein	142
143		144	1D3 variable region of light chain+constant region-nucleic acid
145	1D3 variable region of light chain+constant region-protein	144
145	1D3 variable region of light chain+constant region-protein	146	1F3 variable region of heavy chain+IgG1 constant region-nucleic acid
147	1F3 variable region of heavy chain+IgG1 constant region-protein	146
147		148	1F3 variable region of light chain+constant region-nucleic acid
149	1F3 variable region of light chain+constant region-protein	148
149	1F3 variable region of light chain+constant region-protein	150	3A12 variable region of heavy chain+IgG1 constant region-nucleic acid
151	3A12 variable region of heavy chain+IgG1 constant region-protein	150
151		152	3A12 variable region of light chain+constant region-nucleic acid
153	3A12 variable region of light chain+constant region-protein	152

Embodiment 3-proteinic the preparation of multiple reorganization hHGF

Present embodiment has been described the clone and the expression of the multiple recombinant protein of the antibody that is used for characterizing embodiment 1 and embodiment 14 preparations.Particularly, present embodiment has been described following proteinic clone and expression, and described protein is: reorganization hHGF protein, contain the reorganization hHGF protein (G555E) that is replaced by L-glutamic acid at 555 position glycine, contain the reorganization hHGF protein (C561R) that is replaced by arginine at 561 position halfcystines, recombined small-mouse-people-mouse (mhm) mosaic HGF protein that contains the people V495-L585 HGF sequence that places mouse HGF sequence, the reorganization mhm mosaic HGF protein that contains the people I499-R566 HGF sequence that places mouse HGF sequence, and the reorganization mhm mosaic HGF protein that contains the people W507-L585 HGF sequence that places mouse HGF sequence.

Utilize the molecular engineering of standard to produce following expression construct, and verify the cDNA sequence of gained by dna sequencing.

a.hHGF-Fc

In the first round of PCR, produce 2 eclipsed PCR fragments, wherein between hHGF and hIgFc, introduced the Not I site and the 6xHis label of having encoded.In second takes turns, eclipsed PCR fragment as template with the amplification hHGF-his-IgFc.With the fragment of NheI and BamHI digestion gained, be cloned into again pcDNA5/FRT (Invitrogen, #35-3014) in.Then, amplification hHGF from Invitrogen clone sequence number: IOH29794 (people HGF cDNA).Find that the sequence of gained is corresponding to the sequence of the number of including that stores among the NCBI for NM_000601.4.

(1) 5 ' hHGF NheI primer

ACTGGCTAGCATGTGGGTGACCAAACTCCT(SEQ ID NO.102)

(2) 3 ' hHGF Notl His label primers

GTGATGGTGATGGTGATGGCGGCCGCATGACTGTGGTACCTTATATG (SEQ IDNO.103)

(3) 5 ' HisIgFc primers

ACTGGCGGCCGCCATCACCATCACCATCAC(SEQ ID NO.104)

(4) 3 ' IgFc BamHI primers

ACTGGGATCCTCACTATTTACCCGGGGACAG(SEQ ID NO.105)

B.hHGF-Fc G555E and hHGF-Fc C561R

Use QuikChange II XL rite-directed mutagenesis test kit (Stratagene), the specification sheets according to manufacturers provides produces hHGF-Fc mutant G555E and hHGF-Fc mutant C561R by rite-directed mutagenesis.

(1) hHGF-Fc (G555E) sense primer

CATGATGTCCACGAAAGAGGAGATGAG(SEQ ID NO.106)

(2) hHGF-Fc (G555E) antisense primer

CTCATCTCCTCTTTCGTGGACATCATG(SEQ ID NO.107)

(3) hHGF-Fc (C561R) sense primer

GGAAGAGGAGATGAGAAACGCAAACAGGTTCTCAATG(SEQ ID NO.108)

(4) hHGF-Fc (C561R) antisense primer

CATTGAGAACCTGTTTGCGTTTCTCATCTCCTCTTCC(SEQ ID NO.109)

C. mouse-people-mouse mosaic Fc

Mouse-people-mouse mosaic IgFc construct comprises the β chain amino acid Val495-Leu585 of mHGF α chain-hHGF, people HGF and is the terminal β chain of the mHGFC of 6xHis label and IgG-Fc thereafter.

People HGF cDNA from Invitrogen clone sequence number: IOH29794 (people HGF cDNA) amplification coding amino acid V495-L585.This sequence is corresponding to the sequence of the number of including that stores among the NCBI for NM_000601.4.Use is from the Super Script OneStepRT-PCR test kit (#10928-034) of Invitrogen, and the specification sheets that provides according to manufacturers is from total RNA (Clontech, # 636603) of Mouse Liver, by RT-PCR amplification mouse HGF sequence.The mHGF cDNA sequence of gained is corresponding to the sequence of the number of including that stores among the NCBI for D 10213.1.

Use eclipsed PCR primer to produce 3 fragments (being called as fragment 1,2 and 3 respectively), and in the successive round of pcr amplification, anneal.End product is cut with NheI and NotI enzyme, and be cloned among the pcDNA5/FRT IgGFc.

(1) is used for the primer of the fragment 1 of mHGF α chain 5 ' NheI

5′ATCGGCTAGCATGATGTGGGGGACCAAAC(SEQ ID NO.110)

3′GAATCCCATTTACAACCCGCAGTTGTTTTGTTTTGG(SEQ ID NO.111)

(2) be used for the primer of the fragment 2 of hHGF β chain aa V495-L585

5′CCAAAACAAAACAACTGCGGGTTGTAAATGGGATTC(SEQ ID NO.112)

3′CAGGATTGCAGGTCGAGCAAGCTTCATTAAAACCAGATCT(SEQ ID NO.113)

(3) be used for the primer of the fragment 3 of mHGF β chain C-terminal 3 ' NotI

5′AGATCTGGTTTTAATGAAGCTTGCTCGACCTGCAATCCTG(SEQ ID NO.114)

3′GTAATTTTGACATACAAGTTGTGCGGCCGCCATCACCATCACCATCAC(SEQ ID NO.115)

The chimeric structure of d.hHGF and mhm

Do not contain the carrier Fc-label, that encode hHGF and mhm mosaic (V495-L585), i.e. pcDNA5/FRT hHGF and pcDNA5/FRT-mhm mosaic (V495-L585) by the rite-directed mutagenesis generation.Use QuikChange II XL rite-directed mutagenesis test kit (Stratagene), the specification sheets that provides according to manufacturers is introduced terminator codon at 3 ' end of 6xHis-label.Mutant primer comprises primer 1 and primer 2, and wherein primer 1 is:

CATCACCATCACCATCACTAAGCGGGTCTGGTGCCACG(SEQ ID NO.116)，

Primer 2 is:

CGTGGCACCAGACCCGCTTAGTGATGGTGATGGTGATG(SEQ ID NO.117)。

In addition, use QuikChange II XL rite-directed mutagenesis test kit (Stratagene), the specification sheets according to manufacturers provides produces 2 other mhm mosaics by rite-directed mutagenesis by pcDNA5/FRT-mhm (V495-L585) construct.Wherein 1 mhm construct contains the I499-R556 zone of the hHGF between the mouse sequence.Another mhm construct contains the W507-L585 zone of the hHGF between the mouse sequence.

For mhm mosaic (I499-R556), utilize suitable oligonucleotide sequence, in template pcDNA5/FRT-mhm mosaic (V495-L585) construct, make following point mutation: D558E, C561R, V564I, V567I and M583L successively.For mhm mosaic (W507-L585), utilize suitable oligonucleotide sequence, in template pcDNA5/FRT-mhm mosaic (V495-L585) construct, in a step, introduce following point mutation: Q502R, N504T and I505V.

The proteinic nucleotide sequence of the hHGF-Fc of gained comprises signal sequence (Nucleotide 1-93) and prodomain (prodomain) (Nucleotide 94-162) shown in SEQ ID NO.118.The proteinic aminoacid sequence of hHGF-Fc is shown in SEQ ID NO.119.

The nucleotide sequence of coding mhm (the V495-L585)-Fc chimeric protein of gained comprises signal sequence (Nucleotide 1-96) and prodomain (Nucleotide 97-165) shown in SEQ ID NO.120.The aminoacid sequence of mhm (V495-L585)-Fc chimeric protein is shown in SEQ IDNO.121.

The protein sequence of the nucleotide sequence of coding mhm (V495-L585) construct of gained and qualification mhm (V495-L585) construct is respectively shown in SEQ ID NO.211 and 212.Nucleotide sequence shown in SEQ ID NO.211 comprises signal sequence (Nucleotide 1-96) and prodomain (Nucleotide 97-165), and the protein sequence shown in SEQ ID NO.212 comprises active protein sequence (not containing signal sequence or prodomain).The protein sequence of the nucleotide sequence of coding mhm (I499-R556) construct of gained and qualification mhm (I499-R556) construct is respectively shown in SEQ ID NO.213 and 214.Nucleotide sequence shown in SEQ ID NO.213 comprises signal sequence (Nucleotide 1-96) and prodomain (Nucleotide 97-165), and the protein sequence shown in SEQ ID NO.214 comprises active protein sequence (not containing signal sequence or prodomain).The protein sequence of the nucleotide sequence of the coding mhm (W507-L585) of gained and qualification mhm (W507-L585) is respectively shown in SEQ ID NO.215 and 216.Nucleotide sequence shown in SEQ ID NO.215 comprises signal sequence (Nucleotide 1-96) and prodomain (Nucleotide 97-165), and the protein sequence shown in SEQ ID NO.216 comprises active protein sequence (not containing signal sequence or prodomain).

E. protein expression

(1) cell cultures

At 37 ℃, 5% CO ₂And 100 μ g/mL Zeocin (Invitrogen, CatalogNo.R250-01) under the condition, at F12K substratum (ATCC, Catalog No.30-2004), 10% FCS (Invitrogen, Catalog No.10438026), 1% penicillin (10000units/mL)/Streptomycin sulphate (10,000 μ g/mL) cultivates CHO FlpIn cell (Invitrogen, Catalog No.R758-07) in (Invitrogen, Catalog No.15140-122).

(2) generation of stable CHO FlpIn clone

Using Lipofectamine 2000 (Invitrogen, Catalog No.11668-027), according to the specification sheets that manufacturers provides, is the pOG44:pcDNA5/FRT expression plasmid DNA transfection CHO FlpIn host cell of 9:1 with ratio.PcDNA5/FRT carrier/pOG44 that use is empty and independent pOG44 plasmid (Invitrogen, CatalogNo.35-3018) transfectional cell, in contrast.After the transfection 24 hours, cell separately, and after 48 hours, in this cell, add 0.5mg/mL hygromycin B (Sigma, Catalog No.H0654-SPEC).In F12K, 10%FCS, 1% penicillin/streptomycin, 0.5mg/mL hygromycin B, select polyclonal stable cell.

( 3) protein expression in stable CHO FlpIn clone

With about 2 x 10 ⁶Individual cell inoculation is in the flat board of 15cm, and at 37 ℃, 5% CO ₂In ratio be middle the cultivation 5-6 days of F12K (ATCC, Catalog No.30-2004)/DMEM high sugar (Invitrogen, Catalog No. 11995065) and 5% utmost point low IgG FCS (Invitrogen, # 16250-78) of 1:1 down.Collect supernatant liquor, and analyze the protein of gained by ELISA and surface plasma body resonant vibration.

The binding characteristic of embodiment 4-anti-hHGF monoclonal antibody

Some reorganization HGF protein of preparation can characterize by they abilities in conjunction with hHGF among the monoclonal antibody of embodiment 1 preparation and the embodiment 3,

Use BIAcore T100 device, analyze described antibody and fused protein that some are discussed by surface plasma body resonant vibration in embodiment 3, thereby assess their abilities in conjunction with HGF.Use amine coupling (BIAcore, Catalog No.BR-1000-50), the specification sheets that provides according to manufacturers, the coupling method by standard is fixed in every kind of antibody on the sensor chip (BIAcore, Catalog No.BR-1006-68) of carboxymethylated dextran CM5.

Under 25 ℃, use PBS (GIBCO, Catalog No.14040-133) agent is analyzed as runtime buffer, described PBS contains 0.05% tensio-active agent P20 (BIAcore, Catalog No.R-1000-54), 2mg/mL BSA (EMD, Catalog No.2930) and the sodium salt of 10mg/mLCM-dextran (Fluka, Catalog No.86524).The supernatant liquor that will contain the supernatant liquor of different HGF fused proteins or the empty carrier cells transfected of using by oneself is injected on every kind of antibody time remaining 3 minutes with the flow velocity of 30 μ L/min.When injection finished back 30 seconds, on baseline, measure the combination of gained with resonance units (RU).Will in conjunction with the people HGF (R﹠amp that is diluted in the runtime buffer agent; D Systems, Catalog No.294-HGN-025) relatively.By relatively with the combining of contrast surface, monitor and do not have specific combination, comprise on the wherein said contrast surface and use identical amine coupling method fixed mouse IgG (Rockland, Catalog No.010-0102).

The results are summarized in the table 3 of gained.

Table 3

Antibody	rhHGF(R&D Systems)	rmHGF(R&D Systems)	Mhm mosaic (V495-L585)	People HGF	G555E	C561R
Antibody	rhHGF(R&D Systems)	rmHGF(R&D Systems)	Mhm mosaic (V495-L585)	People HGF	G555E	C561R	1A3	In conjunction with	Debond	Debond	In conjunction with	In conjunction with	In conjunction with
1D3	In conjunction with	Debond	In conjunction with	In conjunction with	In conjunction with	In conjunction with	1A3	In conjunction with	Debond	Debond	In conjunction with	In conjunction with	In conjunction with
1D3	In conjunction with	Debond	In conjunction with	In conjunction with	In conjunction with	In conjunction with	1F3	In conjunction with	In conjunction with	In conjunction with	In conjunction with	In conjunction with	In conjunction with
2B8	In conjunction with	Debond	In conjunction with	In conjunction with	In conjunction with	In conjunction with	1F3	In conjunction with	In conjunction with	In conjunction with	In conjunction with	In conjunction with	In conjunction with
2B8	In conjunction with	Debond	In conjunction with	In conjunction with	In conjunction with	In conjunction with	2F8	In conjunction with	In conjunction with	Debond	In conjunction with	In conjunction with	In conjunction with
3A12	In conjunction with	Debond	Debond	In conjunction with	In conjunction with	In conjunction with	2F8	In conjunction with	In conjunction with	Debond	In conjunction with	In conjunction with	In conjunction with
3A12	In conjunction with	Debond	Debond	In conjunction with	In conjunction with	In conjunction with	3B6	In conjunction with	Debond	Debond	In conjunction with	In conjunction with	In conjunction with
3D11	In conjunction with	Debond	Debond	In conjunction with	In conjunction with	In conjunction with	3B6	In conjunction with	Debond	Debond	In conjunction with	In conjunction with	In conjunction with

Result in the table 3 shows that every kind of antibody all combines with the people HGF of rHGF and purifying.In addition, all antibody all combine with the hHGF that contains point mutation G555E and C561R.In general, all antibody except 1F3 and 2F8 do not combine with mouse HGF, show that antibody 1A3,1D3,2B8,3A12,3B6 and 3D11 are specifically in conjunction with people HGF.Antibody 1D3,1F3 and 2B8 are in conjunction with mouse-people-mouse mosaic, and other antibody are this mosaic of debond then.These results show that antibody 1D3 and 2B8 are at least in part in conjunction with the residue 495-585 of people HGF.Antibody 1A3,3A12,3B6 and 3D11 seem in conjunction with the part except residue 495-585 of people hHGF.Still uncertain at present why 2F8 debond mhm mosaic, and as if it in conjunction with hHGF and mHGF.

The monoclonal antibody of embodiment 5-anti-hHGF is in conjunction with the ability of reductive and non-reducing HGF

In the present embodiment, the monoclonal antibody of the anti-hHGF of embodiment 1 preparation is analyzed in conjunction with the ability of reductive and non-reducing HGF.

Assess the reactivity of serum with the reorganization hHGF of anti-HGF by immunoblotting.At 4-12% Bis-Tris 1.0mmX2D hole gel (well gel) (Invitrogen, Carlsbad, CA) on, to (the 8 μ g reorganization hHGF in the NuPAGE MOPS SDS runtime buffer agent (Invitrogen) of Invitrogen carries out classification containing or do not contain NuPAGE sample reduction buffer reagent.Then, use the method for standard that the fractionated protein transduction is moved on the nitrocellulose membrane.This nitrocellulose membrane is contained 0.1% with being dissolved in

(TBST) sealing of 5% skim-milk solution in the Tris buffer salt solution, then mounting to Mini Protean IIMulti-Screen instrument (BioRad) in so that further seal.

Film with antibody purified pin check gained on the Multi-Screen instrument.Antibody purified is diluted to 5 μ g/mL in the sealing buffer reagent.Then, nitrocellulose membrane is taken out from described instrument, and with the IgG antibody incubation of the anti-mouse of horseradish peroxidase-labeled.The results are summarized in the table 4 of gained, wherein numeral has been reacted the bonded degree ,-represent minimum combination (hardly in conjunction with or debond) and 3+ represents maximum combinations.

Table 4

Antibody	Reductive HGF (exposing 3-5 minute)	Non-reducing HGF (exposing 20 seconds)
Antibody	Reductive HGF (exposing 3-5 minute)	Non-reducing HGF (exposing 20 seconds)	1A3	2+	2+
1D3	2+	2+	1A3	2+	2+
1D3	2+	2+	1F3	2+	2+
2B8	-	1+	1F3	2+	2+
2B8	-	1+	2F8	2+	2+
3A12	-	2+	2F8	2+	2+
3A12	-	2+	3B6	3+	2+
3D11	-	3+	3B6	3+	2+

Data in the table 4 show that all antibody all combines with non-reducing rhHGF.In contrast be that monoclonal antibody 1A3,1D3,1F3,2F8 and 3B6 are in conjunction with reductive rhHGF, but antibody 2B8,3A12 and 3D11 debond reductive rhHGF.

Embodiment 6-binding affinity

Decide binding affinity and the interactional kinetics of the every kind of antibody and the hHGF of embodiment 1 preparation by surface plasmon resonance measurement.

Use amine coupling (BIAcore, Catalog No.BR-1000-50), the specification sheets that provides according to manufacturers, coupling method by standard is with the anti-mouse immuning ball protein (BIAcore of rabbit, Catalog No.BR-1005-14) is fixed on the sensor chip (BIAcore, Catalog No.BR-1006-68) of carboxymethylated dextran CM5.Under 25 ℃, use PBS (GIBCO, Catalog No.14040-133) agent is analyzed as runtime buffer, described PBS contains 0.05% tensio-active agent P20 (BIAcore, Catalog No.BR-1000-54), 2mg/mL BSA (EMD, Catalog No.2930) and the sodium salt of 10mg/mL CM-dextran (Fluka, Catalog No.86524).

With antibody capture is on the single mobile cell of 10 μ L/min at flow velocity.Be variable the inject time that is used for every kind of antibody, thereby make the antibody of catching about 20 RU in each circulation.With the speed of 60 μ L/min, in 2 minutes, on reference to surface (not having captive antibody) and active surface (antibody to be tested is arranged), inject buffer reagent successively or be diluted in HGF (R﹠amp in the runtime buffer agent; D Systems, Catalog No.294-HGN-025).According to concentration, monitor 15 or 90 minutes mutually to dissociating.Then, begin to use glycine-HCl of 10mM before another circulation, it is that the flow velocity with 60 μ L/min injects in 3 minutes that pH1.7 (BIAcore, Catalog No.BR-1003-54) makes described surface regeneration, wherein said glycine-HCl again.The concentration of the HGF that is tested is 0.46nM to 7.5nM.

Utilize the kinetic function of BIAevalutation software and the difference level of recommendation, determine kinetic parameter.Kinetic parameter (the k of every kind of antibody _a(association rate constant), k _d(dissociation rate constant) and K _D(equilibrium dissociation constant)) be summarized in the table 5.

Table 5

Antibody	ka/(1/Ms)	SE(ka)	kd(1/s)	SE(kd)	K _D(pM)	SD
Antibody	ka/(1/Ms)	SE(ka)	kd(1/s)	SE(kd)	K _D(pM)	SD	1A3	1.7 x 10 ⁶	7.3 x 10 ⁴	5.2 x 10 ^-5	8.4 x 10 ^-7	30.1	5.6
1D3	1.7 x 10 ⁶	3.1 x 10 ⁴	8.2 x 10 ^-5	1.7 x 10 ^-6	54.2	27.4	1A3	1.7 x 10 ⁶	7.3 x 10 ⁴	5.2 x 10 ^-5	8.4 x 10 ^-7	30.1	5.6
1D3	1.7 x 10 ⁶	3.1 x 10 ⁴	8.2 x 10 ^-5	1.7 x 10 ^-6	54.2	27.4	1F3	1.5 x 10 ⁶	5.0 x 10 ⁴	2.6 x 10 ^-5	6.6 x 10 ^-7	18.1	8.2
2B8	1.6 x 10 ⁶	2.9 x 10 ⁴	2.1 x 10 ^-5	1.4 x 10 ^-7	13.5	4.4	1F3	1.5 x 10 ⁶	5.0 x 10 ⁴	2.6 x 10 ^-5	6.6 x 10 ^-7	18.1	8.2
2B8	1.6 x 10 ⁶	2.9 x 10 ⁴	2.1 x 10 ^-5	1.4 x 10 ^-7	13.5	4.4	3A12	1.6 x 10 ⁶	3.7 x 10 ⁴	1.6 x 10 ^-4	1.6 x 10 ^-6	103.0	10.4
3B6	2.0 x 10 ⁶	6.5 x 10 ⁴	3.9 x 10 ^-5	3.2 x 10 ^-7	17.0	3.4	3A12	1.6 x 10 ⁶	3.7 x 10 ⁴	1.6 x 10 ^-4	1.6 x 10 ^-6	103.0	10.4

Data in the table 5 show, the K of described antibodies hHGF _DBe about 100pM or lower, about 50pM or lower or 20pM or lower.

The neutralization activity of the antibody of embodiment 7-anti-hHGF

In the present embodiment, characterize the antibody that embodiment 1 prepares with antibody (a) inhibition hHGF and c-Met bonded ability with the ability that (b) suppresses the BrdU introducing that HGF stimulates in the 4MBr-5 cell.

A.HGF-Met is in conjunction with inhibition analysis (neutralizing effect analysis)

Use the ELISA test antibody to suppress hHGF and c-Met bonded ability.

Particularly, under 4 ℃, be cushioned agent (15mM Na with the carbonate bag ₂CO ₃And 34mMNaHCO ₃, pH9.0) 100 μ L, the 6.25 μ g/mL HGF (R﹠amp in; D Systems, Catalog No.294-HGN-025) wrap by Wallac 96 hole DELFIA and analyze dull and stereotyped (Wallac company, Catalog No.AAAND-0001) 16 hours.Then, at room temperature will analyze dull and stereotyped non-skim-milk with 200 μ L 5% among the PBS sealed 1 hour.Join the 2nMc-Met (R﹠amp among the PBS that contains 5% non-skim-milk by the antibody (0.033-667nM, 3 times of serial dilutions) that is in the research with rising concentration; D Systems, Catalog No.358-MT/CF) in, this antibody of preparation in the plate that separates.The sample transfer of 100 μ L in each hole to analyzing in the flat board, and is incubated overnight under 4 ℃.Then, should analyze dull and stereotyped with PBS-0.1%Tween 20 washings 3 times, and at room temperature, with the antibody (R﹠amp of the biotin labeled anti-people c-Met of 2 μ g/mL in 100 μ L/ holes; D Systems, Catalog No.BAF358) incubation, the antibody of wherein said anti-people c-Met is middle preparation in the PBS that contains 5% non-skim-milk.

Then, gained is dull and stereotyped with PBS-0.1%Tween 20 washings 3 times, and at room temperature, and at DELFIA analysis buffer (Wallac, Streptavidin (Wallac, the Catalog No.1244-360) incubation of the Eu mark that dilutes with 1:1000 Catalog No.4002-0010) 1 hour.Gained is dull and stereotyped with DELFIA washings (Wallac, Catalog No.4010-0010) washing 3 times, and at room temperature, the DELFIA with 100 μ L/ holes under agitation condition strengthened solution (Wallac #4001-0010) incubation 15 minutes.

Use the europium tracer method, at Victor ³Read described flat board on the V instrument (Perkin Elmer).Calculate IC ₅₀Value, and be summarized in the table 6.

Table 6

Antibody	IC ₅₀(nM)	SD
Antibody	IC ₅₀(nM)	SD	1A3	5.65	0.91
1D3	4.43	2.27	1A3	5.65	0.91
1D3	4.43	2.27	1F3	6.57	0.28
2B8	5.57	1.19	1F3	6.57	0.28
2B8	5.57	1.19	2F8	5.36	0.88
3A12	5.26	2.11	2F8	5.36	0.88
3A12	5.26	2.11	3B6	-	-
3D11	5.66	2.75	3B6	-	-

These results show, all antibody except 3B6 (that is, 1D3,1A3,2B8,3A12,1F3,3D11 and 2F8) effectively in and the combining of HGF and c-Met.

B. in and the introducing of the BrdU that HGF stimulate to produce in the 4MBr-5 cell

The hHGF of 10 μ L 12.5nM is assigned in each hole of 96 hole tissue culture microwell plates (CostarCatalog No.3903).With 10 μ L concentration be 6667,2222,740,247,82,27,9.1,3.0,1.0 and the antibody of the serial dilution of 0.33nM add in each hole.Then at room temperature, with HGF-mixtures of antibodies incubation 30 minutes.To cultivate at F-12K (ATCC5 30-2004), 15% FBS (Gibco 10438-026), 30ng/mL EGF (SigmaE9644) and 1% penicillin/streptomycin (PS, Gibco Catalog No.15140-122) the monkey bronchial epithelial cell 4MBr-5 (ATCC in, CCL208) with trypsin GibcoCatalog No.25200-056) dissociate, and with 75,000 cell/mL is resuspended in and analyzes in the substratum (F-12K, 2.5% FBS, 1% PS), then 80 μ L cell suspending liquids is assigned in the HGF-mixtures of antibodies.

With the cell of gained at 37 ℃, 5% CO ₂Following incubation.After 48 hours, add the BrdU (Roche Catalog No.1669915) of 10 μ L, 100 μ M.After 72 hours, remove substratum, use the dry described flat board of hair dryer, and handle according to the specification sheets that manufacturers provides with BrdU ELISA (Roche Catalog No.1669915).

Use the flat reader of Synergy HT (Bio-Tek) that fluorescent signal is carried out quantitatively.Gained data and middle variable contrary flexure dose response (sigmoidal dose response) the curve match mutually of slope of GraphPad Prism (GraphPad Software), the equation of described curve is: y=floors+(top value-floors)/(1+10^ (log (EC50-x) * rate of curve)).In identical sample, each test all repeats 3 times at least, and average EC ₅₀Value is shown in Table 7.

Table 7

Antibody	IC ₅₀(nM)
Antibody	IC ₅₀(nM)	1A3	4.69
1D3	4.99	1A3	4.69
1D3	4.99	1F3	1.94
2B8	1.41	1F3	1.94
2B8	1.41	2F8	19.24
3A12	30.30	2F8	19.24
3A12	30.30	3B6	36.08
3D11	51.12	3B6	36.08

Result in the table 7 shows that all antibody (1A3,1D3,1F3,2B8,2F8,3A12,3B6 and 3D11) all suppresses HGF inductive 4MBr-5 cell proliferation.

The anti-diffusion activity of embodiment 8-anti-hHGF antibody

The antibody that present embodiment has been described embodiment 1 preparation suppresses the feature of the ability of HGF inductive diffusion activity.HGF has induced " diffusion " (mobility) of mdck cell (ATCC, Manassas, VA, Catalog No.CCL-34) bunch.

With mdck cell with every hole 4 x 10 ³The density of individual cell is inoculated in 96 hole Costar tissue culture plate (Corning Incorporated, Corning, NY3 Catalog No.3595) in, wherein MEM (the ATCC Manassas that comprises 10% foetal calf serum (InvitrogenCatalog No.10438026) and 1% penicillin-Streptomycin sulphate (Invitrogen Catalog No.15140122) of 80 μ L is equipped with in each hole, VA, CatalogNo.30-2003).Every kind of antibody to be studied is diluted to 6,667 nM in the MEM that contains 10% foetal calf serum and 1% penicillin-Streptomycin sulphate.Then, with every kind of different antibody diluent and contain 10% foetal calf serum and 1% penicillin-Streptomycin sulphate but the MEM that do not contain antibody respectively with isopyknic MEM and 100ng/ml HGF (R﹠amp that contains 10% foetal calf serum and 1% penicillin-Streptomycin sulphate; DSystems Catalog No.294-HGN-025) mixes.Under 25 ℃, with antibody/HGF diluent incubation 30 minutes.Every kind of antibody/HGF diluent of 20 μ L is joined respectively in each hole, make that final antibody concentration is 666.7nM, and final HGF concentration is 10ng/ml.Then, at 37 ℃, 5% CO ₂Down, with mdck cell incubation 24 hours.

Behind the incubation 24 hours, use the ice-cold PBS (Invitrogen Catalog No.14190144) of every hole 100 μ L to wash carefully 1 time mdck cell, and fix, under 25 ℃, shook 10 minutes simultaneously with the ice-cold methyl alcohol of every hole 100L.Then flat board is washed 1 time carefully with distilled water.The crystal violet solution that will be in each hole adds 100 μ L, it is by the ethanol formation in the distilled water of being dissolved in of 0.5% Viola crystallina (Sigma, St.Louis, MO, Catalog No.C3886) and 50%, and cell was shaken incubation 20 minutes under 25 ℃.

After will using crystal violet solution dyeing, the cell of gained is washed 3 times carefully with distilled water.Then, in each hole, add PBS to prevent sample drying.Use Leica DMIRB microscope (Leica Microsystems GmbH, Wetzler, Germany), DC500 photographic camera (Leica Microsystems GmbH, Wetzler, Germany) and MagnaFire 2.1C software (Optronics, Goleta, CA) pair cell imaging, and the diffusion levels of evaluation sample.The results are summarized in the table 8 of gained.

Table 8

-do not suppress

+++extremely strong is near suppressing fully

++ strongly inhibited

But the inhibition of+detection level

Result shown in the table 8 shows that antibody 2B8 more can suppress the diffusion of HGF inductive than other antibody.Antibody 1D3 and 3B6 demonstrate the inhibition of medium level; Antibody 1A3 demonstrates the inhibition that is lower than medium level; Antibody 1F3 and 2F8 demonstrate low-level inhibition; And antibody 3A12 and 3D11 produce inhibition hardly or detect less than inhibition.

The inhibition of the c-Met phosphorylation of embodiment 9-HGF is stimulated

The antibody that present embodiment has been described embodiment 1 preparation suppresses the feature of the ability of the c-Met phosphorylation that HGF stimulates in the PC-3 cell.HGF has induced the phosphorylation of Met in the PC-3 cell (ATCC No.CRL-1435).

With the PC-3 cell with every hole 4.5 x 10 ⁴The density of individual cell is inoculated in each hole of 96 hole Costar tissue culture plate (Corning Catalog No.3595), wherein F-12K (the ATCC that comprises 10% foetal calf serum (Invitrogen Catalog No.10438026) and 1% penicillin-Streptomycin sulphate (Invitrogen Catalog No.15140122) of 100 μ L is equipped with in each hole, Manassas, VA, Catalog No.30-2004).At 37 ℃, 5% CO ₂Down, remove substratum through after 24 hours, and with not containing serum but contain the F-12K rinsing cell 1 time of 1% penicillin-Streptomycin sulphate.Then with cell incubation 24 hours in the serum-free F-12K that contains 1% penicillin-Streptomycin sulphate of 100 μ L.

Following 10 kinds of different diluents: 6667nM, 2222nM, 741nM, 247nM, 82.3nM, 27.4nM, 9.1nM, 3.0nM, 1.0nM and the 0.3nM that in containing the serum-free F-12K of 1% penicillin-Streptomycin sulphate, prepare every kind of antibody to be studied.With every kind of antibody diluent and the serum-free F-12K that contains 1% penicillin-Streptomycin sulphate that do not contain antibody respectively with isopyknic serum-free F-12K and 500ng/mL HGF (R﹠amp that contains 1% penicillin-Streptomycin sulphate; D Systems Catalog No.294-HGN-025) mixes.Under 25 ℃, with the diluent incubation of these antibody/HGF 30 minutes.Obtain the HGF that ultimate density is 1.25nM thus.

Then, with the PC-3 cell with the serum-free F-12K rinsing 1 time that contains 1% penicillin-Streptomycin sulphate.Then in cell, add 70 μ L and contain the serum-free F-12K of 1% penicillin-Streptomycin sulphate, and then add 10 μ L and contain 10mM Na ₃VO ₄The serum-free F-12K that contains 1% penicillin-Streptomycin sulphate of (Sigma Catalog No.S6508).Then, with cell at 37 ℃, 5% CO ₂Following incubation 60 minutes.Behind the incubation, every kind of antibody/HGF of 20 μ L is added respectively in each hole, the ultimate density that obtains HGF is 50ng/mL, and the ultimate density of every kind of antibody is as follows: 666.7nM, 222.2nM, 74.1nM, 24.7nM, 8.23nM, 2.74nM, 0.91nM, 0.30nM, 0.10nM and 0.03nM.Then, with cell at 37 ℃, 5% CO ₂Following incubation 10 minutes is removed the mixture of substratum/antibody/HGF thereafter, and flat board is placed on ice.Then, contain 1mM Na with every hole 100 μ L ₃VO ₄Ice-cold PBS (InvitrogenCatalog No.14190144) rinsing cell 1 time.Then, with cell under 4 ℃, incubation is 30 minutes in the ice-cold cracking buffer reagent of every hole 100 μ L, described cracking buffer reagent is by 1% OmniPur Triton X-100 (MERCK KGaA, Darmstadt, Germany, Catalog No.9410), 50mM Tris-HCl pH8.0,100mM NaCl, 0.3mMNa ₃VO ₄, 1x protease inhibitor cocktail (Sigma Catalog No.P8340) and 1x inhibitors of phosphatases mixture 2 (Sigma Catalog No.5726) constitute.

Antibody (R﹠amp with biotin labeled anti-people HGF-R (c-met); D Systems CatalogNo.BAF 358) at the DELFIA analysis buffer (PerkinElmer that comprises 1% foetal calf serum (Sigma Catalog No.A2153), Turku, Finland, Catalog No.4002-0010) being diluted to concentration in is 2 μ g/mL, and this diluent of 50 μ L is joined in each hole of yellow Streptavidin micropore flat board (PerkinElmer Catalog No.AAAND-0005).Then, under 25 ℃, described flat board and antibody were shaken incubation 30 minutes.Behind the incubation, (PerkinElmer Catalog No.4010-0010) washs described flat board with the DELFIA washings, and every kind of different PC-3 cell lysate of 80 μ L added respectively in each hole of the Streptavidin micropore flat board through washing.

The Streptavidin micropore flat board that will contain the PC-3 cell lysate shook incubation 60 minutes under 25 ℃, then with the washing of DELFIA washings.100 μ L dilution is had before adding in the DELFIA analysis buffer that contains 1% foetal calf serum of 600ng/mLDELFIA Eu-N1P-Tyr-100 antibody (PerkinElmer Catalog No.AD0159) with PC-3 cell lysate incubation in the Streptavidin micropore flat board of washing.Should under 25 ℃, shake incubation 60 minutes by flat board.Described flat board is washed last 1 time with the DELFIA washings.Then, the DELFIA of 200 μ L is strengthened solution (PerkinElmer Catalog No.4001-0010) join in each hole of the Streptavidin micropore flat board of washing, and this plate was shaken incubation 5 minutes in the dark under 25 ℃.

Then, use the europium tracer method, go up measured signal at Victor3V instrument (PerkinElmer).(San Diego CA) calculates EC with contrary flexure dose response equation for GraphPad Software, Inc. to use Prism 4 for Windows ₅₀Value.

In nM as EC ₅₀The results are shown in Table 9 and sum up.

Table 9

Antibody	The mean value of 2 tests	Standard deviation
Antibody	The mean value of 2 tests	Standard deviation	1A3	0.684	0.242
1D3	0.984	0.129	1A3	0.684	0.242
1D3	0.984	0.129	1F3	1.19	1.01
2B8	0.287	0.104	1F3	1.19	1.01
2B8	0.287	0.104	2F8	1.39	2.12
3A12	2.00	0.553	2F8	1.39	2.12
3A12	2.00	0.553	3B6	1.01	1.11
3D11	2.28	N/A	3B6	1.01	1.11

Data in the table 9 show that 8 all antibody all are effective inhibitor of HGF inductive c-Met phosphorylation in the PC-3 cell.

Tumor suppression in the embodiment 10-U87MG xenograft models

Test mouse monoclonal antibody of the present invention suppresses the ability of tumor growth in the U87MG xenograft models.Under 37 ℃, containing 5% CO ₂With the amplification U87MG cell (ATCC) down of culture condition in the atmosphere of 95% air, use therein substratum comprises the Dulbecco ' s ModifiedEagle substratum (DMEM) that contains 10% foetal calf serum, 100 units/mL penicillin and 100 μ g/mL Streptomycin sulphates.By using cell pair cell on trypsinase-EDTA separation and Culture ware wall to carry out secondary cultivation and keeping.

Be used for collecting the approaching cell that converges by tryptic digestion, then with 50%Matrigel (BDBiosciences; Catalog no.356237) cell of the 5x106 in is subcutaneously injected in the zone of back upside between the shoulder blades of 7 week female ICR SCID mouse in age (Taconic Labs).The long diameter (L) of use kind of calliper tumour and short diameter (W) are (mm).Following calculating tumor size (vol.): volume (mm3)=L x W2/2.When tumor growth arrived about 200mm3, the mouse that will have tumour was divided into 5 groups at random, every group of 10 mouse.One group of mouse is wherein accepted PBS.Every group of mouse in other 4 groups accepted a kind of antibody among antibody 1A3,1D3,1F3 or the 2B8.The dosage of all antibody is the 1mg/kg body weight, and 2 times weekly, and by 5 dosage of peritoneal injection.The body weight of 2 tumor size of per week record and mouse.Utilize the inhibition of student t check analysis tumor growth.The results are summarized among Fig. 6 and the table 10.

Table 10

In 2B8 treatment group, obtained partly decline (referring to Fig. 6).In 1A3 treatment group and 1F3 treatment group, observe the significant growth-inhibiting of statistics (referring to table 10).It is 51% that the tumor growth of 1D3 suppresses, and its p value is 0.075.Do not observe tangible body weight loss.

The inhibition of tumour in embodiment 11-U118 xenograft models

Test antibody 1A3,1D3,1F3 and 2B8 suppress the ability of tumor growth in the U118 xenograft models.According among the embodiment 10 (the foregoing description) at the described increment of U87MG cell U118 cell (ATCC).

Embodiment 10 described Subcutaneous tumor models of setting up as mentioned, difference are that used mouse is the female NCr nude mice (Taconic) in 7 ages in week, and when tumor growth to about 80mm ³The time begin treatment.As in the U87MG model, the dosage of all antibody is the 1mg/kg body weight, and 2 times weekly, and by 4 dosage of peritoneal injection.The body weight of 2 tumor size of per week record and mouse.Utilize the inhibition of student t check analysis tumor growth.The results are summarized among Fig. 7 and the table 11.

Table 11

In 2B8 and 1A3 treatment group, observe the significant growth-inhibiting of statistics (referring to Fig. 7).Have the growth of tumor of appropriateness to suppress in 1F3 and 1D3 group, its p value is less than 0.05 (in this research, it is remarkable that this situation is defined as statistics) (referring to table 11).Do not observe tangible body weight loss.

The humanization of embodiment 12-mouse monoclonal antibody

Present embodiment has been described the humanization of virtuous mouse 2B8 antibody, and the feature of gained humanized antibody.Make mouse 2B8 variable region of heavy chain and variable region of light chain " humanization " by two kinds of methods.

A. the humanization method 1

In first method, according at people such as Hwang (2005) METHODS 36:35-42; People such as Tan (2002) J.IMMUNOL.169:1119-1125; And United States Patent (USP) sequence number 6,881, " the super humanization " described in 557 designs 3 humanized variable region of heavy chain and 2 humanized kappa variable region of light chain.

The Chothia regular structure type (Chothia canonical structural class) of determining each mouse 2B8 CDR according to length and the amino acid whose composition of CDR.According to the known ethnic group of describing in International Immunogentics Information System (IMGT) network address (can get on the imgt.cines.fr of World Wide Web andbiochem.unizh.ch/antibody/Sequences/index.html) is that the variable region identifies that with reference to allelotrope by the variable region of light chain of identical Chothia regular structure type and the ethnic group that variable region of heavy chain constitutes be the variable region.By calculating percentage identity or the similarity between the cdr amino acid residue, be variable region and the comparison of mouse 2B8 variable region with the ethnic group of these same structure types.Those ethnic groups that selection and mouse 2B8 CDR residue identity and/or similarity are the highest are the variable region, are used for CDR and transplant.Mouse 2B8 CDR residue being used to replace in mouse 2B8 CDR and ethnic group is corresponding race different between the CDR when being the variable region residue, and keeping ethnic group is the framework residue of variable region.Then, the carboxyl terminal that will the people J district the most similar joins the variable region of " super humanization " to 2B8 mouse J district.Then, signal sequence is joined the aminoterminal of the variable region of " super humanization ", and these aminoacid sequences are converted into nucleotide sequence.

Utilize gene to synthesize PCR method (people such as Young, (2004) NUCL.ACIDS RES.32:e59) make up complete variable region nucleotide sequence, and the Protocols in Molecular Biology of the standard of use is cloned into it in mammalian expression vector (based on pcDNA3.2 DEST (Invitrogen)) that contains constant IgG1 of people (Glm (17,1) allotype) or Kappa (Km (3) allotype (allelotrope 2)) district (downstream of variable region).4 all heavy chain IgG1 antibody (mosaic 2B8 and 3 humanized heavy chains (Hu2B8 Hv1-f.1, Hu2B8 Hv5 one a.1 and Hu2B8Hv5-51.1)) and the possible combination of all 3 kappa chain antibodies (mosaic 2B8 and 2 humanized light chains (Hu2B8 Kv1 1 and Hu2B8 Kv3-15.1)) are expressed, thereby produced 12 kinds of different antibody proteins.Then, measure combining of mosaic, chimeric/humanized antibody and humanized antibody and people HGF according to the following stated, and will the results are summarized among Fig. 8.Each of variable region that table has been listed heavy chain immunoglobulin and light chain immunoglobulin among the 12A may make up.

Table 12A

Variable region of heavy chain	Variable region of light chain
Variable region of heavy chain	Variable region of light chain	Mosaic 2B8 (SEQ ID NO:12)	Mosaic 2B8 (SEQ ID NO:14)
Mosaic 2B8 (SEQ ID NO:12)	Hu2B8 Kv1-39.1 (SEQ ID NO：173)	Mosaic 2B8 (SEQ ID NO:12)	Mosaic 2B8 (SEQ ID NO:14)
Mosaic 2B8 (SEQ ID NO:12)	Hu2B8 Kv1-39.1 (SEQ ID NO：173)	Mosaic 2B8 (SEQ ID NO:12)	Hu2B8 Kv3-15.1(SEQ ID NO：179)
Hu2B8 Hy1-f.1(SEQ ID NO：159)	Mosaic 2B8 (SEQ ID NO:14)	Mosaic 2B8 (SEQ ID NO:12)	Hu2B8 Kv3-15.1(SEQ ID NO：179)
Hu2B8 Hy1-f.1(SEQ ID NO：159)	Mosaic 2B8 (SEQ ID NO:14)	Hu2B8 Hy1-f.1(SEQ ID NO：159)	Hu2B8 Kv1-39.1(SEQ ID NO：173)
Hu2B8 Hy1-f.1(SEQ ID NO：159)	Hu2B8 Kv3-15.1(SEQ ID NO：179)	Hu2B8 Hy1-f.1(SEQ ID NO：159)	Hu2B8 Kv1-39.1(SEQ ID NO：173)
Hu2B8 Hy1-f.1(SEQ ID NO：159)	Hu2B8 Kv3-15.1(SEQ ID NO：179)	Hu2B8 Hv5-a.1(SEQ ID NO：165)	Mosaic 2B8 (SEQ ID NO:14)
Hu2B8 Hv5-a.1(SEQ ID NO：165)	Hu2B8 Kv1-39.1(SEQ ID NO：173)	Hu2B8 Hv5-a.1(SEQ ID NO：165)	Mosaic 2B8 (SEQ ID NO:14)
Hu2B8 Hv5-a.1(SEQ ID NO：165)	Hu2B8 Kv1-39.1(SEQ ID NO：173)	Hu2B8 Hv5-a.1(SEQ ID NO：165)	Hu2B8 Kv3-15.1(SEQ ID NO：179)
Hu2B8 Hv5-51.1(SEQ ID NO：169)	Mosaic 2B8 (SEQ ID NO:14)	Hu2B8 Hv5-a.1(SEQ ID NO：165)	Hu2B8 Kv3-15.1(SEQ ID NO：179)
Hu2B8 Hv5-51.1(SEQ ID NO：169)	Mosaic 2B8 (SEQ ID NO:14)	Hu2B8 Hv5-51.1(SEQ ID NO：169)	Hu2B8 Kv1-39.1(SEQ ID NO：173)
Hu2B8 Hv5-51.1(SEQ ID NO：169)	Hu2B8 Kv3-15.1(SEQ ID NO：179)	Hu2B8 Hv5-51.1(SEQ ID NO：169)	Hu2B8 Kv1-39.1(SEQ ID NO：173)

Table listed heavy chain immunoglobulin and light chain immunoglobulin among the 12B each may make up.

Table 12B

Heavy chain immunoglobulin	Light chain immunoglobulin
Heavy chain immunoglobulin	Light chain immunoglobulin	Mosaic 2B8 IgG1 (SEQ ID NO:155)	Mosaic 2B8 Kappa (Km (3)) (SEQ ID NO:157)
Mosaic 2B8 IgG1 (SEQ ID NO:155)	Hu2B8 Kv1-39.1+Kappa constant region (Km (3) allotype) (allelotrope 2) (SEQ ID NO:177)	Mosaic 2B8 IgG1 (SEQ ID NO:155)	Mosaic 2B8 Kappa (Km (3)) (SEQ ID NO:157)
Mosaic 2B8 IgG1 (SEQ ID NO:155)		Mosaic 2B8 IgG1 (SEQ ID NO:155)	Hu2B8 Kv3-15.1+Kappa constant region (Km (3) allotype) (allelotrope 2) (SEQ ID NO:181)
Hu2B8 Hv1-f.1+IgG1 constant region (Glm (17,1)) allotype (SEQ ID NO:163)	Mosaic 2B8 Kappa (Km (3)) (SEQ ID NO:157)	Mosaic 2B8 IgG1 (SEQ ID NO:155)
	Mosaic 2B8 Kappa (Km (3)) (SEQ ID NO:157)	Hu2B8 Hv1-f.1+IgG1 constant region (Glm (17,1)) allotype (SEQ ID NO:163)	Hu2B8 Kv1-39.1+Kappa constant region (Km (3) allotype) (allelotrope 2) (SEQ ID NO:177)
Hu2B8 Hv1-f.1+IgG1 constant region (Glm (17,1)) allotype (SEQ ID NO:163)	Hu2B8 Kv3-15.1+Kappa constant region (Km (3) allotype) (allelotrope 2) (SEQ ID NO:181)
		Hu2B8 Hv5-a.1+IgG1 constant region (Glm (17,1)) allotype (SEQ ID NO:167)	Mosaic 2B8 Kappa (Km (3)) (SEQ ID NO:157)
Hu2B8 Hv5-a.1+IgG1 constant region (Glm (17,1)) allotype (SEQ ID NO:167)	Hu2B8 Kv1-39.1+Kappa constant region (Km (3) allotype) (allelotrope 2) (SEQ ID NO:177)		Mosaic 2B8 Kappa (Km (3)) (SEQ ID NO:157)
		Hu2B8 Hv5-a.1+IgG1 constant region (Glm (17,1)) allotype (SEQ ID NO:167)	Hu2B8 Kv3-15.1+Kappa constant region (Km (3) allotype) (allelotrope 2) (SEQ ID NO:181)
Hu2B8 Hv5-51.1+IgG1 constant region (Glm (17,1)) allotype (SEQ ID NO:171)	Mosaic 2B8 Kappa (Km (3)) (SEQ ID NO:157)
	Mosaic 2B8 Kappa (Km (3)) (SEQ ID NO:157)	Hu2B8 Hv5-51.1+IgG1 constant region (Glm (17,1)) allotype (SEQ ID NO:171)	Hu2B8 Kv1-39.1+Kappa constant region (Km (3) allotype) (allelotrope 2) (SEQ ID NO:177)
Hu2B8 Hv5-51.1+IgG1 constant region (Glm (17,1)) allotype (SEQ ID NO:171)	Hu2B8 Kv3-15.1+Kappa constant region (Km (3) allotype) (allelotrope 2) (SEQ ID NO:181)

Two kinds of possible antibody construction designs that contain total length heavy chain immunoglobulin and light chain (containing humanized variable region) are as follows:

Sh2B8-9 (Glm (17,1))=hu2B8 Hv5-51.1 (+IgG1 constant region (Glm (17,1)

Allotype) (SEQ ID NO.171)+hu2B8 Kv1-39.1 (+

Kappa constant region (Km (3) allotype (allelotrope 2)))

(SEQ ID NO.177)；

Sh2B8-12 (Glm (17,1))=hu2B8 Hv5-51.1 (+IgG1 constant region (Glm (17,1)

Allotype)) (SEQ ID NO.171)+hu2B8 Kv 3-15.1 (+

Kappa constant region (Km (3) allotype (allelotrope

2)))(SEQ ID NO.181)。

The encode nucleotide sequence of each humanized antibody and the protein sequence that limits each humanized antibody is summarized as follows.In this part, last Nucleotide of each variable region is first base by Next Password of variable region/constant region linker generation.This Nucleotide is included in the variable region, and this is because it is the part of exon.The aminoacid sequence of the constant region of below listing has comprised the translation product of this linker codon.

(1) coding total length mosaic 2B8 heavy chain (mouse variable region and human IgG1's constant region) (together Kind of special-shaped Glm (17,1)) nucleotide sequence (underscore be signal sequence)(SEQ ID NO.154)

1 atgggatgga gctatatcat cctctttttg gtagcaacag ctacagatgt ccactcccag

61 gtccaactgc agcagcctgg ggctgaactg gtgaagcctg ggacttcagt gaagctgtcc

121 tgcaaggctt ctggctacac cttcaccacc tactggatgc actgggtgaa tcagaggcct

181 ggacaaggcc ttgagtggat tggagagatt aatcctacca acggtcatac taactacaat

241 gagaagttca agagcaaggc cacactgact gtagacaaat cctccagcac agcctacatg

301 caactcagca gcctgacatc tgaggactct gcggtctatt actgtgcaag aaactatgtt

361 ggtagcatct ttgactactg gggccaaggc accactctca ccgtctcctc agcctccacc

421 aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg

481 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca

541 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac

601 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc

661 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt

721 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc

781 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca

841 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac

901 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac

961 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag

1021 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa

1081 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag

1141 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag

1201 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc

1261 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg

1321 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc

1381 ctctccctgt ctccgggtaa atga

(2) define total length mosaic 2B8 heavy chain (mosaic 2B8 IgG1 (Glm (17,1) Allotype) protein sequence (not having signal sequence)(SEQ ID NO.155)

1 qvqlqqpgae lvkpgtsvkl sckasgytft tywmhwvnqr pgqglewigeinptnghtny

61 nekfkskatl tvdkssstay mqlssltsed savyycarny vgsifdywgq gttltvssas

121 tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht fpavlqssgl

181 yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkkvepks cdkthtcppc papellggps

241 vflfppkpkd tlmisrtpev tcvvvdvshe dpevkfnwyv dgvevhnakt kpreeqynst

301 yrvvsvltvl hqdwlngkey kckvsnkalp apiektiska kgqprepqvy tlppsrdelt

361 knqvsltclv kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq

421 gnvfscsvmh ealhnhytqk slslspgk

(3) coding total length mosaic 2B8 light chain (mouse variable region and human constant region) (mosaic 2B8 Kappa (Km (3))) nucleotide sequence (underscore be signal sequence)(SEQID NO.156)

1 atggaatcac agactctggt cttcatatcc atactgctct ggttatatgg tgctgatggg

61 aacattgtaa tgacccaatc tcccaaatcc atgtccatgt cagtaggaga gagggtcacc

121 ttgagctgca aggccagtga gaatgtggtt tcttatgtat cctggtatca acagaaacca

181 gcgcagtctc ctaaactgctgatatacggg gcatccaacc ggaacactgg ggtccccggat

241 cgcttcacag gcagtggatc tgcaacagat ttcactctga ccatcagcag tgtgcgggct

301 gaagaccttg cagattatca ctgtgggcag agttacaact atccgtacac gttcggaggg

361 gggaccaggc tggaaataaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca

421 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat

481 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag

541 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg

601 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcacc catcagggc

661 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttga

(4) define the egg of total length mosaic 2B8 light chain (mosaic 2B8 Kappa (Km (3))) White matter sequence (not having signal sequence)(SEQ ID NO.157)

1 nivmtqspks msmsvgervt lsckasenvv syvswyqqkp aqspklliyg asnrntgvpd

61 rftgsgsatd ftltissvra edladyhcgq synypytfgg gtrleikrtv aapsvfifpp

121 sdeqlksgta svvcllnnfy preakvqwkv dnalqsgnsq esvteqdskd styslsstlt

181 l skadyekhk vyacevthqg lsspvtksfn rgec

(5) nucleotide sequence (underscore of coding humanization Hu2B8 Hv1-f.1 variable region of heavy chain Be signal sequence)(SEQ ID NO.158)

1 atggactgca cctggaggat cctcctcttg gtggcagca gctacaggcac ccacgccgag

61 gtccagctgg tacagtctgg ggctgaggtg aagaagcctg gggctacagt gaaaatctcc

121 tgcaaggttt ctggatacac cttcaccacctactggatgc actgggtgca acaggcccct

181 ggaaaagggc ttgagtggat gggagagatt aatcctacca acggtcatac taactacaat

241 gagaagttcc agggcagagt caccataacc gcggacacgt ctacagacac agcctacatg

301 gagctgagca gcctgagatc tgaggacacg gccgtgtatt actgtgcaac aaactatgtt

361 ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc ag

(6) protein sequence that defines humanization Hu2B8 Hv1-f.1 variable region of heavy chain (does not have Signal sequence is arranged)(SEQ ID NO.159)

1 evqlvqsgae vkkpgatvki sckvsgytft tywmhwvqqa pgkglewmge inptnghtny

61 nekfqgrvti tadtstdtay melsslrsed tavyycatny vgsifdywgq gtlvtvss

(7) nucleotide sequence of coding human IgG1's CH (Glm (17,1) allotype) (SEQ ID NO.160)

1 cctccaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc acctctgggg

61 gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg acggtgtcgt

121 ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta cagtcctcag

181 gactctactc cctcagcagcgtggtgaccg tgccctccag cagcttgggc acccagacct

241 acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaaa gttgagccca

301 aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc ctggggggac

361 cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc cggacccctg

421 aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt

481 acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag cagtacaaca

541 gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg aatggcaagg

601 agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa accatctcca

661 aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc cgggatgagc

721 tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc agcgacatcg

781 ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg cctcccgtgc

841 tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag agcaggtggc

901 agcaggggaa cgtcttctcatgctccgtga tgcatgaggc tctgcacaac cactacacgc

961 agaagagcct ctccctgtct ccgggtaaat ga

(8) define the protein of human IgG1's CH (Glm (17,1) allotype) Sequence(SEQ ID NO.161).First amino acid derives from the translation product of 2 Nucleotide of beginning of last Nucleotide of variable region and IgG1 sequence of heavy chain.

1 astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss

61 glyslssvvt vpssslgtqt yicnvnhkps ntkvdkkvep kscdkthtcp pcpapellgg

121 psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna ktkpreeqyn

181 styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsrde

241 ltknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw

301 qqgnvfscsv mhealhnhyt qkslslspgk

(9) coding total length heavy chain humanization Hu2B8 Hylf.1 variable region and people The nucleotide sequence of IgG1 (Glm (17,1) allotype) CH (underscore be the signal preface Row)(SEQ ID NO.162)

1 atggactgca cctggaggat cctcctcttggt ggcagcag ctacaggcac ccacgccgag

61 gtccagctgg tacagtctgg ggctgaggtg aagaagcctg gggctacagt gaaaatctcc

121 tgcaaggttt c tggatacac cttcaccacc tactggatgc actgggtgca acaggcccct

181 ggaaaagggc ttgagtggat gggagagatt aatcctacca acggtcatac taactacaat

241 gagaagttcc agggcagagt caccataacc gcggacacgt ctacagacac agcctacatg

301 gagctgagca gcctgagatc tgaggacacg gccgtgtatt actgtgcaac aaactatgtt

361 ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc agcctccacc

421 aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg

481 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca

541 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac

601 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc

661 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt

721 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc

781 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca

841 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac

901 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac

961 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag

1021 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa

1081 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag

1141 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag

1201 tgggagagca atgggcagcc ggagaacaactacaagacca cgcctcccgt gctggactcc

1261 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg

1321 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc

1381 ctctccctgt ctccgggtaa atga

(10) defining total length heavy chain humanization Hu2B8 Hv1f.1 variable region and human IgG1 weighs The protein sequence (not having signal sequence) of chain constant region (Glm (17,1) allotype)(SEQ IDNO.163)

1 evqlvqsgae vkkpgatvki sckvsgytft tywmhwvqqa pgkglewmge inptnghtny

61 nekfqgrvti tadtstdtay melsslrsed tavyycatny vgsifdywgq gtlvtvssas

121 tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht fpavlqssgl

181 yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkkvepks cdkthtcppc papellggps

241 vflfppkpkd tlmisrtpev tcvvvdvshe dpevkfnwyv dgvevhnakt kpreeqynst

301 yrvvsvltvl hqdwlngkey kckvsnkalp apiektiska kgqprepqvy tlppsrdelt

361 knqvsltclv kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq

421 gnvfscsvmh ealhnhytqk slslspgk

(11) nucleotide sequence (underscore of coding humanization Hu2B8 Hv5a.1 variable region of heavy chain Be signal sequence)(SEQ ID NO.164)

1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgaa

61 gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaggatctcc

121 tgtaagggtt ctggatacag ctttaccacc tactggatgc actgggtgcg ccagatgccc

181 gggaaaggcc tggagtggat gggggagatt aatcctacca acggtcatac taactacaat

241 ccgtccttcc aaggccacgt caccatctca gctgacaagt ccatcagcac tgcctacctg

301 cagtggagca gcctgaaggc ctcggacacc gccatgtatt actgtgcgag aaactatgtt

361 ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc ag

(12) protein sequence that defines humanization Hu2B8 Hv5a.1 variable region of heavy chain (does not have Signal sequence is arranged)(SEQ ID NO.165)

1 evqlvqsgae vkkpgeslri sckgsgysft tywmhwvrqm pgkglewmge inptnghtny

61 npsfqghvti sadksistay lqwsslkasd tamyycarny vgsifdywgq gtlvtvss

(13) coding total length humanization Hu2B8 Hv5a.1 variable region of heavy chain and human IgG1 The nucleotide sequence of (Glm (17,1) allotype) CH (underscore be the signal preface Row)(SEQ ID NO.166)

1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgaa

61 gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaggatctcc

121 tgtaagggtt ctggatacag ctttaccacc tactggatgc actgggtgcg ccagatgccc

181 gggaaaggcc tggagtggatgggggagatt aatcctacca acggtcatac taactacaat

241 ccgtccttcc aaggccacgt caccatctca gctgacaagt ccatcagcac tgcctacctg

301 cagtggagca gcctgaaggc ctcggacacc gccatgtatt actgtgcgag aaactatgtt

361 ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc agcctccacc

421 aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg

481 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca

541 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac

601 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc

661 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt

721 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc

781 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca

841 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac

901 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac

961 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag

1021 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa

1081 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag

1141 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag

1201 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc

1261 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg

1321 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc

1381 ctctccctgt ctccgggtaa atga

(14) define total length humanization Hu2B8 Hv5a.1 variable region of heavy chain and human IgG1 The protein sequence (not having signal sequence) of (Glm (17,1) allotype) CH(SEQID NO.167)

1 evqlvqsgae vkkpgeslri sckgsgysft tywmhwvrqm pgkglewmge inptnghtny

61 npsfqghvti sadksistay lqwsslkasd tamyycarny vgsifdywgq gtlvtvssas

121 tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht fpavlqssgl

181 yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkkvepks cdkthtcppc papellggps

241 vflfppkpkd tlmisrtpev tcvvvdvshe dpevkfnwyv dgvevhnakt kpreeqynst

301 yrvvsvltvl hqdwlngkey kckvsnkalp apiektiska kgqprepqvy tlppsradelt

361 knqvsltclv kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq

421 gnvfscsvmh ealhnhytqk slslspgk

(15) nucleotide sequence (underscore of coding humanization Hu2B8 Hv5-51.1 variable region of heavy chain Be signal sequence)(SEQ ID NO.168)

1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgaa

61 gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaagatctcc

121 tgtaagggtt ctggatacag ctttaccacc tactggatgc actgggtgcg ccagatgccc

181 gggaaaggcc tggagtggat gggggagatt aatcctacca acggtcatac taactacaat

241 ccgtccttcc aaggccaggt caccatctca gctgacaagt ccatcagcac tgcctacctg

301 cagtggagca gcctgaaggc ctcggacacc gccatgtatt actgtgcgag aaactatgtt

361 ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc ag

(16) define the protein preface of humanization Hu2B8 Hv5-51.1 weight chain variabl area sequence Row (not having signal sequence)(SEQ ID NO.169)

1 evqlvqsgae vkkpgeslki sckgsgysft tywmhwvrqm pgkglewmge inptnghtny

61 npsfqgqvti sadksistay lqwsslkasd tamyycarny vgsifdywgq gtlvtvss

(17) coding total length humanization Hu2B8Hv5-51.1 variable region of heavy chain and human IgG1 The nucleotide sequence of (Glm (17,1) allotype) CH (underscore be the signal preface Row)(SEQ ID NO.170)

1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgaa

61 gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaagatctcc

121 tgtaagggtt ctggatacag ctttaccacc tactggatgc actgggtgcg ccagatgccc

181 gggaaaggcc tggagtggat gggggagatt aatcctacca acggtcatac taactacaat

241 ccgtccttcc aaggccaggt caccatctca gctgacaagt ccatcagcac tgcctacctg

301 cagtggagca gcctgaaggc ctcggacacc gccatgtatt actgtgcgag aaactatgtt

361 ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc agcctccacc

421 aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg

481 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca

541 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac

601 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc

661 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt

721 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc

781 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca

841 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac

901 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac

961 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag

1021 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa

1081 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag

1141 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag

1201 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc

1261 gacggctcct tcttcctctacagcaagctc accgtggaca agagcaggtg gcagcagggg

1321 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc

1381 ctctccctgt ctccgggtaa atga

(18) define total length humanization Hu2B8 Hv5-51.1 variable region of heavy chain and human IgG1 The protein sequence (not having signal sequence) of (Glm (17,1) allotype) CH(SEQID NO.171)

1 evqlvqsgae vkkpgeslki sckgsgysft tywmhwvrqm pgkglewmge inptnghtny

61 npsfqgqvti sadksistay lqwsslkasd tamyycarny vgsifdywgq gtlvtvssas

121 tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht fpavlqssgl

181 yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkkvepks cdkthtcppc papellggps

241 vflfppkpkd tlmisrtpev tcvvvdvshe dpevkfnwyv dgvevhnakt kpreeqynst

301 yrvvsvltvl hqdwlngkey kckvsnkalp apiektiska kgqprepqvy tlppsrdelt

361 knqvsltclv kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq

421 gnvfscsvmh ealhnhytqk slslspgk

(19) nucleotide sequence of coding humanization Hu2B8 Kv1-39.1 Kappa chain variable region (down What rule is signal sequence)(SEQ ID NO.172).Two possible initial ATG illustrate with bigger letters.

1 ATGgacATGa gggtccccgc tcagctcctg gggctcctgc tactctggct ccgaggtgcc

61 agatgtgaca tccagatgac ccagtctcca tcctccctgt ctgcatctgt aggagacaga

121 gtcaccatca cttgcaaggc cagtgagaat gtggtttctt atgtatcctg gtatcagcag

181 aaaccaggga aagcccctaa gctcctgatc tatggggcat ccaaccggaa cactggggtc

241 ccatcaaggt tcagtggcag tggatctggg acagatttca ctctcaccat cagcagtctg

301 caacctgaag attttgcaac ttactactgt gggcagagtt acaactatcc gtacacgttt

361 ggccagggga ccaagctgga gatcaaac

(20) define the protein of humanization Hu2B8 Kv1-39.1Kappa chain variable regionSequence (not having signal sequence) (SEQ ID NO.173)

1 diqmtqspss lsasvgdrvt itckasenvv syvswyqqkp gkapklliyg asnrntgvps

61 rfsgsgsgtd ftltisslqp edfatyycgq synypytfgq gtkleik

(21) nuclear of coding people's Kappa chain constant region (Km (3) allotype) (allelotrope 2) Acid sequence(SEQ ID NO.174)

1 gaactgtggc tgcaccatct gtcttcatct tcccgccatc tgatgagcag ttgaaatctg

61 gaactgcctc tgttgtgtgc ctgctgaata acttctatcc cagagaggcc aaagtacagt

121 ggaaggtgga taacgccctc caatcgggta actcccagga gagtgtcaca gagcaggaca

181 gcaaggacag cacctacagc ctcagcagca ccctgacgct gagcaaagca gactacgaga

241 aacacaaagt ctacgcctgc gaagtcaccc atcagggcct gagctcgccc gtcacaaaga

301 gcttcaacag gggagagtgt tga

(22) define people Kappa chain constant region (Km (3) allotype) (allelotrope 2) Protein sequence(SEQ ID NO.175).First amino acid derives from the translation product of two Nucleotide of beginning of last Nucleotide of variable region and Kappa sequence of light chain.

1 rtvaapsvfi fppsdeqlks gtasvvclln nfypreakvq wkvdnalqsg nsqesvteqd

61 skdstyslss tltlskadye khkvyacevt hqglsspvtk sfnrgec

(23) coding total length humanization Hu2B8 KvI-39.1 variable region of light chain and people Kappa chain The nucleotide sequence of constant region (Km (3) allotype) (allelotrope 2) (underscore be the signal preface Row)(SEQ ID NO.176)

1 atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggct ccgaggtgcc

61 agatgtgaca tccagatgac ccagtctcca tcctccctgt ctgcatctgt aggagacaga

121 gtcaccatca cttgcaaggc cagtgagaat gtggtttctt atgtatcctg gtatcagcag

181 aaaccaggga aagcccctaa gctcctgatc tatggggcat ccaaccggaa cactggggtc

241 ccatcaaggt tcagtggcag tggatctggg acagatttca ctctcaccat cagcagtctg

301 caacctgaag attttgcaac ttactactgt gggcagagtt acaactatcc gtacacgttt

361 ggccagggga ccaagctgga gatcaaacga actgtggctg caccatctgt cttcatcttc

421 ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac

481 ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac

541 tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc

601 ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat

661 cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgttg a

(24) define total length humanization Hu2B8 KvI-39.1 variable region of light chain and people Kappa The protein sequence of chain constant region (Km (3) allotype) (allelotrope 1)(SEQ IDNO.177)

1 diqmtqspss lsasvgdrvt itckasenvv syvswyqqkp gkapklliyg asnrntgvps

61 rfsgsgsgtd ftltisslqp edfatyycgq synypytfgq gtkleikrtv aapsvfifpp

121 sdeqlksgta svvcllnnfy preakvqwkv dnalgsgnsq esvteqdskd styslsstlt

181 lskadyekhk vyacevthqg lsspvtksfn rgec

(25) nucleotide sequence of coding humanization Hu2B8Kv 3-15.1 variable region of light chain is (following stroke Line be signal sequence)(SEQ ID NO.178)

1 atggaagccc cagcgcagct tctcttcctcct gctactct ggctcccaga taccactgga

61 gaaatagtga tgacgcagtc tccagccacc ctgtctgtgtctccagggga aagagccacc

121 ctctcctgca aggccagtga gaatgtggtt tcttatgtat cctggtacca gcagaaacct

181 ggccaggctc ccaggctcct catctatggg gcatccaacc ggaacactggtatcccagcc

241 aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct

301 gaagattttg cagtttatta ctgtgggcag agttacaact atccgtacac gtttggccag

361 gggaccaagc tggagatcaa ac

(26) define the protein sequence of humanization Hu2B8 Kv3-15.1 variable region of light chain (not having signal sequence)(SEQ ID NO.179)

1 eivmtqspat lsvspgerat lsckasenvv syvswyqqkp gqaprlliyg asnrntgipa

61 rfsgsgsgte ftltisslqs edfavyycgq synypytfgq gtkleik

(27) coding total length humanization Hu2B8Kv 3-15.1 variable region of light chain and people Kappa chain The nucleic acid of constant region (Km (3) allotype) (allelotrope 2) (underscore be signal sequence)(SEQ ID NO.180)

1 atggaagccc cagcgcagct tctcttcctc ctgctactct ggctcccaga taccactgga

61 gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc

121 ctctcctgca aggccagtga gaatgtggtt tcttatgtat cctggtacca gcagaaacct

181 ggccaggctc ccaggctcct catctatggg gcatccaacc ggaacactgg tatcccagcc

241 aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct

301 gaagattttg cagtttatta ctgtgggcag agttacaact atccgtacac gtttggccag

361 gggaccaagc tggagatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca

421 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat

481 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag

541 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg

601 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc

661 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttga

(28) define humanization Hu2B8 Kv3-15.1 variable region of light chain and people Kappa chain perseverance The protein sequence in fixed district (Km (3) allotype) (allelotrope 2) (does not have the signal preface Row)(SEQ ID NO.181)

1 eivmtqspat lsvspgerat lsckasenvv syvswyqqkp gqaprlliyg asnrntgipa

61 rfsgsgsgte ftltisslqs edfavyycgq synypytfgq gtkleikrtv aapsvfifpp

121 sdeqlksgta svvcllnnfy preakvqwkv dnalqsgnsq esvteqdskd styslsstlt

181 lskadyekhk vyacevthqg lsspvtksfn rgec

For convenience, table 13 provides the concordance list of the corresponding relation between those sequences of listing in the full length sequence of the antibody of discussing in this part and the sequence table.

Table 13

SEQ ID NO.	Protein or nucleic acid
SEQ ID NO.	Protein or nucleic acid	154	Mosaic 2B8 IgG1 (Glm (17,1))-nucleic acid
155	Mosaic 2B8 IgG1 (Glm (17,1))-protein	154	Mosaic 2B8 IgG1 (Glm (17,1))-nucleic acid
155	Mosaic 2B8 IgG1 (Glm (17,1))-protein	156	Mosaic 2B8 Kappa (Km (3))-nucleic acid
157	Mosaic 2B8 Kappa (Km (3))-protein	156	Mosaic 2B8 Kappa (Km (3))-nucleic acid
157	Mosaic 2B8 Kappa (Km (3))-protein	158	Hu2B8 Hv1f.1 variable region of heavy chain-nucleic acid
159	Hu2B8 Hv1f.1 variable region of heavy chain-protein	158	Hu2B8 Hv1f.1 variable region of heavy chain-nucleic acid
159	Hu2B8 Hv1f.1 variable region of heavy chain-protein	160	Human IgG1's CH (Glm (17,1)) allotype-nucleic acid
161	Human IgG1's CH (Glm (17,1)) allotype-protein	160	Human IgG1's CH (Glm (17,1)) allotype-nucleic acid
161	Human IgG1's CH (Glm (17,1)) allotype-protein	162	Hu2B8 Hv1f.1+IgG1 constant region (Glm (17,1) allotype-nucleic acid
163	Hu2B8 Hv1f.1+IgG1 constant region (Glm (17,1) allotype-protein	162
163		164	Hu2B8 Hv5a.1 variable region of heavy chain-nucleic acid
165	Hu2B8 Hv5a.1 variable region of heavy chain-protein	164	Hu2B8 Hv5a.1 variable region of heavy chain-nucleic acid
165	Hu2B8 Hv5a.1 variable region of heavy chain-protein	166	Hu2B8 Hv5a.1+IgG1 constant region (Glm (17,1) allotype)-nucleic acid
167	Hu2B8 Hv5a.1+IgG1 constant region (Glm (17,1) allotype)-protein	166
167		168	Hu2B8 Hv5-51.1 variable region of heavy chain-nucleic acid

169	Hu2B8 Hv5-51.1 variable region of heavy chain-protein
169	Hu2B8 Hv5-51.1 variable region of heavy chain-protein	170	Hu2B8 Hv5-51.1+IgG1 constant region (Glm (17,1) allotype)-nucleic acid
171	Hu2B8 Hv5-51.1+IgG1 constant region (Glm (17,1) allotype)-protein	170
171		172	Hu2B8 Kv1-39.1 Kappa chain variable region-nucleic acid
173	Hu2B8 Kv1-39.1 Kappa chain variable region-protein	172	Hu2B8 Kv1-39.1 Kappa chain variable region-nucleic acid
173	Hu2B8 Kv1-39.1 Kappa chain variable region-protein	174	People Kappa chain constant region (Km (3) allotype) (allelotrope 2)-nucleic acid
175	People Kappa chain constant region (Km (3) allotype) (allelotrope 2)-protein	174
175		176	Hu2B8 Kv1-39.1+Kappa constant region (Km (3) allotype) (allelotrope 2)-nucleic acid
177	Hu2B8 Kv1-39.1+Kappa constant region (Km (3) allotype) (allelotrope 2)-protein	176
177		178	Hu2B8 Kv3-15.1 variable region of light chain-nucleic acid
179	Hu2B8 Kv3-15.1 variable region of light chain-protein	178	Hu2B8 Kv3-15.1 variable region of light chain-nucleic acid
179	Hu2B8 Kv3-15.1 variable region of light chain-protein	180	Hu2B8 Kv3-15.1+Kappa constant region (Km (3) allotype) (allelotrope 2)-nucleic acid
1181	Hu2B8 Kv3-15.1+Kappa constant region (Km (3) allotype) (allelotrope 2)-protein	180

B. the humanization method 2

The second kind of humanization method that is used to reduce mouse 2B8 antibody mediated immunity originality is based on the method described in people such as Studnicka (1994) the PROTEIN ENG.7:805-814.The heavy chain and the kappa chain ethnic group that identify with the identity the highest (on amino acid whose level) of the variable region of heavy chain of mouse 2B8 and kappa chain variable region are the variable region.According to the residue variable effect in conjunction with or immunogenic possible risk, will be between mouse and people different residue conversion adults' sequence.At variable region of heavy chain (produce LR2B8HC) and kappa variable region (generation LR2B8LC), low-risk residue (that is, may not influence antigenic combination when it changes but also can reduce the immunogenic residue of potential) is become people's amino acid.In addition, in variable region of heavy chain (producing LRMR2B8HC) and kappa variable region (producing LRMR2B8LC), the residue (that is, might influence antigenic combination slightly when it changes and still also can reduce the immunogenic residue of potential) of low risk and medium risk is become people's amino acid.Human IgG1's CH (Glm (3) allotype (allelotrope 1)) is joined the C-terminal of two people's variable region of heavy chain through transforming, and people Kappa constant region (Km (3) allotype (allelotrope 1)) joined the C-terminals of two people's variable region of light chain through transforming, produce four people's antibody chains thus through transforming.At first, by gene synthetic method synthetic variable region nucleotide sequence, then this sequence is joined in the human constant region sequence.These people's antibody clonings through transforming in mammalian proteins matter expression vector, are then added the combination marking protein of light chain with four kinds of possible heavy chains.As described below, use technical measurement mosaic, mosaic/humanized antibody or the humanized antibody of routine and combining of people HGF.

The encode nucleotide sequence of each humanized antibody and the protein sequence that limits each humanized antibody is summarized as follows.In this part, last Nucleotide of each variable region is first base by variable region/Next Password that the constant region linker is produced.This Nucleotide is included in the variable region, and this is because it is the part of exon.Below the aminoacid sequence of listed constant region comprise the translation product of this linker codon.

(1) nucleotide sequence of coding humanization LR2B8HC variable region of heavy chain (underscore be signal Sequence)(SEQ ID NO.182)

1 atgggctggt catatattat tctctttctt gttgctaccg ctaccgatgt gcactctcaa

61 gtccaactcg tacaaccagg cgctgaagtc gtaaaacccg gaacatctgt taaactctca

121 tgcaaagcct caggatacac tttcacaact tactggatgc attgggtcaa tcaagccccc

181 ggacaaggcc tcgaatggat tggcgaaatt aacccaacta acggacatac taattataat

241 gaaaaattta agggcaaagc tacactcacc gtcgataaat caacctctac agcttatatg

301 gaactttcat ccctgagatc agaagataca gccgtctact attgcgccag aaactacgta

361 ggatcaatat tcgattactg gggtcaaggc actctcctca cagtcagctc ag

(2) protein sequence that defines humanization LR2B 8HC variable region of heavy chain (does not have the signal preface Row)(SEQ ID NO.183)

1 qvqlvqpgae vvkpgtsvkl sckasgytft tywmhwvnqa pgqglewige inptnghtny

61 nekfkgkatl tvdkststay melsslrsed tavyycarny vgsifdywgq gtlltvss

(3) coding human IgG1 CH (Glm (3) allotype) (allelotrope 1) Nucleotide sequence(SEQ ID NO.184)

1 ccagcacaaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc acctctgggg

61 gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg acggtgtcgt

121 ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta cagtcctcag

181 gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc acccagacct

241 acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaga gttgagccca

301 aatcttgtga caaaactcac acatgtccac cgtgcccagc acctgaactc ctggggggac

361 cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc cggacccctg

421 aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt

481 acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag cagtacaaca

541 gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg aatggcaagg

601 agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa accatctcca

661 aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc cgggaggaga

721 tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc agcgacatcg

781 ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg cctcccgtgc

841 tggactccga cggctccttc ttcctctata gcaagctcac cgtggacaag agcaggtggc

901 agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac cactacacgc

961 agaagagcct ctccctgtcc ccgggtaaat ga

(4) define human IgG1's CH (Glm (3) allotype) (allelotrope 1 or 2) protein sequence(SEQ ID NO.185).First amino acid derives from the translation product of two Nucleotide of beginning of last Nucleotide of variable region and IgG1 sequence of heavy chain.

1 astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss

61 glyslssvvt vpssslgtqt yicnvnhkps ntkvdkrvep kscdkthtcp pcpapellgg

121 psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna ktkpreeqyn

181 styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsree

241 mtknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw

301 qqgnvfscsv mhealhnhyt qkslslspgk

(5) coding total length heavy chain humanization LR2B8HC variable region of heavy chain and human IgG1's heavy chain perseverance The nucleotide sequence in fixed district (Glm (3) allotype) (allelotrope 1) (underscore be the signal preface Row)(SEQ ID NO.186)

1 atgggctggt catatattat tctctttctt gttgctaccg ctaccgatgt gcactctcaa

61 gtccaactcg tacaaccagg cgctgaagtc gtaaaacccg gaacatctgt taaactctca

121 tgcaaagcct caggatacac tttcacaact tactggatgc attgggtcaa tcaagccccc

181 ggacaaggcc tcgaatggat tggcgaaatt aacccaacta acggacatact aattataat

241 gaaaaattta agggcaaagc tacactcacc gtcgataaat caacctctac agcttatatg

301 gaactttcat ccctgagatc agaagataca gccgtctact attgcgccag aaactacgta

361 ggatcaatat tcgattactg gggtcaaggc actctcctca cagtcagctc agccagcaca

421 aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg

481 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca

541 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac

601 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc

661 aacgtgaatc acaagcccag caacaccaag gtggacaaga gagttgagcc caaatcttgt

721 gacaaaactc acacatgtcc accgtgccca gcacctgaac tcctgggggg accgtcagtc

781 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca

841 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac

901 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac

961 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag

1021 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa

1081 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag

1141 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag

1201 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc

1261 gacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg

1321 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc

1381 ctctccctgt ccccgggtaa atga

(6) define total length heavy chain humanization LR2B8HC variable region of heavy chain and human IgG1's heavy chain The protein sequence of constant region (Glm (3) allotype) (allelotrope 1) (does not have the signal preface Row)(SEQ ID NO.187)

1 qvqlvqpgae vvkpgtsvkl sckasgytft tywmhwvnqa pgqglewige inptnghtny

61 nekfkgkatl tvdkststay melsslrsed tavyycarny vgaifdywgq gtlltvssas

121 tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht fpavlqssgl

181 yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkrvepks cdkthtcppc papellggps

241 vflfppkpkd tlmisrtpev tcvvvdvshe dpevkfnwyv dgvevhnakt kpreeqynst

301 yrvvsvltvl hqdwlngkey kckvsnkalp apiektiska kgqprepqvyt lppsreemt

361 knqvsltclv kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq

421 gnvfscsvmh ealhnhytqk slslspgk

(7) nucleotide sequence of coding humanization LRMR2B8HC variable region of heavy chain (underscore be letter Number sequence)(SEQ ID NO.188)

1 atgggttggt catatattat actctttctc gtagccaccg ccaccgacgt acactctcag

61 gttcaactcg tacaacccgg cgccgaagtc aagaaaccag gaacatcagt caaactctca

121 tgtaaagcaa gcggatacac ctttactact tattggatgc attgggtaag acaagccccc

181 ggacaaggac tcgaatggat aggcgaaata aatcccacta atggacatac aaattataat

241 caaaaatttc aaggacgcgc tacactcacc gtcgataaat caacctcaac cgcatacatg

301 gaactcagct ccctccgatc cgaagacact gccgtttatt attgtgccag aaactatgta

361 ggatctattt tcgattactg gggacaagga acacttctca ccgtaagctc ag

(8) protein sequence that defines humanization LRMR2B8HC variable region of heavy chain (is not believed Number sequence)(SEQ ID NO.189)

1 qvqlvqpgae vkkpgtsvkl sckasgytft tywmhwvrqa pgqglewige inptnghtny

61 nqkfqgratl tvdkststay melsslrsed tavyycarny vgsifdywgq gtlltvss

(9) coding total length heavy chain humanization LRMR2B8HC variable region of heavy chain and human IgG1's heavy chain The nucleotide sequence of constant region (Glm (3) allotype) (allelotrope 1) (underscore be signal Sequence)(SEQ ID NO.190)

1 atgggttggt catatattat actctttctc gtagccaccg ccaccgacgt acactctcag

61 gttcaactcg tacaacccgg cgccgaagtc aagaaaccag gaacatcagt caaactctca

121 tgtaaagcaa gcggatacac ctttactact tattggatgc attgggtaag acaagccccc

181 ggacaaggac tcgaatggat aggcgaaata aatcccacta atggacatac aaattataat

241 caaaaatttc aaggacgcgc tacactcacc gtcgataaat caacctcaac cgcatacatg

301 gaactcagct ccctccgatc cgaagacact gccgtttatt attgtgccag aaactatgta

361 ggatctattt tcgattactg gggacaagga acacttctca ccgtaagctc agccagcaca

421 aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg

481 gccctgggct gcctggtcaa ggacta cttc cccgaaccgg tgacggtgtc gtggaactca

541 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac

601 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc

661 aacgtgaatc acaagcccag caacaccaag gtggacaaga gagttgagcc caaatcttgt

721 gacaaaactc acacatgtcc accgtgccca gcacctgaac tcctgggggg accgtcagtc

781 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca

841 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac

901 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac

961 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag

1021 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa

1081 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag

1141 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag

1201 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc

1261 gacggctcct tcttcctcta tagc aagctc accgtggaca agagcaggtg gcagcagggg

1321 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc

1381 ctctccctgt ccccgggtaa atga

(10) defining total length heavy chain humanization LRMR2B8HC variable region of heavy chain and human IgG1 weighs The protein sequence of chain constant region (Glm (3) allotype) (allelotrope 1) (does not have the signal preface Row)(SEQ ID NO.191)

1 qvqlvqpgae vkkpgtsvkl sckasgytft tywmhwvrqa pgqglewige inptnghtny

61 nqkfqgratl tvdkststay melsslrsed tavyycamy vgsifdywgq gtlltvssas

12 ltkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht fpavlqssgl

181 yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkrvepks cdkthtcppc papellggps

241 vflfppkpkd tlmisrtpev tcvvvdvshe dpevkfnwyv dgvevhnakt kpreeqynst

301 yrvvsvltvl hqdwlngkey kckvsnkalp apiektiska kgqprepqvy tlppsreemt

361 knqvsltclv kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq

421 gnvfscsvmh ealhnhytqk slslspgk

(11) nucleotide sequence of coding humanization LR2B8LC variable region of light chain (underscore be letterNumber sequence) (SEQ ID NO.192)

1 atggaaagtc agacccttgt attcatctct attcttcttt ggttgtatgg agcagacggc

61 gacattgtga tgacccaatc ccccgatagt atggccatga gtgtaggaga aagagtcacc

121 cttaattgca aagcctccga aaatgtcgtt tcatatgtgt cttggtatca acaaaaaccc

181 ggccaatcac ccaaacttct catatacggc gcttcaaaca gaaacacagg cgttcccgac

241 agatttagtg gatccggatc agctacagat ttcaccctta ccatcagttc agttcaagca

301 gaagacgttg cagactatca ttgcggacaa tcttataact acccttacac attcggacaa

(12) protein sequence that defines humanization LR2B8LC variable region of light chain (does not have signal Sequence)(SEQ ID NO.193)

1 divmtqspds mamsvgervt lnckasenvv syvswyqqkp gqspklliyg asnrntgvpd

61 rfsgsgsatd ftltissvqa edvadyhcgq synypytfgq gtkleik

(13) nuclear of coding people's Kappa chain constant region (Km (3) allotype) (allelotrope 1) Acid sequence(SEQ ID NO.194)

1 gtacggtggc tgcaccatct gtcttcatct tcccgccatc tgatgagcag ttgaaatctg

61 gaactgcctc tgttgtgtgc ctgctgaata acttctatcc cagagaggcc aaagtacagt

121 ggaaggtgga taacgccctc caatcgggta actcccagga gagtgtcaca gagcaggaca

181 gcaaggacag catcctacagc ctcagcagca ccctgacgct gagcaaagca gactacgaga

241 aacacaaagt ctacgcctgc gaagtcaccc atcagggcct gagctcgccc gtcacaaaga

301 gcttcaacag gggagagtgt tag

(14) define people Kappa chain constant region (Km (3) allotype) (allelotrope 1) Protein sequence(SEQ ID NO.195).First amino acid derives from the translation product of two Nucleotide of beginning of last Nucleotide of variable region and Kappa sequence of light chain.

1 rtvaapsvfi fppsdeqlks gtasvvclln nfypreakvq wkvdnalqsg nsqesvteqd

61 skdstyslss tltlskadye khkvyacevt hqglsspvtk sfnrgec

(15) coding total length humanization LR2B8LC variable region of light chain and people Kappa chain constant region The nucleotide sequence of (Km (3) allotype) (allelotrope 1)(SEQ ID NO.196)

1 atggaaagtc agacccttgt attcatctct attcttcttt ggttgtatgg agcagacggc

61 gacattgtga tgacccaatc ccccgatagt atggccatga gtgtaggaga aagagtcacc

121 cttaattgca aagcctccga aaatgtcgtt tcatatgtgt cttggtatca acaaaaaccc

181 ggccaatcac ccaaacttct catatacggc gcttcaaaca gaaacacagg cgttcccgac

241 agatttagtg gatccggatc agctacagat ttcaccctta ccatcagttc agttcaagca

301 gaagacgttg cagactatca ttgcggacaa tcttataact acccttacac attcggacaa

361 ggaaccaaac tcgaaattaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca

421 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat

481 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag

541 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg

601 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc

661 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag

(16) coding humanization LR2B8LC variable region of light chain and people Kappa chain constant region (Km (3) Allotype) protein sequence of (allelotrope 1)(SEQ ID NO.197)

1 divmtqspds mamsvgervt lnckasenvv syvswyqqkp gqspklliyg asnrntgvpd

61 rfsgsgsatd ftltissvqa edvadyhcgq synypytfgq gtkleikrtv aapsvfifpp

121 sdeqlksgta svvcllnnfy preakvqwkv dnalqsgnsq esvteqdskd styslsstlt

181 lskadyekhk vyacevthqg lsspvtksfn rgec

(17) nucleotide sequence of coding humanization LRMR2B8LC variable region of light chain (underscore be Signal sequence)(SEQ ID NO.198)

1 atggaatccc aaacccttgt tttcatctct atccttctct ggctttatgg cgccgacgga

61 gacatcgtaa tgacacaatc ccctgactct cttgctatga gcttgggcga acgagtaaca

121 cttaactgca aagcatccga aaatgtcgta tcttacgtat cctggtatca gcaaaaacct

181 ggtcaaagtc ctaaacttct tatatatggt gcaagtaatc gtgaaagtgg cgtcccagac

241 agatttagcg gttcaggttc agcaactgac tttacactta caatttctag cgttcaggcc

301 gaagacgttg cagactatca ttgtggacaa tcttataact atccttatac tttcggacaa

361 ggcactaaac ttgaaattaa ac

(18) protein sequence that defines humanization LRMR2B8LC variable region of light chain (is not believed Number sequence)(SEQ ID NO.199)

1 divmtqspds lamslgervt lnckasenvv syvswyqqkp gqspkdlliyg asnresgvpd

61 rfsgsgsatd ftltissvqa edvadyhcgq synypytfgq gtkleik

(19) coding total length humanization LRMR2B8LC variable region of light chain and people Kappa chain constant region The nucleotide sequence of (Km (3) allotype) (allelotrope 1) (underscore be the signal preface Row)(SEQ ID NO.200)

1 atggaatccc aaacccttgt tttcatctct atccttctct ggctttatgg cgccgacgga

61 gacatcgtaa tgacacaatc ccctgactct cttgctatga gcttgggcga acgagtaaca

121 cttaactgca aagcatccga aaatgtcgta tcttacgtat cctggtatca gcaaaaacct

181 ggtcaaagtc ctaaacttct tatatatggt gcaagtaatc gtgaaagtgg cgtcccagac

241 agatttagcg gttcaggttc agcaactgac tttacactta caatttctag cgttcaggcc

301 gaagacgttg cagactatca ttgtggacaa tcttataact atccttatac tttcggacaa

361 ggcactaaac ttgaaattaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca

421 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat

481 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag

541 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg

601 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc

661 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag

(20) define total length humanization LRMR2B8LC variable region of light chain and people Kappa chain perseverance The protein sequence in fixed district (Km (3) allotype) (allelotrope 1)(SEQ ID NO.201)

1 divmtqspds lamslgervt lnckasenvv syvswyqqkp gqspklliyg asnresgvpd

61 rfsgsgsatd ftltissvqa edvadyhcgq synypytfgq gtkleikrtv aapsvfifpp

121 sdeqlksgta svvcllnnfy preakvqwkv dnalqsgnsq esvteqdskd styslsstlt

181lskadyekhk vyacevthqg lsspvtksfn rgec

For convenience, table 14 provides the concordance list of the corresponding relation between those sequences of listing in the full length sequence of the antibody of discussing in this part and the sequence table.

Table 14

SEQ ID NO.	Protein or nucleic acid
SEQ ID NO.	Protein or nucleic acid	182	LR2B8HC variable region of heavy chain-nucleic acid
183	LR2B8HC variable region of heavy chain-protein	182	LR2B8HC variable region of heavy chain-nucleic acid
183	LR2B8HC variable region of heavy chain-protein	184	Human IgG1's CH (Glm (3) allotype (allelotrope 1)-nucleic acid
185	Human IgG1's CH (Glm (3) allotype (allelotrope 1)-protein	184
185	Human IgG1's CH (Glm (3) allotype (allelotrope 1)-protein	186	LR2B8HC+IgG1 constant region (Glm (3) allotype (allelotrope 1)-nucleic acid
187	LR2B8HC+IgG1 constant region (Glm (3) allotype (allelotrope 1)-protein	186
187		188	LRMR2B8HC variable region of heavy chain-nucleic acid
189	LRMR2B8HC variable region of heavy chain-protein	188	LRMR2B8HC variable region of heavy chain-nucleic acid
189	LRMR2B8HC variable region of heavy chain-protein	190	LRMR2B8HC+IgG1 constant region (Glm (3) allotype (allelotrope 1)-nucleic acid
191	LRMR2B8HC+IgG1 constant region (Glm (3) allotype (allelotrope 1)-protein	190
191		192	LR2B8LC variable region of light chain-nucleic acid
193	LR2B8LC variable region of light chain-protein	192	LR2B8LC variable region of light chain-nucleic acid
193	LR2B8LC variable region of light chain-protein	194	People Kappa chain constant region (Km (3) allotype) (allelotrope 1)-nucleic acid
195	People Kappa chain constant region (Km (3) allotype) (allelotrope 1)-protein	194
195		196	LR2B8LC+Kappa constant region (Km (3) allotype) (allelotrope 1)-nucleic acid
197	LR2B8LC+Kappa constant region (Km (3) allotype) (allelotrope 1)-egg	196

	White matter
	White matter	198	LRMR2B8LC variable region of light chain-nucleic acid
199	LRMR2B8LC variable region of light chain-protein	198	LRMR2B8LC variable region of light chain-nucleic acid
199	LRMR2B8LC variable region of light chain-protein	200	LRMR2B8LC+Kappa constant region (Km (3) allotype) (allelotrope 1)-nucleic acid
201	LRMR2B8LC+Kappa constant region (Km (3) allotype) (allelotrope 1)-protein	200

Table 15 has been summed up the heavy chain CDR sequence (Kabat definition) by the humanization 2B8 antibody of the humanization method 1 of the present embodiment of describing hereinbefore and 2 preparations of humanization method.

Table 15

Antibody	CDR1	CDR2	CDR3	The total length variable region of heavy chain
Antibody	CDR1	CDR2	CDR3	The total length variable region of heavy chain	Mouse 2B8 heavy chain	TYWMH (SEQ ID NO：15)	EINPTNGHTNYNEKFKS (SEQ ID NO：16)	NYVGSIFDY (SEQ ID NO：17)	SEQ ID NO：12
Hu2B8 Hv1f.1	TYWMH (SEQ ID NO：15)	EINPTNGHTNYNEKFQG (SEQ ID NO：202)	NYVGSIFDY (SEQ ID NO：17)	SEQ ID NO：159	Mouse 2B8 heavy chain	TYWMH (SEQ ID NO：15)	EINPTNGHTNYNEKFKS (SEQ ID NO：16)	NYVGSIFDY (SEQ ID NO：17)	SEQ ID NO：12
Hu2B8 Hv1f.1	TYWMH (SEQ ID NO：15)	EINPTNGHTNYNEKFQG (SEQ ID NO：202)	NYVGSIFDY (SEQ ID NO：17)	SEQ ID NO：159	Hu2B8 Hv5a.1	TYWMH (SEQ ID NO：15)	EINPTNGHTNYNPSFQG (SEQ ID NO：203)	NYVGSIFDY (SEQ ID NO：17)	SEQ ID NO：165
Hu2B8 Hv5-51.1	TYWMH (SEQ ID NO：15)	EINPTNGHTNYNPSFQG (SEQ ID NO：203)	NYVGSIFDY (SEQ ID NO：17)	SEQ ID NO：169	Hu2B8 Hv5a.1	TYWMH (SEQ ID NO：15)	EINPTNGHTNYNPSFQG (SEQ ID NO：203)	NYVGSIFDY (SEQ ID NO：17)	SEQ ID NO：165
Hu2B8 Hv5-51.1	TYWMH (SEQ ID NO：15)	EINPTNGHTNYNPSFQG (SEQ ID NO：203)	NYVGSIFDY (SEQ ID NO：17)	SEQ ID NO：169	LR2B8HC	TYWMH (SEQ ID NO：15)	EINPTNGHTNYNEKFKG (SEQ ID NO：204)	NYVGSIFDY (SEQ ID NO：17)	SEQ ID NO：183
LRMR2B8HC	TYWMH (SEQ ID NO：15)	EINPTNGHTNYNQKFQG (SEQ ID NO：205)	NYVGSIFDY (SEQ ID NO：17)	SEQ ID NO：189	LR2B8HC	TYWMH (SEQ ID NO：15)	EINPTNGHTNYNEKFKG (SEQ ID NO：204)	NYVGSIFDY (SEQ ID NO：17)	SEQ ID NO：183

Table 16 has been summed up the light chain CDR sequence (Kabat definition) by the humanization 2B8 antibody of the humanization method 1 of the present embodiment of describing hereinbefore and 2 preparations of humanization method.

Table 16

Antibody	CDR1	CDR2	CDR3	The full-length light chains variable region
Antibody	CDR1	CDR2	CDR3	The full-length light chains variable region	Mouse 2B8 light chain	KASENVVSYVS (SEQ ID NO：18)	GASNRNT (SEQ ID NO：19)	GQSYNYPYT (SEQ ID NO：20)	SEQ ID NO：14
Hu2B8 Kv1-39.1	KASENVVSYVS (SEQ ID NO：18)	GASNRNT (SEQ ID NO：19)	GQSYNYPYT (SEQ ID NO：20)	SEQ ID NO：173	Mouse 2B8 light chain	KASENVVSYVS (SEQ ID NO：18)	GASNRNT (SEQ ID NO：19)	GQSYNYPYT (SEQ ID NO：20)	SEQ ID NO：14
Hu2B8 Kv1-39.1	KASENVVSYVS (SEQ ID NO：18)	GASNRNT (SEQ ID NO：19)	GQSYNYPYT (SEQ ID NO：20)	SEQ ID NO：173	Hu2B8 Kv3-15.1	KASENVVSYVS (SEQ ID NO：18)	GASNRNT (SEQ ID NO：19)	GQSYNYPYT (SEQ ID NO：20)	SEQ ID NO：179
LR2B8LC	KASENVVSYVS (SEQ ID NO：18)	GASNRNT (SEQ ID NO：19)	GQSYNYPYT (SEQ ID NO：20)	SEQ ID NO：193	Hu2B8 Kv3-15.1	KASENVVSYVS (SEQ ID NO：18)	GASNRNT (SEQ ID NO：19)	GQSYNYPYT (SEQ ID NO：20)	SEQ ID NO：179
LR2B8LC	KASENVVSYVS (SEQ ID NO：18)	GASNRNT (SEQ ID NO：19)	GQSYNYPYT (SEQ ID NO：20)	SEQ ID NO：193	LRMR2B8LC	KASENVVSYVS (SEQ ID NO：18)	GASNRES (SEQ ID NO：206)	GQSYNYPYT (SEQ ID NO：20)	SEQ ID NO：199

C. the binding affinity of humanization 2B8 antibody

Use BIAcore T100 device to assess antibody binding affinity and interactional kinetics by surface plasma body resonant vibration.Use amine coupling (BIAcore, Catalog No.BR-1000-50), according to manufacturer's recommendation, coupling method by standard is with mouse anti human immunoglobulin (Ig) (Jackson ImmunoResearch Labs, 209-005-098) be fixed on the sensor chip (BIAcore, Catalog No.BR-1005-34) of carboxymethylated dextran CM4.Under 25 ℃, use PBS (GIBCO, Catalog No.14040-133) agent is analyzed as runtime buffer, described PBS contains 0.05% tensio-active agent P20 (BIAcore, Catalog No.BR-1000-54), 2mg/mL BSA (EMD, Catalog No.2930) and the sodium salt of 10mg/mL CM-dextran (Fluka, Catalog No.86524).

Antibody is trapped on the single mobile cell that flow velocity is 10 μ L/min.Be variable the inject time that is used for every kind of antibody, thereby make the antibody of catching about 20RU in each circulation.On with reference to surface (not having captive antibody) and active surface (having caught antibody to be tested), inject buffer reagent successively or be diluted in HGF (R﹠amp in the runtime buffer agent with the speed of 60 μ L/min; D Systems, Catalog No.294-HGN-025), the time is 2 minutes.According to concentration, monitor 15 or 90 minutes mutually to dissociating.Before another circulation of beginning, use glycine-HCl (BIAcore, Catalog No.BR-1003-55) of 10mM pH2.0 to make described surface regeneration then, wherein said glycine-HCl flow velocity with 60 μ L/min in 3 minutes injects.The concentration of the HGF that is tested is 1.88,3.75 and 7.5nM.Utilize the kinetic function of BIAevalutation software and the difference level of recommendation, determine kinetic parameter.Kinetic parameter (the k of every kind of antibody _a(association rate constant), k _d(dissociation rate constant) and K _D(equilibrium dissociation constant)) be summarized among Fig. 8.

The result that Fig. 8 sums up shows, some combination of super humanization heavy chain (Hu2B8 Hv5a.1, Hu2B8Hv5-51.1 or Hu2B8 Hv1-f.1) and light chain (Hu2B8 Kv1-39.1 or Hu2B8Kv3-15.1) has kept and binding affinity (K HGF identical with 2B8 with mosaic 2B8 (mouse variable region and human constant region) _D) (table 5).

D. mutual exclusion binding analysis

Using BIAcore T100 device to assess with the mutual exclusion of HGF by the surface plasma body resonant vibration technology combines.Use amine coupling (BIAcore, Catalog No.BR-1000-50), according to manufacturer's recommendation, coupling method by standard is with mouse anti human immunoglobulin (Ig) (Jackson ImmunoResearch Labs, 209-005-098) be fixed on the sensor chip (BIAcore, Catalog No.BR-1006-68) of carboxymethylated dextran CM5.Under 25 ℃, use PBS (GIBCO, Catalog No.14040-133) agent is analyzed as runtime buffer, described PBS contains 0.05% tensio-active agent P20 (BIAcore, #BR-1000-54), 2mg/mL BSA (EMD, Catalog No.2930) and the sodium salt of 10mg/mL CM-dextran (Fluka, Catalog No.86524).

Humanized antibody is trapped on the single mobile cell that flow velocity is 30 μ L/min.Be variable the inject time that is used for every kind of antibody, thereby make the antibody of catching about 150RU in each circulation.Injecting the ultimate density that is diluted in the runtime buffer agent with the speed of 30 μ L/min on the humanized antibody of catching is the HGF (R﹠amp of 7.5 μ g/mL; D Systems, Catalog No.294-HGN-025), time remaining 90 seconds.The combination of monitoring HGF is subsequently with the speed injection mouse 2B8 antibody of 30 μ L/min or the antibody (R﹠amp of the anti-HGF of polyclone goat; D Systems, AF294), time remaining 3 minutes.Before another circulation of beginning, it is that the flow velocity with 60 μ L/min injects in 3 minutes that glycine-HCl (BIAcore, Catalog No.BR-1003-55) of use 10mM pH2.0 makes described surface regeneration, wherein said glycine-HCl then.Gained the results are summarized among Fig. 9.

The result who sums up among Fig. 9 shows, the two has all suppressed combining of mouse 2B8 and HGF humanized 2B8 antibody and mosaic 2B8 antibody.These results prove that humanized antibody is still as the identical HGF epi-position of primary 2B8 antibodies.

The preparation of the humanized 2B8 variant of embodiment 13-

A. HUMAN ENGINEERED ^TM Antibody

Low-risk and the low risk of codon and expression optimization is cloned in the instantaneous antibody expression vector of XOMA synchronously to the people's light chain (being respectively LR2B8LC and LRMR2B8LC) and the heavy chain (being respectively LR2B8HC and LRMR2B8HC) through transforming of medium risk, and this carrier contains people Kappa and Gamma-1 constant region construction module (module).In the HEK293E cell, produce 4 kinds of people 2B8 variants by transient transfection through transforming.4 kinds of antibody that produced are as follows: HE2B8-1=LR2B8HC (+IgG1 constant region (Glm (3) allotype (allelotrope 1)) (SEQID NO.187)+LR2B8LC (+Kappa constant region (Km (3) allotype (allelotrope 1))) (SEQ ID NO.197)

HE2B8-2=LR2B8HC (+IgG1 constant region (Glm (3) allotype (allelotrope 1)) (SEQID NO.187)+LRMR2B8 LC (+Kappa constant region (Km (3) allotype (allelotrope 1))) (SEQ ID NO.201)

HE2B8-3=LRMR2B8HC (+IgG1 constant region (Glm (3) allotype (allelotrope 1)) (SEQ ID NO.191)+LR2B8LC (+Kappa constant region (Km (3) allotype (allelotrope 1))) (SEQ ID NO.197)

HE2B8-4=LRMR2B8HC (+IgG1 constant region (Glm (3) allotype (allelotrope 1)) (SEQ ID NO.191)+LRMR2B8LC (+Kappa constant region (Km (3) allotype (allelotrope 1))) (SEQ ID NO.201)

The suspension to XOMA of light chain and heavy chain cotransfection is adapted in the HEK293E cell, described cell cultures 2 liters the IS293 substratum that shakes bottle (Irvine Scientific, Irvine, CA) in.After 24 hours, centrifugal in shaking bottle to the transfectional cell of 200mL, be resuspended in the fresh culture of 40mL then and be transferred to the Integra flask (Wilson WolfManufacturing company, MN) in, be used for producing.Behind the incubation 7 days, cell suspending liquid is taken out from the Integra bottle, centrifugal, and keep culture supernatants.Antibody in the last purifying culture supernatants of the centrifugal post of a-protein (Pro-Chem) with the PBS dialysis, concentrates and Sterile Filtration then again.

B.SUPERHUMANIZED ^TM Antibody

Utilize HindIII and EcoRI restriction site, with total length Hu2B8_Hv5-51.1+ human IgG1 constant region (Glm (3) allotype) cDNA be cloned into pEE6.4 (Lonza Biologics, Berkshire, UK) in.Utilize HindIII and EcoRI restriction site, total length Hu2B8_Kv1-39.1 variable region+people Kappa constant region cDNA and total length Hu2B8_Kv3-15.1 variable region+people Kappa constant region cDNA are cloned into respectively among the pEE14.4 (Lonza Biologies).Digest by NotI/SalI, pipette hCMV-MIE promotor+total length Hu2B8_Hv5-51.1+ human IgG1 constant region structural domain (Glm (3) allotype) cDNA+SV40 poly A fragment (in pEE6.4), and by the NotI/SalI site, be inserted in any Kappa chain pEE14.4 carrier, produce 2 different expression vectors thus, each passes through and all expresses heavy chain and light chain simultaneously, thereby produces following antibody:

((Glm (3) is of the same race different for+IgG1 constant region for sh2B8-9 (Glm (3))=hu2B8 Hv5-51.1

Type) (allelotrope 2)) (SEQ ID NO.210)+hu2B8 Kv

1-39.1 (+Kappa constant region (Km (3) allotype (allelotrope

2)))(SEQ ID NO：177)

((Glm (3) is of the same race different for+IgG1 constant region for sh2B8-12 (Glm (3))=hu2B8 Hv5-51.1

Type) (allelotrope 2)) (SEQ ID NO.210)+hu2B8 Kv

3-15.1 (+Kappa constant region (Km (3) allotype (allelotrope

2)))(SEQ ID No.181)

Below list coding human IgG1 CH Glm (3) allotype (allelotrope 2) and the nucleotide sequence of every kind of total length sequence of heavy chain and the protein sequence that defines human IgG1's CH Glm (3) allotype (allelotrope 2) and every kind of total length sequence of heavy chain.Sequence of light chain identical with described in the embodiment 12.

(1) nuclear of coding human IgG1's CH (Glm (3) allotype) (allelotrope 2) Acid sequence(SEQ ID NO.207)

1 cctccaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc acctctgggg

61 gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg acggtgtcgt

121 ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta cagtcctcag

181 gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc acccagacct

241 acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaga gttgagccca

301 aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc ctggggggac

361 cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc cggacccctg

421 aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt

481 acgtggacgg cgtggaggtgcataatgcca agacaaagcc gcgggaggag cagtacaaca

541 gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg aatggcaagg

601 agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaag accatctcca

661 aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc cgggaggaga

721 tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc agcgacatcg

781 ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg cctcccgtgc

841 tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag agcaggtggc

901 agcaggggaa cgtcttctca tgctccgtga tcatgaggc tctgcacaac cactacacgc

961 agaagagcct ctccctgtct ccgggtaaat ga

(2) define human IgG1's CH (Glm (3) allotype) (allelotrope 1 or 2) protein sequence(SEQ ID NO.208).First amino acid derives from the translation product of two Nucleotide of beginning of last Nucleotide of variable region and IgG1 sequence of heavy chain.

1 astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss

61 glyslssvvt vpssslgtqt yicnvnhkps ntkvdkrvep kscdkthtcp pcpapellgg

121 psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna ktkpreeqyn

181 styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsree

241 mtknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw

301 qqgnvfscsv mhealhnhyt qkslslspgk

(3) coding contains humanized Hu2B8 Hv5-51.1 variable region of heavy chain and human IgG1's weight Nucleotide sequence (the underscore of the total length chain of chain constant region Glm (3) allotype (allelotrope 2) Be signal sequence)(SEQ ID NO.209)

1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgaa

61 gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaagatctcc

121 tgtaagggtt ctggatacag ctttaccacc tactggatgc actgggtgcg ccagatgccc

181 gggaaaggcc tggagtggat gggggagatt aatcctacca acggtcatac taactacaat

241 ccgtccttcc aaggccaggt caccatctca gctgacaagt ccatcagcac tgcctacctg

301 cagtggagca gcctgaaggc ctcggacacc gccatgtattactgtgcgag aaactatgtt

361 ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc agcctccacc

421 aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg

481 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca

541 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac

601 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc

661 aacgtgaatc acaagcccag caacaccaag gtggacaaga gagttgagcc caaatcttgt

721 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc

781 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca

841 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac

901 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac

961 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag

1021 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga agaccatctc caaagccaaa

1081 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag

1141 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag

1201 tgggagagca atgggcagcc ggagaacaactacaagacca cgcctcccgt gctggactcc

1261 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg

1321 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc

1381 ctctccctgt ctccgggtaa atga

(4) define and contain humanized Hu2B8 Hv5-51.1 variable region of heavy chain and human IgG1 The protein sequence of the total length heavy chain of CH Glm (3) allotype (allelotrope 2) (not having signal sequence)(SEQ ID NO.210)

1 evqlvqsgae vkkpgeslki sckgsgysft tywmhwvrqm pgkglewmge inptnghtny

61 npsfqgqvti sadksistay lqwsslkasd tamyycarny vgsifdywgq gtlvtvssas

121 tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht fpavlqssgl

181 yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkrvepks cdkthtcppc papellggps

241 vflfppkpkd tlmisrtpev tcvvvdvshe dpevkfnwyv dgvevhnakt kpreeqynst

301 yrvvsvltvl hqdwlngkey kckvsnkalp apiektiska kgqprepqvy tlppsreemt

361 knqvsltclv kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq

421 gnvfscsvmh ealhnhytqk slslspgk

With each dual-expression vector all transfection in the 293T cell, and use the DMEM contain 10% foetal calf serum to carry out transient expression.After the transfection 48 hours, cell is used the serum free medium IS GRO that contains the 4mM L-glutaminate ^TM(Irvine Scientific, Santa Ana CA) wash, and replace original substratum with this substratum.Collect supernatant liquor every day, and, so continue 10 days with the supernatant liquor that new substratum replacement is collected.Culture supernatants is centrifugal, filter (0.45 μ m) and concentrate 10-100 doubly.Go up antibody purification at ProSep vA resin (Millipore), and, concentrate again and Sterile Filtration with the PBS dialysis.

The binding characteristic of embodiment 14-humanization 2B8 variant

The reorganization HGF protein for preparing among the humanized antibody of preparation and the embodiment 3 among the embodiment 13 characterizes by their abilities in conjunction with hHGF.

Use BIAcore T100 device to analyze antibody to assess its ability in conjunction with the fused protein of discussing among hHGF and the embodiment 3 by surface plasma body resonant vibration.Use amine coupling (BIAcore, Catalog No.BR-1000-50), the specification sheets that provides according to manufacturers is fixed in every kind of antibody on the sensor chip (BIAcore, Catalog No.BR-1006-68) of carboxymethylated dextran CM5 by the coupling method of standard.

Under 25 ℃, use PBS (GIBCO, Catalog No.14040-133) agent is analyzed as runtime buffer, described PBS contains 0.05% tensio-active agent P20 (BIAcore, Catalog No.R-1000-54), 2mg/mL BSA (EMD, Catalog No.2930) and the sodium salt of 10mg/mL CM-dextran (Fluka, Catalog No.86524).Maybe must the hang oneself supernatant liquor of empty carrier cells transfected of the supernatant liquor that will contain different HGF fused proteins is expelled on every kind of antibody time remaining 3 minutes with the flow velocity of 30 μ L/min.When injection finished back 30 seconds, on baseline, measure the combination of gained with resonance units (RU).Will in conjunction with the people HGF (R﹠amp that is diluted in the runtime buffer agent; D Systems, Catalog No.294-HGN-025) compare.By will there not being specific combination in conjunction with comparing with contrast surface, monitoring.The results are summarized in the table 17.

Table 17

Antibody	rhHGF(R&D Systems)	rmHGF(R&D Systems)	MHM mosaic (495-585)	MHM mosaic (507-585)	MHM mosaic (499-556)
Antibody	rhHGF(R&D Systems)	rmHGF(R&D Systems)	MHM mosaic (495-585)	MHM mosaic (507-585)	MHM mosaic (499-556)	2B8	In conjunction with	Debond	In conjunction with	In conjunction with	In conjunction with
HE2B8-1	In conjunction with	Debond	In conjunction with	In conjunction with	In conjunction with	2B8	In conjunction with	Debond	In conjunction with	In conjunction with	In conjunction with
HE2B8-1	In conjunction with	Debond	In conjunction with	In conjunction with	In conjunction with	HE2B8-2	In conjunction with	Debond	In conjunction with	In conjunction with	In conjunction with
HE2B8-3	In conjunction with	Debond	In conjunction with	In conjunction with	In conjunction with	HE2B8-2	In conjunction with	Debond	In conjunction with	In conjunction with	In conjunction with
HE2B8-3	In conjunction with	Debond	In conjunction with	In conjunction with	In conjunction with	HE2B8-4	In conjunction with	Debond	In conjunction with	In conjunction with	In conjunction with
sh2B8-9 (Glm(3))	In conjunction with	Debond	In conjunction with	In conjunction with	In conjunction with	HE2B8-4	In conjunction with	Debond	In conjunction with	In conjunction with	In conjunction with
sh2B8-9 (Glm(3))	In conjunction with	Debond	In conjunction with	In conjunction with	In conjunction with	sh2B8-12 (Glm(3))	In conjunction with	Debond	In conjunction with	In conjunction with	In conjunction with

Result in the table 17 shows, every kind based on mouse-people-mouse mosaic in the antibody of humanized 2B8 and all 3 all in conjunction with rhHGF.

The binding affinity of embodiment 15-humanization 2B8 variant

The binding affinity and the interactional kinetics of the antibody that the use surface plasmon resonance measurement is decided to list in the table 15.

Use amine coupling (BIAcore, Catalog No.BR-1000-50), the specification sheets that provides according to manufacturers passes through the coupling method of standard with mouse anti human immunoglobulin (Ig) (JacksonLabs, Catalog No.209-005) is fixed on the sensor chip (BIAcore, Catalog No.BR-1006-68) of carboxymethylated dextran CM4.Under 25 ℃, use the PBS (GIBCO, Catalog No.14040-133) that contains 0.05% tensio-active agent P20 (BIAcore, Catalog No.BR-1000-54) and 2mg/mLBSA (EMD, Catalog No.2930) to analyze.

Antibody is trapped on the single mobile cell that flow velocity is 10 μ L/min.Be variable the inject time that is used for every kind of antibody, thereby make the antibody of catching about 20RU in each circulation.On with reference to surface (not having captive antibody) and active surface (having caught antibody to be tested), inject buffer reagent successively or be diluted in HGF (R﹠amp in the runtime buffer agent with the speed of 60 μ L/min; D Systems, Catalog No.294-HGN-025), time remaining 2 minutes.According to concentration, monitor 15 or 90 minutes mutually to dissociating.Before another circulation of beginning, it is that the flow velocity with 60 μ L/min injects in 3 minutes that glycine-HCl (BIAcore, Catalog No.BR-1003-54) of use 10mM pH2.2 makes described surface regeneration, wherein said glycine-HCl then.The concentration of the HGF that is tested is 0.46nM to 7.5nM.

Utilize BIAevalutation ^TMThe difference level of the kinetic function of software and recommendation is determined kinetic parameter.Kinetic parameter (the k of every kind of antibody _a(association rate constant), k _d(dissociation rate constant) and K _D(equilibrium dissociation constant)) be summarized in the table 18.

Table 18

Antibody	k _a(1/Ms)	k _d(1/s)	K _D(pM)	SD
Antibody	k _a(1/Ms)	k _d(1/s)	K _D(pM)	SD	2B8	1.4 x 10 ⁶	1.0 x 10 ^-5	7.3	-
HE2B8-1	2.2 x 10 ⁶	1.4 x 10 ^-5	7.1	5.2	2B8	1.4 x 10 ⁶	1.0 x 10 ^-5	7.3	-
HE2B8-1	2.2 x 10 ⁶	1.4 x 10 ^-5	7.1	5.2	HE2B8-2	1.8 x 10 ⁶	9.6 x 10 ^-5	5.2	2.7
HE2B8-3	2.0 x 10 ⁶	4.1 x 10 ^-5	2.0	1.1	HE2B8-2	1.8 x 10 ⁶	9.6 x 10 ^-5	5.2	2.7
HE2B8-3	2.0 x 10 ⁶	4.1 x 10 ^-5	2.0	1.1	HE2B8-4	1.7 x 10 ⁶	1.1 x 10 ^-5	6.5	1.3
sh2B8-9 (Glm17，1)	2.0 x 10 ⁶	1.7 x 10 ^-5	8.1	5.3	HE2B8-4	1.7 x 10 ⁶	1.1 x 10 ^-5	6.5	1.3
sh2B8-9 (Glm17，1)	2.0 x 10 ⁶	1.7 x 10 ^-5	8.1	5.3	sh2B8-12 (Glm17，1)	1.9 x 10 ⁶	2.3 x 10 ^-5	12	0.4

These data show that humanized antibody has fast association rate (k _a), extremely slow dissociation rate (k _d) and high avidity (K _D).Particularly, the avidity of described antibody is 2.0-12pM.

25 ℃ of embodiment 16-comparison down and the binding affinity under 37 ℃

Use surface plasma body resonant vibration, under different conditions, measure binding affinity and the interactional kinetics of antibody HE2B8-4, sh2B8-9, sh2B8-12 and mouse 2B8.

Use amine coupling (BIAcore, Catalog No.BR-1000-50), the specification sheets that provides according to manufacturers passes through the coupling method of standard with mouse anti human immunoglobulin (Ig) (JacksonLabs, Catalog No.209-005) or the anti-mouse immuning ball protein (BIAcore of rabbit, Catalog No.BR-1005-14) is fixed on the sensor chip (BIAcore, Catalog No.BR-1006-68) of carboxymethylated dextran CM4.When measuring antibody sh2b8-9 and sh2B8-12 down for 25 ℃, use CM5 sensor chip (BIAcore, Catalog No.BR-1006-68).Under 25 ℃ and 37 ℃, use contains 0.05% tensio-active agent P20 (BIAcore, Catalog No.BR-1000-54) and 2mg/mL BSA (EMD, PBS CatalogNo.2930) (GIBCO, Catalog No.14040-133) as runtime buffer agent analyze.

Antibody is trapped on the single mobile cell that flow velocity is 10 μ L/min.Be variable the inject time that is used for every kind of antibody, thereby make the antibody of catching about 20RU in each circulation.On with reference to surface (not having captive antibody) and active surface (having caught antibody to be tested), inject buffer reagent successively or be diluted in HGF (R﹠amp in the runtime buffer agent with the speed of 60 μ L/min; D Systems, Catalog No.294-HGN-025), time remaining 2 minutes.According to concentration, monitor 15 or 90 minutes mutually to dissociating.Then before another circulation of beginning, use glycine-HCl (BIAcore of 10mM pH2.2, Catalog No.BR-1003-54) make the surface regeneration of mouse anti human immunoglobulin (Ig) sensor chip, wherein said glycine-HCl is that the flow velocity with 60 μ L/min injects in 3 minutes.Before another circulation of beginning, use glycine-HCl (BIAcore of 10mM pH1.7, Catalog No.BR-1003-54) make the surface regeneration of the anti-mouse immuning ball protein sensor chip of rabbit, wherein said glycine-HCl is that the flow velocity with 60 μ L/min injects in 3 minutes.The concentration of the HGF that is tested is 0.46nM to 7.5nM.

Utilize the kinetic function of BIAevaluation software and the difference level of recommendation, determine kinetic parameter.Kinetic parameter (the k of every kind of antibody _a(association rate constant), k _d(dissociation rate constant) and K _D(equilibrium dissociation constant)) be summarized in the table 19.

Table 19

Antibody	Temperature (℃)	k _a(1/Ms)	k _d(1/s)	K _D(pM)
Antibody	Temperature (℃)	k _a(1/Ms)	k _d(1/s)	K _D(pM)	2B8	25	1.6 x 10 ⁶	2.1 x 10 ^-5	13.5
2B8	37	2.8 x 10 ⁶	1.3 x 10 ^-5	4.5	2B8	25	1.6 x 10 ⁶	2.1 x 10 ^-5	13.5
2B8	37	2.8 x 10 ⁶	1.3 x 10 ^-5	4.5	HE2B8-4	25	2.0 x 10 ⁶	1.2 x 10 ^-5	5.6
HE2B8-4	37	3.1 x 10 ⁶	1.0 x 10 ^-5	3.3	HE2B8-4	25	2.0 x 10 ⁶	1.2 x 10 ^-5	5.6
HE2B8-4	37	3.1 x 10 ⁶	1.0 x 10 ^-5	3.3	sh2B8-9 (Glm(17，1))	25	2.0 x 10 ⁶	1.7 x 10 ^-5	8.1
sh2B8-9 (Glm(3))	37	2.5 x 10 ⁶	1.4 x 10 ^-5	5.8	sh2B8-9 (Glm(17，1))	25	2.0 x 10 ⁶	1.7 x 10 ^-5	8.1
sh2B8-9 (Glm(3))	37	2.5 x 10 ⁶	1.4 x 10 ^-5	5.8	sh2B8-12 (Glm(17，1))	25	1.9 x 10 ⁶	2.3 x 10 ^-5	12.0
sh2B8-12 (Glm(3))	37	2.4 x 10 ⁶	1.1 x 10 ^-5	4.8	sh2B8-12 (Glm(17，1))	25	1.9 x 10 ⁶	2.3 x 10 ^-5	12.0

As expected, association rate constant raises along with the rising of temperature.The people is in surprise in addition, and dissociation constant does not obviously change along with the corresponding rising of temperature.Therefore, overall equilibrium dissociation constant (K _D) than little about 1.4 to 3 times (avidity is higher) of the equilibrium dissociation constant under the physiological temp (37 ℃).

The neutralization activity of embodiment 17-humanized 2B8 variant

(a) that characterizes the antibody of embodiment 14 preparations suppresses hHGF and with c-Met bonded ability and (b) suppresses in the 4MBr-5 cell ability by the HGF of the generation that BrdU stimulates of introducing.

According to the following stated carry out HGF-Met in conjunction with inhibition analysis (neutralization analysis).Detect antibody by ELISA and suppress hHGF and c-Met bonded ability.Particularly, under 4 ℃, Wallac 96 hole DELFIA are analyzed dull and stereotyped (Wallac company, Catalog No.AAAND-0001) be cushioned agent (15mM Na with 100 μ L carbonate bags ₂CO ₃And 34mMNaHCO ₃, pH9.0) the 6.25 μ g/mL HGF (R﹠amp in; D Systems, Catalog No.294-HGN-025) wrap by 16 hours.Then, at room temperature will analyze flat board sealed 1 hour with 5% non-skim-milk among the 200 μ LPBS.In independent analysis flat board, by the antibody (0.033-250nM in the research of being in that concentration is raise, 2 times of serial dilutions) join among the biotin labeled c-Met of 2nM and prepare this antibody, wherein said c-Met is dissolved in and contains among 5% the PBS by non-skim-milk.The specification sheets that provides according to manufacturers, with the ratio (Pierce, Catalog No.21335) of the vitamin H of 10:1: c-Met to c-Met (R﹠amp; D Systems, Catalog No.358-MT/CF) carries out biotin labeling.With 100 μ L sample transfer in every hole to analyzing in the flat board, and incubation 2 hours at room temperature.Then, income analysis is dull and stereotyped with PBS-0.1% Tween 20 washings 3 times, and at room temperature, Streptavidin (Wallac with the Eu mark, Catalog No.1244-360) incubation is 1 hour, wherein the Streptavidin of Eu mark with the dilution proportion of 1:1000 in the DELFIA analysis buffer (Wallac, CatalogNo.4002-0010) in., and at room temperature strengthen solution (Wallac#4001-0010) and stirred incubation 15 minutes with DELFIA washings (Wallac, Catalog No.4010-0010) washing 3 times sheets thus obtained with the DELFIA in 100 L/ holes.Use the europium tracer method, at Victor ³Read described flat board on the V instrument (Perkin Elmer).Use Prism to calculate IC ₅₀Value.

The IC of gained ₅₀Value is shown in Table 20.

Table 20

Antibody	IC ₅₀(nM)	SD
Antibody	IC ₅₀(nM)	SD	2B8	9.2	1.2
HE2B8-1	6.0	1.2	2B8	9.2	1.2
HE2B8-1	6.0	1.2	HE2B8-2	5.7	1.1
HE2B8-3	5.9	1.1	HE2B8-2	5.7	1.1
HE2B8-3	5.9	1.1	HE2B8-4	6.5	1.2
sh2B8-9(Glm(3))	4.2	-	HE2B8-4	6.5	1.2
sh2B8-9(Glm(3))	4.2	-	sh2B8-12(Glm(3))	6.8	-

The result of table 20 shows, the humanized antibody of being tested effectively in and the combining of HGF and c-Met.

In addition, in the described analysis of cell proliferation of embodiment 7 (b), the antibody in the his-and-hers watches 17 is also tested.The results are summarized in the table 21.

Table 21

Antibody	IC ₅₀(nM)	SD
Antibody	IC ₅₀(nM)	SD	2B8	0.86	0.35
HE2B8-1	0.47	0.15	2B8	0.86	0.35
HE2B8-1	0.47	0.15	HE2B8-2	0.66	0.13
HE2B8-3	0.55	0.28	HE2B8-2	0.66	0.13
HE2B8-3	0.55	0.28	HE2B8-4	0.58	0.26
sh2B8-9(Glm(3))	0.52	0.11	HE2B8-4	0.58	0.26
sh2B8-9(Glm(3))	0.52	0.11	sh2B8-12(Glm(3))	0.81	0.22

The result of table 212 shows that the humanized antibody of all tests all suppresses the propagation of HGF inductive 4MBr-5 cell.

The anti-diffusion activity of embodiment 18-humanization 2B8 variant

In embodiment 8 described anti-diffusion analyses, the antibody in the his-and-hers watches 17 is tested.The results are summarized in the table 22.

Table 22

-do not suppress

+++extremely strong is near suppressing fully

++ strongly inhibited

But the inhibition of+detection level

Result in the table 22 shows that all the inhibition degree with mouse monoclonal antibody 2B8 is identical for the inhibition degree that the humanized antibody of all tests spreads the HGF inductive.

The inhibition of the c-Met phosphorylation of embodiment 19-HGF is stimulated

In embodiment 9 described c-Met analysis of Phosphorylation, the antibody in the his-and-hers watches 17 is tested.The results are summarized in the table 23.

Table 23

Antibody	The mean value of 2 tests	Standard error
Antibody	The mean value of 2 tests	Standard error	2B8	0.91	0.02
HE2B8-1	0.80	0.04	2B8	0.91	0.02
HE2B8-1	0.80	0.04	HE2B8-2	0.88	0.15
HE2B8-3	0.79	0.05	HE2B8-2	0.88	0.15
HE2B8-3	0.79	0.05	HE2B8-4	0.75	0.14
sh2B8-9(Glm(3))	0.93	0.03	HE2B8-4	0.75	0.14
sh2B8-9(Glm(3))	0.93	0.03	sh2B8-12(Glm(3))	0.81	0.07

The result of table 23 shows that the humanized antibody of all tests all is effective inhibitor of HGF inductive c-Met phosphorylation in the PC-3 cell.

Tumor suppression in embodiment 20-U87MG xenograft models

In the U87MG xenograft models, test the ability that Humanized monoclonal antibodies of the present invention suppresses tumor growth.Under 37 ℃, containing 5% CO ₂With amplification U87MG cell (ATCC) in the cultivation of the atmosphere of 95% air, comprising the substratum of the Dulbecco ' s Modified Eagle substratum (DMEM) that contains 10% foetal calf serum, 100 units/mL penicillin and 100 μ g/mL Streptomycin sulphates.Pair cell carries out secondary cultivation, and keeps by the cell that uses trypsinase-EDTA to pipette on the culture dish wall.

Collect near the cell that converges, subsequently with 50% Matrigel (BDBiosciences by tryptic digestion; Catalog no.356237) 5x10 in ⁶Individual cell is subcutaneously injected in the zone of back upside between the shoulder blades of 7 week female ICR SCID mouse in age (Taconic Labs).The long diameter (L) of use kind of calliper tumour and short diameter (W) are (mm).Following calculating tumor size (vol.): volume (mm ³)=L x W ²/ 2.When tumor growth arrives about 200mm ³The time, the mouse that will have tumour is divided into 5 groups at random, every group of 10 mouse.One group of mouse is accepted PBS, and one group of mouse is accepted the human IgG contrast.Every group of mouse in other 4 groups accepted a kind of in the humanized antibody (HE2B8-1, HE2B8-2, HE2B8-3 and HE2B8-4).The dosage of all antibody is the 0.25mg/kg body weight, and 2 times weekly, and by 5 dosage of peritoneal injection.The body weight of 2 tumor size of per week record and mouse.Utilize the inhibition of student t check analysis tumor growth.

The humanized antibody of being tested is activated in vivo.It is 57% that the tumor growth of HE2B8-1 suppresses, and its p value is 0.02; It is 61% that the tumor growth of HE2B8-2 suppresses, and its p value is 0.02; It is 85% that the tumor growth of HE2B8-3 suppresses, and its p value is 0.0004; And the inhibition of the tumor growth of HE2B8-4 is 74%, and its p value is 0.001.Do not observe tangible body weight loss.

In the female NCR nude mice (Taconic Labs) of rib side subcutaneous vaccination U87MG tumour, carry out research subsequently as mentioned above.Every group of mouse (10 every group) all accepted a kind of in the following treatment with the amount of 0.5mg/kg: PBS vehicle Control, huIgG contrast, HE2B8-4 or sh2B8-9.In minimum 5 week, 2 intraperitoneal are treated weekly.Each treatment group all shows as similar tumour decline, and wherein the inhibition of the tumor growth of sh2B8-9 is 113%, and it is 115% that the tumor growth of HE2B8-4 suppresses, and the shortest tumor growth delay is 30 days.The tolerance of described two kinds of treatments is all good, and significantly not loss of body weight.

Incorporate this paper by reference into

Be all purposes, every part of patent documentation that this paper is mentioned and whole disclosures of science article are all incorporated this paper into way of reference.

Equivalent

The present invention can show as other specific forms, and does not break away from spirit of the present invention or essential characteristic.Therefore, above-mentioned embodiment under any circumstance all is considered to schematically, rather than limits invention as herein described.Therefore, scope of the present invention shows by appending claims rather than above-mentioned explanation, and all is contemplated as falling with in the scope of the present invention changing with the meaning of claim equivalence and the institute in the scope.

Sequence table

＜110〉Korea Spro M.

S.K. rely special

W.M. Winston

L. Breault

Lin Jie

B. Yi Temaide-Gilbertson

C. Ke Nihe

J. for a long time in this

＜120〉conjugated protein of pHGF (HGF)

<130>AVO-001A

<150>60/810，714

<151>2006-06-02

<150>60/860，461

<151>2006-11-21

<160>216

<170>PatentIn version 3.3

<210>1

<211>424

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic variable region of heavy chain 1A3

<400>1

<210>2

<211>141

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic variable region of heavy chain 1A3

<400>2

<210>3

<211>382

<212>DNA

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain variable region 1A3 of synthetic

<400>3

<210>4

<211>127

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain variable region 1A3 of synthetic

<400>4

<210>5

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic heavy chain CDR1 1A3

<400>5

<210>6

<211>17

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic heavy chain CDR2 1A3

<400>6

<210>7

<211>13

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic heavy chain CDR3 1A3

<400>7

<210>8

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain CDR1 1A3 of synthetic

<400>8

<210>9

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain CDR2 1A3 of synthetic

<400>9

<210>10

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain CDR3 1A3 of synthetic

<400>10

<210>11

<211>412

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic variable region of heavy chain 2B8

<400>11

<210>12

<211>137

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic variable region of heavy chain 2B8

<400>12

<210>13

<211>382

<212>DNA

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain variable region 2B8 of synthetic

<400>13

<210>14

<211>127

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain variable region 2B8 of synthetic

<400>14

<210>15

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic heavy chain CDR1 2B8

<400>15

<210>16

<211>17

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic heavy chain CDR2 2B8

<400>16

<210>17

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic heavy chain CDR3 2B8

<400>17

<210>18

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain CDR1 2B8 of synthetic

<400>18

<210>19

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain CDR2 2B8 of synthetic

<400>19

<210>20

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain CDR3 2B8 of synthetic

<400>20

<210>21

<211>412

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic variable region of heavy chain 2F8

<400>21

<210>22

<211>137

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic variable region of heavy chain 2F8

<400>22

<210>23

<211>394

<212>DNA

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain variable region 2F8 of synthetic

<400>23

<210>24

<211>131

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain variable region 2F8 of synthetic

<400>24

<210>25

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic heavy chain CDR1 2F8

<400>25

<210>26

<211>17

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic heavy chain CDR2 2F8

<400>26

<210>27

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic heavy chain CDR3 2F8

<400>27

<210>28

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain CDR1 2F8 of synthetic

<400>28

<210>29

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain CDR2 2F8 of synthetic

<400>29

<210>30

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain CDR3 2F8 of synthetic

<400>30

<210>31

<211>418

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic variable region of heavy chain 3B6

<400>31

<210>32

<211>139

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic variable region of heavy chain 3B6

<400>32

<210>33

<211>388

<212>DNA

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain variable region 3B6 of synthetic

(2 possible ATG initiator codons)

<400>33

<210>34

<211>129

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain variable region 3B6 of synthetic

(2 possible initial methionines)

<400>34

<210>35

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic heavy chain CDR1 3B6

<400>35

<210>36

<211>17

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic heavy chain CDR2 3B6

<400>36

<210>37

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic heavy chain CDR3 3B6

<400>37

<210>38

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain CDR1 3B6 of synthetic

<400>38

<210>39

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain CDR2 3B6 of synthetic

<400>39

<210>40

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain CDR3 3B6 of synthetic

<400>40

<210>41

<211>397

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic variable region of heavy chain 3D11

<400>41

<210>42

<211>132

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic variable region of heavy chain 3D11

<400>42

<210>43

<211>385

<212>DNA

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain variable region 3D11 of synthetic

<400>43

<210>44

<211>128

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain variable region 3D11 of synthetic

<400>44

<210>45

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic heavy chain CDR1 3D11

<400>45

<210>46

<211>16

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic heavy chain CDR2 3D11

<400>46

<210>47

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic heavy chain CDR3 3D11

<400>47

<210>48

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain CDR1 3D11 of synthetic

<400>48

<210>49

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain CDR2 3D11 of synthetic

<400>49

<210>50

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain CDR3 3D11 of synthetic

<400>50

<210>51

<211>424

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic variable region of heavy chain 1D3

<400>51

<210>52

<211>141

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic variable region of heavy chain 1D3

<400>52

<210>53

<211>382

<212>DNA

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain variable region 1D3 of synthetic

<400>53

<210>54

<211>127

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain variable region 1D3 of synthetic

<400>54

<210>55

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic heavy chain CDR1 1D3

<400>55

<210>56

<211>17

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic heavy chain CDR2 1D3

<400>56

<210>57

<211>13

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic heavy chain CDR3 1D3

<400>57

<210>58

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain CDR1 1D3 of synthetic

<400>58

<210>59

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain CDR2 1D3 of synthetic

<400>59

<210>60

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain CDR3 1D3 of synthetic

<400>60

<210>61

<211>424

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic variable region of heavy chain 1F3

<400>61

<210>62

<211>141

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic variable region of heavy chain 1F3

<400>62

<210>63

<211>382

<212>DNA

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain variable region 1F3 of synthetic

<400>63

<210>64

<211>127

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain variable region 1F3 of synthetic

<400>64

<210>65

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic heavy chain CDR1 1F3

<400>65

<210>66

<211>17

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic heavy chain CDR2 1F3

<400>66

<210>67

<211>13

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic heavy chain CDR3 1F3

<400>67

<210>68

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain CDR1 1F3 of synthetic

<400>68

<210>69

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain CDR2 1F3 of synthetic

<400>69

<210>70

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain CDR3 1F3 of synthetic

<400>70

<210>71

<211>424

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic variable region of heavy chain 3A12

<400>71

<210>72

<211>141

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic variable region of heavy chain 3A12

<400>72

<210>73

<211>382

<212>DNA

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain variable region 3A12 of synthetic

<400>73

<210>74

<211>127

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain variable region 3A12 of synthetic

<400>74

<210>75

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic heavy chain CDR1 3A12

<400>75

<210>76

<211>17

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic heavy chain CDR2 3A12

<400>76

<210>77

<211>13

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic heavy chain CDR3 3A12

<400>77

<210>78

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain CDR1 3A12 of synthetic

<400>78

<210>79

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain CDR2 3A12 of synthetic

<400>79

<210>80

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉light (kappa) chain CDR3 3A12 of synthetic

<400>80

<210>81

<211>974

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with reference to mouse IgG1 CH (J00453)

<400>81

<210>82

<211>974

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be defined as the mouse IgG1 CH (is mouse from the AJ kind) of 1A3,1D3,1F3 and 2B8

<400>82

<210>83

<211>323

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be defined as 1D3,1F3 and 2B8 with reference to mouse Kappa constant region of light chain (V00807) and mouse Kappa constant region of light chain (is mouse from the AJ kind)

<400>83

<210>84

<211>323

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be defined as the synthetic mouse Kappa constant region of light chain of 1A3, it comprises the Nucleotide of 1 change of 207 positions of comparing with 2B8 with 1D3,1F3

<400>84

<210>85

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic Oligonucleolide primers BD SMART II A

<400>85

<210>86

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic Oligonucleolide primers RACE CDS

<220>

<221>misc feature

<222>(27)..(27)

<223>n is a，c，g，t or u

<400>86

<210>87

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic Oligonucleolide primers Universal Primer Mix A

<400>87

<210>88

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic Oligonucleolide primers Universal Primer Mix A

<400>88

<210>89

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic IgG1 constant region special primer

<400>89

<210>90

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic IgG1 constant region special primer

<400>90

<210>91

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic Oligonucleolide primers

<400>91

<210>92

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic Oligonucleolide primers

<400>92

<210>93

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic Oligonucleolide primers T7

<400>93

<210>94

<211>17

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic Oligonucleolide primers M13 forward

<400>94

<210>95

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic Oligonucleolide primers M13 is reverse

<400>95

<210>96

<211>63

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide forward primer

<400>96

<210>97

<211>54

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide reverse primer

<400>97

<210>98

<211>62

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide forward primer

<400>98

<210>99

<211>53

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide reverse primer

<400>99

<210>100

<211>62

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide forward primer

<400>100

<210>101

<211>54

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide reverse primer

<400>101

<210>102

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide 5 primer hHGF NheI primers

<400>102

<210>103

<211>46

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide 3 primer hHGF NotI his label primers

<400>103

<210>104

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide 5 primer His IgFc primers

<400>104

<210>105

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide 3 primer I gFc BamHI primers

<400>105

<210>106

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic hHGF-Fc (G555E) sense primer

<400>106

<210>107

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic hHGF-Fc (G555E) antisense primer

<400>107

<210>108

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic hHGF-Fc (C561R) sense primer

<400>108

<210>109

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic hHGF-Fc (C561R) antisense primer

<400>109

<210>110

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be used for the primer of the synthetic fragment 1 of mHGF a chain 5 primer NheI

<400>110

<210>111

<211>36

<212>DNA

＜213〉artificial sequence

<220>

<400>111

<210>112

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be used for the primer of the synthetic fragment 2 of hHGF β chain aa V495-L585

<400>112

<210>113

<211>40

<212>DNA

＜213〉artificial sequence

<220>

<400>113

<210>114

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be used for the primer of the fragment 3 of the terminal 3 primer NotI of mHGF β chain C-

<400>114

<210>115

<211>48

<212>DNA

＜213〉artificial sequence

<220>

<400>115

<210>116

<211>38

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer 1 of synthetic sudden change

<400>116

<210>117

<211>38

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer 2 of synthetic sudden change

<400>117

<210>118

<211>2922

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the proteinic nucleotide sequence of synthetic hHGF-Fc

<400>118

<210>119

<211>919

<212>PRT

＜213〉artificial sequence

<220>

＜223〉aminoacid sequence of synthetic hHGF-Fc protein (not having signal sequence and prodomain)

<400>119

<210>120

<211>2901

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic mhm (V495-L585)-Fc chimeric protein

<400>120

<210>121

<211>911

<212>PRT

＜213〉artificial sequence

<220>

＜223〉aminoacid sequence of synthetic mhm-Fc activity form (removing signal sequence and prodomain)

<400>121

<210>122

<211>1398

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic total length 1A3 sequence of heavy chain (1A3 variable region of heavy chain and IgG1 constant region)

<400>122

<210>123

<211>446

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic total length 1A3 sequence of heavy chain (1A3 variable region of heavy chain and IgG1 constant region)

<400>123

<210>124

<211>705

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic total length 1A3 sequence of light chain (1A3 Kappa variable region and constant region)

<400>124

<210>125

<211>214

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic total length 1A3 sequence of light chain (1A3 Kappa variable region and constant region)

<400>125

<210>126

<211>1386

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic total length 2B8 sequence of heavy chain (2B8 variable region of heavy chain and IgC1 constant region)

<400>126

<210>127

<211>442

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic total length 2B8 sequence of heavy chain (2B8 variable region of heavy chain and IgG1 constant region)

<400>127

<210>128

<211>705

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic total length 2B8 sequence of light chain (2B8 Kappa variable region of light chain and constant region)

<400>128

<210>129

<211>214

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic total length 2B8 sequence of light chain (2B8 Kappa variable region and constant region)

<400>129

<210>130

<211>1386

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic total length 2F8 sequence of heavy chain (2F8 variable region of heavy chain and IgG1 constant region)

<400>130

<210>131

<211>442

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic total length 2F8 sequence of heavy chain (2F8 variable region of heavy chain and IgG1 constant region)

<400>131

<210>132

<211>717

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic total length 2F8 sequence of light chain (2F8 Kappa variable region and constant region)

<400>132

<210>133

<211>218

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic total length 2F8 sequence of light chain (2F8 Kappa variable region and constant region)

<400>133

<210>134

<211>1392

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic total length 3B6 sequence of heavy chain (3B6 variable region of heavy chain and IgG1 constant region)

<400>134

<210>135

<211>444

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic total length 3B6 sequence of heavy chain (3B6 variable region of heavy chain and IgG1 constant region)

<400>135

<210>136

<211>711

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic total length 3B6 sequence of light chain (3B6 Kappa variable region and constant region)

<400>136

<210>137

<211>214

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic total length 3B6 sequence of light chain (3B6 Kappa variable region and constant region)

<400>137

<210>138

<211>1361

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic total length 3D11 sequence of heavy chain (3D11 variable region of heavy chain and IgG1 constant region)

<400>138

<210>139

<211>437

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic total length 3D11 sequence of heavy chain (3D11 variable region of heavy chain and IgG1 constant region)

<400>139

<210>140

<211>708

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic total length 3D11 sequence of light chain (3D11 Kappa variable region and constant region)

<400>140

<210>141

<211>213

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic total length 3D11 sequence of light chain (3D11 Kappa variable region and constant region)

<400>141

<210>142

<211>1398

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic total length 1D3 sequence of heavy chain (1D3 variable region of heavy chain and IgG1 constant region)

<400>142

<210>143

<211>446

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic total length 1D3 sequence of heavy chain (1D3 variable region of heavy chain and IgG1 constant region)

<400>143

<210>144

<211>705

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic total length 1D3 sequence of light chain (1D3 Kappa variable region and constant region)

<400>144

<210>145

<211>214

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic total length 1D3 sequence of light chain (1D3 Kappa variable region and constant region)

<400>145

<210>146

<211>1398

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic total length 1F3 sequence of heavy chain (1F3 variable region of heavy chain and IgG1 constant region)

<400>146

<210>147

<211>446

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of total length 1F3 sequence of heavy chain (1F3 variable region of heavy chain and IgG1 constant region)

<400>147

<210>148

<211>705

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic total length 1F3 sequence of light chain (1F3 Kappa variable region and constant region)

<400>148

<210>149

<211>214

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic total length 1F3 sequence of light chain (1F3 Kappa variable region and constant region)

<400>149

<210>150

<211>1398

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic total length 3A12 sequence of heavy chain (3A12 variable region of heavy chain and Ig61 constant region)

<400>150

<210>151

<211>446

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic total length 3A12 sequence of heavy chain (3A12 variable region of heavy chain and IgG1 constant region)

<400>151

<210>152

<211>705

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic total length 3A12 sequence of light chain (3A12 Kappa variable region and constant region)

<400>152

<210>153

<211>214

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of total length 3A12 sequence of light chain (3A12 Kappa variable region and constant region)

<400>153

<210>154

<211>1404

<212>DNA

＜213〉artificial sequence

<220>

＜223〉coding synthetic total length mosaic 2B8 heavy chain (mouse variable region and human IgG1's constant region) (allotype Glm (and 17, nucleotide sequence 1V)

<400>154

<210>155

<211>448

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the synthetic total length mosaic 2B8 heavy chain (protein sequence (not having signal sequence) of mosaic 2B8 IgG1 (Glm (17,1) allotype)

<400>155

<210>156

<211>705

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic total length mosaic 2B8 light chain (mouse variable region and human constant region) (mosaic 2B8 Kappa (Km (3)))

<400>156

<210>157

<211>214

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic total length mosaic 2B8 light chain (mosaic 2B8 Kappa (Km (3)))

<400>157

<210>158

<211>412

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic humanization Hu2B8 Hv1-f.1 variable region of heavy chain

<400>158

<210>159

<211>118

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic humanization Hu2B8 Hv1-f.1 variable region of heavy chain

<400>159

<210>160

<211>992

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic human IgG1's CH (Glm (17,1) allotype)

<400>160

<210>161

<211>330

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence of synthetic human IgG1 CH (Glm (17,1) allotype)

<400>161

<210>162

<211>1404

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic total length heavy chain humanization Hu2B8 Hv1f.1 variable region and human IgG1's (Glm (17,1) allotype) CH

<400>162

<210>163

<211>448

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic total length heavy chain humanization Hu2B8 Hv1f.1 variable region and human IgG1's CH (Glm (17,1) allotype)

<400>163

<210>164

<211>412

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic humanization Hu2B8 Hv5a.1 variable region of heavy chain

<400>164

<210>165

<211>118

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic humanization Hu2B8 Hv5a.1 variable region of heavy chain

<400>165

<210>166

<211>1404

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic total length humanization Hu2B8 Hv5a.1 variable region of heavy chain and human IgG1's (Glm (17,1) allotype) CH

<400>166

<210>167

<211>448

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic total length humanization Hu2B8 Hv5a.1 variable region of heavy chain and human IgG1 (Glm (17,1) allotype) CH

<400>167

<210>168

<211>412

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic humanization Hu2B8 Hv5-51.1 variable region of heavy chain

<400>168

<210>169

<211>118

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic humanization Hu2B8 Hv5-51.1 weight chain variabl area sequence

<400>169

<210>170

<211>1404

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic total length humanization Hu2B8 Hv551.1 variable region of heavy chain and human IgG1's (Glm (17,1) allotype) CH

<400>170

<210>171

<211>448

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define synthetic total length humanization Hu2B8 Hv5-51.1 variable region of heavy chain and human IgG1 (Glm (17,1) allotype)

The protein sequence of CH (not having signal sequence)

<400>171

<210>172

<211>388

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic humanization Hu2B8 Kv1-39.1 Kappa chain variable region

<400>172

<210>173

<211>107

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic humanization Hu2B8 Kv1-39.1 Kappa chain variable region

<400>173

<210>174

<211>323

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic people's Kappa chain constant region (Km (3) allotype) (allelotrope 2)

<400>174

<210>175

<211>107

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence of synthetic people Kappa chain constant region (Km (3) allotype) (allelotrope 2)

<400>175

<210>176

<211>711

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic total length humanization Hu2B8 Kv1-39.1 variable region of light chain and people Kappa chain constant region (Km (3) allotype) (allelotrope 2)

<400>176

<210>177

<211>214

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence of synthetic total length humanization Hu2B8 Kv1-39.1 variable region of light chain and people Kappa chain constant region (Km (3) allotype) (allelotrope 1)

<400>177

<210>178

<211>382

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic humanization Hu2B8 Kv3-15.1 variable region of light chain

<400>178

<210>179

<211>107

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic humanization Hu2B8 Kv3-15.1 variable region of light chain

<400>179

<210>180

<211>705

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleic acid of coding synthetic total length humanization Hu2B8 Kv3-15.1 variable region of light chain and people Kappa chain constant region (Km (3) allotype) (allelotrope 2)

<400>180

<210>181

<211>214

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic humanization Hu2B8 Kv3-15.1 variable region of light chain and people Kappa chain constant region (Km (3) allotype) (allelotrope 2)

<400>181

<210>182

<211>412

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic humanization LR2B8HC variable region of heavy chain

<400>182

<210>183

<211>118

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic humanization LR2B8HC variable region of heavy chain

<400>183

<210>184

<211>992

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic human IgG1's CH (Glm (3) allotype) (allelotrope 1)

<400>184

<210>185

<211>330

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence of synthetic human IgG1 CH (Glm (3) allotype) (allelotrope 1 or 2)

<400>185

<210>186

<211>1404

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic total length heavy chain humanization LR2B8HC variable region of heavy chain and human IgG1's CH (Glm (3) allotype) (allelotrope 1)

<400>186

<210>187

<211>448

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic total length heavy chain humanization LR2B8HC variable region of heavy chain and human IgG1's CH (Glm (3) allotype) (allelotrope 1)

<400>187

<210>188

<211>412

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic humanization LRMR2B8HC variable region of heavy chain

<400>188

<210>189

<211>118

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic humanization LRMR2B8HC variable region of heavy chain

<400>189

<210>190

<211>1404

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic total length heavy chain humanization LRMR2B8HC variable region of heavy chain and human IgG1's CH (Glm (3) allotype) (allelotrope 1)

<400>190

<210>191

<211>448

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic total length heavy chain humanization LRMR2B8HC variable region of heavy chain and human IgG1's CH (Glm (3) allotype) (allelotrope 1)

<400>191

<210>192

<211>360

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic humanization LR2B8LC variable region of light chain

<400>192

<210>193

<211>107

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic humanization LR2B8LC variable region of light chain

<400>193

<210>194

<211>323

<212>DNA

＜213〉artificial sequence

<220>

＜223〉coding synthetic people's Kappa chain constant region (Km (3) allotype) (allelotrope " nucleotide sequence

<400>194

<210>195

<211>107

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence of synthetic people Kappa chain constant region (Km (3) allotype) (allelotrope 1)

<400>195

<210>196

<211>705

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic total length humanization LR2B8LC variable region of light chain and people Kappa chain constant region (Km (3) allotype) (allelotrope 1)

<400>196

<210>197

<211>214

<212>PRT

＜213〉artificial sequence

<220>

＜223〉protein sequence of coding synthetic humanization LR2B8LC variable region of light chain and people Kappa chain constant region (Km (3) allotype) (allelotrope 1)

<400>197

<210>198

<211>382

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic humanization LRMR2B8LC variable region of light chain

<400>198

<210>199

<211>107

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of synthetic humanization LRMR2B8LC variable region of light chain

<400>199

<210>200

<211>705

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic total length humanization LRMR2B8LC variable region of light chain and people Kappa chain constant region (Km (3) allotype) (allelotrope 1)

<400>200

<210>201

<211>214

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence of synthetic total length humanization LRMR2B8LC variable region of light chain and people Kappa chain constant region (Km (3) allotype) (allelotrope 1)

<400>201

<210>202

<211>17

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the heavy chain CDR2 sequence of synthetic humanization Hu2B8 Hv1f.1 antibody (Kabat definition)

<400>202

<210>203

<211>17

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the heavy chain CDR2 sequence of synthetic humanization Hu2B8 Hv5a.1 antibody (Kabat definition)

<400>203

<210>204

<211>17

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the heavy chain CDR2 sequence of synthetic humanization LR2B8HC antibody (Kabat definition)

<400>204

<210>205

<211>17

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the heavy chain CDR2 sequence of synthetic humanization LRMR2B8HC antibody (Kabat definition)

<400>205

<210>206

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the light chain CDR2 sequence of synthetic humanization LRMR2BLC antibody (Kabat definition)

<400>206

<210>207

<211>992

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of coding synthetic human IgG1's CH (Glm (3) allotype) (allelotrope 2)

<400>207

<210>208

<211>330

<212>PRT

＜213〉artificial sequence

<220>

<400>208

<210>209

<211>1404

<212>DNA

＜213〉artificial sequence

<220>

＜223〉coding contains the nucleotide sequence of the synthetic total length chain of humanized Hu2B8 Hv5-51.1 variable region of heavy chain and human IgG1's CH Glm (3) allotype (allelotrope 2)

<400>209

<210>210

<211>448

<212>PRT

＜213〉artificial sequence

<220>

＜223〉define the protein sequence (not having signal sequence) of the synthetic total length heavy chain that contains humanized Hu2B8 Hv5-51.1 variable region of heavy chain and human IgG1's CH Glm (3) allotype (allelotrope 2)

<400>210

<210>211

<211>2209

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic HGF mhm (V495-L585)

<400>211

<210>212

<211>680

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic HGF mhm495-585 is activated

<400>212

<210>213

<211>2194

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic HGF mhm (I499-R556)

<400>213

<210>214

<211>675

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic HGF mhm 499-556 is activated

<400>214

<210>215

<211>2193

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic HGF mhm (W507-L585)

<400>215

<210>216

<211>675

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic HGF mhm507-585 is activated

<400>216

Claims

1. isolating conjugated protein in conjunction with human hepatocyte growth factor (HGF), this conjugated protein comprises:

(a) comprise structure C DR _L1-CDR _L2-CDR _L3Immunoglobulin light chain variable region, wherein

(i) CDR _L1Comprise aminoacid sequence X ₁X ₂SerX ₄X ₅X ₆X ₇X ₈X ₉X ₁₀X ₁₁X ₁₂X ₁₃X ₁₄X ₁₅, amino acid X wherein ₁Be Arg, Lys or Ser, X ₂Be Ala or Thr, X ₄Is Glu, Gln or Ser, X ₅Be Asn, Asp or Ser, X ₆Be Ile or Val, X ₇Be Asp, Lys, Ser, Val or Tyr, X ₈Be peptide bond or Tyr, X ₉Be peptide bond or Asp, X ₁₀Be peptide bond or Gly, X ₁₁Be peptide bond or Asn, X ₁₂Be peptide bond, Ile or Ser, X ₁₃Be Asn or Tyr, X ₁₄Be Ile, Leu, Met or Val, X ₁₅Be Ala, Asn, His or Ser,

(ii) CDR _L2Comprise aminoacid sequence X ₁₆X ₁₇X ₁₈X ₁₉X ₂₀X ₂₁X ₂₂, amino acid X wherein ₁₆Be Ala, Asp, Arg, Gly or Val, X ₁₇Be Ala, Thr or Val, X ₁₈Be Asn, Ser or Thr, X ₁₉Be Arg, Asn, Lys or His, X ₂₀Be Leu or Arg, X ₂₁Be Ala, Asn, Glu, Val or Pro, X ₂₂Be Asp, Ser or Thr, and

(iii) CDR _L3Comprise aminoacid sequence X ₂₃X ₂₄X ₂₅X ₂₆X ₂₇X ₂₈ProX ₃₀Thr, wherein amino acid X ₂₃Be Leu, Gly or Gln, X ₂₄Be His or Gln, X ₂₅Be Phe, Ser, Trp or Tyr, X ₂₆Be Asp, Ile, Ser, Trp or Tyr, X ₂₇Be Gly, Glu, Asn or Ser, X ₂₈Be Asp, Asn, Phe, Thr or Tyr, X ₃₀Be Leu, Phe, Pro or Tyr; And

(b) comprise the immunoglobulin heavy chain variable region of 3 complementarity-determining regions,

The complementarity-determining region of wherein said light chain immunoglobulin and described heavy chain immunoglobulin defines the binding site in conjunction with people HGF.

2. isolating conjugated protein in conjunction with human hepatocyte growth factor (HGF), this conjugated protein comprises:

(a) comprise structure C DR _H1-CDR _H2-CDR _H3Immunoglobulin heavy chain variable region, wherein

(i) CDR _H1Comprise aminoacid sequence X ₁TyrX ₃X ₄X ₅, amino acid X wherein ₁Be Asp, Asn, Ser or Thr, X ₃Be Phe, Ser, Trp or Tyr, X ₄Be Ile, Leu or Met, X ₅Be Asn, His or Ser,

(ii) CDR _H2Comprise aminoacid sequence X ₆IleX ₈X ₉X ₁₀X ₁₁GlyX ₁₃X ₁₄X ₁₅TyrX ₁₇X ₁₈X ₁₉X ₂₀X ₂₁X ₂₂, amino acid X wherein ₆Be Lys, Gln, Glu, Val or Tyr, X ₈Be Asn, Gly, Ser, Trp or Tyr, X ₉Be Ala, Pro or Ser, X ₁₀Be Gly or Thr, X ₁₁Be peptide bond, Asp, Asn, Gly or Ser, X ₁₃Be Asp, Asn, His or Ser, X ₁₄Be Ser or Thr, X ₁₅Be Asn or Tyr, X ₁₇Be Asn or Pro, X ₁₈Be Ala, Asp, Gly, Glu, Pro or Ser, X ₁₉Be Asn, Lys, Met or Ser, X ₂₀Be Leu, Phe or Val, X ₂₁Be Lys, Met or Gln, X ₂₂Be Asp, Gly or Ser,

(iii) CDR _H3Comprise aminoacid sequence X ₂₃X ₂₄X ₂₅X ₂₆X ₂₇X ₂₈X ₂₉X ₃₀X ₃₁X ₃₂X ₃₃X ₃₄Tyr, wherein amino acid X ₂₃Be Arg, Asn, Gln or Glu, X ₂₄Be Gly, Leu, Arg or Tyr, X ₂₅Be peptide bond, Asp or Gly, X ₂₆Be peptide bond or Gly, X ₂₇Be peptide bond or Tyr, X ₂₈Be peptide bond, Leu or Tyr, X ₂₉Be peptide bond, Gly, Leu, Arg or Val, X ₃₀Be peptide bond, Asp, Gly or Glu, X ₃₁Be peptide bond, Asn, Arg, Ser or Tyr, X ₃₂Be peptide bond, Ala, Gly, Ile or Tyr, X ₃₃Be Met or Phe, X ₃₄Be Ala or Asp; And

(b) comprise the immunoglobulin light chain variable region of 3 complementarity-determining regions,

3. the described isolated antibody of claim 2, wherein said immunoglobulin light chain variable region comprises structure C DR _L1-CDR _L2-CDR _L3, wherein

(iii) CDR _L3Comprise aminoacid sequence X ₂₃X ₂₄X ₂₅X ₂₆X ₂₇X ₂₈ProX ₃₀Thr, wherein amino acid X ₂₃Be Leu, Gly or Gln, X ₂₄Be His or Gln, X ₂₅Be Phe, Ser, Trp or Tyr, X ₂₆Be Asp, Ile, Ser, Trp or Tyr, X ₂₇Be Gly, Glu, Asn or Ser, X ₂₈Be Asp, Asn, Phe, Thr or Tyr, X ₃₀Be Leu, Phe, Pro or Tyr.

4. claim 1,2 or 3 described conjugated proteins, wherein said complementarity-determining region is inserted between the framework region.

5. the described conjugated protein of claim 4, wherein said CDR sequence is inserted between people or the humanized framework region.

6. isolating nucleic acid, it comprises the nucleotide sequence of the described immunoglobulin light chain variable region of coding claim 1.

7. expression vector, it comprises the described nucleotide sequence of claim 6.

8. host cell, it comprises the described expression vector of claim 7.

9. method for preparing conjugated protein, this method comprises:

(a) cultivate the described host cell of claim 8 under certain condition, make the described immunoglobulin light chain variable region of this host cell expression; And

(b) collect described immunoglobulin light chain variable region.

10. the described method of claim 9 is wherein after step (b), covalently bound with described immunoglobulin light chain variable region and described immunoglobulin heavy chain variable region, make described light chain with variable region of heavy chain in conjunction with people HGF.

11. an isolating nucleic acid, it comprises the nucleotide sequence of the described immunoglobulin heavy chain variable region of coding claim 2.

12. an expression vector, it comprises the described nucleotide sequence of claim 11.

13. a host cell, it comprises the described expression vector of claim 12.

14. a method for preparing conjugated protein, this method comprises:

(a) cultivate the described host cell of claim 13 under certain condition, make the described immunoglobulin heavy chain variable region of this host cell expression; And

(b) collect described immunoglobulin heavy chain variable region.

15. the described method of claim 14 is wherein after step (b), covalently bound with described immunoglobulin heavy chain variable region and described immunoglobulin light chain variable region, makes described light chain define binding site in conjunction with people HGF with variable region of heavy chain.

16. the isolating conjugated protein in conjunction with human hepatocyte growth factor (HGF), this conjugated protein comprises:

(a) comprise structure C DR _L1-CDR _L2-CDR _L3Immunoglobulin light chain variable region; Wherein

(i) CDR _L1Comprise the sequence that is selected from SEQ ID NO.8 (1A3), SEQ ID NO.18 (2B8), SEQ ID NO.28 (2F8), SEQ ID NO.38 (3B6), SEQ ID NO.48 (3D11), SEQ ID NO.58 (1D3), SEQ ID NO.68 (1F3) and SEQ ID NO.78 (3A12)

(ii) CDR _L2Comprise the sequence that is selected from SEQ ID NO.9 (1A3), SEQ ID NO.19 (2B8), SEQ ID NO.29 (2F8), SEQ ID NO.39 (3B6), SEQ ID NO.49 (3D11), SEQ ID NO.59 (1D3), SEQ ID NO.69 (1F3) and SEQ ID NO.79 (3A12)

(iii) CDR _L3Comprise the sequence that is selected from SEQ ID NO.10 (1A3), SEQ ID NO.20 (2B8), SEQ ID NO.30 (2F8), SEQ ID NO.40 (3B6), SEQ ID NO.50 (3D11), SEQ ID NO.60 (1D3), SEQ ID NO.70 (1F3) and SEQ ID NO.80 (3A12); And

(b) immunoglobulin heavy chain variable region, wherein said immunoglobulin light chain variable region and described immunoglobulin heavy chain variable region define the single binding site that is used in conjunction with people HGF together.

17. the described conjugated protein of claim 16, wherein said immunoglobulin light chain variable region comprises:

(i) comprise the CDR of sequence SEQ ID NO.8 (1A3) _L1,

The CDR that (ii) comprises sequence SEQ ID NO.9 (1A3) _L2, and

The CDR that (iii) comprises sequence SEQ ID NO.10 (1A3) _L3

18. the described conjugated protein of claim 16, wherein said immunoglobulin light chain variable region comprises:

(i) comprise the CDR of sequence SEQ ID NO.18 (2B8) _L1,

The CDR that (ii) comprises sequence SEQ ID NO.19 (2B8) _L2, and

The CDR that (iii) comprises sequence SEQ ID NO.20 (2B8) _L3

19. the described conjugated protein of claim 16, wherein said immunoglobulin light chain variable region comprises:

(i) comprise the CDR of sequence SEQ ID NO.28 (2F8) _L1,

The CDR that (ii) comprises sequence SEQ ID NO.29 (2F8) _L2, and

The CDR that (iii) comprises sequence SEQ ID NO.30 (2F8) _L3

20. the described conjugated protein of claim 16, wherein said immunoglobulin light chain variable region comprises:

(i) comprise the CDR of sequence SEQ ID NO.38 (3B6) _L1,

The CDR that (ii) comprises sequence SEQ ID NO.39 (3B6) _L2, and

The CDR that (iii) comprises sequence SEQ ID NO.40 (3B6) _L3

21. the described conjugated protein of claim 16, wherein said immunoglobulin light chain variable region comprises:

(i) comprise the CDR of sequence SEQ ID NO.48 (3D11) _L1,

The CDR that (ii) comprises sequence SEQ ID NO.49 (3D11) _L2, and

The CDR that (iii) comprises sequence SEQ ID NO.50 (3D11) _L3

22. the described conjugated protein of claim 16, wherein said immunoglobulin light chain variable region comprises:

(i) comprise the CDR of sequence SEQ ID NO.58 (1D3) _L1,

The CDR that (ii) comprises sequence SEQ ID NO.59 (1D3) _L2, and

The CDR that (iii) comprises sequence SEQ ID NO.60 (1D3) _L3

23. the described conjugated protein of claim 16, wherein said immunoglobulin light chain variable region comprises:

(i) comprise the CDR of sequence SEQ ID NO.68 (1F3) _L1,

The CDR that (ii) comprises sequence SEQ ID NO.69 (1F3) _L2, and

The CDR that (iii) comprises sequence SEQ ID NO.70 (1F3) _L3

24. the described conjugated protein of claim 16, wherein said immunoglobulin light chain variable region comprises:

(i) comprise the CDR of sequence SEQ ID NO.78 (3A12) _L1,

The CDR that (ii) comprises sequence SEQ ID NO.79 (3A12) _L2, and

The CDR that (iii) comprises sequence SEQ ID NO.80 (3A12) _L3

25. the described conjugated protein of claim 16, wherein CDR _L1, CDR _L2And CDR _L3Be inserted between people or the humanized immunoglobulin (Ig) framework region.

26. the described conjugated protein of claim 16, wherein said conjugated protein are antibody or its Fab.

27. the described conjugated protein of claim 26, wherein said antibody are monoclonal antibody.

28. the isolating conjugated protein in conjunction with human hepatocyte growth factor (HGF), this conjugated protein comprises:

(a) comprise structure C DR _H1-CDR _H2-CDR _H3Immunoglobulin heavy chain variable region; Wherein

(i) CDR _H1Comprise the sequence that is selected from SEQ ID NO.5 (1A3), SEQ ID NO.15 (2B8), SEQ ID NO.25 (2F8), SEQ ID NO.35 (3B6), SEQ ID NO.45 (3D11), SEQ ID NO.55 (1D3), SEQ ID NO.65 (1F3) and SEQ ID NO.75 (3A12)

(ii) CDR _H2Comprise the sequence that is selected from SEQ ID NO.6 (1A3), SEQ ID NO.16 (2B8), SEQ ID NO.26 (2F8), SEQ ID NO.36 (3B6), SEQ ID NO.46 (3D11), SEQ ID NO.56 (1D3), SEQ ID NO.66 (1F3), SEQ ID NO.76 (3A12), SEQ ID NO.202 (Hu2B8 Hv1f.1) and SEQ IDNO.203 (Hu2B8 Hv5a.1 and Hu2B8Hv5-51.1)

(iii) CDR _H3Comprise the sequence that is selected from SEQ ID NO.7 (1A3), SEQ ID NO.17 (2B8), SEQ ID NO.27 (2F8), SEQ ID NO.37 (3B6), SEQ ID NO.47 (3D11), SEQ ID NO.57 (1D3), SEQ ID NO.67 (1F3) and SEQ ID NO.77 (3A12), and

(b) immunoglobulin light chain variable region, wherein said immunoglobulin heavy chain variable region and described immunoglobulin light chain variable region define the single binding site that is used in conjunction with people HGF together.

29. the described conjugated protein of claim 28, wherein said immunoglobulin heavy chain variable region comprises:

(i) comprise the CDR of sequence SEQ ID NO.5 (1A3) _H1,

The CDR that (ii) comprises sequence SEQ ID NO.6 (1A3) _H2, and

The CDR that (iii) comprises sequence SEQ ID NO.7 (1A3) _H3

30. the described conjugated protein of claim 28, wherein said immunoglobulin heavy chain variable region comprises:

(i) comprise the CDR of sequence SEQ ID NO.15 (2B8) _H1,

The CDR that (ii) comprises sequence SEQ ID NO.16 (2B8), SEQ ID NO.202 (Hu2B8Hv lf.1) or SEQ ID NO.203 (Hu2B8Hv5a.1 and Hu2B8Hv5-51.1) _H2, and

The CDR that (iii) comprises sequence SEQ ID NO.17 (2B8) _H3

31. the described conjugated protein of claim 28, wherein said immunoglobulin heavy chain variable region comprises:

(i) comprise the CDR of sequence SEQ ID NO.25 (2F8) _H1,

The CI that (ii) comprises sequence SEQ ID NO.26 (2F8)) R _H2, and

The CDR that (iii) comprises sequence SEQ ID NO.27 (2F8) _H3

32. the described conjugated protein of claim 28, wherein said immunoglobulin heavy chain variable region comprises:

(i) comprise the CDR of sequence SEQ ID NO.35 (3B6) _H1,

The CDR that (ii) comprises sequence SEQ ID NO.36 (3B6) _H2, and

The CDR that (iii) comprises sequence SEQ ID NO.37 (3B6) _H3

33. the described conjugated protein of claim 28, wherein said immunoglobulin heavy chain variable region comprises:

(i) comprise the CDR of sequence SEQ ID NO.45 (3D11) _H1,

The CDR that (ii) comprises sequence SEQ ID NO.46 (3D11) _H2, and

The CDR that (iii) comprises sequence SEQ ID NO.47 (3D11) _H3

34. the described conjugated protein of claim 28, wherein said immunoglobulin heavy chain variable region comprises:

(i) comprise the CDR of sequence SEQ ID NO.55 (1D3) _H1,

The CDR that (ii) comprises sequence SEQ ID NO.56 (1D3) _H2, and

The CDR that (iii) comprises sequence SEQ ID NO.57 (1D3) _H3

35. the described conjugated protein of claim 28, wherein said immunoglobulin heavy chain variable region comprises:

(i) comprise the CDR of sequence SEQ ID NO.65 (1F3) _H1,

The CDR that (ii) comprises sequence SEQ ID NO.66 (1F3) _H2, and

The CDR that (iii) comprises sequence SEQ ID NO.67 (1F3) _H3

36. the described conjugated protein of claim 28, wherein said immunoglobulin heavy chain variable region comprises:

(i) comprise the CDR of sequence SEQ ID NO.75 (3A12) _H1,

The CDR that (ii) comprises sequence SEQ ID NO.76 (3A12) _H2, and

The CDR that (iii) comprises sequence SEQ ID NO.77 (3A12) _H3

37. the described conjugated protein of claim 28, wherein CDR _H1, CDR _H2And CDR _H3Be inserted between people or the humanized immunoglobulin (Ig) framework region.

38. the described conjugated protein of claim 28, wherein said conjugated protein are antibody or its Fab.

39. claim 38 is described in conjunction with framework, wherein said antibody is monoclonal antibody.

40. an isolating nucleic acid, it comprises the nucleotide sequence of the described immunoglobulin light chain variable region of coding claim 16.

41. an expression vector, it comprises the described nucleotide sequence of claim 40.

42. a host cell, it comprises the described expression vector of claim 41.

43. a method for preparing conjugated protein, this method comprises:

(i) cultivate the described host cell of claim 42 under certain condition, make the described immunoglobulin light chain variable region of this host cell expression; And

(ii) collect described immunoglobulin light chain variable region.

44. the described method of claim 43 is wherein after step (b), covalently bound with described immunoglobulin light chain variable region and described immunoglobulin heavy chain variable region, make described light chain with variable region of heavy chain in conjunction with people HGF.

45. an isolating nucleic acid, it comprises the nucleotide sequence of the described immunoglobulin heavy chain variable region of coding claim 28.

46. an expression vector, it comprises the described nucleic acid of claim 45.

47. a host cell, it comprises the described expression vector of claim 46.

48. a method for preparing conjugated protein, this method comprises:

(i) cultivate the described host cell of claim 47 under certain condition, make the described immunoglobulin heavy chain variable region of this host cell expression; And

(ii) collect described immunoglobulin heavy chain variable region.

49. the described method of claim 48, wherein after step (b), described immunoglobulin heavy chain variable region and described immunoglobulin light chain variable region is covalently bound, and making that described light chain defines with variable region of heavy chain can be in conjunction with the binding site of people HGF.

50. the isolating conjugated protein in conjunction with human hepatocyte growth factor (HGF), this conjugated protein comprises:

Immunoglobulin light chain variable region, it is selected from: the residue 21-127 of SEQ ID NO.4 (1A3), the residue 21-127 of SEQ ID NO.14 (2B8), the residue 20-131 of SEQ ID NO.24 (2F8), the residue 23-129 of SEQ ID NO.34 (3B6), the residue 23-128 of SEQ ID NO.44 (3D11), the residue 21-127 of SEQ ID NO.54 (1D3), the residue 21-127 of SEQ ID NO.64 (1F3), the residue 21-127 of SEQ ID NO.74 (3A12), SEQ ID NO.173 (Hu2B8 Kv1-39.1 Kappa chain variable region) and SEQ ID NO.179 (Hu2B8 Kv3-15.1Kappa chain variable region); And

Immunoglobulin heavy chain variable region, it is selected from: the residue 20-141 of SEQ ID NO.2 (1A3), the residue 20-137 of SEQ ID NO.12 (2B8), the residue 20-137 of SEQ ID NO.22 (2F8), the residue 20-139 of SEQ ID NO.32 (3B6), the residue 20-132 of SEQ ID NO.42 (3D11), the residue 20-141 of SEQ ID NO.52 (1D3), the residue 20-141 of SEQ ID NO.62 (1F3), the residue 20-141 of SEQ ID NO.72 (3A12), SEQ ID NO.159 (Hu2B8Hv1f.1 variable region of heavy chain), SEQ ID NO.165 (Hu2B8Hv5a.1 variable region of heavy chain) and SEQ ID NO.169 (Hu2B8 Hv5-51.1 variable region of heavy chain).

51. the described conjugated protein of claim 50, wherein said immunoglobulin light chain variable region comprises the aminoacid sequence of the residue 21-127 of SEQ ID NO.4 (1A3), and described immunoglobulin heavy chain variable region comprises the aminoacid sequence of the residue 20-141 of SEQ ID NO.2 (1A3).

52. the described conjugated protein of claim 50, wherein said immunoglobulin light chain variable region comprises the aminoacid sequence of the residue 21-127 of SEQ ID NO.14 (2B8), and described immunoglobulin heavy chain variable region comprises the aminoacid sequence of the residue 20-137 of SEQ ID NO.12 (2B8).

53. the described conjugated protein of claim 50, wherein said immunoglobulin light chain variable region comprises the aminoacid sequence of the residue 20-131 of SEQ ID NO.24 (2F8), and described immunoglobulin heavy chain variable region comprises the aminoacid sequence of the residue 20-137 of SEQ ID NO.22 (2F8).

54. the described conjugated protein of claim 50, wherein said immunoglobulin light chain variable region comprises the aminoacid sequence of the residue 23-129 of SEQ ID NO.34 (3B6), and described immunoglobulin heavy chain variable region comprises the aminoacid sequence of the residue 20-139 of SEQ ID NO.32 (3B6).

55. the described conjugated protein of claim 50, wherein said immunoglobulin light chain variable region comprises the aminoacid sequence of the residue 23-128 of SEQ ID NO.44 (3D11), and described immunoglobulin heavy chain variable region comprises the aminoacid sequence of the residue 20-132 of SEQ ID NO.42 (3D11).

56. the described conjugated protein of claim 50, wherein said immunoglobulin light chain variable region comprises the aminoacid sequence of the residue 21-127 of SEQ ID NO.54 (1D3), and described immunoglobulin heavy chain variable region comprises the aminoacid sequence of the residue 20-141 of SEQ ID NO.52 (1D3).

57. the described conjugated protein of claim 50, wherein said immunoglobulin light chain variable region comprises the aminoacid sequence of the residue 21-127 of SEQ ID NO.64 (1F3), and described immunoglobulin heavy chain variable region comprises the aminoacid sequence of the residue 20-141 of SEQ ID NO.62 (1F3).

58. the described conjugated protein of claim 50, wherein said immunoglobulin light chain variable region comprises the aminoacid sequence of the residue 21-127 of SEQ ID NO.74 (3A12), and described immunoglobulin heavy chain variable region comprises the aminoacid sequence of the residue 20-141 of SEQ ID NO.72 (3A12).

59. the isolating conjugated protein in conjunction with human hepatocyte growth factor (HGF), this conjugated protein comprises:

Immunoglobulin light chain variable region, it is selected from: SEQ ID NO.173 (Hu2B8Kv1-39.1 variable region of light chain) and SEQ ID NO.179 (Hu2B8 Kv3-15.1 variable region of light chain); And

Immunoglobulin heavy chain variable region, it is selected from: SEQ ID NO.159 (Hu2B8 Hv1f.1 variable region of heavy chain), SEQ ID NO.165 (Hu2B8 Hv5a.1 variable region of heavy chain) and SEQ IDNO.169 (Hu2B8 Hv5-51.1 variable region of heavy chain).

60. the isolating conjugated protein in conjunction with human hepatocyte growth factor (HGF), this conjugated protein comprises:

Immunoglobulin light chain variable region, it is selected from: SEQ ID NO.177 (Hu2B8Kv1-39.1+kappa constant region (Km (3) allotype (allelotrope 2)) and SEQ ID NO.181 (Hu2B8 Kv3-15.1+Kappa constant region (Km (3) allotype (allelotrope 2)); And

Immunoglobulin heavy chain variable region, it is selected from: ((Glm (17 for the Hu2B8Hv1f.1+IgG1 constant region for SEQ ID NO.163,1) allotype)), ((Glm (17 for the Hu2B8Hv5a.1+IgG1 constant region for SEQ ID NO.167,1) allotype)), SEQ ID NO.171 (Hu2B8Hv5-51.1+IgG1 constant region (Glm (17,1) allotype)) and SEQ ID NO.210 (Hu2B8Hv5-51.1+IgG1 constant region Glm (3) allotype (allelotrope 2)).

61. claim 50,59 or 60 described conjugated proteins, wherein said conjugated protein are antibody or its Fab.

62. the described conjugated protein of claim 61, wherein said antibody are monoclonal antibody.

63. the isolating conjugated protein in conjunction with human hepatocyte growth factor (HGF), this conjugated protein comprises:

(i) comprise the immunoglobulin light chain variable region of 3 complementarity-determining regions; And

The immunoglobulin heavy chain variable region that (ii) comprises 3 complementarity-determining regions,

The complementarity-determining region of wherein said light chain immunoglobulin and described heavy chain immunoglobulin defines the binding site in conjunction with reductive people HGF together.

64. the described conjugated protein of claim 63, wherein said conjugated protein are antibody or its Fab.

65. the described conjugated protein of claim 64, wherein said antibody are monoclonal antibody.

66. the described conjugated protein of claim 63, wherein said complementarity-determining region is inserted between the framework region.

67. the described conjugated protein of claim 63, wherein said heavy chain immunoglobulin are IgGl.

68. the described conjugated protein of claim 63, wherein said conjugated protein are replaced by arginine with the halfcystine that is included in 561 positions or the glycine in 555 positions is combined by the people HGF that L-glutamic acid replaces.

69. the described conjugated protein of claim 63, wherein said conjugated protein is in conjunction with the α chain of people HGF.

70. the described conjugated protein of claim 63, wherein said immunoglobulin light chain variable region comprise at least 1 complementarity-determining region (CDR), this complementarity-determining region is selected from CDR _L1, CDR _L2And CDR _L3,

CDR wherein _L1Comprise aminoacid sequence X ₁X ₂SerX ₄X ₅X ₆X ₇X ₈X ₉X ₁₀X ₁₁X ₁₂X ₁₃X ₁₄X ₁₅, amino acid X wherein ₁Be Arg or Lys, X ₂Be Ala or Thr, X ₄Is Glu or Gln, X ₅Be Asn, Ser or Asp, X ₆Be Ile or Val, X ₇Be Tyr, Asp or Lys, X ₈Be peptide bond or Tyr, X ₉Be peptide bond or Asp, X ₁₀Be peptide bond or Gly, X ₁₁Be peptide bond or Asn, X ₁₂Be peptide bond or Ser, X ₁₃Be Asn or Tyr, X ₁₄Be Ile or Leu, X ₁₅Be Ala, Asn or Ser,

CDR wherein _L2Comprise aminoacid sequence X ₁₆X ₁₇X ₁₈X ₁₉LeuX ₂₁X ₂₂, amino acid X wherein ₁₆Be Ala, Asp, Val or Arg, X ₁₇Be Ala or Val, X ₁₈Be Asn, Ser or Thr, X ₁₉Be Arg, Asn or His, X ₂₁Be Ala, Glu, Val or Pro, X ₂₂Be Asp or Ser, and

CDR wherein _L3Comprise aminoacid sequence X ₂₃X ₂₄X ₂₅X ₂₆X ₂₇X ₂₈ProX ₃₀Thr, wherein amino acid X ₂₃Be Leu or Gln, X ₂₄Be His or Gln, X ₂₅Be Phe, Ser or Tyr, X ₂₆Be Asp, Ile or Trp, X ₂₇Be Gly or Glu, X ₂₈Be Asp, Phe or Thr, X ₃₀Be Phe, Pro or Tyr.

71. claim 63 or 70 described conjugated proteins, wherein said immunoglobulin heavy chain variable region comprises at least 1 CDR, and this CDR is selected from CDR _L1, CDR _L2And CDR _L3,

CDR wherein _H1Comprise aminoacid sequence X ₁TyrX ₃X ₄X ₅, amino acid X wherein ₁Be Asp, Asn, Ser or Thr, X ₃Be Phe, Trp or Tyr, X ₄Be Ile or Met, X ₅Be Asn, His or Ser,

CDR wherein _H2Comprise aminoacid sequence X ₆IleX ₈X ₉GlyX ₁₁GlyX ₁₃X ₁₄X ₁₅TyrX ₁₇X ₁₈X ₁₉X ₂₀LysX ₂₂, amino acid X wherein ₆Be Lys, Gln or Tyr, X ₈Be Gly, Ser or Tyr, X ₉Be Pro or Ser, X ₁₁Be Asp, Gly or Ser, X ₁₃Be Asp or Ser, X ₁₄Be Ser or Thr, X ₁₅Be Asn or Tyr, X ₁₇Be Asn or Pro, X ₁₈Be Ala, Asp, Gly or Glu, X ₁₉Be Asn, Met or Ser, X ₂₀Be Phe or Val, X ₂₂Be Asp or Gly, and

CDR wherein _H3Comprise aminoacid sequence X ₂₃X ₂₄X ₂₅X ₂₆X ₂₇X ₂₈X ₂₉X ₃₀X ₃₁X ₃₂X ₃₃AspTyr, wherein amino acid X ₂₃Be Arg or Gln, X ₂₄Be Gly or Leu, X ₂₅Be Asp, Gly or peptide bond, X ₂₆Be Gly or peptide bond, X ₂₇Be peptide bond or Tyr, X ₂₈Be Leu, peptide bond or Tyr, X ₂₉Be Gly, Arg or Leu, X ₃₀Be Asp, Gly or Glu, X ₃₁Be Tyr, Arg or Asn, X ₃₂Be Ala, Gly or Tyr, X ₃₃Be Met or Phe.

72. the described conjugated protein of claim 70, wherein said light chain immunoglobulin comprises:

(i) CDR _L1, it comprises the sequence that is selected from SEQ ID NO.8 (1A3), SEQ ID NO.28 (2F8), SEQ ID NO.38 (3B6), SEQ ID NO.58 (1D3) and SEQ ID NO.68 (1F3),

(ii) CDR _L2, it comprises the sequence that is selected from SEQ ID NO.9 (1A3), SEQ ID NO.29 (2F8), SEQ ID NO.39 (3B6), SEQ ID NO.59 (1D3) and SEQ ID NO.69 (1F3), and

(iii) CDR _L3, it comprises the sequence that is selected from SEQ ID NO.10 (1A3), SEQ ID NO.30 (2F8), SEQ ID NO.40 (3B6), SEQ ID NO.60 (1D3) and SEQ ID NO.70 (1F3).

73. the described conjugated protein of claim 72, wherein said CDR sequence is inserted between people or the humanized framework region.

74. the described conjugated protein of claim 72, wherein said light chain immunoglobulin comprise the aminoacid sequence of the residue 21-127 of the residue 21-127 of residue 23-129, SEQ ID NO.54 (1D3) of residue 20-131, SEQ ID NO.34 (3B6) of the residue 21-127, the SEQ ID NO.24 (2F8) that are selected from SEQ ID NO.4 (1A3) and SEQ ID NO.64 (1F3).

75. the described conjugated protein of claim 71, wherein said immunoglobulin heavy chain variable region comprises:

(i) CDR _H1, it comprises the sequence that is selected from SEQ ID NO.5 (1A3), SEQ ID NO.25 (2F8), SEQ ID NO.35 (3B6), SEQ ID NO.55 (1D3) and SEQ ID NO.65 (1F3),

(ii) CDR _H2, it comprises the sequence that is selected from SEQ ID NO.6 (1A3), SEQ ID NO.26 (2F8), SEQ ID NO.36 (3B6), SEQ ID NO.56 (1D3) and SEQ ID NO.66 (1F3), and

(iii) CDR _H3, it comprises the sequence that is selected from SEQ ID NO.7 (1A3), SEQ ID NO.27 (2F8), SEQ ID NO.37 (3B6), SEQ ID NO.57 (1D3) and SEQ ID NO.67 (1F3).

76. the described conjugated protein of claim 75, wherein said CDR sequence is inserted between people or the humanized framework region.

77. the described conjugated protein of claim 75, wherein said immunoglobulin heavy chain variable region comprise the aminoacid sequence of the residue 20-141 of the residue 20-141 of residue 20-139, SEQ I DNO.52 (1D3) of residue 20-137, SEQ ID NO.32 (3B6) of the residue 20-141, the SEQ ID NO.22. (2F8) that are selected from SEQ ID NO.2 (1A3) and SEQ ID NO.62 (1F3).

78. an isolating nucleic acid, it comprises the nucleotide sequence of coding claim 63,70,72 or 74 described immunoglobulin light chain variable regions.

79. an expression vector, it comprises the described nucleotide sequence of claim 78.

80. a host cell, it comprises the described expression vector of claim 79.

81. an isolating nucleic acid, it comprises the nucleotide sequence of coding claim 63,71,75 or 77 described immunoglobulin heavy chain variable regions.

82. an expression vector, it comprises the described nucleotide sequence of claim 81.

83. a host cell, it comprises the described expression vector of claim 82.

84. isolating conjugated protein in conjunction with human hepatocyte growth factor (HGF), this conjugated protein comprises immunoglobulin light chain variable region and immunoglobulin heavy chain variable region, wherein said isolating conjugated protein with at least a kind with reference to antibody competition combine HGF, describedly be selected from reference to antibody:

(i) have the antibody of immunoglobulin heavy chain variable region of the residue 20-137 of the immunoglobulin light chain variable region of residue 20-131 of SEQ IDNO.24 (2F8) and SEQ ID NO.22 (2F8),

The antibody of immunoglobulin heavy chain variable region that (ii) has the residue 20-139 of the immunoglobulin light chain variable region of residue 23-129 of SEQ ID NO.34 (3B6) and SEQ ID NO.32 (3B6), and

The antibody of immunoglobulin heavy chain variable region that (iii) has the residue 20-132 of the immunoglobulin light chain variable region of residue 23-128 of SEQ ID NO.44 (3D11) and SEQ ID NO.42 (3D11).

85. the described conjugated protein of claim 84, wherein said conjugated protein is in conjunction with the epi-position of the HGF identical with one of reference antibody.

86. an isolating conjugated protein, itself and human hepatocyte growth factor (HGF) bonded k _dBe 4.0 x 10 ^-5s ^-1Or it is lower.

87. the described conjugated protein of claim 86, wherein k _dBe 3.0 x 10 ^-5s ^-1Or it is lower.

88. the described conjugated protein of claim 87, wherein k _dBe 2.0 x 10 ^-5s ^-1Or it is lower.

89. an isolating conjugated protein, itself and human hepatocyte growth factor (HGF) specificity bonded K _DBe 20pM or lower.

90. the described conjugated protein of claim 89, wherein K _DBe 10pM or lower.

91. the described conjugated protein of claim 90, wherein K _DBe 5pM or lower.

92. an isolating conjugated protein, wherein said antibody in conjunction with human hepatocyte growth factor (HGF) under 37 ℃ in conjunction with the K of people HGF _DThan this antibody K in conjunction with people HGF under 25 ℃ _DLower.

93. the described conjugated protein of claim 92, the K of wherein said conjugated protein under 37 ℃ _DLess than 5pM.

94. method that suppresses or reduce tumor cell proliferation, this method comprises described cellular exposure in the claim 1,2,3,16,28,50,59,60,63,70,71,72,74,75,77,84 of significant quantity, 86 or 89 described conjugated proteins, thereby suppresses or reduce the propagation of described tumour cell.

95. method that suppresses or reduce tumor cell proliferation, this method comprises described cellular exposure in the conjugated protein of significant quantity, thereby suppress or reduce the propagation of described tumour cell, wherein said conjugated protein is specifically in conjunction with people HGF but do not reduce people HGF and c-Met bonded ability substantially.

96. the described method of claim 95, wherein said conjugated protein comprise claim 22,23,24,34,35,36,56,57,58,84,86 or 89 described conjugated proteins.

97. claim 94 or 95 described methods, wherein said tumour cell is a human tumor cells.

98. method that suppresses or reduce tumor growth in the Mammals, this method comprises described Mammals is exposed in the claim 1,2,3,16,28,50,59,60,63,84,86,89 or 92 described conjugated proteins of significant quantity, thereby suppresses or reduce the propagation of tumour.

99. a method for the treatment of tumour in the Mammals, this method comprise claim 1,2,3,16,28,50,59,60,63,84,86, the 89 or 92 described conjugated proteins of using significant quantity.

100. claim 98 or 99 described methods, wherein said Mammals is behaved.