CN101460520B - Hepatocyte growth factor (hgf) binding proteins - Google Patents

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; 509 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); Also be called as spreading factor (Scatter Factor; SF); Be a kind of multi-functional heterodimer protein that mainly produces, 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 by mesenchymal cell.People Met acceptor also is called as " c-Met ".Sophisticated HGF comprises two polypeptied 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, it is active that HGF has regulated various kinds of cell.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 USP sequence number 5,997,868 and 5,707,624.The investigator has developed many HGF regulators (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 substruction of all antibody.Antibody is the polymer protein that contains 4 polypeptied chains.2 polypeptied chains wherein are called as heavy chain or H chain, and other 2 polypeptied chains are called as light chain or L chain.The heavy chain and the light chain that connect Tegeline through the interchain disulfide linkage.The heavy chain that connects Tegeline through 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 respectively 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.

USP sequence number 5,707,624 has been described the purposes of antibody in the treatment Kaposi sarcoma of anti-HGF.Similarly, USP sequence number 5,997,868 has been described through using the antibody of anti-HGF to patient to be treated, thereby block endogenous HGF promotes the oncotherapy of 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 needs can be used as other HGF regulators of therapeutical agent and diagnostic reagent.

Summary of the invention

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

Therefore the neutralized activity of HGF of said conjugated protein can be used as therapeutical agent.In certain embodiments, described conjugated protein suppresses HGF and 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 said 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 through hereinafter described, show clearly of the present invention these with other aspects and advantage.

Description of drawings

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

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 in square frame, defines definition signal peptide, CDR 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 in square frame, defines definition signal peptide, CDR1, 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 result's 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 summary view 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 result's 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 summary view 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 people 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 that for summing up the histogram of testing data Hu2B8 combines the epi-position of repelling each other with mouse monoclonal antibody 2B8.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 polyclonal goat anti HGF) 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 combine specifically HGF and in and the discovery of the active conjugated protein family of HGF (particularly people HGF).Said conjugated protein can be used for many diagnosis and treatment is used.Described conjugated protein based on combine with regard to it HGF and in the active ability 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 the growth and/or the propagation of HGF responsive cell (for example cancer cells).When described conjugated protein is used as therapeutical agent, can transforms it, thereby minimize or eliminate the risk of when being applied to the recipient, inducing to the immunne response of said conjugated protein.In addition, according to concrete application, comprise said conjugated protein and other parts (for example detectable label (like radioactive labels) and effector molecule (like other protein with based on micromolecular therapeutical agent)) are puted together.Hereinafter will carry out detailed explanation in these characteristics of the present invention and the aspect each.

I-combines the conjugated protein of HGF

In one aspect, the present invention provides the isolating conjugated protein of a kind of people of combination HGF, and 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 to combine people HGF together.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.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 present invention provides the isolating conjugated protein of a kind of people of combination HGF, and 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 to combine people 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 said conjugated protein comprises aforesaid light chain immunoglobulin and heavy chain immunoglobulin or its fragment.In addition, should be appreciated that said 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 present invention provides a kind of isolating conjugated protein that combines people HGF specifically.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 to combine 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 to be 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, said 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, said 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, said 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, said 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, said 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, said 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, said 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, said 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 said CDR _L1, CDR _L2And CDR _L3Sequence is inserted between people or the humanized IgF R.Should be appreciated that said conjugated protein can be complete antibody, its Fab or biosynthetic antibody sites.

On the other hand, the present invention provides the isolating conjugated protein of a kind of people of combination 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 to combine 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 Hv1f.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, said 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, said conjugated protein comprises immunoglobulin heavy chain variable region, and this immunoglobulin heavy chain variable region comprises CDR _H1, CDR _H2And CDR _H2, 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, said 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, said 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, said 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, said 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, said 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, said 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 said CDR _H1, CDR _H2And CDR _H3Sequence is inserted between people or the humanized IgF R.Should be appreciated that said conjugated protein can be complete antibody, its Fab or biosynthetic antibody sites.

On the other hand, the present invention provides the conjugated protein of a kind of people of combination 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 the residue 20-139 of the residue 20-137 of the residue 20-141 of SEQ ID NO.2 (1A3), SEQID NO.12 (2B8), the residue 20-137 of SEQ ID NO.22 (2F8), SEQ IDNO.32 (3B6), the residue 20-132 of SEQ ID NO.42 (3D11), SEQ ID NO.52 (1D3), the residue 20-141 of SEQ ID NO.62 (1F3) and the residue 20-141 of SEQ ID NO.72 (3A12), described immunoglobulin light chain variable region is selected from: the residue 21-127 of the residue 23-129 of the residue 21-127 of the residue 21-127 of SEQ ID NO.4 (1A3), SEQ IDNO.14 (2B8), the residue 20-131 of SEQ ID NO.24 (2F8), SEQ ID NO.34 (3B6), the residue 23-128 of SEQ ID NO.44 (3D11), 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; Said 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; Said 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; Said 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; Said 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; Said 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; Said 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; Said 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; Said 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 ID NO.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, said conjugated protein is complete antibody, its Fab or biosynthetic antibody sites.

On the other hand, the present invention provides the isolating conjugated protein of a kind of people of combination HGF.This conjugated protein comprises: (i) immunoglobulin light chain variable region, and it is selected from: SEQ ID NO.173 (Hu2B8Kv1-39.1 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 (Hu2B8Hv5a.1 variable region of heavy chain); SEQ ID NO.169 (Hu2B8Hv5-51.1 variable region of heavy chain), SEQ ID NO.183 (LR2B8HC variable region of heavy chain) and SEQ ID NO.189 (LRMR2B8HC variable region of heavy chain).Said conjugated protein can be complete antibody, its Fab or biosynthetic antibody sites.

On the other hand, the present invention provides the isolating conjugated protein of a kind of people of combination HGF.This conjugated protein comprises: (i) light chain immunoglobulin, and it is selected from: SEQ ID NO.177 (Hu2B8Kv1-39.1+Kappa constant region (Km (3) allotype (allelotrope 2)), SEQ ID NO.181 (Hu2B8Kv 3-15.1+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 Hvlf.1+IgG1 constant region for SEQ IDNO.163; 1) allotype)), ((Glm (17 for the Hu2B8Hv5a.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)).Said conjugated protein can be complete antibody, its Fab or biosynthetic antibody sites.

On the other hand, the present invention provides a kind of isolating conjugated protein that combines 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 the binding site (the for example α chain of reductive HGF) that combines reductive people HGF together.Reductive HGF is meant to use is enough to reduce the HGF that the reductive agent (for example WR 34678 (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, said 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, said conjugated protein comprises 2 CDR, for example CDR _L1And CDR _L2, or CDR _L1And CDR _L3, or CDR _L1And CDR _L3Alternatively, said 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.CDR _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 Gln, X ₂₈Be Asp, Phe or Thr, X ₃₀Be Phe, Pro or Tyr.

In another embodiment, said 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, said conjugated protein comprises 2 CDR, for example CDR _H1And CDR _H2, or CDR _H1And CDR _H3, or CDR _H1And CDR _H3Alternatively, said 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 said conjugated protein comprises aforesaid heavy chain immunoglobulin and light chain immunoglobulin or its fragment.In addition, should be appreciated that said conjugated protein is complete antibody or its Fab or biosynthetic antibody sites.

In certain embodiments, said conjugated protein comprises immunoglobulin light chain variable region, and this immunoglobulin light chain variable region comprises: (i) CDR _L1, 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).Can the CDR sequence be inserted between people or the humanized FR.In other embodiments; Said 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 residue 21-127 and SEQ ID NO.64 (1F3) of residue 23-129, SEQ ID NO.54 (1D3) of residue 20-131, the SEQ ID NO.34 (3B6) of the residue 21-127 that is selected from SEQ ID NO.4 (1A3), SEQ ID NO.24 (2F8).

In certain embodiments, said 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).Can the CDR sequence 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 residue 20-141 and SEQ ID NO.62 (1F3) of residue 20-139, SEQ ID NO.52 (1D3) of residue 20-137, the SEQ ID NO.32 (3B6) of the residue 20-141 that is selected from SEQ ID NO.2 (1A3), SEQID NO.22. (2F8).

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

Should be appreciated that every kind of conjugated protein that preceding text are discussed can be complete antibody, for example monoclonal antibody.Alternatively, said 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 USP sequence number 5,476, and 786 is said.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 for combining the antibody or the antibody fragment of at least two kinds of different antigens.For example, the dual specific conjugated protein can combine 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 through merging hybridoma or through 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 l-arginine with the halfcystine that is included in 561 positions or the glycocoll in 555 positions is combined by the substituted hHGF of L-glutamic acid.

In another aspect, this law provides a kind of isolating conjugated protein, itself and people HGF bonded k _dBe 4.0x10 ^-5s ^-1Or lower, 3.0x10 ^-5s ^-1Or lower or 2.0x10 ^-5s ^-1Or it is lower.Said isolating protein and people HGF bonded k _dBe 5.0x10 ^-5s ^-1To 0.5x10 ^-5s ^-1, 4.0x10 ^-5s ^-1To 1.0x10 ^-5s ^-1, or 3.0x10 ^-5s ^-1To 1.5x10 ^-5s ^-1In another aspect, the present 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.Said 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 through method and condition described in the embodiment 6 _DValue.

On the other hand, this law provides the conjugated protein of a kind of isolating combination 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.Said conjugated protein is randomly under 37 ℃, to be lower than the K of 5pM _DCombine with people HGF.

In other respects with embodiment in, said 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 said conjugated protein ₅₀(50% o'clock concentration of largest inhibition) is at least about 4.0,4.5,5.0,5.5,6.0,6.5 and 7.0nM.In other the embodiment, through using the described method of embodiment 7 (b), said 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 said 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, through using the analysis described in the embodiment 9, said 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, said 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, through 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.Can this synthetic dna molecular 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 hybridization technique or the round pcr of synthetic nucleic probe through routine by cloning the sequence that this paper provides in the hybridoma, the sequence of wherein said nucleic 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 through 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, MK cells (COS), human liver cancer cell (for example Hep G2) and myeloma cell, these cells can not produce Tegeline 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.This process is realized in downstream through engineered gene being placed suitable bacterium promotor (for example Trp or Tac) and signal sequence (the 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 behind smudge cells, gather in the crops through French press (French press) or supersonic method.Then, the dissolving refractile body, and use the method refolding that is directed against other recombinant proteins foundation and shear expressed protein.

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, Tegeline 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 said conjugated protein, can modify, thereby optimize its performance this conjugated protein.For example, when said conjugated protein is used as therapeutical agent, can modify this conjugated protein, to reduce its immunogenicity in the purpose recipient.Alternatively or additionally, can said conjugated protein and another protein or peptide (for example growth factor, cytokine or cytotoxin) 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 said 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, the constant region for immunoglobulin that is wherein 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 USP sequence number 6,893,625 (Robinson), 5,500,362 (Robinson) and 4,816,567 (Cabilly).

In another 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 confirm total aminoacid sequence.CDR is implanted in for example 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; Among 332 (Hoogenboom), 5,585,089 (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 through 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 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) " that is called that is used to produce humanized antibody.For example referring to people such as Vaswami (1998) ANNALSOF ALLERGY, ASTHMA, & IMMUNOL.81:105; People such as Roguska (1996) PROT.ENGINEER 9:895-904; And 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 that same position place in people's antibody more frequently occurs.This antibody is reinvented technology and is existed, and for example the 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), what this technology related in mammalian cell expressing antibodies is the carrier on basis with the vaccinia virus to technology for Vaccinex company, Rochester.It is said, with the high-caliber combination variety that produces heavy chain immunoglobulin and light chain.For example referring to USP sequence number 6,706,477 (Zauderer), 6,800,442 (Zauderwer) 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 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 in mouse, producing complete people's antibody.In the method, use transgenic mice to prepare people's antibody, the substantive part that the gene that produces mouse antibodies in the said 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 said 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 said 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 said conjugated protein chemical coupling.On the other hand; If described effector molecule is protein or peptide (for example enzyme, acceptor, toxin, growth factor, cytokine or other immunomodulators); Can use the interior coupling chemical method of body to make said conjugated protein and effector chemical coupling so, perhaps can make said conjugated protein and effector coupling form fused protein.Can use with part II in the similar technology of method discussed make up and expressed fusion protein matter.

The purposes of IV-conjugated protein

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

(1) treatment is used

Owing to the activity of HGF, use in the conjugated protein of the present invention 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 (the for example cancer of various ways).

Can use said 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, said conjugated protein inhibition tumor cell proliferation is at least 50%, 60%, 70%, 80%, 90%, 95% or 100%.

In certain embodiments, said 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, even when said conjugated protein combines hHGF but do not have to suppress the combining of hHGF and c-Met substantively, said conjugated protein is used to suppress or reduce the propagation of tumour cell, shown in the antibody 3B6 in the table 5 and 6.

In addition, said 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 said conjugated protein of administration significant quantity, thus growth of tumor in the inhibition or the Mammals that slows down.Therefore, said conjugated protein can be used for treatment, the for example tumour in the Mammals.Said method comprises the said conjugated protein to administration treatment significant quantity.Said 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 level or tissue level fast, 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 said conjugated protein (for example monoclonal antibody) freeze-drying, and when using, with its reconstruct in BS.

Said 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 said conjugated protein, perhaps can before or after said conjugated protein, use.

The preparation that comprises the conjugated protein of therepic use contains the conjugated protein with pharmaceutically useful carrier combinations usually.Use like this paper; " 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 compatible 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 through any suitable method (comprising known any method in the pharmaceutics field).Should pharmaceutical composition of the present invention be processed compatible mutually with the route of administration of its plan.The instance of route of administration comprises that parenteral administration or parenteral use outward, and for example intravenous administration, intradermal administration, suction are used, transdermal administration (part), pass through mucosal administration and rectal administration.Can be through 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 compsn; Solution in waterborne liquid or non-aqueous liquid or suspension-s; 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, Ucar 35 or other synthetic solvents injected; Antiseptic-germicide, for example phenylcarbinol or para methyl paraben; Inhibitor, for example xitix or sodium sulfite anhy 96; Sequestrant, for example YD 30; 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 processed by glass or plastics in.

Substantially, the compsn 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 intravenous administration, suitable carriers comprise saline water, 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 preserved 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, Ucar 35 and liquid polyethylene glycol) and their suitable mixture.

Pharmaceutical prepn is preferably aseptic.For example sterilize through the filtration of aseptic filter membrane.Under the situation of the said compsn 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-s, 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 said 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 ri, as ³Hydrogen ( ³H), ¹⁴Carbon ( ¹⁴C), ³²Phosphorus ( ³²P), ³⁵Sulphur ( ³⁵S) or ¹²⁵Iodine ( ¹²⁵I); Fluorescent chemicals or chemiluminescence compound are like fluorescein isothiocyanate, rhodamine or resorcinolphthalein; Enzyme is like SEAP, beta galactosidase enzyme or horseradish peroxidase; Spin probe is like spin labeling; Or coloured particle, like 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.Perhaps detect described mark through for example range estimation through spectrophotometer or other detectors.

Use said 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 through 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 through 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 in vivo partly comprises nitroxide spin labeling and lanthanon and transition metal ion, and all these materials can both be induced the original position proton relaxation.Except immune video picture, can in the RIT method of standard, use the compound radioactive segment, thereby destroy target cell.The preferred Nucleotide that is used for the high dosage RIT comprises radioactive atom ⁹⁰Yttrium ( ⁹⁰Yt), ¹³¹Iodine ( ¹³¹I) or ¹¹¹Indium ( ¹¹¹In).Can use known coupling technology in the video picture field, use ¹³¹I, ¹¹¹In with ^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 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 USP sequence number 5,534,254.

In whole specification sheets, have, comprise or when containing concrete composition, comprise that compsn also is grouped into by described one-tenth basically or is grouped into by described one-tenth when compsn is described as.Similarly, have, comprise or when comprising concrete treatment step, this method also is made up of described treatment step basically 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.

According to multidigit point repetition immunity (RIMMS) method, (Portland ME) carries out immunity, fusion and primary dcreening operation by MBS company.Use recombinant human HGF (R&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 in (ELISA) its serum and demonstrates active 2 mouse of the highest anti-HGF and be used for fusion subsequently.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.Through ELISA, just combine hHGF that the supernatant from the fusions of gained is screened.Be accredited as the supernatant that comprises to the antibody of HGF, through further characterizing like function test in the described body of following examples.Select one group of hybridoma, and to this hybridoma subclone and amplification.Then, through 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 mensuration of nucleotide sequence of immunoglobulin heavy chain variable region and variable region of light chain

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 through PCR (polymerase chain reaction) the increase variable region of Tegeline 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 ' cgactgaggcacctccagatgtt 3 ' (SEQ ID NO.92)) amplification Kappa chain.

Separate independent PCR product through 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 TOPO TA Cloning

test kit (having pCR

2.1-TOPO

carrier) (Invitrogen subsequently based on topoisomerase; Carlsbad; CA); The specification sheets that provides according to manufacturers with the PCR product cloning in pCR2.1 TOPO plasmid; And the transformation technology of the standard of use, the gained plasmid is transformed in the DH5 bacterium.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 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 mensuration of nucleotide sequence of heavy chain immunoglobulin and constant region of light chain sequence of Kappa and IgG1 chain of 1A3,1D3,1F3 and 2B8

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 AtgGgatggagctatatcatcctcttt 3 ' (the line part is an initiator codon) (SEQ ID NO.98) and reverse primer 5 ' ggggaccactttgtacaagaaagctgggt TcaTttaccaggagagtgggagag 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.Through 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 at the upper reaches 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 Next Password that the variable region/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 the variable region sequences of mark 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 atgagtggtgc 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 aggactctggt 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 tgcaacagat ttcactctga 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 ttactacaat

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

81 caacagaaac caggacagcc acccaaagtc ctcatctatg ttgcatccaa tctagaatct

241 gggatcccag ccaggtttag tggcagtggg tctgggacag acttcaccct caacatccat

301 ctgtggagg aggaggatgc tgcaacctat tactgtcagc 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 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 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 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 tcag

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

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 ac

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

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 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 ggagttatta ctgtcaacat 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 (is mouse derived from AJ) of the mensuration of 1A3,1D3,1F3 and 2B8(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 the mouse Kappa constant region of light chain (is mouse derived from AJ) of the mensuration of mouse Kappa constant region of light chain (V00807) and 1D3,1F3 and 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) compare the Nucleotide (representing) that contains 1 change with the situation of 1D3,1F3 and 2B8, the mouse Kappa constant region of light chain of the mensuration of 1A3 with underscore(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, confirmed 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, confirmed 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 ease, 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
		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
		5	Heavy chain CDR 11A3
6	Heavy chain CDR 21A3
		7	Heavy chain CDR 31A3
8	Light (kappa) CDR 11A3

[0328]

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

[0329]

38	Light (kappa) CDR 13B6
		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
		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
		47	Heavy chain CDR 33D11
48	Light (kappa) CDR 13D11
		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
		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
		57	Heavy chain CDR 31D3
58	Light (kappa) CDR 11D3
		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
		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

[0330]

67	Heavy chain CDR 31F3
		68	Light (kappa) CDR 11F3
69	Light (kappa) CDR 21F3
		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
		74	Light (kappa) chain variable region 3A12-protein
75	Heavy chain CDR 13A12
		76	Heavy chain CDR 23A12
77	Heavy chain CDR 33A12
		78	Light (kappa) CDR 13A12
79	Light (kappa) CDR 23A12
		80	Light (kappa) CDR 33A12

In addition, for ease, following sequence has been represented total length sequence of heavy chain reality or that estimate and the sequence of light chain (that is, comprise variable region sequences and constant region sequence the two) of described each antibody of present embodiment.Notice that the constant region of mouse antibodies 2F8,3A12,3B6 and 3D11 is order-checking not, but suppose that they comprise and the identical constant region sequence of 1D3,1F3 and 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.

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

1 atgaactttg ggctcagatt gattttccttgtccttgttt 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 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

(2) define the protein sequence (not having signal sequence) of total length 1A3 sequence of heavy chain (1A3 variable region of heavy chain and IgG1 constant region)(SEQ ID NO.123)

1 evqlvesggg lvqpggslkl scaaseftfs nyymswvrqt pekrlqwvay ispgggssyy

61 pasvkgrfti srdnakntly lqmsslksed tamyycargg 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

(3) nucleotide sequence (underscore be signal sequence) of coding total length 1A3 sequence of light chain (1A3 Kappa variable region and constant region)(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 ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcatg

601 ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca

661 tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag

(4) define the protein sequence (not having signal sequence) of total length 1A3 sequence of light chain (1A3 Kappa variable region and constant region)(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) nucleotide sequence (underscore be signal sequence) of coding total length 2B8 sequence of heavy chain (2B8 variable region of heavy chain and IgG1 constant region)(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 qagaagttca agagcaaggc cacactgact gtagacaaat cctccagcac agcctacatg

301 caactcagca gcctgacatc tgaggactct qcggtctatt 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 cagtgactgt 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 aatgcagggt 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 cagatggctc ttacttcgtc

1261 tacagcaagc tcaatgtgca gaagagcaac tgggaggcag gaaatacttt cacctgctct

1321 gtgttacatg agggcctgca caaccaccat actgagaaga gcctctccca ctctcctggt

1381 aaatga

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

1 qvqlqqpgae lvkpgtsvkl sckasgytft tywmhwvnqr pgqglewige inptnghtny

61 nekfkskatl tvdkssstay mqlssltsed savyycarny vgsifdywgq 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

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

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 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 protein sequence (not having signal sequence) of total length 2B8 sequence of light chain (2B8 Kappa variable region and constant region)(SEQ ID NO.129)

1 nivmtqspks msmsvgervt lsckasenw syvswyqqkp aqspklliyg asnrntgvpd

61 rftgsgsatd ftltissvra edladyhcgq synypytfgg gtrleikrad aaptvsifpp

121 sseqltsgga svvcflnnfy pkdinvkwki dgserqngvl nswtdqdskd stysmsstlt

181 ltkdeyerhn sytceathkt stspivksfn rnec

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

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 ttactacaat

241 gagatgttca aagacaaggc cacattgact gtagacacat cctccagcac agcctacatg

301 cagctcagca gcctgacatc tgacgactct gcggtctatt tctgtgcaag aaggggactg

361 ggacgtggct 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 cagtgactgt 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 aatgcagggt 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 cagatggctc ttacttcgtc

1261 tacagcaagc tcaatgtgca gaagagcaac tgggaggcag gaaatacttt cacctgctct

1321 gtgttacatg agggcctgca caaccaccat actgagaaga gcctctccca ctctcctggt

1381 aaatga

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

1 qvqlkqsgae lvrpgtsvkm sckasgytft tyyihwvnqr pgqglewigk igpgsgstyy

61 nemfkdkatl tvdtssstay mqlssltsdd savyfcarrg 1grgfdywgq 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) nucleotide sequence (underscore be signal sequence) of coding total length 2F8 sequence of light chain (2F8 Kappa variable region and constant region)(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 protein sequence (not having signal sequence) of total length 2F8 sequence of light chain (2F8 Kappa variable region and constant region)(SEQ ID NO.133)

1 divltqspas lavslgqrat isckasqsvd ydgnsyinwy qqkpgqppkv liyvasnlcs

61 giparfsgsg sgtdftlnih pveeedaaty ycqqsiedpp tfgagtklel kradaaptvs

121 ifppsseqlt sggasvvcfl nnfypkdinv kwkidgserq ngvlnswtdq dskdstysms

181 stltltkdey erhnsytcea thktstspiv ksfnrnec

(13) nucleotide sequence (underscore be signal sequence) of coding total length 3B6 sequence of heavy chain (3B6 variable region of heavy chain and IgG1 constant region)(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 the protein sequence (not having signal sequence) of total length 3B6 sequence of heavy chain (3B6 variable region of heavy chain and IgG1 constant region)(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 ngqpaenykn tqpimdtdgs yfvysklnvq ksnweagntf

421 tcsvlheglh nhhtekslsh spgk

(15) nucleotide sequence (underscore be signal sequence) of coding total length 3B6 sequence of light chain (3B6 Kappa variable region and constant region)(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 gtcctgaata gttggactga tcaggacagc aaagacagca cctacagcat gagcagcacc

601 ctcacgttga ccaaggacga gtatgaacga cataacagct atacctgtga ggccactcac

661 aagacatcaa cttcacccattgtcaagagc ttcaacagga atgagtgtta g

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

1 dikmtqspss myaslgervt itckasqdik sylswfqqkp gkspktliyr vnrlvdgvps

61 rfsgsgsgqd ssltitslen edmgiyyclq ydefpftfgg gtkleikrad aaptvsifpp

121 sseqltsgga svvcflnnfy pkdinvkwki dgserqngvl nswtdqdskd stysmsstlt

181 ltkdeyerhn sytceathkt stspivksfn rnec

(17) nucleotide sequence (underscore be signal sequence) of coding total length 3D11 sequence of heavy chain (3D11 variable region of heavy chain and IgG1 constant region)(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 the protein sequence (not having signal sequence) of total length 3D11 sequence of heavy chain (3D11 variable region of heavy chain and IgG1 constant region)(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 ykntqpimdt dgsyfvyskl nvqksnweag ntftcsvlhe

421 glhnhhteks lshspgk

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

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

1 qivltqspai msaypgekvt mtcsasssvs ymhwyqqksg tspkrwiydt sklasgvpar

61 fsgsgsgtsy sltissmeae daatyycqqw ssnpltfgag tklelkrada aptvsifpps

121 seqltsggas vvcflnnfyp kdinvkwkid gserqngvln swtdqdskds tysmsstltl

181 tkdeyerhns ytceathkts tspivksfnr nec

(21) nucleotide sequence (underscore be signal sequence) of coding total length 1D3 sequence of heavy chain (1D3 variable region of heavy chain and IgG1 constant region)(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 the protein sequence (not having signal sequence) of total length 1D3 sequence of heavy chain (1D3 variable region of heavy chain and IgG1 constant region)(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 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

(23) nucleotide sequence (underscore be signal sequence) of coding total length 1D3 sequence of light chain (1D3 Kappa variable region and constant region)(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 the protein sequence (not having signal sequence) of total length 1D3 sequence of light chain (1D3 Kappa variable region and constant region)(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) nucleotide sequence (underscore be signal sequence) of coding total length 1F3 sequence of heavy chain (1F3 variable region of heavy chain and IgG1 constant region)(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 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)(SEQ ID NO.147)

1 evqlvesggg lvqsggslkl scaasgftfs nyfmswvrqt pekrlewvay issgggstyy

61 pdsvkgrfti srdnakntly lqmsslksed 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

(27) nucleotide sequence (underscore be signal sequence) of coding total length 1F3 sequence of light chain (1F3 Kappa variable region and constant region)(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 taccagatgg tgtgccatca

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 aacgacaaaa tggcgtcctg

541 aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg

601 ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca

661 tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag

(28) define the protein sequence (not having signal sequence) of total length 1F3 sequence of light chain (1F3 Kappa variable region and constant region)(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) nucleotide sequence (underscore be signal sequence) of coding total length 3A12 sequence of heavy chain (3A12 variable region of heavy chain and IgG1 constant region)(SEQ ID NO.150)

1 atgaacttg 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 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 acsttactgt 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 the protein sequence (not having signal sequence) of total length 3A12 sequence of heavy chain (3A12 variable region of heavy chain and IgG1 constant region)(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) nucleotide sequence (underscore be signal sequence) of coding total length 3A12 sequence of light chain (3A12 Kappa variable region and constant region)(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 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

(32) define the protein sequence (not having signal sequence) of total length 3A12 sequence of light chain (3A12 Kappa variable region and constant region)(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 ease, table 2 provides the concordance list of the corresponding relation between those sequences of listing in full length sequence and the sequence table of the antibody of being discussed in the present embodiment.

Table 2

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
		124	1A3 variable region of light chain+constant region-nucleic acid
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
		128	2B8 variable region of light chain+constant region-nucleic acid
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
		132	2F8 variable region of light chain+constant region-nucleic acid
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
		136	3B6 variable region of light chain+constant region-nucleic acid
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
		140	3D11 variable region of light chain+constant region-nucleic acid
141	3D11 variable region of light chain+constant region-protein

[0750]

142	1D3 variable region of heavy chain+IgG1 constant region-nucleic acid
		143	1D3 variable region of heavy chain+IgG1 constant region-protein
144	1D3 variable region of light chain+constant region-nucleic acid
		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
148	1F3 variable region of light chain+constant region-nucleic acid
		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
152	3A12 variable region of light chain+constant region-nucleic acid
		153	3A12 variable region of light chain+constant region-protein

Embodiment 3-proteinic the preparation of multiple reorganization hHGF

Present embodiment has been described clone and the expression that is used for characterizing the embodiment 1 and the multiple recombinant protein of the antibody of embodiment 14 preparations.Particularly; Proteinic clone and expression below present embodiment has been described, described protein is: reorganization hHGF protein, contain 555 position glycocoll by the substituted reorganization of L-glutamic acid hHGF protein (G555E), contain 561 position halfcystines by the substituted reorganization of l-arginine hHGF protein (C561R), contain recombined small-mouse-people-mouse (mhm) mosaic HGF protein of the people V495-L585 HGF sequence that places mouse HGF sequence, the reorganization mhm mosaic HGF protein that contains the reorganization mhm mosaic HGF protein of the people I499-R566 HGF sequence that places mouse HGF sequence and contain 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 through 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 two mutants G555E and hHGF-Fc two mutants C561R through 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 One StepRT-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, through 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) through 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 through 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; Catalog No.R250-01) under the condition, at F12K substratum (ATCC, Catalog No.30-2004), 10% FCS (Invitrogen; Catalog No.10438026), 1% penicillium mould (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

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

(3) protein expression in stable CHO FlpIn clone

With about 2x10 ⁶Individual cell inoculation is in the flat board of 15cm, and at 37 ℃, 5% CO ₂In ratio be the high sugar of F12K (ATCC, Catalog No.30-2004)/DMEM (Invitrogen, Catalog No.11995065) and 5% utmost point low IgG FCS (Invitrogen, #16250-78) the middle cultivation 5-6 days of 1:1 down.Collect supernatant, and analyze the protein of gained through ELISA and surface plasma body resonant vibration.

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

Some reorganization HGF protein of preparation can combine the ability of hHGF to characterize through them 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 through surface plasma body resonant vibration in embodiment 3, thereby assess the ability that they combine HGF.Use amine coupling (BIAcore; Catalog No.BR-1000-50); The specification sheets that provides according to manufacturers, the coupling method through standard is fixed in every kind of antibody on the sensor chip (BIAcore, Catalog No.BR-1006-68) of carboxymethylated VISOSE CM5.

Under 25 ℃; Use PBS (GIBCO; Catalog No.14040-133) agent is analyzed as runtime buffer, and 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-VISOSE (Fluka, Catalog No.86524).The supernatant that will contain the supernatant 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).With combining people HGF (R&D Systems, Catalog No.294-HGN-025) to compare with being diluted in the runtime buffer agent.Through 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 result of gained is summarized in the table 3.

Table 3

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

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

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

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 combine people HGF specifically.Antibody 1D3,1F3 and 2B8 combine mouse-people-mouse mosaic, and other antibody are this mosaic of debond then.These results show that antibody 1D3 and 2B8 combine the residue 495-585 of people HGF at least in part.As if antibody 1A3,3A12,3B6 and 3D11 combine the part except residue 495-585 of people hHGF.Still uncertain at present why 2F8 debond mhm mosaic, and as if it combine hHGF and mHGF.

The monoclonal antibody of embodiment 5-anti-hHGF combines the ability of reductive and non-reducing HGF

In the present embodiment, the monoclonal antibody to the anti-hHGF of embodiment 1 preparation combines the ability of reductive and non-reducing HGF to analyze.

Assess the reactivity of serum with the reorganization hHGF of anti-HGF through 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.With this nitrocellulose membrane with being dissolved in the 5% skim-milk solution sealing that contains in 0.1% Tween-

the Tris buffer salt solution (TBST), 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 said instrument, and with the IgG antibody incubation of the anti-mouse of horseradish peroxidase-labeled.The result of gained is summarized in the table 4, and wherein numeral has been reacted the bonded degree ,-represent minimum combination (combining hardly or debond) and 3+ representative combination at most.

Table 4

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

Data in the table 4 show that all antibody all combines with non-reducing rhHGF.Form therewith and correlatedly be, monoclonal antibody 1A3,1D3,1F3,2F8 and 3B6 combine 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 through surface plasmon resonance measurement.

Use amine coupling (BIAcore; Catalog No.BR-1000-50); The specification sheets that provides according to manufacturers; Coupling method through standard is fixed in the anti-mouse immuning ball protein of rabbit (BIAcore, Catalog No.BR-1005-14) on the sensor chip (BIAcore, Catalog No.BR-1006-68) of carboxymethylated VISOSE CM5.Under 25 ℃; Use PBS (GIBCO; Catalog No.14040-133) agent is analyzed as runtime buffer, and 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-VISOSE (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 20RU in each circulation.Speed with 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 the HGF (R&D Systems, Catalog No.294-HGN-025) in the runtime buffer agent.According to concentration, monitor 15 or 90 minutes mutually to dissociating.Then, begin to use glycocoll-HCl of 10mM before another circulation, it is that the flow velocity with 60 μ L/min injects in 3 minutes that pH 1.7 (BIAcore, Catalog No.BR-1003-54) makes described surface regeneration, wherein said glycocoll-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, confirm 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

1A3

1.7x10 6

7.3x10 4

5.2x10 -5

8.4x10 -7

30.1

5.6

1D3

1.7x10 6

3.1x10 4

8.2x10 -5

1.7x10 -6

54.2

27.4

1F3

1.5x10 6

5.0x10 4

2.6x10 -5

6.6x10 -7

18.1

8.2

2B8

1.6x10 6

2.9x10 4

2.1x10 -5

1.4x10 -7

13.5

4.4

3A12

1.6x10 6

3.7x10 4

1.6x10 -5

1.6x10 -6

103.0

10.4

3B6

2.0x10 6

6.5x10 4

3.9x10 -5

3.2x10 -7

17.0

3.4

[0826]Data in the table 5 show, the K of said antibodies hHGF _DBe about 100pM or lower, about 50pM or lower or 20pM or lower.

The neutralization of the antibody of embodiment 7-anti-hHGF is active

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 combines inhibition analysis (neutralizing effect analysis)

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

Particularly, under 4 ℃, encapsulate buffer reagent (15mM Na with carbonate ₂CO ₃And 34mMNaHCO ₃, pH 9.0) in 100 μ L, 6.25 μ g/mL HGF (R&D Systems, Catalog No.294-HGN-025) encapsulate 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.The antibody (0.033-667nM, 3 times of serial dilutions) that is in the research through with rising concentration joins among the 2nM c-Met (R&D Systems, Catalog No.358-MT/CF) among the PBS that contains 5% non-skim-milk, 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 antibody (R&D Systems, the Catalog No.BAF358) incubation of the biotin labeled anti-people c-Met of 2 μ g/mL in 100 μ L/ holes, the antibody of wherein said anti-people c-Met in the PBS that contains 5% non-skim-milk in preparation.

Then; Wash 3 times with PBS-0.1% Tween 20 gained is dull and stereotyped, 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 said 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
			1A3	5.65	0.91
1D3	4.43	2.27
			1F3	6.57	0.28
2B8	5.57	1.19
			2F8	5.36	0.88
3A12	5.26	2.11
			3B6	-	-
3D11	5.66	2.75

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

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

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,, and handle according to the specification sheets that manufacturers provides with BrdU ELISA (Roche Catalog No.1669915) with the dry said flat board of hair dryer.

Use the flat reader (Bio-Tek) of Synergy HT 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 equality of said curve is: y=floors+(top value-floors)/(1+10^ (1og (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)
		1A3	4.69
1D3	4.99
		1F3	1.94
2B8	1.41
		2F8	19.24
3A12	30.30
		3B6	36.08
3D11	51.12

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 characteristic 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 4x10 ³The density of individual cell is inoculated in 96 hole Costar tissue culture plate (Corning Incorporated; Corning; NY3 Catalog No.3595) in; Wherein the MEM (ATCC Manassas, VA, Catalog No.30-2003) that comprises 10% foetal calf serum (InvitrogenCatalog No.10438026) and 1% penicillium mould-Streptomycin sulphate (Invitrogen Catalog No.15140122) of 80 μ L is equipped with in each hole.Every kind of antibody to be studied is diluted to 6,667nM in the MEM that contains 10% foetal calf serum and 1% penicillium mould-Streptomycin sulphate.Then, with every kind of different antibody diluent and contain 10% foetal calf serum and 1% penicillium mould-Streptomycin sulphate but the MEM that do not contain antibody mix with isopyknic MEM and 100ng/ml HGF (R&DSystems Catalog No.294-HGN-025) that contains 10% foetal calf serum and 1% penicillium mould-Streptomycin sulphate respectively.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 AC 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 100 μ L.Then flat board is washed 1 time with zero(ppm) water carefully.With the crystal violet solution that adds 100 μ L in each hole, it is by 0.5% the Viola crystallina (Sigma, St.Louis, MO, Catalog No.C3886) and 50% the ethanol formation in the zero(ppm) water that is dissolved in, and cell was shaken incubation 20 minutes under 25 ℃.

After will using crystal violet solution dyeing, the cell of gained is washed 3 times with zero(ppm) water carefully.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 result of gained is summarized in the table 8.

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 characteristic 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 4.5x10 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% penicillium mould-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% penicillium mould-Streptomycin sulphate.Then with cell incubation 24 hours in the serum-free F-12K that contains 1% penicillium mould-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% penicillium mould-Streptomycin sulphate, prepare every kind of antibody to be studied.Every kind of antibody diluent and the serum-free F-12K that contains 1% penicillium mould-Streptomycin sulphate that do not contain antibody are mixed with isopyknic serum-free F-12K and 500ng/mL HGF (R&D Systems Catalog No.294-HGN-025) that contains 1% penicillium mould-Streptomycin sulphate respectively.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% penicillium mould-Streptomycin sulphate.Then in cell, add 70 μ L and contain the serum-free F-12K of 1% penicillium mould-Streptomycin sulphate, and then add 10 μ L and contain 10mM Na ₃VO ₄The serum-free F-12K that contains 1% penicillium mould-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 following: 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, and described cracking buffer reagent is by 1% OmniPur Triton X-100 (MERCK KGaA, Darmstadt; Germany, Catalog No.9410), 50mM Tris-HCl pH 8.0,100mM 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.

With the antibody (R&D Systems CatalogNo.BAF358) of biotin labeled anti-people HGF-R (c-met) 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 dull and stereotyped (PerkinElmer Catalog No.AAAND-0005).Then, under 25 ℃, said flat board and antibody were shaken incubation 30 minutes.Behind the incubation, (PerkinElmer Catalog No.4010-0010) washs said flat board with the DELFIA washings, and every kind of different PC-3 cell lysate of 80 μ L is added respectively in dull and stereotyped each hole of the Streptavidin micropore of washing.

The Streptavidin micropore flat board that will contain the PC-3 cell lysate shook incubation 60 minutes under 25 ℃, wash with the DELFIA washings then.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.Said 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 dull and stereotyped each hole of the Streptavidin micropore 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 equality for GraphPad Software, Inc. to use Prism 4 for Windows ₅₀Value.

In nM as EC ₅₀And the result who sums up lists in the table 9.

Table 9

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

[0867]Data in the table 9 show that 8 all antibody all are effective suppressor factor of HGF inductive c-Met phosphorylation in the PC-3 cell.

Tumor suppression in 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 penicillium mould and 100 μ g/mL Streptomycin sulphates.Through using cell pair cell on trypsinase-EDTA separation and Culture ware wall to carry out secondary cultivation and keeping.

Be used for collecting cell through tryptic digestion, then with 50%Matrigel (BD Biosciences near converging; 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).The following tumor size (vol.) of calculating: 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 through 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 result is 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 to being directed against the said increment of U87MG cell U118 cell (ATCC) among the embodiment 10 (the foregoing description).

Like the preceding text embodiment 10 said Subcutaneous tumor models of setting up, difference is that used mouse is the female NCr nude mice (Taconic) in 7 ages in week, and as the extremely about 80mm of tumor growth ³The time begin treatment.As such in the U87MG model, the dosage of all antibody is the 1mg/kg body weight, and 2 times weekly, and through 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 result is 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).In 1F3 and 1D3 group, have the growth of tumor of appropriateness to suppress, 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 characteristic of gained humanized antibody.Make mouse 2B8 variable region of heavy chain and variable region of light chain " humanization " through 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 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 confirming each mouse 2B8CDR according to length and the amino acid whose composition of CDR.Known ethnic group according in International Immunogentics Information System (IMGT) network address (on the imgt.cines.fr of World Wide Web andbiochem.unizh.ch/antibody/Sequences/index.html, can get), describing 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.Through 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.Selection is the variable region with mouse 2B8CDR residue identity and/or those the highest ethnic groups of similarity, is used for CDR and transplants.Mouse 2B8CDR residue being used to replace in mouse 2B8CDR 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, will join the carboxyl terminal of the variable region of " super humanization " with the most similar people J district, 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).Make the possible combination expression of 4 all heavy chain IgG1 antibody (mosaic 2B8 and 3 humanized heavy chains (Hu2B8 Hv1-f.1, Hu2B8 Hv5-a.1 and Hu2B8Hv5-51.1)) and all 3 kappa chain antibodies (mosaic 2B8 and 2 humanized light chains (Hu2B8 Kv1-39.1 and Hu2B8 Kv3-15.1)), thereby produce 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 the result is summarized among Fig. 8.Each of variable region that table has been listed heavy chain immunoglobulin and light chain immunoglobulin among the 12A possibly make up.

Table 12A

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)	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 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 Hv5-51.1(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)

[0888]Table listed heavy chain immunoglobulin and light chain immunoglobulin among the 12B each possibly make up.

Table 12B

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)	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)
		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)
		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)
		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 constructions that contain total length heavy chain immunoglobulin and light chain (containing humanized variable region) design as follows:

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

Allotype) (SEQ ID NO.171)+hu2B8 Kv 1-39.1 (+

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 summed up as follows.In this part, last Nucleotide of each variable region is first the sub base of Next Password that is produced by variable region/constant region linker.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) nucleotide sequence (underscore be signal sequence) of coding total length mosaic 2B8 heavy chain (mouse variable region and human IgG1's constant region) (allotype Glm (17,1))(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 the total length mosaic 2B8 heavy chain (protein sequence (not having signal sequence) of mosaic 2B8 IgG1 (Glm (17,1) allotype)(SEQ ID NO.155)

1 qvqlqqpgae lvkpgtsvkl sckasgytft tywmhwvnqr pgqglewige inptnghtny

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) nucleotide sequence (underscore be signal sequence) of coding total length mosaic 2B8 light chain (mouse variable region and human constant region) (mosaic 2B8 Kappa (Km (3)))(SEQIDNO.156)

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 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 gcgaagtcac ccatcagggc

661 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttga

(4) define the protein sequence (not having signal sequence) of total length mosaic 2B8 light chain (mosaic 2B8 Kappa (Km (3)))(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 lskadyekhk vyacevthqg lsspvtksfn rgec

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

1 atggactgca cctggaggat cctcctcttg gtggcagcag ctacaggcac ccacgccgag

61 gtccagctgg tacagtctgg ggctgaggtg aagaagcctg gggctacagt gaaaatctcc

121 tgcaaggttt ctggatacac 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 ag

(6) define the protein sequence (not having signal sequence) of humanization Hu2B8 Hv1-f.1 variable region of heavy chain(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 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 cctcagcagc gtggtgaccg 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 cgtcttctca tgctccgtga tgcatgaggc tctgcacaac cactacacgc

961 agaagagcct ctccctgtct ccgggtaaat ga

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

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) nucleotide sequence of coding total length heavy chain humanization Hu2B8 Hy1f.1 variable region and human IgG1's (Glm (17,1) allotype) CH (underscore be signal sequence)(SEQ ID NO.162)

1 atggactgca cctggaggat cctcctcttg gtggcagcag ctacaggcac ccacgccgag

61 gtccagctgg tacagtctgg ggctgaggtg aagaagcctg gggctacagt gaaaatctcc

121 tgcaaggttt ctggatacac 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 ggagaacaac tacaagacca cgcctcccgt gctggactcc

1261 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg

1321 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc

1381 ctctccctgt ctccgggtaa atga

(10) define the protein sequence (not having signal sequence) of total length heavy chain humanization Hu2B8 Hv1f.1 variable region and human IgG1's CH (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 kgqprepgvy tlppsrdelt

361 knqvsltclv kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq

421 gnvfscsvmh ealhnhytqk slslspgk

(11) nucleotide sequence of coding humanization Hu2B8 Hv5a.1 variable region of heavy chain (underscore 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) define the protein sequence (not having signal sequence) of humanization Hu2B8 Hv5a.1 variable region of heavy chain(SEQ ID NO.165)

1 evqlvqsgae vkkpgeslri sckgsgysft tywmhwvrqm pgkglewmge inptnghtny

61 npsfqghvti sadksistay lqwsslkasd tamyycarny vgsifdywgq gtlvtvss

(13) nucleotide sequence of coding total length humanization Hu2B8 Hv5a.1 variable region of heavy chain and human IgG1's (Glm (17,1) allotype) CH (underscore be signal sequence)(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 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 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 the protein sequence (not having signal sequence) of total length humanization Hu2B8 Hv5a.1 variable region of heavy chain and human IgG1 (Glm (17,1) allotype) CH(SEQID NO.167)

1 evqlvqsgae vkkpgeslri sckgsgysft tywmhwvrgm 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 tlppsrdelt

361 knqvsltclv kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq

421 gnvfscsvmh ealhnhytqk slslspgk

(15) nucleotide sequence of coding humanization Hu2B8 Hv5-51.1 variable region of heavy chain (underscore 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 sequence (not having signal sequence) of humanization Hu2B8 Hv5-51.1 weight chain variabl area sequence(SEQ ID NO.169)

1 evqlvqsgae vkkpgeslki sckgsgysft tywmhwvrqm pgkglewmge inptnghtny

61 npsfqgqvti sadksistay lqwsslkasd tamyycarny vgsifdywgq gtlvtvss

(17) nucleotide sequence of coding total length humanization Hu2B8 Hv5-51.1 variable region of heavy chain and human IgG1's (Glm (17,1) allotype) CH (underscore be signal sequence)(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 tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg

1321 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc

1381 ctctccctgt ctccgggtaa atga

(18) define the protein sequence (not having signal sequence) of total length humanization Hu2B8 Hv5-51.1 variable region of heavy chain and human IgG1 (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 (underscore be 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 sequence (not having signal sequence) of humanization Hu2B8 Kv1-39.1 Kappa chain variable region(SEQ ID NO.173)

1 diqmtqspss lsasvgdrvt itckasenvv syvswyqqkp gkapklliyg asnrntgvps

61 rfsgsgsgtd ftltisslqp edfatyycgq synypytfgq gtkleik

(21) nucleotide sequence of coding human Kappa chain constant region (Km (3) allotype) (allelotrope 2)(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 the protein sequence of people Kappa chain constant region (Km (3) allotype) (allelotrope 2)(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) nucleotide sequence of coding total length humanization Hu2B8 KvI-39.1 variable region of light chain and people Kappa chain constant region (Km (3) allotype) (allelotrope 2) (underscore be signal sequence)(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 agtcacccac

661 cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgttg a

(24) define the protein sequence of total length humanization Hu2B8 KvI-39.1 variable region of light chain and people Kappa 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 dnalqsgnsq esvteqdskd styslsstlt

181 lskadyekhk vyacevthqg lsspvtksfn rgec

(25) nucleotide sequence of coding humanization Hu2B8 Kv3-15.1 variable region of light chain (underscore be signal sequence)(SEQ ID NO.178)

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 ac

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

1 eivmtqspat lsvspgerat lsckasenvv syvswyqqkp gqaprlliyg asnrntgipa

61 rfsgsgsgte ftltisslqs edfavyycgq synypytfgq gtkleik

(27) nucleic acid of coding total length humanization Hu2B8 Kv3-15.1 variable region of light chain and people Kappa chain 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 the protein sequence (not having signal sequence) of humanization Hu2B8 Kv3-15.1 variable region of light chain and people Kappa chain constant region (Km (3) allotype) (allelotrope 2)(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 ease, table 13 provides the concordance list of the corresponding relation between those sequences of listing in full length sequence and the sequence table of the antibody of discussing in this part.

Table 13

SEQ ID NO.	Protein or nucleic acid
		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
		158 159	Hu2B8 Hv1f.1 variable region of heavy chain-nucleic acid
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
		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
		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
		168	Hu2B8 Hv5-51.1 variable region of heavy chain-nucleic acid
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

[1184]

171	Hu2B8 Hv5-51.1+IgG1 constant region (Glm (17,1) allotype)-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
		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
		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
181	Hu2B8 Kv3-15.1+Kappa constant region (Km (3) allotype) (allelotrope 2)-protein

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.Combine or immunogenic possible risk according to the residue variable effect, 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, when it changes, possibly not influence antigenic combination 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, when it changes, might influence antigenic combination slightly 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 two C-terminals through people's variable region of heavy chain of transformation; And people Kappa constant region (Km (3) allotype (allelotrope 1)) joined two C-terminals through people's variable region of light chain of transforming, produce four people's antibody chains thus through transforming.At first, through gene synthetic method synthetic variable region nucleotide sequence, then this sequence is joined in the human constant region sequence.With these through people's antibody cloning of transforming in mammalian proteins matter expression vector, then add the combination marking protein of light chain with four kinds of possible heavy chains.Be described below, use technical measurement mosaic, mosaic/humanized antibody or the humanized antibody of routine and combining of people HGF.

The nucleotide sequence of each humanized antibody of encoding is summed up as follows with the protein sequence that limits each humanized antibody.In this part, last Nucleotide of each variable region is first base by Next Password that the variable region/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) define the protein sequence (not having signal sequence) of humanization LR2B8HC variable region of heavy chain(SEQ ID NO.183)

1 qvqlvqpgae vvkpgtsvkl sckasgytft tywmhwvnqa pgqglewige inptnghtny

61 nekfkgkatl tvdkststay melsslrsed tavyycarny vgsifdywgq gtlltvss

(3) nucleotide sequence of coding human IgG1 CH (Glm (3) allotype) (allelotrope 1)(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 the protein sequence of human IgG1's CH (Glm (3) allotype) (allelotrope 1 or 2)(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) nucleotide sequence of coding total length heavy chain humanization LR2B8HC variable region of heavy chain and human IgG1's CH (Glm (3) allotype) (allelotrope 1) (underscore be signal sequence)(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 acggacatac taattataat

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 the protein sequence (not having signal sequence) of total length heavy chain humanization LR2B8HC variable region of heavy chain and human IgG1's CH (Glm (3) allotype) (allelotrope 1)(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 ytvvsvltvl hqdwlngkey kckvsnkalp apiektiska kgqprepqvy tlppsreemt

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 signal 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) define the protein sequence (not having signal sequence) of humanization LRMR2B8HC variable region of heavy chain(SEQ ID NO.189)

1 qvqlvqpgae vkkpgtsvkl sckasgytft tywmhwvrqa pgqglewige inptnghtny

61 nqkfqgratl tvdkststay melsslrsed tavyycarny vgsifdywgq gtlltvss

(9) nucleotide sequence of coding total length heavy chain humanization LRMR2B8HC variable region of heavy chain and human IgG1's CH (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 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

(10) define the protein sequence (not having signal sequence) of total length heavy chain humanization LRMR2B8HC variable region of heavy chain and human IgG1's CH (Glm (3) allotype) (allelotrope 1)(SEQ ID NO.191)

1 qvqlvqpgae vkkpgtsvkl sckasgytft tywmhwvrqa pgqglewige inptnghtny

61 nqkfqgratl tvdkststay melsslrsed tavyycarny vgsifdywgq 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 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 signal 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) define the protein sequence (not having signal sequence) of humanization LR2B8LC variable region of light chain(SEQ ID NO.193)

1 divmtqspds mamsvgervt lnckasenvv syvswyqqkp gqspklliyg asnrntgvpd

61 rfsgsgsatd ftltissvqa edvadyhcgq synypytfgq gtkleik

(13) nucleotide sequence of coding human Kappa chain constant region (Km (3) allotype) (allelotrope 1)(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 cacctacagc ctcagcagca ccctgacgct gagcaaagca gactacgaga

241 aacacaaagt ctacgcctgc gaagtcaccc atcagggcct gagctcgccc gtcacaaaga

301 gcttcaacag gggagagtgt tag

(14) define the protein sequence of people Kappa chain constant region (Km (3) allotype) (allelotrope 1)(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) nucleotide sequence of coding total length humanization LR2B8LC variable region of light chain and people Kappa chain constant region (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) protein sequence of coding humanization LR2B8LC variable region of light chain and people Kappa chain constant region (Km (3) allotype) (allelotrope 1)(SEQ ID NO.197)

1 divmtqspds mamsvgervt lnckasenvv syvswyqqkp ggspklliyg asnrntgvpd

61 rfsgsgsatd ftltissvga 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) define the protein sequence (not having signal sequence) of humanization LRMR2B8LC variable region of light chain(SEQ ID NO.199)

1 divmtqspds lamslgervt lnckasenvv syvswyqqkp gqspklliyg asaresgvpd

61 rfsgsgsatd ftltissvqa edvadyhcgq synypytfgq gtkleik

(19) nucleotide sequence of coding total length humanization LRMR2B8LC variable region of light chain and people Kappa chain constant region (Km (3) allotype) (allelotrope 1) (underscore be signal sequence)(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 the protein sequence of total length humanization LRMR2B8LC variable region of light chain and people Kappa chain constant region (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

181 lskadyekhk vyacevthqg lsspvtksfn rgec

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

Table 14

SEQ ID NO.	Protein or nucleic acid
		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
		186	LR2B8HC+IgG1 constant region (Glm (3) allotype (allelotrope 1)-nucleic acid
187	LR2B8HC+IgG1 constant region (Glm (3) allotype (allelotrope 1)-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
		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
		196	LR2B8LC+Kappa constant region (Km (3) allotype) (allelotrope 1)-nucleic acid
197	LR2B8LC+Kappa constant region (Km (3) allotype) (allelotrope 1)-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

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

Table 15

Antibody	CDR1	CDR2	CDR3	The total length variable region of heavy chain
					Mouse 2B8 heavy chain	TYWMH (SEQ ID N0：15)	EINPTNGHTNYNEKFKS (SEQ ID NO：16)	NYVGSIFDY (SEQ ID NO：17)	SEQ ID N0：12
Hu2B8 Hv1f.1	TYWMH (SEQ ID N0：15)	EINPTNGHTNYNEKFQG (SEQ ID NO：202)	NYVGSIFDY (SEQ ID N0：17)	SEQ ID N0：159
					Hu2B8 Hv5a.1	TYWMH (SEQ ID NO：15)	EINPTNGHTNYNPSFQG (SEQ ID NO：203)	NYVGSIFDY (SEQ ID N0：17)	SEQ ID NO：165
Hu2B8 Hv5-51.1	TYWMH (SEQ ID N0：15)	EINPTNGHTNYNPSFQG (SEQ ID N0：203)	NYVGSIFDY (SEQ ID NO：17)	SEQ ID NO：169
					LR2B8HC	TYWMH (SEQ ID N0：15)	EINPTNGHTNYNEKFKG (SEQ ID NO：204)	NYVGSIFDY (SEQ ID NO：17)	SEQ ID N0：183
LRMR2B8HC	TYWMH (SEQ ID NO：15)	EINPTNGHTNYNQKFQG (SEQ ID NO：205)	NYVGSIFDY (SEQ ID NO：17)	SEQ ID N0：189

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

Table 16

Antibody	CDR1	CDR2	CDR3	The full-length light chains variable region
					Mouse 2B8 light chain	KASENVVSYVS (SEQ ID N0：18)	GASNRNT (SEQ ID NO：19)	GQSYNYPYT (SEQ ID N0：20)	SEQ ID NO：14
Hu2B8 Kv1-39.1	KASENVVSYVS (SEQ ID NO：18)	GASNRNT (SEQ ID NO：19)	GQSYNYPYT (SEQ ID N0：2O)	SEQ ID NO：173
					Hu2B8 Kv3-15.1	KASENVVSYVS (SEQ ID NO：18)	GASNRNT (SEQ ID NO：19)	GQSYNYPYT (SEQ ID N0：20)	SEQ ID N0：179
LR2B8LC	KASENVVSYVS (SEQ ID N0：18)	GASNRNT (SEQ ID NO：19)	GQSYNYPYT (SEQ ID N0：2O)	SEQ ID N0：193
					LRMR2B8LC	KASENVVSYVS (SEQ ID NO：18)	GASNRES (SEQ ID N0：206)	GQSYNYPYT (SEQ ID NO：2O)	SEQ ID N0：199

C. the binding affinity of humanization 2B8 antibody

Use BIAcore T100 device to assess antibody binding affinity and interactional kinetics through surface plasma body resonant vibration.Use amine coupling (BIAcore; Catalog No.BR-1000-50); According to manufacturer's recommendation; (Jackson ImmunoResearch Labs 209-005-098) is fixed on the sensor chip (BIAcore, Catalog No.BR-1005-34) of carboxymethylated VISOSE CM4 coupling method through standard with the mouse anti human Tegeline.Under 25 ℃; Use PBS (GIBCO; Catalog No.14040-133) agent is analyzed as runtime buffer, and said 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-VISOSE (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.Speed with 60 μ L/min is injected buffer reagent successively or is diluted in HGF (the R&D Systems in the runtime buffer agent on reference to surface (not having captive antibody) and active surface (having caught antibody to be tested); 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 glycocoll-HCl (BIAcore, Catalog No.BR-1003-55) of 10mM pH 2.0 to make described surface regeneration then, wherein said glycocoll-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, confirm 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 through the surface plasma body resonant vibration technology combines.Use amine coupling (BIAcore; Catalog No.BR-1000-50); According to manufacturer's recommendation; (Jackson ImmunoResearch Labs 209-005-098) is fixed on the sensor chip (BIAcore, Catalog No.BR-1006-68) of carboxymethylated VISOSE CM5 coupling method through standard with the mouse anti human Tegeline.Under 25 ℃; Use PBS (GIBCO; Catalog No.14040-133) agent is analyzed as runtime buffer, said PBS contain 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-VISOSE (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 150 RU in each circulation.On the humanized antibody of catching, inject the HGF that the ultimate density that is diluted in the runtime buffer agent is 7.5 μ g/mL (R&D Systems, Catalog No.294-HGN-025), time remaining 90 seconds with the speed of 30 μ L/min.The combination of monitoring HGF, subsequently with the speed of 30 μ L/min inject mouse 2B8 antibody or polyclone goat anti HGF antibody (R& 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 glycocoll-HCl (BIAcore, Catalog No.BR-1003-55) of use 10mM pH 2.0 makes described surface regeneration, wherein said glycocoll-HCl then.Gained is the result be 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

Codon and the people's light chain (being respectively LR2B8LC and LRMR2B8LC) and the heavy chain (being respectively LR2B8HC and LRMR2B8HC) through transforming of expressing low-risk and low risk to the medium risk of optimizing are cloned in the instantaneous antibody expression vector of XOMA synchronously, 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 through transient transfection through transforming.4 kinds of antibody that produced are following:

HE2B8-1=LR2B8HC (+IgG1 constant region (Glm (3) allotype (allelotrope

1)) (SEQ ID 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)) (SEQ ID NO.187)+LRMR2B8LC (+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, said 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.Go up the antibody in the purifying culture supernatants at the centrifugal post of a-protein (Pro-Chem), with the PBS dialysis, concentrate again and Sterile Filtration then.

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 through 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 through 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)

The protein sequence of below listing the nucleotide sequence of coding human IgG1 CH Glm (3) allotype (allelotrope 2) and every kind of total length sequence of heavy chain and defining 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) nucleotide sequence of coding human IgG1 CH (Glm (3) allotype) (allelotrope 2)(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 cgtggaggtg cataatgcca 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 tgcatgaggc tctgcacaac cactacacgc

961 agaagagcct ctccctgtct ccgggtaaat ga

(2) define the protein sequence of human IgG1's CH (Glm (3) allotype) (allelotrope 1 or 2)(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 ennykttpv ldsdgsffly skltvdksrw

301 qqgnvfscsv mhealhnhyt qkslslspgk

(3) coding contains the nucleotide sequence (underscore be signal sequence) of the total length chain of humanized Hu2B8 Hv5-51.1 variable region of heavy chain and human IgG1's CH Glm (3) allotype (allelotrope 2)(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 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 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 ggagaacaac tacaagacca cgcctcccgt gctggactcc

1261 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg

1321 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc

1381 ctctccctgt ctccgggtaa atga

(4) define the protein sequence (not having signal sequence) of the 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)(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) washing, and with the original substratum of this substratum replacement.Collect supernatant every day, and, so continue 10 days with the supernatant 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 combines the ability of hHGF to characterize through them.

Use BIAcore T100 device to analyze antibody to assess the ability that it combines the fused protein of discussion among hHGF and the embodiment 3 through 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 VISOSE CM5 through the coupling method of standard.

Under 25 ℃; Use PBS (GIBCO; Catalog No.14040-133) agent is analyzed as runtime buffer, and said 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-VISOSE (Fluka, Catalog No.86524).Maybe must the hang oneself supernatant of empty carrier cells transfected of the supernatant 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).With combining people HGF (R&D Systems, Catalog No.294-HGN-025) to compare with being diluted in the runtime buffer agent.Compare with contrast surface through will combining, monitor and do not have specific combination.The result is 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)
						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
						HE2B8-4	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 all combines rhHGF based on mouse-people-mouse mosaic in the antibody of humanized 2B8 and all 3.

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 coupling method that the specification sheets that provides according to manufacturers passes through standard is with mouse anti human Tegeline (JacksonLabs; Catalog No.209-005) is fixed on the sensor chip (BIAcore, Catalog No.BR-1006-68) of carboxymethylated VISOSE 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.Speed with 60 μ L/min is injected buffer reagent successively or is diluted in HGF (the R&D Systems in the runtime buffer agent on reference to surface (not having captive antibody) and active surface (having caught antibody to be tested); Catalog No.294-HGN-025), time remaining is 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 glycocoll-HCl (BIAcore, Catalog No.BR-1003-54) of use 10mM pH 2.2 makes described surface regeneration, wherein said glycocoll-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 confirmed 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
					2B8	1.4x10 6	1.0x10 -5	7.3	-
HE2B8-1	2.2x10 6	1.4x10 -5	7.1	5.2
					HE2B8-2	1.8x10 6	9.6x10 -5	5.2	2.7
HE2B8-3	2.0x10 6	4.1x10 -5	2.0	1.1
					HE2B8-4	1.7x10 6	1.1x10 -5	6.5	1.3
sh2B8-9 (Glm17，1)	2.0x10 6	1.7x10 -5	8.1	5.3
					sh2B8-12 (Glm17，1)	1.9x10 6	2.3x10 -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 said antibody is 2.0-12pM.

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

Use surface plasma body resonant vibration, under various 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 coupling method that the specification sheets that provides according to manufacturers passes through standard is with mouse anti human Tegeline (JacksonLabs; Catalog No.209-005) or the anti-mouse immuning ball protein of rabbit (BIAcore, Catalog No.BR-1005-14) be fixed on the sensor chip (BIAcore, Catalog No.BR-1006-68) of carboxymethylated VISOSE 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.Speed with 60 μ L/min is injected buffer reagent successively or is diluted in HGF (the R&D Systems in the runtime buffer agent on reference to surface (not having captive antibody) and active surface (having caught antibody to be tested); Catalog No.294-HGN-025), time remaining is 2 minutes.According to concentration, monitor 15 or 90 minutes mutually to dissociating.Then before another circulation of beginning; Use glycocoll-HCl (BIAcore of 10mM pH 2.2; Catalog No.BR-1003-54) make the surface regeneration of mouse anti human Tegeline sensor chip, wherein said glycocoll-HCl is that the flow velocity with 60L/min injects in 3 minutes.Before another circulation of beginning; Use glycocoll-HCl (BIAcore of 10mM pH 1.7; Catalog No.BR-1003-54) make the surface regeneration of the anti-mouse immuning ball protein sensor chip of rabbit, wherein said glycocoll-HCl is that the flow velocity with 60L/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, confirm 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)
					2B8	25	1.6x10 6	2.1x10 -5	13.5
2B8	37	2.8x10 6	1.3x10 -5	4.5
					HE2B8-4	25	2.0x10 6	1.2x10 -5	5.6
HE2B8-4	37	3.1x10 6	1.0x10 -5	3.3
					sh2B8-9 (Glm(17，1))	25	2.0x10 6	1.7x10 -5	8.1
sh2B8-9 (Glm(3))	37	2.5x10 6	1.4x10 -5	5.8
					sh2B8-12 (Glm(17，1))	25	1.9x10 6	2.3x10 -5	12.0
sh2B8-12 (Glm(3))	37	2.4x10 6	1.1x10 -5	4.8

As estimate, 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 of embodiment 17-humanized 2B8 variant is active

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

Carry out the combination inhibition analysis (neutralization analysis) of HGF-Met according to the following stated.Detect antibody through 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) encapsulate buffer reagent (15mM Na with 100 μ L carbonate ₂CO ₃And 34mMNaHCO ₃, pH 9.0) in 6.25 μ g/mL HGF (R&D Systems, Catalog No.294-HGN-025) encapsulated 16 hours.Then, at room temperature will analyze flat board with 5% non-skim-milk sealing among the 200 μ LPBS 1 hour.In independent analysis flat board; Through the antibody (0.033-250nM in the research that is 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.Specification sheets according to manufacturers provides carries out biotin labeling with the ratio (Pierce, Catalog No.21335) of the vitamin H of 10:1: c-Met to c-Met (R&D Systems, Catalog No.358-MT/CF).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, with Streptavidin (Wallac, the Catalog No.1244-360) incubation of Eu mark 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 μ L/ holes.Use the europium tracer method, at Victor ³Read said 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	I 50(nM)	SD
			2B8	9.2	1.2
HE2B8-1	6.0	1.2
			HE2B8-2	5.7	1.1
HE2B8-3	5.9	1.1
			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 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 result is summarized in the table 21.

Table 21

Antibody	IC 50(nM)	SD
			2B8	0.86	0.35
HE2B8-1	0.47	0.15
			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
			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 result is 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 result is summarized in the table 23.

Table 23

Antibody	The MV of 2 tests	Standard error
			2B8	0.91	0.02
HE2B8-1	0.80	0.04
			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
			sh2B8-12(Glm(3))	0.81	0.07

The result of table 23 shows that the humanized antibody of all tests all is effective suppressor factor 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 penicillium mould and 100 μ g/mL Streptomycin sulphates.Pair cell carries out secondary cultivation, and keeps through the cell that uses trypsinase-EDTA to pipette on the petridish wall.

Collect near the cell that converges, subsequently with 50% Matrigel (BD Biosciences through 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).The following tumor size (vol.) of calculating: volume (mm ³)=L x W ²/ 2.When tumor growth arrives about 200 mm 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 through 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 stated.A kind of below every group of mouse (10 every group) all accepted with the amount of 0.5mg/kg in the treatment: 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 TGD is 30 days.The tolerance of said 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 with claim meaning of equal value and scope in change and all be contemplated as falling with in the scope of the present invention.

Sequence table

< 110>W.M. Winston

S.K. rely special

M. Korea Spro

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-001B

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<151>2006-06-02

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1 5

<210>31

<211>418

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic variable region of heavy chain 3B6

<400>31

atggaatggc cttgtatctt tctcttcctc ctgtcagtaa ctgaaggtgt ccactcccag 60

gttcagctgc agcagtctgg ggctgaactg gtgaggcctg ggtcctcagt gaagatttcc 120

tgcaaggctt ctggctatgt attcagtagc tactggatga actgggtgaa gcagaggcct 180

ggacagggtc ttgagtggat tggacagatt tatcctggag atggtgatag taactacaat 240

ggaaacttca agggtaaagc cacactgact gcagacaaat cctccagtac agcctacatg 300

cagctcagca gcctaacatc tgaggactct gcggtctatt tctgtgcatc ccagctcggg 360

ctacgtgaga actactttga ctactggggc caaggcacca ctctcacagt ctcctcag 418

<210>32

<211>139

<212>PRT

< 213>artificial sequence

<220>

< 223>synthetic variable region of heavy chain 3B6

<400>32

Met Glu Trp Pro Cys Ile Phe Leu Phe Leu Leu Ser Val Thr Glu Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg

20 25 30

Pro Gly Ser Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Val Phe

35 40 45

Ser Ser Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Gln Ile Tyr Pro Gly Asp Gly Asp Ser Asn Tyr Asn

65 70 75 80

Gly Asn Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Phe Cys Ala Ser Gln Leu Gly Leu Arg Glu Asn Tyr Phe Asp Tyr

115 120 125

Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

130 135

<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

atggacatga ggacccctgc tcagtttctt ggaatcttgt tgctctggtt tccaggtatc 60

aaatgtgaca tcaagatgac ccagtctcca tcttccatgt atgcatctct aggagagaga 120

gtcacaatca cttgcaaggc gagtcaggac attaaaagct atttaagctg gttccagcag 180

aaaccaggga aatctcctaa gaccctgatc tatcgtgtaa acagattggt agatggggtc 240

ccatcaaggt tcagtggcag tggatctggg caagattctt ctctcaccat caccagcctg 300

gagaatgaag atatgggaat ttattattgt ctacagtatg atgagtttcc gttcacgttc 360

ggagggggga ccaagctgga aataaagc 388

<210>34

<21l>129

<212>PRT

< 213>artificial sequence

<220>

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

(2 possible initial methionines)

<400>34

Met Asp Met Arg Thr Pro Ala Gln Phe Leu Gly Ile Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ile Lys Cys Asp Ile Lys Met Thr Gln Ser Pro Ser Ser

20 25 30

Met Tyr Ala Ser Leu Gly Glu Arg Val Thr Ile Thr Cys Lys Ala Ser

35 40 45

Gln Asp Ile Lys Ser Tyr Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys

50 55 60

Ser Pro Lys Thr Leu Ile Tyr Arg Val Asn Arg Leu Val Asp Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp Ser Ser Leu Thr

85 90 95

Ile Thr Ser Leu Glu Asn Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln

100 105 110

Tyr Asp Glu Phe Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

115 120 125

Lys

<210>35

<211>5

<212>PRT

< 213>artificial sequence

<220>

< 223>synthetic heavy chain CDR1 3B6

<400>35

Ser Tyr Trp Met Asn

1 5

<210>36

<211>17

<212>PRT

< 213>artificial sequence

<220>

< 223>synthetic heavy chain CDR2 3B6

<400>36

Gln Ile Tyr Pro Gly Asp Gly Asp Ser Asn Tyr Asn Gly Asn Phe Lys

1 5 10 15

Gly

<210>37

<211>11

<212>PRT

< 213>artificial sequence

<220>

< 223>synthetic heavy chain CDR3 3B6

<400>37

Gln Leu Gly Leu Arg Glu Asn Tyr Phe Asp Tyr

1 5 10

<210>38

<211>11

<212>PRT

< 213>artificial sequence

<220>

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

<400>38

Lys Ala Ser Gln Asp Ile Lys Ser Tyr Leu Ser

1 5 10

<210>39

<211>7

<212>PRT

< 213>artificial sequence

<220>

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

<400>39

Arg Val Asn Arg Leu Val Asp

1 5

<210>40

<211>9

<212>PRT

< 213>artificial sequence

<220>

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

<400>40

Leu Gln Tyr Asp Glu Phe Pro Phe Thr

1 5

<210>41

<211>397

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic variable region of heavy chain 3D11

<400>41

atggctgtcc cggtgctgtt cctctgcctg gttgcatttc caagctgtgt cctgtcccag 60

gtacagctga aggagtcagg acctggcctg gtggcgccct cacagagcct gtccatcact 120

tgcactgtct ctgggttttc attaaccagc tatagtttac actgggttcg ccagcctcca 180

ggaaagggtc tggaatggct gggagtaata tgggctggtg gaaacacaaa ttataattcg 240

tctctcatgt ccagactgac catcaggaaa gacaactcca agagccaagt tttcttaaaa 300

atgaacagtc tgcaaactga tgacacagcc atgtactact gtgccagaga gaggtttgct 360

tactggggcc aagggactct ggtcactgtc tctgcag 397

<210>42

<211>132

<212>PRT

< 213>artificial sequence

<220>

< 223>synthetic variable region of heavy chain 3D11

<400>42

Met Ala Val Pro Val Leu Phe Leu Cys Leu Val Ala Phe Pro Ser Cys

1 5 10 15

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

20 25 30

Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu

35 40 45

Thr Ser Tyr Ser Leu His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu

50 55 60

Glu Trp Leu Gly Val Ile Trp Ala Gly Gly Asn Thr Asn Tyr Asn Ser

65 70 75 80

Ser Leu Met Ser Arg Leu Thr Ile Arg Lys Asp Asn Ser Lys Ser Gln

85 90 95

Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr

100 105 110

Tyr Cys Ala Arg Glu Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

115 120 125

Thr Val Ser Ala

130

<210>43

<211>385

<212>DNA

< 213>artificial sequence

<220>

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

<400>43

atggattttc aagtgcagat tttcagcttc ctgctaatca gtgcctcagt caaaatatcc 60

agaggacaaa ttgttctcac ccagtctcca gcaatcatgt ctgcatatcc aggggagaag 120

gtcaccatga cctgcagtgc cagctcaagt gtaagttaca tgcactggta ccagcagaag 180

tcaggcacct cccccaaaag atggatttat gacacatcca aactggcttc tggagtccct 240

gctcgcttca gtggcagtgg gtctgggacc tcttactccc tcacaatcag tagtatggag 300

gctgaagatg ctgccactta ttactgccag cagtggagta gtaacccact cacgttcggt 360

gctgggacca agctggagct gaaac 385

<210>44

<211>128

<212>PRT

< 213>artificial sequence

<220>

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

<400>44

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Lys Ile Ser Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile

20 25 30

Met Ser Ala Tyr Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser

35 40 45

Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly Thr Ser

50 55 60

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

65 70 75 80

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

85 90 95

Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp

100 105 110

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

115 120 125

<210>45

<211>5

<212>PRT

< 213>artificial sequence

<220>

< 223>synthetic heavy chain CDR1 3D11

<400>45

Ser Tyr Ser Leu His

1 5

<210>46

<211>16

<212>PRT

< 213>artificial sequence

<220>

< 223>synthetic heavy chain CDR2 3D11

<400>46

Val Ile Trp Ala Gly Gly Asn Thr Asn Tyr Asn Ser Ser Leu Met Ser

1 5 10 15

<210>47

<211>5

<212>PRT

< 213>artificial sequence

<220>

< 223>synthetic heavy chain CDR3 3D11

<400>47

Glu Arg Phe Ala Tyr

1 5

<210>48

<211>10

<212>PRT

< 213>artificial sequence

<220>

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

<400>48

Ser Ala Ser Ser Ser Val Ser Tyr Met His

1 5 10

<210>49

<211>7

<212>PRT

< 213>artificial sequence

<220>

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

<400>49

Asp Thr Ser Lys Leu Ala Ser

1 5

<210>50

<211>9

<212>PRT

< 213>artificial sequence

<220>

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

<400>50

Gln G1n Trp Ser Ser Asn Pro Leu Thr

1 5

<210>51

<211>424

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic variable region of heavy chain 1D3

<400>51

atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgaa 60

gtgcagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaactctcc 120

tgtgcagcct ctggattcac tttcagtgac tattacatgt cttgggttcg ccagactcca 180

gagaagaggc tggagtgggt cgcatacatt agtagtggtg gtggtagcac ctactatcca 240

gacagtgtga agggtcgatt caccatctcc cgagacaatg ccaagaacac cctgtacctg 300

caaatgagca gtctgaagtc tgaggacaca gccatatatt actgtgtgag acaaggggat 360

ggttattacg gggactatgc tatggactac tggggtcaag gaacctcagt catcgtctcc 420

tcag 424

<210>52

<211>141

<212>PRT

< 213>artificial sequence

<220>

< 223>synthetic variable region of heavy chain 1D3

<400>52

Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Val Leu Lys Gly

1 5 10 15

Val Lys Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Asp Tyr Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Tyr Tyr Cys Val Arg Gln Gly Asp Gly Tyr Tyr Gly Asp Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Ser Val Ile Val Ser Ser

130 135 140

<210>53

<211>382

<212>DNA

< 213>artificial sequence

<220>

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

<400>53

atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgtcagatgt 60

gacatccaga tgactcagtc tccagcctcc ctatctgtat ctgtgggaga aactgtcacc 120

atcacatgtc gaacaagtga gaatatttac agtaatttag cgtggtatca gcagaaacag 180

ggaaaatctc ctcagctcct aatctatgct gcaacaaact tagcagatgg tgtgccatca 240

aggttcagtg gcagtggatc aggcacacag ttttccctca ggatcaacag cctgcagtct 300

gaagattttg ggaggtatta ctgtcaacat ttttggggga ctccgtacac gttcggaggg 360

gggaccaaac tggaaataaa ac 382

<210>54

<211>127

<212>PRT

< 213>artificial sequence

<220>

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

<400>54

Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr

1 5 10 15

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

20 25 30

Val Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg Thr Ser Glu Asn

35 40 45

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

50 55 60

Gln Leu Leu Ile Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Ser Leu Arg Ile Asn

85 90 95

Ser Leu Gln Ser Glu Asp Phe Gly Arg Tyr Tyr Cys Gln His Phe Trp

100 105 110

Gly Thr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

115 120 125

<210>55

<211>5

<212>PRT

< 213>artificial sequence

<220>

< 223>synthetic heavy chain CDR1 1D3

<400>55

Asp Tyr Tyr Met Ser

1 5

<210>56

<211>17

<212>PRT

< 213>artificial sequence

<220>

< 223>synthetic heavy chain CDR2 1D3

<400>56

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

1 5 10 15

Gly

<210>57

<211>13

<212>PRT

< 213>artificial sequence

<220>

< 223>synthetic heavy chain CDR3 1D3

<400>57

Gln Gly Asp Gly Tyr Tyr Gly Asp Tyr Ala Met Asp Tyr

1 5 10

<210>58

<211>11

<212>PRT

< 213>artificial sequence

<220>

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

<400>58

Arg Thr Ser Glu Asn Ile Tyr Ser Asn Leu Ala

1 5 10

<210>59

<211>7

<212>PRT

< 213>artificial sequence

<220>

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

<400>59

Ala Ala Thr Asn Leu Ala Asp

1 5

<210>60

<211>9

<212>PRT

< 213>artificial sequence

<220>

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

<400>60

Gln His Phe Trp Gly Thr Pro Tyr Thr

1 5

<210>61

<211>424

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic variable region of heavy chain 1F3

<400>61

atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgag 60

gtgcagctgg tggagtctgg gggaggctta gtgcagtctg gagggtccct gaaactctcc 120

tgtgcggcct ctggattcac tttcagtaac tatttcatgt cttgggttcg ccagactcca 180

gagaagaggc tggagtgggt cgcatatatt agtagtggtg gtggtagcac ctactatcca 240

gacagtgtga agggtcgatt caccatctct agagacaatg ccaagaacac cctgtacctg 300

caaatgagca gtctgaagtc tgaggacaca gccatgtatt actgtgtaag acaaggggat 360

ggttactacg gggactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc 420

tcag 424

<210>62

<211>141

<212>PRT

< 213>artificial sequence

<220>

< 223>synthetic variable region of heavy chain 1F3

<400>62

Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Val Leu Lys Gly

1 5 10 15

Val Lys Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

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

35 40 45

Ser Asn Tyr Phe Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Tyr Tyr Cys Val Arg Gln Gly Asp Gly Tyr Tyr Gly Asp Tyr Ala Met

115 120 125

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

130 135 140

<210>63

<211>382

<212>DNA

< 213>artificial sequence

<220>

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

<400>63

atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt 60

gacatccaga tgactcagtc tccagcctcc ctatctgtat ctgtgggaga aactgtcacc 120

atcacatgtc gagcaagtga gaatatttac agtaatttag catggtatca gcagaaacag 180

ggaaaatctc ctcagctcct ggtctatgat gcaacacact taccagatgg tgtgccatca 240

aggttcagtg gcagtggatc aggcacacag ttttccctca agatcaacag cctgcagtct 300

gaagattttg ggagttatta ctgtcaacat ttttggggta ctccgtacac gtttggaggg 360

gggaccagac tggaaattaa ac 382

<210>64

<211>127

<212>PRT

< 213>artificial sequence

<220>

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

<400>64

Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr

1 5 10 15

Asp Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser

20 25 30

Val Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn

35 40 45

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

50 55 60

Gln Leu Leu Val Tyr Asp Ala Thr His Leu Pro Asp Gly Val Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn

85 90 95

Ser Leu Gln Ser Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp

100 105 110

Gly Thr Pro Tyr Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys

115 120 125

<210>65

<211>5

<212>PRT

< 213>artificial sequence

<220>

< 223>synthetic heavy chain CDR1 1F3

<400>65

Asn Tyr Phe Met Ser

1 5

<210>66

<211>17

<212>PRT

< 213>artificial sequence

<220>

< 223>synthetic heavy chain CDR2 1F3

<400>66

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

1 5 10 15

Gly

<210>67

<211>13

<212>PRT

< 213>artificial sequence

<220>

< 223>synthetic heavy chain CDR3 1F3

<400>67

Gln Gly Asp Gly Tyr Tyr Gly Asp Tyr Ala Met Asp Tyr

1 5 10

<210>68

<211>11

<212>PRT

< 213>artificial sequence

<220>

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

<400>68

Arg Ala Ser Glu Asn Ile Tyr Ser Asn Leu Ala

1 5 10

<210>69

<211>7

<212>PRT

< 213>artificial sequence

<220>

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

<400>69

Asp Ala Thr His Leu Pro Asp

1 5

<210>70

<211>9

<212>PRT

< 213>artificial sequence

<220>

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

<400>70

Gln His Phe Trp Gly Thr Pro Tyr Thr

1 5

<210>71

<211>424

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic variable region of heavy chain 3A12

<400>71

atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgaa 60

gtgcagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaaatctcc 120

tgtgcagcct ctggatttac tttcagtaac tatttcatgt cttgggttcg ccagactcca 180

gagaagaggc tggagtgggt cgcatacatt agtagtggtg gtggtagcac ctactatcca 240

gacagtgtga agggtcgatt caccatctcc agagacaatg ccaagaacac cctgtacctg 300

caaatgaaca gtctgaagtc tgaggacaca gccatgtatt actgtgtaag acaaggagat 360

ggttactatg gggactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc 420

tcag 424

<210>72

<211>141

<212>PRT

< 213>artificial sequence

<220>

< 223>synthetic variable region of heavy chain 3A12

<400>72

Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Val Leu Lys Gly

1 5 10 15

Val Lys Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Lys Ile Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Asn Tyr Phe Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Tyr Tyr Cys Val Arg Gln Gly Asp Gly Tyr Tyr Gly Asp Tyr Ala Met

115 120 125

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

130 135 140

<210>73

<211>382

<212>DNA

< 213>artificial sequence

<220>

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

<400>73

atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt 60

gacatccaga tgactcagtc gccagcctcc ctatctgtat ctgtgggaga aactgtcacc 120

atcacatgtc gagcaagtga gaatatttac attaatttag catggtatca gcagaaacag 180

ggaaaatctc ctcagctcct ggtccatgct gcaacaaagt tagcagatgg tgtgccatca 240

aggttcagtg gcagtggatc aggcacacag tattccctca agatcaacag cctgcagtct 300

gaagattttg ggagttatta ctgtcaacat ttttggggta ctccgtacac gttcggaggg 360

gggaccaaac tagaaataaa ac 382

<210>74

<211>127

<212>PRT

< 213>artificial sequence

<220>

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

<400>74

Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr

1 5 10 15

Asp Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser

20 25 30

Val Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn

35 40 45

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

50 55 60

Gln Leu Leu Val His Ala Ala Thr Lys Leu Ala Asp Gly Val Pro Ser

65 70 75 80

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

85 90 95

Ser Leu Gln Ser Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp

100 105 110

Gly Thr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

115 120 125

<210>75

<211>5

<212>PRT

< 213>artificial sequence

<220>

< 223>synthetic heavy chain CDR1 3A12

<400>75

Asn Tyr Phe Met Ser

1 5

<210>76

<211>17

<212>PRT

< 213>artificial sequence

<220>

< 223>synthetic heavy chain CDR2 3A12

<400>76

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

1 5 10 15

Gly

<210>77

<211>13

<212>PRT

< 213>artificial sequence

<220>

< 223>synthetic heavy chain CDR3 3A12

<400>77

Gln Gly Asp Gly Tyr Tyr Gly Asp Tyr Ala Met Asp Tyr

1 5 10

<210>78

<211>11

<212>PRT

< 213>artificial sequence

<220>

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

<400>78

Arg Ala Ser Glu Asn Ile Tyr Ile Asn Leu Ala

1 5 10

<210>79

<211>7

<212>PRT

< 213>artificial sequence

<220>

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

<400>79

Ala Ala Thr Lys Leu Ala Asp

1 5

<210>80

<211>9

<212>PRT

< 213>artificial sequence

<220>

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

<400>80

Gln His Phe Trp Gly Thr Pro Tyr Thr

1 5

<210>81

<211>974

<212>DNA

< 213>artificial sequence

<220>

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

<400>81

ccaaaacgac acccccatct gtctatccac tggcccctgg atctgctgcc caaactaact 60

ccatggtgac cctgggatgc ctggtcaagg gctatttccc tgagccagtg acagtgacct 120

ggaactctgg atccctgtcc agcggtgtgc acaccttccc agctgtcctg gagtctgacc 180

tctacactct gagcagctca gtgactgtcc cctccagccc tcggcccagc gagaccgtca 240

cctgcaacgt tgcccacccg gccagcagca ccaaggtgga caagaaaatt gtgcccaggg 300

attgtggttg taagccttgc atatgtacag tcccagaagt atcatctgtc ttcatcttcc 360

ccccaaagcc caaggatgtg ctcaccatta ctctgactcc taaggtcacg tgtgttgtgg 420

tagacatcag caaggatgat cccgaggtcc agttcagctg gtttgtagat gatgtggagg 480

tgcacacagc tcagacgcaa ccccgggagg agcagttcaa cagcactttc cgctcagtca 540

gtgaacttcc catcatgcac caggactggc tcaatggcaa ggagttcaaa tgcagggtca 600

acagtgcagc tttccctgcc cccatcgaga aaaccatctc caaaaccaaa ggcagaccga 660

aggctccaca ggtgtacacc attccacctc ccaaggagca gatggccaag gataaagtca 720

gtctgacctg catgataaca gacttcttcc ctgaagacat tactgtggag tggcagtgga 780

atgggcagcc agcggagaac tacaagaaca ctcagcccat catgaacacg aatggctctt 840

acttcgtcta cagcaagctc aatgtgcaga agagcaactg ggaggcagga aatactttca 900

cctgctctgt gttacatgag ggcctgcaca accaccatac tgagaagagc ctctcccact 960

ctcctggtaa atga 974

<210>82

<211>974

<212>DNA

< 213>artificial sequence

<220>

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

<400>82

ccaaaacgac acccccatct gtctatccac tggcccctgg atctgctgcc caaactaact 60

ccatggtgac cctgggatgc ctggtcaagg gctatttccc tgagccagtg acagtgacct 120

ggaactctgg atccctgtcc agcggtgtgc acaccttccc agctgtcctg cagtctgacc 180

tctacactct gagcagctca gtgactgtcc cctccagcac ctggcccagc gagaccgtca 240

cctgcaacgt tgcccacccg gccagcagca ccaaggtgga caagaaaatt gtgcccaggg 300

attgtggttg taagccttgc atatgtacag tcccagaagt atcatctgtc ttcatcttcc 360

ccccaaagcc caaggatgtg ctcaccatta ctctgactcc taaggtcacg tgtgttgtgg 420

tagacatcag caaggatgat cccgaggtcc agttcagctg gtttgtagat gatgtggagg 480

tgcacacagc tcagacgcaa ccccgggagg agcagttcaa cagcactttc cgctcagtca 540

gtgaacttcc catcatgcac caggactggc tcaatggcaa ggagttcaaa tgcagggtca 600

acagtgcagc tttccctgcc cccatcgaga aaaccatctc caaaaccaaa ggcagaccga 660

aggctccaca ggtgtacacc attccacctc ccaaggagca gatggccaag gataaagtca 720

gtctgacctg catgataaca gacttcttcc ctgaagacat tactgtggag tggcagtgga 780

atgggcagcc agcggagaac tacaagaaca ctcagcccat catggacaca gatggctctt 840

acttcgtcta cagcaagctc aatgtgcaga agagcaactg ggaggcagga aatactttca 900

cctgctctgt gttacatgag ggcctgcaca accaccatac tgagaagagc ctctcccact 960

ctcctggtaa atga 974

<210>83

<211>323

<212>DNA

< 213>artificial sequence

<220>

< 223>confirm as 1D3,1F3 and 2B8 with reference to mouse Kappa constant region of light chain (V00807)

With mouse Kappa constant region of light chain (is mouse from the AJ kind)

<400>83

gggctgatgc tgcaccaact gtatccatct tcccaccatc cagtgagcag ttaacatctg 60

gaggtgcctc agtcgtgtgc ttcttgaaca acttctaccc caaagacatc aatgtcaagt 120

ggaagattga tggcagtgaa cgacaaaatg gcgtcctgaa cagttggact gatcaggaca 180

gcaaagacag cacctacagc atgagcagca ccctcacgtt gaccaaggac gagtatgaac 240

gacataacag ctatacctgt gaggccactc acaagacatc aacttcaccc attgtcaaga 300

gcttcaacag gaatgagtgt tag 323

<210>84

<211>323

<212>DNA

< 213>artificial sequence

<220>

< 223>confirm as the synthetic mouse Kappa constant region of light chain of 1A3, it comprises with 1D3,1F3 and 2B8 compares

The Nucleotide of 1 change of 207 positions

<400>84

gggctgatgc tgcaccaact gtatccatct tcccaccatc cagtgagcag ttaacatctg 60

gaggtgcctc agtcgtgtgc ttcttgaaca acttctaccc caaagacatc aatgtcaagt 120

ggaagattga tggcagtgaa cgacaaaatg gcgtcctgaa cagttggact gatcaggaca 180

gcaaagacag cacctacagc atgagcagca ccctcatgtt gaccaaggac gagtatgaac 240

gacataacag ctatacctgt gaggccactc acaagacatc aacttcaccc attgtcaaga 300

gcttcaacag gaatgagtgt tag 323

<210>85

<211>30

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic Oligonucleolide primers BD SMART II A

<400>85

aagcagtggt atcaacgcag agtacgcggg 30

<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

tttttttttt tttttttttt tttttvn 27

<210>87

<211>45

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic Oligonucleolide primers Universal Primer Mix A

<400>87

ctaatacgac tcactatagg gcaagcagtg gtatcaacgc agagt 45

<210>88

<211>22

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic Oligonucleolide primers Universal Primer Mix A

<400>88

ctaatacgac tcactatagg gc 22

<210>89

<211>21

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic IgG1 constant region special primer

<400>89

tatgcaaggc ttacaaccac a 21

<210>90

<211>28

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic IgG1 constant region special primer

<400>90

gccagtggat agacagatgg gggtgtcg 28

<210>91

<211>27

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic Oligonucleolide primers

<400>91

ctcattcctg ttgaagctct tgacaat 27

<210>92

<211>23

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic Oligonucleolide primers

<400>92

cgactgaggc acctccagat gtt 23

<210>93

<211>20

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic Oligonucleolide primers T7

<400>93

taatacgact cactataggg 20

<210>94

<211>17

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic Oligonucleolide primers M13 forward

<400>94

gtaaaacgac ggccagt 17

<210>95

<211>18

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic Oligonucleolide primers M13 is reverse

<400>95

caggaaacag ctatgacc 18

<210>96

<211>63

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic oligonucleotide forward primer

<400>96

ggggacaagt ttgtacaaaa aagcaggctg ccaccatgaa ctttgggctc agattgattt 60

tcc 63

<210>97

<211>54

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic oligonucleotide reverse primer

<400>97

ggggaccact ttgtacaaga aagctgggtt catttaccag gagagtggga gagg 54

<210>98

<211>62

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic oligonucleotide forward primer

<400>98

ggggacaagt ttgtacaaaa aagcaggctg ccaccatggg atggagctat atcatcctct 60

tt 62

<210>99

<211>53

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic oligonucleotide reverse primer

<400>99

ggggaccact ttgtacaaga aagctgggtt catttaccag gagagtggga gag 53

<210>100

<211>62

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic oligonucleotide forward primer

<400>100

ggggacaagt ttgtacaaaa aagcaggctg ccaccatgga atcacagact ctggtcttca 60

ta 62

<210>101

<211>54

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic oligonucleotide reverse primer

<400>101

ggggaccact ttgtacaaga aagctgggtc taacactcat tcctgttgaa gctc 54

<210>102

<211>30

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic oligonucleotide 5 primer hHGF NheI primers

<400>102

actggctagc atgtgggtga ccaaactcct 30

<210>103

<211>46

<212>DNA

< 213>artificial sequence

<220>

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

<400>103

gtgatggtga tggtgatggc ggccgcatga ctgtggtacc ttatat 46

<210>104

<211>30

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic oligonucleotide 5 primer His IgFc primers

<400>104

actggcggcc gccatcacca tcaccatcac 30

<210>105

<211>31

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic oligonucleotide 3 primer I gFc BamHI primers

<400>105

actgggatcc tcactattta cccggggaca g 31

<210>106

<211>27

<212>DNA

< 213>artificial sequence

<220>

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

<400>106

catgatgtcc acgaaagagg agatgag 27

<210>107

<211>27

<212>DNA

< 213>artificial sequence

<220>

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

<400>107

ctcatctcct ctttcgtgga catcatg 27

<210>108

<211>37

<212>DNA

< 213>artificial sequence

<220>

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

<400>108

ggaagaggag atgagaaacg caaacaggtt ctcaatg 37

<210>109

<211>37

<212>DNA

< 213>artificial sequence

<220>

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

<400>109

cattgagaac ctgtttgcgt ttctcatctc ctcttcc 37

<210>110

<211>29

<212>DNA

< 213>artificial sequence

<220>

< 223>be used for the primer of the synthetic fragment 1 of mHGF α chain 5 primer NheI

<400>110

atcggctagc atgatgtggg ggaccaaac 29

<210>111

<211>36

<212>DNA

< 213>artificial sequence

<220>

< 223>be used for the primer of the synthetic fragment 1 of mHGF α chain 5 primer NheI

<400>111

ggttttgttt tgttgacgcc caacatttac cctaag 36

<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

ccaaaacaaa acaactgcgg gttgtaaatg ggattc 36

<210>113

<211>40

<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>113

tctagaccaa aattacttcg aacgagctgg acgttaggac 40

<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

agatctggtt ttaatgaagc ttgctcgacc tgcaatcctg 40

<210>115

<211>48

<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>115

cactaccact accactaccg ccggcgtgtt gaacatacag ttttaatg 48

<210>116

<211>38

<212>DNA

< 213>artificial sequence

<220>

< 223>primer 1 of synthetic sudden change

<400>116

catcaccatc accatcacta agcgggtctg gtgccacg 38

<210>117

<211>38

<212>DNA

< 213>artificial sequence

<220>

< 223>primer 2 of synthetic sudden change

<400>117

cgtggcacca gacccgctta gtgatggtga tggtgatg 38

<210>118

<211>2922

<212>DNA

< 213>artificial sequence

<220>

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

<400>118

atgtgggtga ccaaactcct gccagccctg ctgctgcagc atgtcctcct gcatctcctc 60

ctgctcccca tcgccatccc ctatgcagag ggacaaagga aaagaagaaa tacaattcat 120

gaattcaaaa aatcagcaaa gactacccta atcaaaatag atccagcact gaagataaaa 180

accaaaaaag tgaatactgc agaccaatgt gctaatagat gtactaggaa taaaggactt 240

ccattcactt gcaaggcttt tgtttttgat aaagcaagaa aacaatgcct ctggttcccc 300

ttcaatagca tgtcaagtgg agtgaaaaaa gaatttggcc atgaatttga cctctatgaa 360

aacaaagact acattagaaa ctgcatcatt ggtaaaggac gcagctacaa gggaacagta 420

tctatcacta agagtggcat caaatgtcag ccctggagtt ccatgatacc acacgaacac 480

agctttttgc cttcgagcta tcggggtaaa gacctacagg aaaactactg tcgaaatcct 540

cgaggggaag aagggggacc ctggtgtttc acaagcaatc cagaggtacg ctacgaagtc 600

tgtgacattc ctcagtgttc agaagttgaa tgcatgacct gcaatgggga gagttatcga 660

ggtctcatgg atcatacaga atcaggcaag atttgtcagc gctgggatca tcagacacca 720

caccggcaca aattcttgcc tgaaagatat cccgacaagg gctttgatga taattattgc 780

cgcaatcccg atggccagcc gaggccatgg tgctatactc ttgaccctca cacccgctgg 840

gagtactgtg caattaaaac atgcgctgac aatactatga atgacactga tgttcctttg 900

gaaacaactg aatgcatcca aggtcaagga gaaggctaca ggggcactgt caataccatt 960

tggaatggaa ttccatgtca gcgttgggat tctcagtatc ctcacgagca tgacatgact 1020

cctgaaaatt tcaagtgcaa ggacctacga gaaaattact gccgaaatcc agatgggtct 1080

gaatcaccct ggtgttttac cactgatcca aacatccgag ttggctactg ctcccaaatt 1140

ccaaactgtg atatgtcaca tggacaagat tgttatcgtg ggaatggcaa aaattatatg 1200

ggcaacttat cccaaacaag atctggacta acatgttcaa tgtgggacaa gaacatggaa 1260

gacttacatc gtcatatctt ctgggaacca gatgcaagta agctgaatga gaattactgc 1320

cgaaatccag atgatgatgc tcatggaccc tggtgctaca cgggaaatcc actcattcct 1380

tgggattatt gccctatttc tcgttgtgaa ggtgatacca cacctacaat agtcaattta 1440

gaccatcccg taatatcttg tgccaaaacg aaacaattgc gagttgtaaa tgggattcca 1500

acacgaacaa acataggatg gatggttagt ttgagataca gaaataaaca tatctgcgga 1560

ggatcattga taaaggagag ttgggttctt actgcacgac agtgtttccc ttctcgagac 1620

ttgaaagatt atgaagcttg gcttggaatt catgatgtcc acggaagagg agatgagaaa 1680

tgcaaacagg ttctcaatgt ttcccagctg gtatatggcc ctgaaggatc agatctggtt 1740

ttaatgaagc ttgccaggcc tgctgtcctg gatgattttg ttagtacgat tgatttacct 1800

aattatggat gcacaattcc tgaaaagacc agttgcagtg tttatggctg gggctacact 1860

ggattgatca actatgatgg cctattacga gtggcacatc tctatataat gggaaatgag 1920

aaatgcagcc agcatcatcg agggaaggtg actctgaatg agtctgaaat atgtgctggg 1980

gctgaaaaga ttggatcagg accatgtgag ggggattatg gtggcccact tgtttgtgag 2040

caacataaaa tgagaatggt tcttggtgtc attgttcctg gtcgtggatg tgccattcca 2100

aatcgtcctg gtatttttgt ccgagtagca tattatgcaa aatggataca caaaattatt 2160

ttaacatata aggtaccaca gtcatgcggc cgccatcacc atcaccatca ctccgcgggt 2220

ctggtgccac gcggtagtga caaaactcac acatgcccac cgtgcccagc acctgaactc 2280

ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 2340

cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 2400

ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 2460

cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 2520

aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 2580

accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 2640

cgggaggaga tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 2700

agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 2760

cctcccgtgc tggactccga cggctccttc ttcctctata gcaagctcac cgtggacaag 2820

agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 2880

cactacacgc agaagagcct ctccctgtcc ccgggtaaat ag 2922

<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

Pro Ala Leu Lys Ile Lys Thr Lys Lys Val Asn Thr Ala Asp Gln Cys

1 5 10 15

Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu Pro Phe Thr Cys Lys Ala

20 25 30

Phe Val Phe Asp Lys Ala Arg Lys Gln Cys Leu Trp Phe Pro Phe Asn

35 40 45

Ser Met Ser Ser Gly Val Lys Lys Glu Phe Gly His Glu Phe Asp Leu

50 55 60

Tyr Glu Asn Lys Asp TyrIle Arg Asn Cys Ile Ile Gly Lys Gly Arg

65 70 75 80

Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys Ser GlyIle Lys Cys Gln

85 90 95

Pro Trp Ser Ser Met Ile Pro His Glu His Ser Phe Leu Pro Ser Ser

100 105 110

Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg Gly

115 120 125

Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser Asn Pro Glu Val Arg Tyr

130 135 140

Glu Val Cys Asp Ile Pro Gln Cys Ser Glu Val Glu Cys Met Thr Cys

145 150 155 160

Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp His Thr Glu Ser Gly Lys

165 170 175

Ile Cys Gln Arg Trp Asp His Gln Thr Pro His Arg His Lys Phe Leu

180 185 190

Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp Asp Asn Tyr Cys Arg Asn

195 200 205

Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr Thr Leu Asp Pro His Thr

210 215 220

Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys Ala Asp Asn Thr Met Asn

225 230 235 240

Asp Thr Asp Val Pro Leu Glu Thr Thr Glu Cys Ile Gln Gly Gln Gly

245 250 255

Glu Gly Tyr Arg Gly Thr Val Asn Thr Ile Trp Asn Gly Ile Pro Cys

260 265 270

Gln Arg Trp Asp Ser Gln Tyr Pro His Glu His Asp Met Thr Pro Glu

275 280 285

Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn Tyr Cys Arg Asn Pro Asp

290 295 300

Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr Asp Pro Asn Ile Arg Val

305 310 315 320

Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp Met Ser His Gly Gln Asp

325 330 335

Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met Gly Asn Leu Ser Gln Thr

340 345 350

Arg Ser Gly Leu Thr Cys Ser Met Trp Asp Lys Asn Met Glu Asp Leu

355 360 365

His Arg His Ile Phe Trp Glu Pro Asp Ala Ser Lys Leu Asn Glu Asn

370 375 380

Tyr Cys Arg Asn Pro Asp Asp Asp Ala His Gly Pro Trp Cys Tyr Thr

385 390 395 400

Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys Pro Ile Ser Arg Cys Glu

405 410 415

Gly Asp Thr Thr Pro Thr Ile Val Asn Leu Asp His Pro Val Ile Ser

420 425 430

Cys Ala Lys Thr Lys Gln Leu Arg Val Val Asn Gly Ile Pro Thr Arg

435 440 445

Thr Asn Ile Gly Trp Met Val Ser Leu Arg Tyr Arg Asn Lys His Ile

450 455 460

Cys Gly Gly Ser Leu Ile Lys Glu Ser Trp Val Leu Thr Ala Arg Gln

465 470 475 480

Cys Phe Pro Ser Arg Asp Leu Lys Asp Tyr Glu Ala Trp Leu Gly Ile

485 490 495

His Asp Val His Gly Arg Gly Asp Glu Lys Cys Lys Gln Val Leu Asn

500 505 510

Val Ser Gln Leu Val Tyr Gly Pro Glu Gly Ser Asp Leu Val Leu Met

515 520 525

Lys Leu Ala Arg Pro Ala Val Leu Asp Asp Phe Val Ser Thr Ile Asp

530 535 540

Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu Lys Thr Ser Cys Ser Val

545 550 555 560

Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn Tyr Asp Gly Leu Leu Arg

565 570 575

Val Ala His Leu Tyr Ile Met Gly Asn Glu Lys Cys Ser Gln His His

580 585 590

Arg Gly Lys Val Thr Leu Asn Glu Ser Glu Ile Cys Ala Gly Ala Glu

595 600 605

Lys Ile Gly Ser Gly Pro Cys Glu Gly Asp Tyr Gly Gly Pro Leu Val

610 615 620

Cys Glu Gln His Lys Met Arg Met Val Leu Gly Val Ile Val Pro Gly

625 630 635 640

Arg Gly Cys Ala Ile Pro Asn Arg Pro Gly Ile Phe Val Arg Val Ala

645 650 655

Tyr Tyr Ala Lys Trp Ile His Lys Ile Ile Leu Thr Tyr Lys Val Pro

660 665 670

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

675 680 685

Pro Arg Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

690 695 700

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

705 710 715 720

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

725 730 735

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

740 745 750

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

755 760 765

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

770 775 780

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

785 790 795 800

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

805 810 815

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys

820 825 830

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

835 840 845

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

850 855 860

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

865 870 875 880

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

885 890 895

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

900 905 910

Leu Ser Leu Ser Pro Gly Lys

915

<210>120

<211>2901

<212>DNA

< 213>artificial sequence

<220>

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

<400>120

atgatgtggg ggaccaaact tctgccggtc ctgttgctgc agcatgtcct cctgcacctc 60

ctcctgcttc atgtcgccat cccctatgca gaaggacaga agaaaagaag aaatacactt 120

catgaattta aaaagtcagc aaaaactact cttaccaagg aagacccatt actgaagatt 180

aaaaccaaaa aagtgaactc tgcagatgag tgtgccaaca ggtgtatcag gaacaggggc 240

tttacgttca cttgcaaggc cttcgttttt gataagtcaa gaaaacgatg ctactggtat 300

cctttcaata gtatgtcaag tggagtgaaa aaagggtttg gccatgaatt tgacctctat 360

gaaaacaaag actatattag aaactgcatc attggtaaag gaggcagcta taaagggacg 420

gtatccatca ctaagagtgg catcaaatgc cagccttgga attccatgat cccccatgaa 480

cacagctatc gcggtaaaga cctacaggaa aactactgtc gaaatcctcg aggggaagaa 540

gggggaccct ggtgtttcac aagcaatcca gaggtacgct acgaagtctg tgacattcct 600

cagtgttcag aagttgaatg catgacctgc aatggtgaaa gctacagagg tcccatggat 660

cacacagaat caggcaagac ttgtcagcgc tgggaccagc agacaccaca ccggcacaag 720

ttcttgccag aaagatatcc cgacaagggc tttgatgata attattgccg caatcctgat 780

ggcaagccga ggccatggtg ctacactctt gaccctgaca ccccttggga gtattgtgca 840

attaaaacgt gcgctcacag tgctgtgaat gagactgatg tccctatgga aacaactgaa 900

tgcattcaag gccaaggaga aggttacagg ggaaccagca ataccatttg gaatggaatt 960

ccctgtcagc gttgggattc gcagtaccct cacaagcatg atatcactcc cgagaacttc 1020

aaatgcaagg accttagaga aaattattgc cgcaatccag atggggctga atcaccatgg 1080

tgttttacca ctgacccaaa catccgagtt ggctactgct ctcaaattcc caagtgtgac 1140

gtgtcaagtg gacaagattg ttatcgtggc aatgggaaaa attacatggg caacttatcc 1200

aaaacaaggt ctggacttac atgttccatg tgggacaaga atatggagga tttacaccgt 1260

catatcttct gggagccaga tgctagcaaa ttgaataaga attactgccg gaatcctgat 1320

gatgatgccc atggaccttg gtgctacacg gggaatcctc ttattccttg ggattattgc 1380

cctatttccc gttgtgaagg agatactaca cctacaattg tcaatttgga ccatcctgta 1440

atatcctgtg ccaaaacaaa acaactgcgg gttgtaaatg ggattccaac acgaacaaac 1500

ataggatgga tggttagttt gagatacaga aataaacata tctgcggagg atcattgata 1560

aaggagagtt gggttcttac tgcacgacag tgtttccctt ctcgagactt gaaagattat 1620

gaagcttggc ttggaattca tgatgtccac ggaagaggag atgagaaatg caaacaggtt 1680

ctcaatgttt cccagctggt atatggccct gaaggatcag atctggtttt aatgaagctt 1740

gctcgacctg caatcctgga taactttgtc agtacaattg atttacctag ttatggttgt 1800

acaatccctg aaaagaccac ttgcagtatt tacggctggg gctacactgg attgatcaac 1860

gcggatggtt tattacgagt agctcatctg tatattatgg ggaatgagaa atgcagtcag 1920

caccatcaag gcaaggtgac tttgaatgag tctgagttat gtgctggggc tgaaaagatt 1980

ggatcaggac catgtgaggg agattatggt ggcccactca tttgtgaaca acacaaaatg 2040

agaatggttc ttggtgtcat tgttcctggt cgtggatgtg ccatcccaaa tcgtcctggt 2100

atttttgttc gagtagcata ttatgcaaaa tggatacaca aagtaatttt gacatacaag 2160

ttgtgcggcc gccatcacca tcaccatcac tccgcgggtc tggtgccacg cggtagtgac 2220

aaaactcaca catgcccacc gtgcccagca cctgaactcc tggggggacc gtcagtcttc 2280

ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 2340

gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 2400

gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt 2460

gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc 2520

aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg 2580

cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 2640

caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 2700

gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 2760

ggctccttct tcctctatag caagctcacc gtggacaaga gcaggtggca gcaggggaac 2820

gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 2880

tccctgtccc cgggtaaata g 2901

<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

Pro Leu Leu Lys Ile Lys Thr Lys Lys Val Asn Ser Ala Asp Glu Cys

1 5 10 15

Ala Asn Arg Cys Ile Arg Asn Arg Gly Phe Thr Phe Thr Cys Lys Ala

20 25 30

Phe Val Phe Asp Lys Ser Arg Lys Arg Cys Tyr Trp Tyr Pro Phe Asn

35 40 45

Ser Met Ser Ser Gly Val Lys Lys Gly Phe Gly His Glu Phe Asp Leu

50 55 60

Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys Ile Ile Gly Lys Gly Gly

65 70 75 80

Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys Ser Gly Ile Lys Cys Gln

85 90 95

Pro Trp Asn Ser Met Ile Pro His Glu His Ser Tyr Arg Gly Lys Asp

100 105 110

Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro

115 120 125

Trp Cys Phe Thr Ser Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile

130 135 140

Pro Gln Cys Ser Glu Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr

145 150 155 160

Arg Gly Pro Met Asp His Thr Glu Ser Gly Lys Thr Cys Gln Arg Trp

165 170 175

Asp Gln Gln Thr Pro His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro

180 185 190

Asp Lys Gly Phe Asp Asp Asn Tyr Cys Arg Asn Pro Asp Gly Lys Pro

195 200 205

Arg Pro Trp Cys Tyr Thr Leu Asp Pro Asp Thr Pro Trp Glu Tyr Cys

210 215 220

Ala Ile Lys Thr Cys Ala His Ser Ala Val Asn Glu Thr Asp Val Pro

225 230 235 240

Met Glu Thr Thr Glu Cys Ile Gln Gly Gln Gly Glu Gly Tyr Arg Gly

245 250 255

Thr Ser Asn Thr Ile Trp Asn Gly Ile Pro Cys Gln Arg Trp Asp Ser

260 265 270

Gln Tyr Pro His Lys His Asp Ile Thr Pro Glu Asn Phe Lys Cys Lys

275 280 285

Asp Leu Arg Glu Asn Tyr Cys Arg Asn Pro Asp Gly Ala Glu Ser Pro

290 295 300

Trp Cys Phe Thr Thr Asp Pro Asn Ile Arg Val Gly Tyr Cys Ser Gln

305 310 315 320

Ile Pro Lys Cys Asp Val Ser Ser Gly Gln Asp Cys Tyr Arg Gly Asn

325 330 335

Gly Lys Asn Tyr Met Gly Asn Leu Ser Lys Thr Arg Ser Gly Leu Thr

340 345 350

Cys Ser Met Trp Asp Lys Asn Met Glu Asp Leu His Arg His Ile Phe

355 360 365

Trp Glu Pro Asp Ala Ser Lys Leu Asn Lys Asn Tyr Cys Arg Asn Pro

370 375 380

Asp Asp Asp Ala His Gly Pro Trp Cys Tyr Thr Gly Asn Pro Leu Ile

385 390 395 400

Pro Trp Asp Tyr Cys Pro Ile Ser Arg Cys Glu Gly Asp Thr Thr Pro

405 410 415

Thr Ile Val Asn Leu Asp His Pro Val Ile Ser Cys Ala Lys Thr Lys

420 425 430

Gln Leu Arg Val Val Asn Gly Ile Pro Thr Arg Thr Asn Ile Gly Trp

435 440 445

Met Val Ser Leu Arg Tyr Arg Asn Lys His Ile Cys Gly Gly Ser Leu

450 455 460

Ile Lys Glu Ser Trp Val Leu Thr Ala Arg Gln Cys Phe Pro Ser Arg

465 470 475 480

Asp Leu Lys Asp Tyr Glu Ala Trp Leu Gly Ile His Asp Val His Gly

485 490 495

Arg Gly Asp Glu Lys Cys Lys Gln Val Leu Asn Val Ser Gln Leu Val

500 505 510

Tyr Gly Pro Glu Gly Ser Asp Leu Val Leu Met Lys Leu Ala Arg Pro

515 520 525

Ala Ile Leu Asp Asn Phe Val Ser Thr Ile Asp Leu Pro Ser Tyr Gly

530 535 540

Cys Thr Ile Pro Glu Lys Thr Thr Cys Ser Ile Tyr Gly Trp Gly Tyr

545 550 555 560

Thr Gly Leu Ile Asn Ala Asp Gly Leu Leu Arg Val Ala His Leu Tyr

565 570 575

Ile Met Gly Asn Glu Lys Cys Ser Gln His His Gln Gly Lys Val Thr

580 585 590

Leu Asn Glu Ser Glu Leu Cys Ala Gly Ala Glu Lys Ile Gly Ser Gly

595 600 605

Pro Cys Glu Gly Asp Tyr Gly Gly Pro Leu Ile Cys Glu Gln His Lys

610 615 620

Met Arg Met Val Leu Gly Val Ile Val Pro Gly Arg Gly Cys Ala Ile

625 630 635 640

Pro Asn Arg Pro Gly Ile Phe Val Arg Val Ala Tyr Tyr Ala Lys Trp

645 650 655

Ile His Lys Val Ile Leu Thr Tyr Lys Leu Cys Gly Arg His His His

660 665 670

His His His Ser Ala Gly Leu Val Pro Arg Gly Ser Asp Lys Thr His

675 680 685

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

690 695 700

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

705 710 715 720

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

725 730 735

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

740 745 750

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

755 760 765

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

770 775 780

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

785 790 795 800

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

805 810 815

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

820 825 830

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

835 840 845

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

850 855 860

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

865 870 875 880

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

885 890 895

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

900 905 910

<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

atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgaa 60

gtgcagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaactctcc 120

tgtgcagcct ctgaattcac tttcagtaac tattacatgt cttgggttcg ccagactcca 180

gagaagaggc tgcagtgggt cgcatacatt agtcctggtg gtggtagctc ctactatcca 240

gccagtgtga agggtcgatt caccatctcc agagacaatg ccaagaacac cctgtacctg 300

caaatgagca gtctgaagtc tgaggacaca gccatgtatt actgtgcaag acaaggggat 360

ggttactacg gggactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc 420

tcagccaaaa cgacaccccc atctgtctat ccactggccc ctggatctgc tgcccaaact 480

aactccatgg tgaccctggg atgcctggtc aagggctatt tccctgagcc agtgacagtg 540

acctggaact ctggatccct gtccagcggt gtgcacacct tcccagctgt cctgcagtct 600

gacctctaca ctctgagcag ctcagtgact gtcccctcca gcacctggcc cagcgagacc 660

gtcacctgca acgttgccca cccggccagc agcaccaagg tggacaagaa aattgtgccc 720

agggattgtg gttgtaagcc ttgcatatgt acagtcccag aagtatcatc tgtcttcatc 780

ttccccccaa agcccaagga tgtgctcacc attactctga ctcctaaggt cacgtgtgtt 840

gtggtagaca tcagcaagga tgatcccgag gtccagttca gctggtttgt agatgatgtg 900

gaggtgcaca cagctcagac gcaaccccgg gaggagcagt tcaacagcac tttccgctca 960

gtcagtgaac ttcccatcat gcaccaggac tggctcaatg gcaaggagtt caaatgcagg 1020

gtcaacagtg cagctttccc tgcccccatc gagaaaacca tctccaaaac caaaggcaga 1080

ccgaaggctc cacaggtgta caccattcca cctcccaagg agcagatggc caaggataaa 1140

gtcagtctga cctgcatgat aacagacttc ttccctgaag acattactgt ggagtggcag 1200

tggaatgggc agccagcgga gaactacaag aacactcagc ccatcatgga cacagatggc 1260

tcttacttcg tctacagcaa gctcaatgtg cagaagagca actgggaggc aggaaatact 1320

ttcacctgct ctgtgttaca tgagggcctg cacaaccacc atactgagaa gagcctctcc 1380

cactctcctg gtaaatga 1398

<210>123

<211>446

<212>PRT

< 213>artificial sequence

<220>

< 223>protein sequence that defines synthetic total length 1A3 sequence of heavy chain (1A3 variable region of heavy chain and IgG1 constant region) (does not have

Signal sequence)

<400>123

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

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Glu Phe Thr Phe Ser Asn Tyr

20 25 30

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

35 40 45

Ala Tyr Ile Ser Pro Gly Gly Gly Ser Ser Tyr Tyr Pro Ala Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gln Gly Asp Gly Tyr Tyr Gly Asp Tyr Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro

115 120 125

Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met

130 135 140

Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val

180 185 190

Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His

195 200 205

Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys

210 215 220

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

225 230 235 240

Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro

245 250 255

Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val

260 265 270

Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr

275 280 285

Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu

290 295 300

Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys

305 310 315 320

Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro

340 345 350

Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile

355 360 365

Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly

370 375 380

Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp

385 390 395 400

Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp

405 410 415

Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His

420 425 430

Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

435 440 445

<210>124

<211>705

<212>DNA

< 213>artificial sequence

<220>

< 223>nucleotide sequence of coding synthetic total length 1A3 sequence of light chain (1A3Kappa variable region and constant region)

<400>124

atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt 60

gacatccaga tgactcagtc tccagcctcc ctatctgttt ctgtgggaga aactgtcacc 120

atcacatgtc gagcaagtga gaatatttat agtaatttag catggtatca gcagaaacag 180

ggaaaatctc ctcagctcct ggtctatgct gcaacaaact tagcagatgg tgtgccatca 240

aggttcagtg gcagtggatc aggcacacag ttttccctca agatcaacag cctgcagtct 300

gaagattttg ggacttatta ctgtcaacat ttttggggta ctccgtacac gttcggaggg 360

gggaccaagc tggaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca 420

tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac 480

cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg 540

aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcatg 600

ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca 660

tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag 705

<210>125

<211>214

<212>PRT

< 213>artificial sequence

<220>

< 223>protein sequence that defines synthetic total length 1A3 sequence of light chain (1A3Kappa variable region and constant region) (is not believed

Number sequence)

<400>125

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

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Gly Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala

100 105 110

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

115 120 125

Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile

130 135 140

Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu

145 150 155 160

Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

165 170 175

Ser Thr Leu Met Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr

180 185 190

Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

195 200 205

Phe Asn Arg Asn Glu Cys

210

<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 IgG1 constant region)

<400>126

atgggatgga gctatatcat cctctttttg gtagcaacag ctacagatgt ccactcccag 60

gtccaactgc agcagcctgg ggctgaactg gtgaagcctg ggacttcagt gaagctgtcc 120

tgcaaggctt ctggctacac cttcaccacc tactggatgc actgggtgaa tcagaggcct 180

ggacaaggcc ttgagtggat tggagagatt aatcctacca acggtcatac taactacaat 240

gagaagttca agagcaaggc cacactgact gtagacaaat cctccagcac agcctacatg 300

caactcagca gcctgacatc tgaggactct gcggtctatt actgtgcaag aaactatgtt 360

ggtagcatct ttgactactg gggccaaggc accactctca cagtctcctc agccaaaacg 420

acacccccat ctgtctatcc actggcccct ggatctgctg cccaaactaa ctccatggtg 480

accctgggat gcctggtcaa gggctatttc cctgagccag tgacagtgac ctggaactct 540

ggatccctgt ccagcggtgt gcacaccttc ccagctgtcc tgcagtctga cctctacact 600

ctgagcagct cagtgactgt cccctccagc acctggccca gcgagaccgt cacctgcaac 660

gttgcccacc cggccagcag caccaaggtg gacaagaaaa ttgtgcccag ggattgtggt 720

tgtaagcctt gcatatgtac agtcccagaa gtatcatctg tcttcatctt ccccccaaag 780

cccaaggatg tgctcaccat tactctgact cctaaggtca cgtgtgttgt ggtagacatc 840

agcaaggatg atcccgaggt ccagttcagc tggtttgtag atgatgtgga ggtgcacaca 900

gctcagacgc aaccccggga ggagcagttc aacagcactt tccgctcagt cagtgaactt 960

cccatcatgc accaggactg gctcaatggc aaggagttca aatgcagggt caacagtgca 1020

gctttccctg cccccatcga gaaaaccatc tccaaaacca aaggcagacc gaaggctcca 1080

caggtgtaca ccattccacc tcccaaggag cagatggcca aggataaagt cagtctgacc 1140

tgcatgataa cagacttctt ccctgaagac attactgtgg agtggcagtg gaatgggcag 1200

ccagcggaga actacaagaa cactcagccc atcatggaca cagatggctc ttacttcgtc 1260

tacagcaagc tcaatgtgca gaagagcaac tgggaggcag gaaatacttt cacctgctct 1320

gtgttacatg agggcctgca caaccaccat actgagaaga gcctctccca ctctcctggt 1380

aaatga 1386

<210>127

<211>442

<212>PRT

< 213>artificial sequence

<220>

< 223>protein sequence that defines synthetic total length 2B8 sequence of heavy chain (2B8 variable region of heavy chain and IgG1 constant region) (does not have

Signal sequence is arranged)

<400>127

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

1 5 10 15

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

20 25 30

Trp Met His Trp Val Asn Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn Pro Thr Asn Gly His Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Val Gly Ser Ile Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro

115 120 125

Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly

130 135 140

Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn

145 150 155 160

Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr

180 185 190

Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser

195 200 205

Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro

210 215 220

Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys

245 250 255

Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp

260 265 270

Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu

275 280 285

Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser

305 310 315 320

Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly

325 330 335

Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln

340 345 350

Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe

355 360 365

Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu

370 375 380

Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe

385 390 395 400

Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn

405 410 415

Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr

420 425 430

Glu Lys Ser Leu Ser His Ser Pro Gly Lys

435 440

<210>128

<211>705

<212>DNA

< 213>artificial sequence

<220>

< 223>nucleotide sequence of coding synthetic total length 2B8 sequence of light chain (2B8Kappa variable region of light chain and constant region)

<400>128

atggaatcac agactctggt cttcatatcc atactgctct ggttatatgg tgctgatggg 60

aacattgtaa tgacccaatc tcccaaatcc atgtccatgt cagtaggaga gagggtcacc 120

ttgagctgca aggccagtga gaatgtggtt tcttatgtat cctggtatca acagaaacca 180

gcgcagtctc ctaaactgct gatatacggg gcatccaacc ggaacactgg ggtccccgat 240

cgcttcacag gcagtggatc tgcaacagat ttcactctga ccatcagcag tgtgcgggct 300

gaagaccttg cagattatca ctgtgggcag agttacaact atccgtacac gttcggaggg 360

gggaccaggc tggaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca 420

tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac 480

cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg 540

aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg 600

ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca 660

tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag 705

<210>129

<211>214

<212>PRT

< 213>artificial sequence

<220>

< 223>protein sequence that defines synthetic total length 2B8 sequence of light chain (2B8Kappa variable region and constant region) (is not believed

Number sequence)

<400>129

Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser Met Ser Val Gly

1 5 10 15

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

20 25 30

Val Ser Trp Tyr Gln Gln Lys Pro Ala Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Asn Arg Asn Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

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

65 70 75 80

Glu Asp Leu Ala Asp Tyr His Cys Gly Gln Ser Tyr Asn Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys Arg Ala Asp Ala Ala

100 105 110

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

115 120 125

Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile

130 135 140

Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu

145 150 155 160

Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

165 170 175

Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr

180 185 190

Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

195 200 205

Phe Asn Arg Asn Glu Cys

210

<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

atggaatgga gctgggtctt tctcttcctc ctgtcagtaa ctgcaggtgt ccactgccag 60

gtccagctga agcagtctgg agctgagctg gtgaggcctg ggacttcagt gaagatgtcc 120

tgcaaggctt ctggctacac cttcactacc tactatatac actgggtgaa tcagaggcct 180

ggacagggcc ttgagtggat tggaaagatt ggtcctggaa gtggtagtac ttactacaat 240

gagatgttca aagacaaggc cacattgact gtagacacat cctccagcac agcctacatg 300

cagctcagca gcctgacatc tgacgactct gcggtctatt tctgtgcaag aaggggactg 360

ggacgtggct ttgactactg gggccaaggc accactctca cagtctcctc agccaaaacg 420

acacccccat ctgtctatcc actggcccct ggatctgctg cccaaactaa ctccatggtg 480

accctgggat gcctggtcaa gggctatttc cctgagccag tgacagtgac ctggaactct 540

ggatccctgt ccagcggtgt gcacaccttc ccagctgtcc tgcagtctga cctctacact 600

ctgagcagct cagtgactgt cccctccagc acctggccca gcgagaccgt cacctgcaac 660

gttgcccacc cggccagcag caccaaggtg gacaagaaaa ttgtgcccag ggattgtggt 720

tgtaagcctt gcatatgtac agtcccagaa gtatcatctg tcttcatctt ccccccaaag 780

cccaaggatg tgctcaccat tactctgact cctaaggtca cgtgtgttgt ggtagacatc 840

agcaaggatg atcccgaggt ccagttcagc tggtttgtag atgatgtgga ggtgcacaca 900

gctcagacgc aaccccggga ggagcagttc aacagcactt tccgctcagt cagtgaactt 960

cccatcatgc accaggactg gctcaatggc aaggagttca aatgcagggt caacagtgca 1020

gctttccctg cccccatcga gaaaaccatc tccaaaacca aaggcagacc gaaggctcca 1080

caggtgtaca ccattccacc tcccaaggag cagatggcca aggataaagt cagtctgacc 1140

tgcatgataa cagacttctt ccctgaagac attactgtgg agtggcagtg gaatgggcag 1200

ccagcggaga actacaagaa cactcagccc atcatggaca cagatggctc ttacttcgtc 1260

tacagcaagc tcaatgtgca gaagagcaac tgggaggcag gaaatacttt cacctgctct 1320

gtgttacatg agggcctgca caaccaccat actgagaaga gcctctccca ctctcctggt 1380

aaatga 1386

<210>131

<211>442

<212>PRT

< 213>artificial sequence

<220>

< 223>protein sequence that defines synthetic total length 2F8 sequence of heavy chain (2F8 variable region of heavy chain and IgG1 constant region) (does not have

Signal sequence)

<400>131

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

1 5 10 15

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

20 25 30

Tyr Ile His Trp Val Asn Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Lys Ile Gly Pro Gly Ser Gly Ser Thr Tyr Tyr Asn Glu Met Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Arg Gly Leu Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro

115 120 125

Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly

130 135 140

Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn

145 150 155 160

Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr

180 185 190

Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser

195 200 205

Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro

210 215 220

Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys

245 250 255

Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp

260 265 270

Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu

275 280 285

Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser

305 310 315 320

Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly

325 330 335

Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln

340 345 350

Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe

355 360 365

Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu

370 375 380

Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe

385 390 395 400

Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn

405 410 415

Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr

420 425 430

Glu Lys Ser Leu Ser His Ser Pro Gly Lys

435 440

<210>132

<211>717

<212>DNA

< 213>artificial sequence

<220>

< 223>nucleotide sequence of coding synthetic total length 2F8 sequence of light chain (2F8Kappa variable region and constant region)

<400>132

atggagacag acacaatcct gctatgggtg ctgctgctct gggttccagg ctccactggt 60

gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 120

atctcctgca aggccagcca aagtgttgat tatgatggta atagttatat caactggtac 180

caacagaaac caggacagcc acccaaagtc ctcatctatg ttgcatccaa tctagaatct 240

gggatcccag ccaggtttag tggcagtggg tctgggacag acttcaccct caacatccat 300

cctgtggagg aggaggatgc tgcaacctat tactgtcagc aaagtattga ggatcctccc 360

acgttcggtg ctgggaccaa gctggagctg aaacgggctg atgctgcacc aactgtatcc 420

atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg 480

aacaacttct accccaaaga catcaatgtc aagtggaaga ttgatggcag tgaacgacaa 540

aatggcgtcc tgaacagttg gactgatcag gacagcaaag acagcaccta cagcatgagc 600

agcaccctca cgttgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc 660

actcacaaga catcaacttc acccattgtc aagagcttca acaggaatga gtgttag 717

<210>133

<211>218

<212>PRT

< 213>artificial sequence

<220>

< 223>protein sequence that defines synthetic total length 2F8 sequence of light chain (2F8Kappa variable region and constant region) (is not believed

Number sequence)

<400>133

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asn Ser Tyr Ile Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

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

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Ile

85 90 95

Glu Asp Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg

100 105 110

Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln

115 120 125

Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr

130 135 140

Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln

145 150 155 160

Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg

180 185 190

His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro

195 200 205

Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

210 215

<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

atggaatggc cttgtatctt tctcttcctc ctgtcagtaa ctgaaggtgt ccactcccag 60

gttcagctgc agcagtctgg ggctgaactg gtgaggcctg ggtcctcagt gaagatttcc 120

tgcaaggctt ctggctatgt attcagtagc tactggatga actgggtgaa gcagaggcct 180

ggacagggtc ttgagtggat tggacagatt tatcctggag atggtgatag taactacaat 240

ggaaacttca agggtaaagc cacactgact gcagacaaat cctccagtac agcctacatg 300

cagctcagca gcctaacatc tgaggactct gcggtctatt tctgtgcatc ccagctcggg 360

ctacgtgaga actactttga ctactggggc caaggcacca ctctcacagt ctcctcagcc 420

aaaacgacac ccccatctgt ctatccactg gcccctggat ctgctgccca aactaactcc 480

atggtgaccc tgggatgcct ggtcaagggc tatttccctg agccagtgac agtgacctgg 540

aactctggat ccctgtccag cggtgtgcac accttcccag ctgtcctgca gtctgacctc 600

tacactctga gcagctcagt gactgtcccc tccagcacct ggcccagcga gaccgtcacc 660

tgcaacgttg cccacccggc cagcagcacc aaggtggaca agaaaattgt gcccagggat 720

tgtggttgta agccttgcat atgtacagtc ccagaagtat catctgtctt catcttcccc 780

ccaaagccca aggatgtgct caccattact ctgactccta aggtcacgtg tgttgtggta 840

gacatcagca aggatgatcc cgaggtccag ttcagctggt ttgtagatga tgtggaggtg 900

cacacagctc agacgcaacc ccgggaggag cagttcaaca gcactttccg ctcagtcagt 960

gaacttccca tcatgcacca ggactggctc aatggcaagg agttcaaatg cagggtcaac 1020

agtgcagctt tccctgcccc catcgagaaa accatctcca aaaccaaagg cagaccgaag 1080

gctccacagg tgtacaccat tccacctccc aaggagcaga tggccaagga taaagtcagt 1140

ctgacctgca tgataacaga cttcttccct gaagacatta ctgtggagtg gcagtggaat 1200

gggcagccag cggagaacta caagaacact cagcccatca tggacacaga tggctcttac 1260

ttcgtctaca gcaagctcaa tgtgcagaag agcaactggg aggcaggaaa tactttcacc 1320

tgctctgtgt tacatgaggg cctgcacaac caccatactg agaagagcct ctcccactct 1380

cctggtaaat ga 1392

<210>135

<211>444

<212>PRT

< 213>artificial sequence

<220>

< 223>protein sequence that defines synthetic total length 3B6 sequence of heavy chain (3B6 variable region of heavy chain and IgG1 constant region) (does not have

Signal sequence)

<400>135

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Gln Ile Tyr Pro Gly Asp Gly Asp Ser Asn Tyr Asn Gly Asn Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Ser Gln Leu Gly Leu Arg Glu Asn Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val

115 120 125

Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr

130 135 140

Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr

145 150 155 160

Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser

180 185 190

Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala

195 200 205

Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys

210 215 220

Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val

245 250 255

Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe

260 265 270

Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro

290 295 300

Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val

305 310 315 320

Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr

325 330 335

Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys

340 345 350

Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp

355 360 365

Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro

370 375 380

Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser

385 390 395 400

Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala

405 410 415

Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His

420 425 430

His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

435 440

<210>136

<211>711

<212>DNA

< 213>artificial sequence

<220>

< 223>nucleotide sequence of coding synthetic total length 3B6 sequence of light chain (3B6Kappa variable region and constant region)

<400>136

atggacatga ggacccctgc tcagtttctt ggaatcttgt tgctctggtt tccaggtatc 60

aaatgtgaca tcaagatgac ccagtctcca tcttccatgt atgcatctct aggagagaga 120

gtcacaatca cttgcaaggc gagtcaggac attaaaagct atttaagctg gttccagcag 180

aaaccaggga aatctcctaa gaccctgatc tatcgtgtaa acagattggt agatggggtc 240

ccatcaaggt tcagtggcag tggatctggg caagattctt ctctcaccat caccagcctg 300

gagaatgaag atatgggaat ttattattgt ctacagtatg atgagtttcc gttcacgttc 360

ggagggggga ccaagctgga aataaagcgg gctgatgctg caccaactgt atccatcttc 420

ccaccatcca gtgagcagtt aacatctgga ggtgcctcag tcgtgtgctt cttgaacaac 480

ttctacccca aagacatcaa tgtcaagtgg aagattgatg gcagtgaacg acaaaatggc 540

gtcctgaaca gttggactga tcaggacagc aaagacagca cctacagcat gagcagcacc 600

ctcacgttga ccaaggacga gtatgaacga cataacagct atacctgtga ggccactcac 660

aagacatcaa cttcacccat tgtcaagagc ttcaacagga atgagtgtta g 711

<210>137

<211>214

<212>PRT

< 213>artificial sequence

<220>

< 223>protein sequence that defines synthetic total length 3B6 sequence of light chain (3B6Kappa variable region and constant region) (is not believed

Number sequence)

<400>137

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Ser Gly Ser Gly Gln Asp Ser Ser Leu Thr Ile Thr Ser Leu Glu Asn

65 70 75 80

Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala

100 105 110

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

115 120 125

Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile

130 135 140

Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu

145 150 155 160

Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

165 170 175

Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr

180 185 190

Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

195 200 205

Phe Asn Arg Asn Glu Cys

210

<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

atggctgtcc cggtgctgtt cctctgcctg gttgcatttc caagctgtgt cctgtcccag 60

gtacagctga aggagtcagg acctggcctg gtggcgccct cacagagcct gtccatcact 120

tgcactgtct ctgggttttc attaaccagc tatagtttac actgggttcg ccagcctcca 180

ggaaagggtc tggaatggct gggagtaata tgggctggtg gaaacacaaa ttataattcg 240

tctctcatgt ccagactgac catcaggaaa gacaactcca agagccaagt tttcttaaaa 300

atgaacagtc tgcaaactga tgacacagcc atgtactact gtgccagaga gaggtttgct 360

tactggggcc aagggactct ggtcactgtc tctgcagcca aaacgacacc cccatctgtc 420

tatccactgg cccctggatc tgctgcccaa actaactcca tggtgaccct gggatgcctg 480

gtcaagggct atttccctga gccagtgaca gtgacctgga actctggatc cctgtccagc 540

ggtgtgcaca ccttcccagc tgtcctgcag tctgacctct acactctgag cagctcagtg 600

actgtcccct ccagcacctg gcccagcgag accgtcacct gcaacgttgc ccacccggcc 660

agcagcacca aggtggacaa gaaaattgtg cccagggatt gtggttgtaa gccttgcata 720

tgtacagtcc cagaagtatc atctgtcttc atcttccccc caaagcccaa ggatgtgctc 780

accattactc tgactcctaa ggtcacgtgt gttgtggtag acatcagcaa ggatgatccc 840

gaggtccagt tcagctggtt tgtagatgat gtggaggtgc acacagctca gacgcaaccc 900

cgggaggagc agttcaacag cactttccgc tcagtcagtg aacttcccat catgcaccag 960

gactggctca atggcaagga gttcaaatgc agggtcaaca gtgcagcttt ccctgccccc 1020

atcgagaaaa ccatctccaa aaccaaaggc agaccgaagg ctccacaggt gtacaccatt 1080

ccacctccca aggagcagat ggccaaggat aaagtcagtc tgacctgcat gataacagac 1140

ttcttccctg aagacattac tgtggagtgg cagtggaatg ggcagccagc ggagaactac 1200

aagaacactc agcccatcat ggacacagat ggctcttact tcgtctacag caagctcaat 1260

gtgcagaaga gcaactggga ggcaggaaat actttcacct gctctgtgtt acatgagggc 1320

ctgcacaacc accatactga tcccactctc ctggtaaatg a 1361

<210>139

<211>437

<212>PRT

< 213>artificial sequence

<220>

< 223>protein sequence that defines synthetic total length 3D11 sequence of heavy chain (3D11 variable region of heavy chain and IgG1 constant region) (does not have

Signal sequence is arranged)

<400>139

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

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

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

35 40 45

Gly Val Ile Trp Ala Gly Gly Asn Thr Asn Tyr Asn Ser Ser Leu Met

50 55 60

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

65 70 75 80

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

85 90 95

Arg Glu Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser

115 120 125

Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly

130 135 140

Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser

145 150 155 160

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr

165 170 175

Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr

180 185 190

Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys

195 200 205

Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val

210 215 220

Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val

225 230 235 240

Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile

245 250 255

Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val

260 265 270

Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser

275 280 285

Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu

290 295 300

Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala

305 310 315 320

Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro

325 330 335

Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys

340 345 350

Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr

355 360 365

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

370 375 380

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

385 390 395 400

Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser

405 410 415

Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser

420 425 430

His Ser Pro Gly Lys

435

<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

atggattttc aagtgcagat tttcagcttc ctgctaatca gtgcctcagt caaaatatcc 60

agaggacaaa ttgttctcac ccagtctcca gcaatcatgt ctgcatatcc aggggagaag 120

gtcaccatga cctgcagtgc cagctcaagt gtaagttaca tgcactggta ccagcagaag 180

tcaggcacct cccccaaaag atggatttat gacacatcca aactggcttc tggagtccct 240

gctcgcttca gtggcagtgg gtctgggacc tcttactccc tcacaatcag tagtatggag 300

gctgaagatg ctgccactta ttactgccag cagtggagta gtaacccact cacgttcggt 360

gctgggacca agctggagct gaaacgggct gatgctgcac caactgtatc catcttccca 420

ccatccagtg agcagttaac atctggaggt gcctcagtcg tgtgcttctt gaacaacttc 480

taccccaaag acatcaatgt caagtggaag attgatggca gtgaacgaca aaatggcgtc 540

ctgaacagtt ggactgatca ggacagcaaa gacagcacct acagcatgag cagcaccctc 600

acgttgacca aggacgagta tgaacgacat aacagctata cctgtgaggc cactcacaag 660

acatcaactt cacccattgt caagagcttc aacaggaatg agtgttag 708

<210>141

<211>213

<212>PRT

< 213>artificial sequence

<220>

< 223>protein sequence that defines synthetic total length 3D11 sequence of light chain (3D11 Kappa variable region and constant region) (does not have

Signal sequence)

<400>141

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Tyr Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala Pro

100 105 110

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

115 120 125

Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn

130 135 140

Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn

145 150 155 160

Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser

165 170 175

Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr

180 185 190

Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe

195 200 205

Asn Arg Asn Glu Cys

210

<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

atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgaa 60

gtgcagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaactctcc 120

tgtgcagcct ctggattcac tttcagtgac tattacatgt cttgggttcg ccagactcca 180

gagaagaggc tggagtgggt cgcatacatt agtagtggtg gtggtagcac ctactatcca 240

gacagtgtga agggtcgatt caccatctcc cgagacaatg ccaagaacac cctgtacctg 300

caaatgagca gtctgaagtc tgaggacaca gccatatatt actgtgtgag acaaggggat 360

ggttattacg gggactatgc tatggactac tggggtcaag gaacctcagt catcgtctcc 420

tcagccaaaa cgacaccccc atctgtctat ccactggccc ctggatctgc tgcccaaact 480

aactccatgg tgaccctggg atgcctggtc aagggctatt tccctgagcc agtgacagtg 540

acctggaact ctggatccct gtccagcggt gtgcacacct tcccagctgt cctgcagtct 600

gacctctaca ctctgagcag ctcagtgact gtcccctcca gcacctggcc cagcgagacc 660

gtcacctgca acgttgccca cccggccagc agcaccaagg tggacaagaa aattgtgccc 720

agggattgtg gttgtaagcc ttgcatatgt acagtcccag aagtatcatc tgtcttcatc 780

ttccccccaa agcccaagga tgtgctcacc attactctga ctcctaaggt cacgtgtgtt 840

gtggtagaca tcagcaagga tgatcccgag gtccagttca gctggtttgt agatgatgtg 900

gaggtgcaca cagctcagac gcaaccccgg gaggagcagt tcaacagcac tttccgctca 960

gtcagtgaac ttcccatcat gcaccaggac tggctcaatg gcaaggagtt caaatgcagg 1020

gtcaacagtg cagctttccc tgcccccatc gagaaaacca tctccaaaac caaaggcaga 1080

ccgaaggctc cacaggtgta caccattcca cctcccaagg agcagatggc caaggataaa 1140

gtcagtctga cctgcatgat aacagacttc ttccctgaag acattactgt ggagtggcag 1200

tggaatgggc agccagcgga gaactacaag aacactcagc ccatcatgga cacagatggc 1260

tcttacttcg tctacagcaa gctcaatgtg cagaagagca actgggaggc aggaaatact 1320

ttcacctgct ctgtgttaca tgagggcctg cacaaccacc atactgagaa gagcctctcc 1380

cactctcctg gtaaatga 1398

<210>143

<211>446

<212>PRT

< 213>artificial sequence

<220>

< 223>protein sequence that defines synthetic total length 1D3 sequence of heavy chain (1D3 variable region of heavy chain and IgG1 constant region) (does not have

Signal sequence)

<400>143

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Val Arg Gln Gly Asp Gly Tyr Tyr Gly Asp Tyr Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Ser Val Ile Val Ser Ser Ala Lys Thr Thr Pro Pro

115 120 125

Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met

130 135 140

Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val

180 185 190

Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His

195 200 205

Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys

210 215 220

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

225 230 235 240

Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro

245 250 255

Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val

260 265 270

Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr

275 280 285

Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu

290 295 300

Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys

305 310 315 320

Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro

340 345 350

Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile

355 360 365

Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly

370 375 380

Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp

385 390 395 400

Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp

405 410 415

Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His

420 425 430

Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

435 440 445

<210>144

<211>705

<212>DNA

< 213>artificial sequence

<220>

< 223>nucleotide sequence of coding synthetic total length 1D3 sequence of light chain (1D3Kappa variable region and constant region)

<400>144

atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgtcagatgt 60

gacatccaga tgactcagtc tccagcctcc ctatctgtat ctgtgggaga aactgtcacc 120

atcacatgtc gaacaagtga gaatatttac agtaatttag cgtggtatca gcagaaacag 180

ggaaaatctc ctcagctcct aatctatgct gcaacaaact tagcagatgg tgtgccatca 240

aggttcagtg gcagtggatc aggcacacag ttttccctca ggatcaacag cctgcagtct 300

gaagattttg ggaggtatta ctgtcaacat ttttggggga ctccgtacac gttcggaggg 360

gggaccaaac tggaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca 420

tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac 480

cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg 540

aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg 600

ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca 660

tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag 705

<210>145

<211>214

<212>PRT

< 213>artificial sequence

<220>

< 223>protein sequence that defines synthetic total length 1D3 sequence of light chain (1D3Kappa variable region and constant region) (is not believed

Number sequence)

<400>145

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

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Ile Tyr Ser Asn

20 25 30

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

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Gln Phe Ser Leu Arg Ile Asn Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Gly Arg Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala

100 105 110

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

115 120 125

Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile

130 135 140

Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu

145 150 155 160

Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

165 170 175

Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr

180 185 190

Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

195 200 205

Phe Asn Arg Asn Glu Cys

210

<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

atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgag 60

gtgcagctgg tggagtctgg gggaggctta gtgcagtctg gagggtccct gaaactctcc 120

tgtgcggcct ctggattcac tttcagtaac tatttcatgt cttgggttcg ccagactcca 180

gagaagaggc tggagtgggt cgcatatatt agtagtggtg gtggtagcac ctactatcca 240

gacagtgtga agggtcgatt caccatctct agagacaatg ccaagaacac cctgtacctg 300

caaatgagca gtctgaagtc tgaggacaca gccatgtatt actgtgtaag acaaggggat 360

ggttactacg gggactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc 420

tcagccaaaa cgacaccccc atctgtctat ccactggccc ctggatctgc tgcccaaact 480

aactccatgg tgaccctggg atgcctggtc aagggctatt tccctgagcc agtgacagtg 540

acctggaact ctggatccct gtccagcggt gtgcacacct tcccagctgt cctgcagtct 600

gacctctaca ctctgagcag ctcagtgact gtcccctcca gcacctggcc cagcgagacc 660

gtcacctgca acgttgccca cccggccagc agcaccaagg tggacaagaa aattgtgccc 720

agggattgtg gttgtaagcc ttgcatatgt acagtcccag aagtatcatc tgtcttcatc 780

ttccccccaa agcccaagga tgtgctcacc attactctga ctcctaaggt cacgtgtgtt 840

gtggtagaca tcagcaagga tgatcccgag gtccagttca gctggtttgt agatgatgtg 900

gaggtgcaca cagctcagac gcaaccccgg gaggagcagt tcaacagcac tttccgctca 960

gtcagtgaac ttcccatcat gcaccaggac tggctcaatg gcaaggagtt caaatgcagg 1020

gtcaacagtg cagctttccc tgcccccatc gagaaaacca tctccaaaac caaaggcaga 1080

ccgaaggctc cacaggtgta caccattcca cctcccaagg agcagatggc caaggataaa 1140

gtcagtctga cctgcatgat aacagacttc ttccctgaag acattactgt ggagtggcag 1200

tggaatgggc agccagcgga gaactacaag aacactcagc ccatcatgga cacagatggc 1260

tcttacttcg tctacagcaa gctcaatgtg cagaagagca actgggaggc aggaaatact 1320

ttcacctgct ctgtgttaca tgagggcctg cacaaccacc atactgagaa gagcctctcc 1380

cactctcctg gtaaatga 1398

<210>147

<211>446

<212>PRT

< 213>artificial sequence

<220>

< 223>protein sequence that defines total length 1F3 sequence of heavy chain (1F3 variable region of heavy chain and IgG1 constant region) (does not have the signal preface

Row)

<400>147

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Val Arg Gln Gly Asp Gly Tyr Tyr Gly Asp Tyr Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro

115 120 125

Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met

130 135 140

Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val

180 185 190

Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His

195 200 205

Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys

210 215 220

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

225 230 235 240

Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro

245 250 255

Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val

260 265 270

Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr

275 280 285

Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu

290 295 300

Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys

305 310 315 320

Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro

340 345 350

Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile

355 360 365

Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly

370 375 380

Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp

385 390 395 400

Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp

405 410 415

Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His

420 425 430

Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

435 440 445

<210>148

<211>705

<212>DNA

< 213>artificial sequence

<220>

< 223>nucleotide sequence of coding synthetic total length 1F3 sequence of light chain (1F3Kappa variable region and constant region)

<400>148

atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt 60

gacatccaga tgactcagtc tccagcctcc ctatctgtat ctgtgggaga aactgtcacc 120

atcacatgtc gagcaagtga gaatatttac agtaatttag catggtatca gcagaaacag 180

ggaaaatctc ctcagctcct ggtctatgat gcaacacact taccagatgg tgtgccatca 240

aggttcagtg gcagtggatc aggcacacag ttttccctca agatcaacag cctgcagtct 300

gaagattttg ggagttatta ctgtcaacat ttttggggta ctccgtacac gtttggaggg 360

gggaccagac tggaaattaa acgggctgat gctgcaccaa ctgtatccat cttcccacca 420

tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac 480

cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg 540

aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg 600

ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca 660

tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag 705

<210>149

<211>214

<212>PRT

< 213>artificial sequence

<220>

< 223>protein sequence that defines synthetic total length 1F3 sequence of light chain (1F3Kappa variable region and constant region) (is not believed

Number sequence)

<400>149

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

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val

35 40 45

Tyr Asp Ala Thr His Leu Pro Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys Arg Ala Asp Ala Ala

100 105 110

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

115 120 125

Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile

130 135 140

Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu

145 150 155 160

Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

165 170 175

Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr

180 185 190

Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

195 200 205

Phe Asn Arg Asn Glu Cys

210

<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 IgG1 constant region)

<400>150

atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgaa 60

gtgcagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaaatctcc 120

tgtgcagcct ctggatttac tttcagtaac tatttcatgt cttgggttcg ccagactcca 180

gagaagaggc tggagtgggt cgcatacatt agtagtggtg gtggtagcac ctactatcca 240

gacagtgtga agggtcgatt caccatctcc agagacaatg ccaagaacac cctgtacctg 300

caaatgaaca gtctgaagtc tgaggacaca gccatgtatt actgtgtaag acaaggagat 360

ggttactatg gggactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc 420

tcagccaaaa cgacaccccc atctgtctat ccactggccc ctggatctgc tgcccaaact 480

aactccatgg tgaccctggg atgcctggtc aagggctatt tccctgagcc agtgacagtg 540

acctggaact ctggatccct gtccagcggt gtgcacacct tcccagctgt cctgcagtct 600

gacctctaca ctctgagcag ctcagtgact gtcccctcca gcacctggcc cagcgagacc 660

gtcacctgca acgttgccca cccggccagc agcaccaagg tggacaagaa aattgtgccc 720

agggattgtg gttgtaagcc ttgcatatgt acagtcccag aagtatcatc tgtcttcatc 780

ttccccccaa agcccaagga tgtgctcacc attactctga ctcctaaggt cacgtgtgtt 840

gtggtagaca tcagcaagga tgatcccgag gtccagttca gctggtttgt agatgatgtg 900

gaggtgcaca cagctcagac gcaaccccgg gaggagcagt tcaacagcac tttccgctca 960

gtcagtgaac ttcccatcat gcaccaggac tggctcaatg gcaaggagtt caaatgcagg 1020

gtcaacagtg cagctttccc tgcccccatc gagaaaacca tctccaaaac caaaggcaga 1080

ccgaaggctc cacaggtgta caccattcca cctcccaagg agcagatggc caaggataaa 1140

gtcagtctga cctgcatgat aacagacttc ttccctgaag acattactgt ggagtggcag 1200

tggaatgggc agccagcgga gaactacaag aacactcagc ccatcatgga cacagatggc 1260

tcttacttcg tctacagcaa gctcaatgtg cagaagagca actgggaggc aggaaatact 1320

ttcacctgct ctgtgttaca tgagggcctg cacaaccacc atactgagaa gagcctctcc 1380

cactctcctg gtaaatga 1398

<210>151

<211>446

<212>PRT

< 213>artificial sequence

<220>

< 223>protein sequence that defines synthetic total length 3A12 sequence of heavy chain (3A12 variable region of heavy chain and IgG1 constant region) (does not have

Signal sequence is arranged)

<400>151

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

1 5 10 15

Ser Leu Lys Ile Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Val Arg Gln Gly Asp Gly Tyr Tyr Gly Asp Tyr Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro

115 120 125

Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met

130 135 140

Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val

180 185 190

Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His

195 200 205

Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys

210 215 220

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

225 230 235 240

Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro

245 250 255

Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val

260 265 270

Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr

275 280 285

Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu

290 295 300

Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys

305 310 315 320

Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro

340 345 350

Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile

355 360 365

Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly

370 375 380

Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp

385 390 395 400

Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp

405 410 415

Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His

420 425 430

Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

435 440 445

<210>152

<211>705

<212>DNA

< 213>artificial sequence

<220>

< 223>nucleotide sequence of coding synthetic total length 3A12 sequence of light chain (3A12Kappa variable region and constant region)

<400>152

atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt 60

gacatccaga tgactcagtc gccagcctcc ctatctgtat ctgtgggaga aactgtcacc 120

atcacatgtc gagcaagtga gaatatttac attaatttag catggtatca gcagaaacag 180

ggaaaatctc ctcagctcct ggtccatgct gcaacaaagt tagcagatgg tgtgccatca 240

aggttcagtg gcagtggatc aggcacacag tattccctca agatcaacag cctgcagtct 300

gaagattttg ggagttatta ctgtcaacat ttttggggta ctccgtacac gttcggaggg 360

gggaccaaac tagaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca 420

tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac 480

cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg 540

aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg 600

ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca 660

tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag 705

<210>153

<211>214

<212>PRT

< 213>artificial sequence

<220>

< 223>protein sequence that defines total length 3A12 sequence of light chain (3A12Kappa variable region and constant region) (does not have the signal preface

Row)

<400>153

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

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ile Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val

35 40 45

His Ala Ala Thr Lys Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala

100 105 110

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

115 120 125

Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile

130 135 140

Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu

145 150 155 160

Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

165 170 175

Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr

180 185 190

Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

195 200 205

Phe Asn Arg Asn Glu Cys

210

<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 (17, nucleotide sequence 1V)

<400>154

atgggatgga gctatatcat cctctttttg gtagcaacag ctacagatgt ccactcccag 60

gtccaactgc agcagcctgg ggctgaactg gtgaagcctg ggacttcagt gaagctgtcc 120

tgcaaggctt ctggctacac cttcaccacc tactggatgc actgggtgaa tcagaggcct 180

ggacaaggcc ttgagtggat tggagagatt aatcctacca acggtcatac taactacaat 240

gagaagttca agagcaaggc cacactgact gtagacaaat cctccagcac agcctacatg 300

caactcagca gcctgacatc tgaggactct gcggtctatt actgtgcaag aaactatgtt 360

ggtagcatct ttgactactg gggccaaggc accactctca ccgtctcctc agcctccacc 420

aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 480

gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540

ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 600

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 660

aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 720

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 780

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 840

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 900

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 960

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 1020

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1080

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 1140

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1200

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1260

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1320

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1380

ctctccctgt ctccgggtaa atga 1404

<210>155

<211>448

<212>PRT

< 213>artificial sequence

<220>

< 223>define the synthetic total length mosaic 2B8 heavy chain (protein of mosaic 2B8IgG1 (Glm (17,1) allotype)

Sequence (not having signal sequence)

<400>155

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

1 5 10 15

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

20 25 30

Trp Met His Trp Val Asn Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn Pro Thr Asn Gly His Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Val Gly Ser Ile Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

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

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

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

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210>156

<211>705

<212>DNA

< 213>artificial sequence

<220>

< 223>coding synthetic total length mosaic 2B8 light chain (mouse variable region and human constant region) (mosaic 2B8Kappa

(Km (3))) nucleotide sequence

<400>156

atggaatcac agactctggt cttcatatcc atactgctct ggttatatgg tgctgatggg 60

aacattgtaa tgacccaatc tcccaaatcc atgtccatgt cagtaggaga gagggtcacc 120

ttgagctgca aggccagtga gaatgtggtt tcttatgtat cctggtatca acagaaacca 180

gcgcagtctc ctaaactgct gatatacggg gcatccaacc ggaacactgg ggtccccgat 240

cgcttcacag gcagtggatc tgcaacagat ttcactctga ccatcagcag tgtgcgggct 300

gaagaccttg cagattatca ctgtgggcag agttacaact atccgtacac gttcggaggg 360

gggaccaggc tggaaataaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 420

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 480

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 540

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 600

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 660

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttga 705

<210>157

<211>214

<212>PRT

< 213>artificial sequence

<220>

< 223>protein sequence that defines synthetic total length mosaic 2B8 light chain (mosaic 2B8Kappa (Km (3))) (does not have

Signal sequence)

<400>157

Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser Met Ser Val Gly

1 5 10 15

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

20 25 30

Val Ser Trp Tyr Gln Gln Lys Pro Ala Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Asn Arg Asn Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

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

65 70 75 80

Glu Asp Leu Ala Asp Tyr His Cys Gly Gln Ser Tyr Asn Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<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

atggactgca cctggaggat cctcctcttg gtggcagcag ctacaggcac ccacgccgag 60

gtccagctgg tacagtctgg ggctgaggtg aagaagcctg gggctacagt gaaaatctcc 120

tgcaaggttt ctggatacac cttcaccacc tactggatgc actgggtgca acaggcccct 180

ggaaaagggc ttgagtggat gggagagatt aatcctacca acggtcatac taactacaat 240

gagaagttcc agggcagagt caccataacc gcggacacgt ctacagacac agcctacatg 300

gagctgagca gcctgagatc tgaggacacg gccgtgtatt actgtgcaac aaactatgtt 360

ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc ag 412

<210>159

<211>118

<212>PRT

< 213>artificial sequence

<220>

< 223>define the protein sequence (not having signal sequence) of synthetic humanization Hu2B8Hv1-f.1 variable region of heavy chain

<400>159

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

1 5 10 15

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

20 25 30

Trp Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Asn Pro Thr Asn Gly His Thr Asn Tyr Asn Glu Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Thr Asn Tyr Val Gly Ser Ile Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<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

cctccaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc acctctgggg 60

gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg acggtgtcgt 120

ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta cagtcctcag 180

gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc acccagacct 240

acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaaa gttgagccca 300

aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc ctggggggac 360

cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc cggacccctg 420

aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt 480

acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag cagtacaaca 540

gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg aatggcaagg 600

agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa accatctcca 660

aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc cgggatgagc 720

tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc agcgacatcg 780

ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg cctcccgtgc 840

tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag agcaggtggc 900

agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac cactacacgc 960

agaagagcct ctccctgtct ccgggtaaat ga 992

<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

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

1 5 10 15

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

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210>162

<211>1404

<212>DNA

< 213>artificial sequence

<220>

< 223>coding synthetic total length heavy chain humanization Hu2B8Hv1f.1 variable region and human IgG1's (Glm (17,1) allotype) are heavy

The nucleotide sequence of chain constant region

<400>162

atggactgca cctggaggat cctcctcttg gtggcagcag ctacaggcac ccacgccgag 60

gtccagctgg tacagtctgg ggctgaggtg aagaagcctg gggctacagt gaaaatctcc 120

tgcaaggttt ctggatacac cttcaccacc tactggatgc actgggtgca acaggcccct 180

ggaaaagggc ttgagtggat gggagagatt aatcctacca acggtcatac taactacaat 240

gagaagttcc agggcagagt caccataacc gcggacacgt ctacagacac agcctacatg 300

gagctgagca gcctgagatc tgaggacacg gccgtgtatt actgtgcaac aaactatgtt 360

ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc agcctccacc 420

aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 480

gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540

ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 600

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 660

aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 720

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 780

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 840

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 900

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 960

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 1020

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1080

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 1140

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1200

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1260

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1320

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1380

ctctccctgt ctccgggtaa atga 1404

<210>163

<211>448

<212>PRT

< 213>artificial sequence

<220>

< 223>define synthetic total length heavy chain humanization Hu2B8Hv1f.1 variable region and human IgG1's CH (Glm (17,1)

Allotype) protein sequence (not having signal sequence)

<400>163

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

1 5 10 15

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

20 25 30

Trp Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Asn Pro Thr Asn Gly His Thr Asn Tyr Asn Glu Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Thr Asn Tyr Val Gly Ser Ile Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

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

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

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

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210>164

<211>412

<212>DNA

< 213>artificial sequence

<220>

< 223>nucleotide sequence of coding synthetic humanization Hu2B8Hv5a.1 variable region of heavy chain

<400>164

atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgaa 60

gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaggatctcc 120

tgtaagggtt ctggatacag ctttaccacc tactggatgc actgggtgcg ccagatgccc 180

gggaaaggcc tggagtggat gggggagatt aatcctacca acggtcatac taactacaat 240

ccgtccttcc aaggccacgt caccatctca gctgacaagt ccatcagcac tgcctacctg 300

cagtggagca gcctgaaggc ctcggacacc gccatgtatt actgtgcgag aaactatgtt 360

ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc ag 412

<210>165

<211>118

<212>PRT

< 213>artificial sequence

<220>

< 223>define the protein sequence (not having signal sequence) of synthetic humanization Hu2B8Hv5a.1 variable region of heavy chain

<400>165

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

1 5 10 15

Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr

20 25 30

Trp Met His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Asn Pro Thr Asn Gly His Thr Asn Tyr Asn Pro Ser Phe

50 55 60

Gln Gly His Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Asn Tyr Val Gly Ser Ile Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210>166

<211>1404

<212>DNA

< 213>artificial sequence

<220>

< 223>coding synthetic total length humanization Hu2B8Hv5a.1 variable region of heavy chain and human IgG1's (Glm (17,1) allotype) are heavy

The nucleotide sequence of chain constant region

<400>166

atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgaa 60

gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaggatctcc 120

tgtaagggtt ctggatacag ctttaccacc tactggatgc actgggtgcg ccagatgccc 180

gggaaaggcc tggagtggat gggggagatt aatcctacca acggtcatac taactacaat 240

ccgtccttcc aaggccacgt caccatctca gctgacaagt ccatcagcac tgcctacctg 300

cagtggagca gcctgaaggc ctcggacacc gccatgtatt actgtgcgag aaactatgtt 360

ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc agcctccacc 420

aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 480

gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540

ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 600

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 660

aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 720

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 780

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 840

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 900

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 960

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 1020

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1080

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 1140

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1200

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1260

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1320

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1380

ctctccctgt ctccgggtaa atga 1404

<210>167

<211>448

<212>PRT

< 213>artificial sequence

<220>

< 223>define synthetic total length humanization Hu2B8Hv5a.1 variable region of heavy chain and human IgG1 (Glm (17,1) allotype)

The protein sequence of CH (not having signal sequence)

<400>167

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

1 5 10 15

Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr

20 25 30

Trp Met His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Asn Pro Thr Asn Gly His Thr Asn Tyr Asn Pro Ser Phe

50 55 60

Gln Gly His Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Asn Tyr Val Gly Ser Ile Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

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

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

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

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<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

atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgaa 60

gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaagatctcc 120

tgtaagggtt ctggatacag ctttaccacc tactggatgc actgggtgcg ccagatgccc 180

gggaaaggcc tggagtggat gggggagatt aatcctacca acggtcatac taactacaat 240

ccgtccttcc aaggccaggt caccatctca gctgacaagt ccatcagcac tgcctacctg 300

cagtggagca gcctgaaggc ctcggacacc gccatgtatt actgtgcgag aaactatgtt 360

ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc ag 412

<210>169

<211>118

<212>PRT

< 213>artificial sequence

<220>

< 223>define the protein sequence (not having signal sequence) of synthetic humanization Hu2B8Hv5-51.1 weight chain variabl area sequence

<400>169

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

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr

20 25 30

Trp Met His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Asn Pro Thr Asn Gly His Thr Asn Tyr Asn Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Asn Tyr Val Gly Ser Ile Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210>170

<211>1404

<212>DNA

< 213>artificial sequence

<220>

< 223>coding synthetic total length humanization Hu2B8Hv5-51.1 variable region of heavy chain and human IgG1's (Glm (17,1) allotype)

The nucleotide sequence of CH

<400>170

atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgaa 60

gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaagatctcc 120

tgtaagggtt ctggatacag ctttaccacc tactggatgc actgggtgcg ccagatgccc 180

gggaaaggcc tggagtggat gggggagatt aatcctacca acggtcatac taactacaat 240

ccgtccttcc aaggccaggt caccatctca gctgacaagt ccatcagcac tgcctacctg 300

cagtggagca gcctgaaggc ctcggacacc gccatgtatt actgtgcgag aaactatgtt 360

ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc agcctccacc 420

aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 480

gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540

ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 600

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 660

aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 720

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 780

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 840

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 900

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 960

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 1020

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1080

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 1140

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1200

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1260

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1320

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1380

ctctccctgt ctccgggtaa atga 1404

<210>171

<211>448

<212>PRT

< 213>artificial sequence

<220>

< 223>(Glm (17,1) is of the same race different to define synthetic total length humanization Hu2B8Hv5-51.1 variable region of heavy chain and human IgG1

Type)

The protein sequence of CH (not having signal sequence)

<400>171

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

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr

20 25 30

Trp Met His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Asn Pro Thr Asn Gly His Thr Asn Tyr Asn Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Asn Tyr Val Gly Ser Ile Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

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

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val yal Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

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

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210>172

<211>388

<212>DNA

< 213>artificial sequence

<220>

< 223>nucleotide sequence of coding synthetic humanization Hu2B8Kv1-39.1Kappa chain variable region

<400>172

atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggct ccgaggtgcc 60

agatgtgaca tccagatgac ccagtctcca tcctccctgt ctgcatctgt aggagacaga 120

gtcaccatca cttgcaaggc cagtgagaat gtggtttctt atgtatcctg gtatcagcag 180

aaaccaggga aagcccctaa gctcctgatc tatggggcat ccaaccggaa cactggggtc 240

ccatcaaggt tcagtggcag tggatctggg acagatttca ctctcaccat cagcagtctg 300

caacctgaag attttgcaac ttactactgt gggcagagtt acaactatcc gtacacgttt 360

ggccagggga ccaagctgga gatcaaac 388

<210>173

<211>107

<212>PRT

< 213>artificial sequence

<220>

< 223>define the protein sequence (not having signal sequence) of synthetic humanization Hu2B8Kv1-39.1Kappa chain variable region

<400>173

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Gly Ala Ser Asn Arg Asn Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<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

gaactgtggc tgcaccatct gtcttcatct tcccgccatc tgatgagcag ttgaaatctg 60

gaactgcctc tgttgtgtgc ctgctgaata acttctatcc cagagaggcc aaagtacagt 120

ggaaggtgga taacgccctc caatcgggta actcccagga gagtgtcaca gagcaggaca 180

gcaaggacag cacctacagc ctcagcagca ccctgacgct gagcaaagca gactacgaga 240

aacacaaagt ctacgcctgc gaagtcaccc atcagggcct gagctcgccc gtcacaaaga 300

gcttcaacag gggagagtgt tga 323

<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

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

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

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210>176

<211>711

<212>DNA

< 213>artificial sequence

<220>

< 223>(Km (3) is of the same race for coding synthetic total length humanization Hu2B8Kv1-39.1 variable region of light chain and people Kappa chain constant region

Abnormal shape) nucleotide sequence of (allelotrope 2)

<400>176

atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggct ccgaggtgcc 60

agatgtgaca tccagatgac ccagtctcca tcctccctgt ctgcatctgt aggagacaga 120

gtcaccatca cttgcaaggc cagtgagaat gtggtttctt atgtatcctg gtatcagcag 180

aaaccaggga aagcccctaa gctcctgatc tatggggcat ccaaccggaa cactggggtc 240

ccatcaaggt tcagtggcag tggatctggg acagatttca ctctcaccat cagcagtctg 300

caacctgaag attttgcaac ttactactgt gggcagagtt acaactatcc gtacacgttt 360

ggccagggga ccaagctgga gatcaaacga actgtggctg caccatctgt cttcatcttc 420

ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac 480

ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac 540

tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc 600

ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat 660

cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgttg a 711

<210>177

<211>214

<212>PRT

< 213>artificial sequence

<220>

< 223>(Km (3) together to define synthetic total length humanization Hu2B8Kv1-39.1 variable region of light chain and people Kappa chain constant region

The kind abnormal shape) protein sequence of (allelotrope 1)

<400>177

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Gly Ala Ser Asn Arg Asn Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>178

<211>382

<212>DNA

< 213>artificial sequence

<220>

< 223>nucleotide sequence of coding synthetic humanization Hu2B8Kv3-15.1 variable region of light chain

<400>178

atggaagccc cagcgcagct tctcttcctc ctgctactct ggctcccaga taccactgga 60

gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 120

ctctcctgca aggccagtga gaatgtggtt tcttatgtat cctggtacca gcagaaacct 180

ggccaggctc ccaggctcct catctatggg gcatccaacc ggaacactgg tatcccagcc 240

aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct 300

gaagattttg cagtttatta ctgtgggcag agttacaact atccgtacac gtttggccag 360

gggaccaagc tggagatcaa ac 382

<210>179

<211>107

<212>PRT

< 213>artificial sequence

<220>

< 223>define the protein sequence (not having signal sequence) of synthetic humanization Hu2B8Kv3-15.1 variable region of light chain

<400>179

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gly Gln Ser Tyr Asn Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>180

<211>705

<212>DNA

< 213>artificial sequence

<220>

< 223>(Km (3) is of the same race for coding synthetic total length humanization Hu2B8Kv3-15.1 variable region of light chain and people Kappa chain constant region

Abnormal shape) nucleic acid of (allelotrope 2)

<400>180

atggaagccc cagcgcagct tctcttcctc ctgctactct ggctcccaga taccactgga 60

gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 120

ctctcctgca aggccagtga gaatgtggtt tcttatgtat cctggtacca gcagaaacct 180

ggccaggctc ccaggctcct catctatggg gcatccaacc ggaacactgg tatcccagcc 240

aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct 300

gaagattttg cagtttatta ctgtgggcag agttacaact atccgtacac gtttggccag 360

gggaccaagc tggagatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 420

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 480

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 540

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 600

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 660

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttga 705

<210>181

<211>214

<212>PRT

< 213>artificial sequence

<220>

< 223>(Km (3) is of the same race different to define synthetic humanization Hu2B8Kv3-15.1 variable region of light chain and people Kappa chain constant region

Type) protein sequence (not having signal sequence) of (allelotrope 2)

<400>181

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gly Gln Ser Tyr Asn Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<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

atgggctggt catatattat tctctttctt gttgctaccg ctaccgatgt gcactctcaa 60

gtccaactcg tacaaccagg cgctgaagtc gtaaaacccg gaacatctgt taaactctca 120

tgcaaagcct caggatacac tttcacaact tactggatgc attgggtcaa tcaagccccc 180

ggacaaggcc tcgaatggat tggcgaaatt aacccaacta acggacatac taattataat 240

gaaaaattta agggcaaagc tacactcacc gtcgataaat caacctctac agcttatatg 300

gaactttcat ccctgagatc agaagataca gccgtctact attgcgccag aaactacgta 360

ggatcaatat tcgattactg gggtcaaggc actctcctca cagtcagctc ag 412

<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

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

1 5 10 15

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

20 25 30

Trp Met His Trp Val Asn Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn Pro Thr Asn Gly His Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Val Gly Ser Ile Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Leu Thr Val Ser Ser

115

<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

ccagcacaaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc acctctgggg 60

gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg acggtgtcgt 120

ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta cagtcctcag 180

gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc acccagacct 240

acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaga gttgagccca 300

aatcttgtga caaaactcac acatgtccac cgtgcccagc acctgaactc ctggggggac 360

cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc cggacccctg 420

aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt 480

acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag cagtacaaca 540

gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg aatggcaagg 600

agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa accatctcca 660

aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc cgggaggaga 720

tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc agcgacatcg 780

ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg cctcccgtgc 840

tggactccga cggctccttc ttcctctata gcaagctcac cgtggacaag agcaggtggc 900

agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac cactacacgc 960

agaagagcct ctccctgtcc ccgggtaaat ga 992

<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

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

1 5 10 15

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

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210>186

<211>1404

<212>DNA

< 213>artificial sequence

<220>

< 223>(Glm (3) is of the same race different for coding synthetic total length heavy chain humanization LR2B8HC variable region of heavy chain and human IgG1's CH

Type) nucleotide sequence of (allelotrope 1)

<400>186

atgggctggt catatattat tctctttctt gttgctaccg ctaccgatgt gcactctcaa 60

gtccaactcg tacaaccagg cgctgaagtc gtaaaacccg gaacatctgt taaactctca 120

tgcaaagcct caggatacac tttcacaact tactggatgc attgggtcaa tcaagccccc 180

ggacaaggcc tcgaatggat tggcgaaatt aacccaacta acggacatac taattataat 240

gaaaaattta agggcaaagc tacactcacc gtcgataaat caacctctac agcttatatg 300

gaactttcat ccctgagatc agaagataca gccgtctact attgcgccag aaactacgta 360

ggatcaatat tcgattactg gggtcaaggc actctcctca cagtcagctc agccagcaca 420

aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 480

gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540

ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 600

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 660

aacgtgaatc acaagcccag caacaccaag gtggacaaga gagttgagcc caaatcttgt 720

gacaaaactc acacatgtcc accgtgccca gcacctgaac tcctgggggg accgtcagtc 780

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 840

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 900

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 960

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 1020

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1080

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 1140

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1200

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1260

gacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg 1320

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1380

ctctccctgt ccccgggtaa atga 1404

<210>187

<211>448

<212>PRT

< 213>artificial sequence

<220>

< 223>(Glm (3) is of the same race to define synthetic total length heavy chain humanization LR2B8HC variable region of heavy chain and human IgG1's CH

Abnormal shape) protein sequence (not having signal sequence) of (allelotrope 1)

<400>187

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

1 5 10 15

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

20 25 30

Trp Met His Trp Val Asn Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn Pro Thr Asn Gly His Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Val Gly Ser Ile Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

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

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

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

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<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

atgggttggt catatattat actctttctc gtagccaccg ccaccgacgt acactctcag 60

gttcaactcg tacaacccgg cgccgaagtc aagaaaccag gaacatcagt caaactctca 120

tgtaaagcaa gcggatacac ctttactact tattggatgc attgggtaag acaagccccc 180

ggacaaggac tcgaatggat aggcgaaata aatcccacta atggacatac aaattataat 240

caaaaatttc aaggacgcgc tacactcacc gtcgataaat caacctcaac cgcatacatg 300

gaactcagct ccctccgatc cgaagacact gccgtttatt attgtgccag aaactatgta 360

ggatctattt tcgattactg gggacaagga acacttctca ccgtaagctc ag 412

<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

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Glu Ile Asn Pro Thr Asn Gly His Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Val Gly Ser Ile Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Leu Thr Val Ser Ser

115

<210>190

<211>1404

<212>DNA

< 213>artificial sequence

<220>

< 223>(Glm (3) is of the same race for coding synthetic total length heavy chain humanization LRMR2B8HC variable region of heavy chain and human IgG1's CH

Abnormal shape) nucleotide sequence of (allelotrope 1)

<400>190

atgggttggt catatattat actctttctc gtagccaccg ccaccgacgt acactctcag 60

gttcaactcg tacaacccgg cgccgaagtc aagaaaccag gaacatcagt caaactctca 120

tgtaaagcaa gcggatacac ctttactact tattggatgc attgggtaag acaagccccc 180

ggacaaggac tcgaatggat aggcgaaata aatcccacta atggacatac aaattataat 240

caaaaatttc aaggacgcgc tacactcacc gtcgataaat caacctcaac cgcatacatg 300

gaactcagct ccctccgatc cgaagacact gccgtttatt attgtgccag aaactatgta 360

ggatctattt tcgattactg gggacaagga acacttctca ccgtaagctc agccagcaca 420

aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 480

gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540

ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 600

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 660

aacgtgaatc acaagcccag caacaccaag gtggacaaga gagttgagcc caaatcttgt 720

gacaaaactc acacatgtcc accgtgccca gcacctgaac tcctgggggg accgtcagtc 780

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 840

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 900

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 960

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 1020

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1080

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 1140

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1200

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1260

gacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg 1320

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1380

ctctccctgt ccccgggtaa atga 1404

<210>191

<211>448

<212>PRT

< 213>artificial sequence

<220>

< 223>define synthetic total length heavy chain humanization LRMR2B8HC variable region of heavy chain and human IgG1's CH (Glm (3)

Allotype) protein sequence (not having signal sequence) of (allelotrope 1)

<400>191

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Glu Ile Asn Pro Thr Asn Gly His Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Val Gly Ser Ile Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

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

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu MetI le Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

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

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<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

atggaaagtc agacccttgt attcatctct attcttcttt ggttgtatgg agcagacggc 60

gacattgtga tgacccaatc ccccgatagt atggccatga gtgtaggaga aagagtcacc 120

cttaattgca aagcctccga aaatgtcgtt tcatatgtgt cttggtatca acaaaaaccc 180

ggccaatcac ccaaacttct catatacggc gcttcaaaca gaaacacagg cgttcccgac 240

agatttagtg gatccggatc agctacagat ttcaccctta ccatcagttc agttcaagca 300

gaagacgttg cagactatca ttgcggacaa tcttataact acccttacac attcggacaa 360

<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

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

1 5 10 15

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

20 25 30

Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Asn Arg Asn Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Val Ala Asp Tyr His Cys Gly Gln Ser Tyr Asn Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>194

<211>323

<212>DNA

< 213>artificial sequence

<220>

< 223>nucleotide sequence of coding synthetic people's Kappa chain constant region (Km (3) allotype) (allelotrope 1)

<400>194

gtacggtggc tgcaccatct gtcttcatct tcccgccatc tgatgagcag ttgaaatctg 60

gaactgcctc tgttgtgtgc ctgctgaata acttctatcc cagagaggcc aaagtacagt 120

ggaaggtgga taacgccctc caatcgggta actcccagga gagtgtcaca gagcaggaca 180

gcaaggacag cacctacagc ctcagcagca ccctgacgct gagcaaagca gactacgaga 240

aacacaaagt ctacgcctgc gaagtcaccc atcagggcct gagctcgccc gtcacaaaga 300

gcttcaacag gggagagtgt tag 323

<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

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

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

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210>196

<211>705

<212>DNA

< 213>artificial sequence

<220>

< 223>coding synthetic total length humanization LR2B8LC variable region of light chain and people Kappa chain constant region (Km (3) allotype) (etc.

Position gene 1) nucleotide sequence

<400>196

atggaaagtc agacccttgt attcatctct attcttcttt ggttgtatgg agcagacggc 60

gacattgtga tgacccaatc ccccgatagt atggccatga gtgtaggaga aagagtcacc 120

cttaattgca aagcctccga aaatgtcgtt tcatatgtgt cttggtatca acaaaaaccc 180

ggccaatcac ccaaacttct catatacggc gcttcaaaca gaaacacagg cgttcccgac 240

agatttagtg gatccggatc agctacagat ttcaccctta ccatcagttc agttcaagca 300

gaagacgttg cagactatca ttgcggacaa tcttataact acccttacac attcggacaa 360

ggaaccaaac tcgaaattaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca 420

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 480

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 540

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 600

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 660

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 705

<210>197

<211>214

<212>PRT

< 213>artificial sequence

<220>

< 223>coding synthetic humanization LR2B8LC variable region of light chain and people Kappa chain constant region (Km (3) allotype) (equipotential base

Cause 1) protein sequence

<400>197

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

1 5 10 15

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

20 25 30

Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Asn Arg Asn Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Val Ala Asp Tyr His Cys Gly Gln Ser Tyr Asn Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<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

atggaatccc aaacccttgt tttcatctct atccttctct ggctttatgg cgccgacgga 60

gacatcgtaa tgacacaatc ccctgactct cttgctatga gcttgggcga acgagtaaca 120

cttaactgca aagcatccga aaatgtcgta tcttacgtat cctggtatca gcaaaaacct 180

ggtcaaagtc ctaaacttct tatatatggt gcaagtaatc gtgaaagtgg cgtcccagac 240

agatttagcg gttcaggttc agcaactgac tttacactta caatttctag cgttcaggcc 300

gaagacgttg cagactatca ttgtggacaa tcttataact atccttatac tttcggacaa 360

ggcactaaac ttgaaattaa ac 382

<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

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

1 5 10 15

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

20 25 30

Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Asn Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Val Ala Asp Tyr His Cys Gly Gln Ser Tyr Asn Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>200

<211>705

<212>DNA

< 213>artificial sequence

<220>

< 223>(Km (3) is of the same race different for coding synthetic total length humanization LRMR2B8LC variable region of light chain and people Kappa chain constant region

Type) nucleotide sequence of (allelotrope 1)

<400>200

atggaatccc aaacccttgt tttcatctct atccttctct ggctttatgg cgccgacgga 60

gacatcgtaa tgacacaatc ccctgactct cttgctatga gcttgggcga acgagtaaca 120

cttaactgca aagcatccga aaatgtcgta tcttacgtat cctggtatca gcaaaaacct 180

ggtcaaagtc ctaaacttct tatatatggt gcaagtaatc gtgaaagtgg cgtcccagac 240

agatttagcg gttcaggttc agcaactgac tttacactta caatttctag cgttcaggcc 300

gaagacgttg cagactatca ttgtggacaa tcttataact atccttatac tttcggacaa 360

ggcactaaac ttgaaattaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca 420

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 480

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 540

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 600

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 660

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 705

<210>201

<211>214

<212>PRT

< 213>artificial sequence

<220>

< 223>(Km (3) is of the same race different to define synthetic total length humanization LRMR2B8LC variable region of light chain and people Kappa chain constant region

Type) protein sequence of (allelotrope 1)

<400>201

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

1 5 10 15

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

20 25 30

Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Asn Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Val Ala Asp Tyr His Cys Gly Gln Ser Tyr Asn Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<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

Glu Ile Asn Pro Thr Asn Gly His Thr Asn Tyr Asn Glu Lys Phe Gln

1 5 10 15

Gly

<210>203

<211>17

<212>PRT

< 213>artificial sequence

<220>

< 223>the heavy chain CDR2 sequence of synthetic humanization Hu2B8Hv5a.1 antibody (Kabat definition)

<400>203

Glu Ile Asn Pro Thr Asn Gly His Thr Asn Tyr Asn Pro Ser Phe Gln

1 5 10 15

Gly

<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

Glu Ile Asn Pro Thr Asn Gly His Thr Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<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

Glu Ile Asn Pro Thr Asn Gly His Thr Asn Tyr Asn Gln Lys Phe Gln

1 5 10 15

Gly

<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

Gly Ala Ser Asn Arg Glu Ser

1 5

<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

cctccaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc acctctgggg 60

gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg acggtgtcgt 120

ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta cagtcctcag 180

gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc acccagacct 240

acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaga gttgagccca 300

aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc ctggggggac 360

cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc cggacccctg 420

aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt 480

acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag cagtacaaca 540

gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg aatggcaagg 600

agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaag accatctcca 660

aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc cgggaggaga 720

tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc agcgacatcg 780

ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg cctcccgtgc 840

tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag agcaggtggc 900

agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac cactacacgc 960

agaagagcct ctccctgtct ccgggtaaat ga 992

<210>208

<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>208

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

1 5 10 15

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

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210>209

<211>1404

<212>DNA

< 213>artificial sequence

<220>

< 223>compile that coding contains humanized Hu2B8Hv5-51.1 variable region of heavy chain and human IgG1's CH Glm (3) is of the same race

The nucleotide sequence of the synthetic total length chain of special-shaped (allelotrope 2)

<400>209

atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgaa 60

gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaagatctcc 120

tgtaagggtt ctggatacag ctttaccacc tactggatgc actgggtgcg ccagatgccc 180

gggaaaggcc tggagtggat gggggagatt aatcctacca acggtcatac taactacaat 240

ccgtccttcc aaggccaggt caccatctca gctgacaagt ccatcagcac tgcctacctg 300

cagtggagca gcctgaaggc ctcggacacc gccatgtatt actgtgcgag aaactatgtt 360

ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc agcctccacc 420

aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 480

gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540

ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 600

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 660

aacgtgaatc acaagcccag caacaccaag gtggacaaga gagttgagcc caaatcttgt 720

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 780

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 840

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 900

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 960

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 1020

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga agaccatctc caaagccaaa 1080

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 1140

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1200

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1260

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1320

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1380

ctctccctgt ctccgggtaa atga 1404

<210>210

<211>448

<212>PRT

< 213>artificial sequence

<220>

< 223>define and contain humanized Hu2B8Hv5-51.1 variable region of heavy chain and human IgG1's CH Glm (3) is of the same race

The protein sequence (not having signal sequence) of the synthetic total length heavy chain of special-shaped (allelotrope 2)

<400>210

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

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr

20 25 30

Trp Met His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Asn Pro Thr Asn Gly His Thr Asn Tyr Asn Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Asn Tyr Val Gly Ser Ile Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

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

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

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

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210>211

<211>2209

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic HGF mhm (V495-L585)

<400>211

atgatgtggg ggaccaaact tctgccggtc ctgttgctgc agcatgtcct cctgcacctc 60

ctcctgcttc atgtcgccat cccctatgca gaaggacaga agaaaagaag aaatacactt 120

catgaattta aaaagtcagc aaaaactact cttaccaagg aagacccatt actgaagatt 180

aaaaccaaaa aagtgaactc tgcagatgag tgtgccaaca ggtgtatcag gaacaggggc 240

tttacgttca cttgcaaggc cttcgttttt gataagtcaa gaaaacgatg ctactggtat 300

cctttcaata gtatgtcaag tggagtgaaa aaagggtttg gccatgaatt tgacctctat 360

gaaaacaaag actatattag aaactgcatc attggtaaag gaggcagcta taaagggacg 420

gtatccatca ctaagagtgg catcaaatgc cagccttgga attccatgat cccccatgaa 480

cacagctttt tgccttcgag ctatcgcggt aaagacctac aggaaaacta ctgtcgaaat 540

cctcgagggg aagaaggggg accctggtgt ttcacaagca atccagaggt acgctacgaa 600

gtctgtgaca ttcctcagtg ttcagaagtt gaatgcatga cctgcaatgg tgaaagctac 660

agaggtccca tggatcacac agaatcaggc aagacttgtc agcgctggga ccagcagaca 720

ccacaccggc acaagttctt gccagaaaga tatcccgaca agggctttga tgataattat 780

tgccgcaatc ctgatggcaa gccgaggcca tggtgctaca ctcttgaccc tgacaccact 840

tgggagtatt gtgcaattaa aacgtgcgct cacagtgctg tgaatgagac tgatgtccct 900

atggaaacaa ctgaatgcat tcaaggccaa ggagaaggtt acaggggaac cagcaatacc 960

atttggaatg gaattccctg tcagcgttgg gattcgcagt accctcacaa gcatgatatc 1020

actcccgaga acttcaaatg caaggacctt agagaaaatt attgccgcaa tccagatggg 1080

gctgaatcac catggtgttt taccactgac ccaaacatcc gagttggcta ctgctctcag 1140

attcccaagt gtgacgtgtc aagtggacaa gattgttatc gtggcaatgg gaaaaattac 1200

atgggcaact tatccaaaac aaggtctgga cttacatgtt ccatgtggga caagaatatg 1260

gaggatttac accgtcatat cttctgggag ccagatgcta gcaaattgaa taagaattac 1320

tgccggaatc ctgatgatga tgcccatgga ccttggtgct acacggggaa tcctcttatt 1380

ccttgggatt attgccctat ttcccgttgt gaaggagata ctacacctac aattgtcaat 1440

ttggaccatc ctgtaatatc ctgtgccaaa acaaaacaac tgcgggttgt aaatgggatt 1500

ccaacacgaa caaacatagg atggatggtt agtttgagat acagaaataa acatatctgc 1560

ggaggatcat tgataaagga gagttgggtt cttactgcac gacagtgttt cccttctcga 1620

gacttgaaag attatgaagc ttggcttgga attcatgatg tccacggaag aggagatgag 1680

aaatgcaaac aggttctcaa tgtttcccag ctggtatatg gccctgaagg atcagatctg 1740

gttttaatga agcttgctcg acctgcaatc ctggataact ttgtcagtac aattgattta 1800

cctagttatg gttgtacaat ccctgaaaag accacttgca gtatttacgg ctggggctac 1860

actggattga tcaacgcgga tggtttatta cgagtagctc atctgtatat tatggggaat 1920

gagaaatgca gtcagcacca tcaaggcaag gtgactttga atgagtctga gttatgtgct 1980

ggggctgaaa agattggatc aggaccatgt gagggagatt atggtggccc actcatttgt 2040

gaacaacaca aaatgagaat ggttcttggt gtcattgttc ctggtcgtgg atgtgccatc 2100

ccaaatcgtc ctgttatttt tgttcgagta gcatattatg caaaatggat acacaaagta 2160

attttgacat acaagttgtg cggccgccat caccatcacc atcactaag 2209

<210>212

<211>680

<212>PRT

< 213>artificial sequence

<220>

< 223>synthetic HGF mhm495-585 is activated

<400>212

Pro Leu Leu Lys Ile Lys Thr Lys Lys Val Asn Ser Ala Asp Glu Cys

1 5 10 15

Ala Asn Arg Cys Ile Arg Asn Arg Gly Phe Thr Phe Thr Cys Lys Ala

20 25 30

Phe Val Phe Asp Lys Ser Arg Lys Arg Cys Tyr Trp Tyr Pro Phe Asn

35 40 45

Ser Met Ser Ser Gly Val Lys Lys Gly Phe Gly His Glu Phe Asp Leu

50 55 60

Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys Ile Ile Gly Lys Gly Gly

65 70 75 80

Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys Ser Gly Ile Lys Cys Gln

85 90 95

Pro Trp Asn Ser Met Ile Pro His Glu His Ser Phe Leu Pro Ser Ser

100 105 110

Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg Gly

115 120 125

Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser Asn Pro Glu Val Arg Tyr

130 135 140

Glu Val Cys Asp Ile Pro Gln Cys Ser Glu Val Glu Cys Met Thr Cys

145 150 155 160

Asn Gly Glu Ser Tyr Arg Gly Pro Met Asp His Thr Glu Ser Gly Lys

165 170 175

Thr Cys Gln Arg Trp Asp Gln Gln Thr Pro His Arg His Lys Phe Leu

180 185 190

Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp Asp Asn Tyr Cys Arg Asn

195 200 205

Pro Asp Gly Lys Pro Arg Pro Trp Cys Tyr Thr Leu Asp Pro Asp Thr

210 215 220

Thr Trp Glu Tyr Cys Ala Ile Lys Thr Cys Ala His Ser Ala Val Asn

225 230 235 240

Glu Thr Asp Val Pro Met Glu Thr Thr Glu Cys Ile Gln Gly Gln Gly

245 250 255

Glu Gly Tyr Arg Gly Thr Ser Asn Thr Ile Trp Asn Gly Ile Pro Cys

260 265 270

Gln Arg Trp Asp Ser Gln Tyr Pro His Lys His Asp Ile Thr Pro Glu

275 280 285

Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn Tyr Cys Arg Asn Pro Asp

290 295 300

Gly Ala Glu Ser Pro Trp Cys Phe Thr Thr Asp Pro Asn Ile Arg Val

305 310 315 320

Gly Tyr Cys Ser Gln Ile Pro Lys Cys Asp Val Ser Ser Gly Gln Asp

325 330 335

Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met Gly Asn Leu Ser Lys Thr

340 345 350

Arg Ser Gly Leu Thr Cys Ser Met Trp Asp Lys Asn Met Glu Asp Leu

355 360 365

His Arg His Ile Phe Trp Glu Pro Asp Ala Ser Lys Leu Asn Lys Asn

370 375 380

Tyr Cys Arg Asn Pro Asp Asp Asp Ala His Gly Pro Trp Cys Tyr Thr

385 390 395 400

Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys Pro Ile Ser Arg Cys Glu

405 410 415

Gly Asp Thr Thr Pro Thr Ile Val Asn Leu Asp His Pro Val Ile Ser

420 425 430

Cys Ala Lys Thr Lys Gln Leu Arg Val Val Asn Gly Ile Pro Thr Arg

435 440 445

Thr Asn Ile Gly Trp Met Val Ser Leu Arg Tyr Arg Asn Lys His Ile

450 455 460

Cys Gly Gly Ser Leu Ile Lys Glu Ser Trp Val Leu Thr Ala Arg Gln

465 470 475 480

Cys Phe Pro Ser Arg Asp Leu Lys Asp Tyr Glu Ala Trp Leu Gly Ile

485 490 495

His Asp Val His Gly Arg Gly Asp Glu Lys Cys Lys Gln Val Leu Asn

500 505 510

Val Ser Gln Leu Val Tyr Gly Pro Glu Gly Ser Asp Leu Val Leu Met

515 520 525

Lys Leu Ala Arg Pro Ala Ile Leu Asp Asn Phe Val Ser Thr Ile Asp

530 535 540

Leu Pro Ser Tyr Gly Cys Thr Ile Pro Glu Lys Thr Thr Cys Ser Ile

545 550 555 560

Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn Ala Asp Gly Leu Leu Arg

565 570 575

Val Ala His Leu Tyr Ile Met Gly Asn Glu Lys Cys Ser Gln His His

580 585 590

Gln Gly Lys Val Thr Leu Asn Glu Ser Glu Leu Cys Ala Gly Ala Glu

595 600 605

Lys Ile Gly Ser Gly Pro Cys Glu Gly Asp Tyr Gly Gly Pro Leu Ile

610 615 620

Cys Glu Gln His Lys Met Arg Met Val Leu Gly Val Ile Val Pro Gly

625 630 635 640

Arg Gly Cys Ala Ile Pro Asn Arg Pro Val Ile Phe Val Arg Val Ala

645 650 655

Tyr Tyr Ala Lys Trp Ile His Lys Val Ile Leu Thr Tyr Lys Leu Cys

660 665 670

Gly Arg His His His His His His

675 680

<210>213

<211>2194

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic HGF mhm (I499-R556)

<400>213

atgatgtggg ggaccaaact tctgccggtc ctgttgctgc agcatgtcct cctgcacctc 60

ctcctgcttc atgtcgccat cccctatgca gaaggacaga agaaaagaag aaatacactt 120

catgaattta aaaagtcagc aaaaactact cttaccaagg aagacccatt actgaagatt 180

aaaaccaaaa aagtgaactc tgcagatgag tgtgccaaca ggtgtatcag gaacaggggc 240

tttacgttca cttgcaaggc cttcgttttt gataagtcaa gaaaacgatg ctactggtat 300

cctttcaata gtatgtcaag tggagtgaaa aaagggtttg gccatgaatt tgacctctat 360

gaaaacaaag actatattag aaactgcatc attggtaaag gaggcagcta taaagggacg 420

gtatccatca ctaagagtgg catcaaatgc cagccttgga attccatgat cccccatgaa 480

cacagctatc gcggtaaaga cctacaggaa aactactgtc gaaatcctcg aggggaagaa 540

gggggaccct ggtgtttcac aagcaatcca gaggtacgct acgaagtctg tgacattcct 600

cagtgttcag aagttgaatg catgacctgc aatggtgaaa gctacagagg tcccatggat 660

cacacagaat caggcaagac ttgtcagcgc tgggaccagc agacaccaca ccggcacaag 720

ttcttgccag aaagatatcc cgacaagggc tttgatgata attattgccg caatcctgat 780

ggcaagccga ggccatggtg ctacactctt gaccctgaca ccccttggga gtattgtgca 840

attaaaacgt gcgctcacag tgctgtgaat gagactgatg tccctatgga aacaactgaa 900

tgcattcaag gccaaggaga aggttacagg ggaaccagca ataccatttg gaatggaatt 960

ccctgtcagc gttgggattc gcagtaccct cacaagcatg atatcactcc cgagaacttc 1020

aaatgcaagg accttagaga aaattattgc cgcaatccag atggggctga atcaccatgg 1080

tgttttacca ctgacccaaa catccgagtt ggctactgct ctcagattcc caagtgtgac 1140

gtgtcaagtg gacaagattg ttatcgtggc aatgggaaaa attacatggg caacttatcc 1200

aaaacaaggt ctggacttac atgttccatg tgggacaaga atatggagga tttacaccgt 1260

catatcttct gggagccaga tgctagcaaa ttgaataaga attactgccg gaatcctgat 1320

gatgatgccc atggaccttg gtgctacacg gggaatcctc ttattccttg ggattattgc 1380

cctatttccc gttgtgaagg agatactaca cctacaattg tcaatttgga ccatcctgta 1440

atatcctgtg ccaaaacaaa acaactgcgg gttgtaaatg ggattccaac acgaacaaac 1500

ataggatgga tggttagttt gagatacaga aataaacata tctgcggagg atcattgata 1560

aaggagagtt gggttcttac tgcacgacag tgtttccctt ctcgagactt gaaagattat 1620

gaagcttggc ttggaattca tgatgtccac ggaagaggag aggagaaaag aaaacagatt 1680

ctcaatattt cccagctggt atatggccct gaaggatcag atctggtttt actgaagctt 1740

gctcgacctg caatcctgga taactttgtc agtacaattg atttacctag ttatggttgt 1800

acaatccctg aaaagaccac ttgcagtatt tacggctggg gctacactgg attgatcaac 1860

gcggatggtt tattacgagt agctcatctg tatattatgg ggaatgagaa atgcagtcag 1920

caccatcaag gcaaggtgac tttgaatgag tctgagttat gtgctggggc tgaaaagatt 1980

ggatcaggac catgtgaggg agattatggt ggcccactca tttgtgaaca acacaaaatg 2040

agaatggttc ttggtgtcat tgttcctggt cgtggatgtg ccatcccaaa tcgtcctggt 2100

atttttgttc gagtagcata ttatgcaaaa tggatacaca aagtaatttt gacatacaag 2160

ttgtgcggcc gccatcacca tcaccatcac taag 2194

<210>214

<211>675

<212>PRT

< 213>artificial sequence

<220>

< 223>synthetic HGF mhm 499-556 is activated

<400>214

Pro Leu Leu Lys Ile Lys Thr Lys Lys Val Asn Ser Ala Asp Glu Cys

1 5 10 15

Ala Asn Arg Cys Ile Arg Asn Arg Gly Phe Thr Phe Thr Cys Lys Ala

20 25 30

Phe Val Phe Asp Lys Ser Arg Lys Arg Cys Tyr Trp Tyr Pro Phe Asn

35 40 45

Ser Met Ser Ser Gly Val Lys Lys Gly Phe Gly His Glu Phe Asp Leu

50 55 60

Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys Ile Ile Gly Lys Gly Gly

65 70 75 80

Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys Ser Gly Ile Lys Cys Gln

85 90 95

Pro Trp Asn Ser Met Ile Pro His Glu His Ser Tyr Arg Gly Lys Asp

100 105 110

Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro

115 120 125

Trp Cys Phe Thr Ser Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile

130 135 140

Pro Gln Cys Ser Glu Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr

145 150 155 160

Arg Gly Pro Met Asp His Thr Glu Ser Gly Lys Thr Cys Gln Arg Trp

165 170 175

Asp Gln Gln Thr Pro His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro

180 185 190

Asp Lys Gly Phe Asp Asp Asn Tyr Cys Arg Asn Pro Asp Gly Lys Pro

195 200 205

Arg Pro Trp Cys Tyr Thr Leu Asp Pro Asp Thr Pro Trp Glu Tyr Cys

210 215 220

Ala Ile Lys Thr Cys Ala His Ser Ala Val Asn Glu Thr Asp Val Pro

225 230 235 240

Met Glu Thr Thr Glu Cys Ile Gln Gly Gln Gly Glu Gly Tyr Arg Gly

245 250 255

Thr Ser Asn Thr Ile Trp Asn Gly Ile Pro Cys Gln Arg Trp Asp Ser

260 265 270

Gln Tyr Pro His Lys His Asp Ile Thr Pro Glu Asn Phe Lys Cys Lys

275 280 285

Asp Leu Arg Glu Asn Tyr Cys Arg Asn Pro Asp Gly Ala Glu Ser Pro

290 295 300

Trp Cys Phe Thr Thr Asp Pro Asn Ile Arg Val Gly Tyr Cys Ser Gln

305 310 315 320

Ile Pro Lys Cys Asp Val Ser Ser Gly Gln Asp Cys Tyr Arg Gly Asn

325 330 335

Gly Lys Asn Tyr Met Gly Asn Leu Ser Lys Thr Arg Ser Gly Leu Thr

340 345 350

Cys Ser Met Trp Asp Lys Asn Met Glu Asp Leu His Arg His Ile Phe

355 360 365

Trp Glu Pro Asp Ala Ser Lys Leu Asn Lys Asn Tyr Cys Arg Asn Pro

370 375 380

Asp Asp Asp Ala His Gly Pro Trp Cys Tyr Thr Gly Asn Pro Leu Ile

385 390 395 400

Pro Trp Asp Tyr Cys Pro Ile Ser Arg Cys Glu Gly Asp Thr Thr Pro

405 410 415

Thr Ile Val Asn Leu Asp His Pro Val Ile Ser Cys Ala Lys Thr Lys

420 425 430

Gln Leu Arg Val Val Asn Gly Ile Pro Thr Arg Thr Asn Ile Gly Trp

435 440 445

Met Val Ser Leu Arg Tyr Arg Asn Lys His Ile Cys Gly Gly Ser Leu

450 455 460

Ile Lys Glu Ser Trp Val Leu Thr Ala Arg Gln Cys Phe Pro Ser Arg

465 470 475 480

Asp Leu Lys Asp Tyr Glu Ala Trp Leu Gly Ile His Asp Val His Gly

485 490 495

Arg Gly Glu Glu Lys Arg Lys Gln Ile Leu Asn Ile Ser Gln Leu Val

500 505 510

Tyr Gly Pro Glu Gly Ser Asp Leu Val Leu Leu Lys Leu Ala Arg Pro

515 520 525

Ala Ile Leu Asp Asn Phe Val Ser Thr Ile Asp Leu Pro Ser Tyr Gly

530 535 540

Cys Thr Ile Pro Glu Lys Thr Thr Cys Ser Ile Tyr Gly Trp Gly Tyr

545 550 555 560

Thr Gly Leu Ile Asn Ala Asp Gly Leu Leu Arg Val Ala His Leu Tyr

565 570 575

Ile Met Gly Asn Glu Lys Cys Ser Gln His His Gln Gly Lys Val Thr

580 585 590

Leu Asn Glu Ser Glu Leu Cys Ala Gly Ala Glu Lys Ile Gly Ser Gly

595 600 605

Pro Cys Glu Gly Asp Tyr Gly Gly Pro Leu Ile Cys Glu Gln His Lys

610 615 620

Met Arg Met Val Leu Gly Val Ile Val Pro Gly Arg Gly Cys Ala Ile

625 630 635 640

Pro Asn Arg Pro Gly Ile Phe Val Arg Val Ala Tyr Tyr Ala Lys Trp

645 650 655

Ile His Lys Val Ile Leu Thr Tyr Lys Leu Cys Gly Arg His His His

660 665 670

His His His

675

<210>215

<211>2193

<212>DNA

< 213>artificial sequence

<220>

< 223>synthetic HGF mhm (W507-L585)

<400>215

atgatgtggg ggaccaaact tctgccggtc ctgttgctgc agcatgtcct cctgcacctc 60

ctcctgcttc atgtcgccat cccctatgca gaaggacaga agaaaagaag aaatacactt 120

catgaattta aaaagtcagc aaaaactact cttaccaagg aagacccatt actgaagatt 180

aaaaccaaaa aagtgaactc tgcagatgag tgtgccaaca ggtgtatcag gaacaggggc 240

tttacgttca cttgcaaggc cttcgttttt gataagtcaa gaaaacgatg ctactggtat 300

cctttcaata gtatgtcaag tggagtgaaa aaagggtttg gccatgaatt tgacctctat 360

gaaaacaaag actatattag aaactgcatc attggtaaag gaggcagcta taaagggacg 420

gtatccatca ctaagagtgg catcaaatgc cagccttgga attccatgat cccccatgaa 480

cacagctatc gcggtaaaga cctacaggaa aactactgtc gaaatcctcg aggggaagaa 540

gggggaccct ggtgtttcac aagcaatcca gaggtacgct acgaagtctg tgacattcct 600

cagtgttcag aagttgaatg catgacctgc aatggtgaaa gctacagagg tcccatggat 660

cacacagaat caggcaagac ttgtcagcgc tgggaccagc agacaccaca ccggcacaag 720

ttcttgccag aaagatatcc cgacaagggc tttgatgata attattgccg caatcctgat 780

ggcaagccga ggccatggtg ctacactctt gaccctgaca ccccttggga gtattgtgca 840

attaaaacgt gcgctcacag tgctgtgaat gagactgatg tccctatgga aacaactgaa 900

tgcattcaag gccaaggaga aggttacagg ggaaccagca ataccatttg gaatggaatt 960

ccctgtcagc gttgggattc gcagtaccct cacaagcatg atatcactcc cgagaacttc 1020

aaatgcaagg accttagaga aaattattgc cgcaatccag atggggctga atcaccatgg 1080

tgttttacca ctgacccaaa catccgagtt ggctactgct ctcagattcc caagtgtgac 1140

gtgtcaagtg gacaagattg ttatcgtggc aatgggaaaa attacatggg caacttatcc 1200

aaaacaaggt ctggacttac atgttccatg tgggacaaga atatggagga tttacaccgt 1260

catatcttct gggagccaga tgctagcaaa ttgaataaga attactgccg gaatcctgat 1320

gatgatgccc atggaccttg gtgctacacg gggaatcctc ttattccttg ggattattgc 1380

cctatttccc gttgtgaagg agatactaca cctacaattg tcaatttgga ccatcctgta 1440

atatcctgtg ccaaaacaaa acaactgcgg gttgtaaatg ggattccaac acaaacaaca 1500

gtaggatgga tggttagttt gagatacaga aataaacata tctgcggagg atcattgata 1560

aaggagagtt gggttcttac tgcacgacag tgtttccctt ctcgagactt gaaagattat 1620

gaagcttggc ttggaattca tgatgtccac ggaagaggag atgagaaatg caaacaggtt 1680

ctcaatgttt cccagctggt atatggccct gaaggatcag atctggtttt aatgaagctt 1740

gctcgacctg caatcctgga taactttgtc agtacaattg atttacctag ttatggttgt 1800

acaatccctg aaaagaccac ttgcagtatt tacggctggg gctacactgg attgatcaac 1860

gcggatggtt tattacgagt agctcatctg tatattatgg ggaatgagaa atgcagtcag 1920

caccatcaag gcaaggtgac tttgaatgag tctgagttat gtgctggggc tgaaaagatt 1980

ggatcaggac catgtgaggg agattatggt ggcccactca tttgtgaaca acacaaaatg 2040

agaatggttc ttggtgtcat tgttcctggt cgtggatgtg ccatcccaaa tcgtcctggt 2100

atttttgttc gagtagcata ttatgcaaaa tggatacaca aagtaatttt gacatacaag 2160

ttgtgcggcc gccatcacca tcaccatcac taag 2194

<210>216

<211>675

<212>PRT

< 213>artificial sequence

<220>

< 223>synthetic HGF mhm507-585 is activated

<400>216

Pro Leu Leu Lys Ile Lys Thr Lys Lys Val Asn Ser Ala Asp Glu Cys

1 5 10 15

Ala Asn Arg Cys Ile Arg Asn Arg Gly Phe Thr Phe Thr Cys Lys Ala

20 25 30

Phe Val Phe Asp Lys Ser Arg Lys Arg Cys Tyr Trp Tyr Pro Phe Asn

35 40 45

Ser Met Ser Ser Gly Val Lys Lys Gly Phe Gly His Glu Phe Asp Leu

50 55 60

Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys Ile Ile Gly Lys Gly Gly

65 70 75 80

Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys Ser Gly Ile Lys Cys Gln

85 90 95

Pro Trp Asn Ser Met Ile Pro His Glu His Ser Tyr Arg Gly Lys Asp

100 105 110

Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro

115 120 125

Trp Cys Phe Thr Ser Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile

130 135 140

Pro Gln Cys Ser Glu Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr

145 150 155 160

Arg Gly Pro Met Asp His Thr Glu Ser Gly Lys Thr Cys Gln Arg Trp

165 170 175

Asp Gln Gln Thr Pro His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro

180 185 190

Asp Lys Gly Phe Asp Asp Asn Tyr Cys Arg Asn Pro Asp Gly Lys Pro

195 200 205

Arg Pro Trp Cys Tyr Thr Leu Asp Pro Asp Thr Pro Trp Glu Tyr Cys

210 215 220

Ala Ile Lys Thr Cys Ala His Ser Ala Val Asn Glu Thr Asp Val Pro

225 230 235 240

Met Glu Thr Thr Glu Cys Ile Gln Gly Gln Gly Glu Gly Tyr Arg Gly

245 250 255

Thr Ser Asn Thr Ile Trp Asn Gly Ile Pro Cys Gln Arg Trp Asp Ser

260 265 270

Gln Tyr Pro His Lys His Asp Ile Thr Pro Glu Asn Phe Lys Cys Lys

275 280 285

Asp Leu Arg Glu Asn Tyr Cys Arg Asn Pro Asp Gly Ala Glu Ser Pro

290 295 300

Trp Cys Phe Thr Thr Asp Pro Asn Ile Arg Val Gly Tyr Cys Ser Gln

305 310 315 320

Ile Pro Lys Cys Asp Val Ser Ser Gly Gln Asp Cys Tyr Arg Gly Asn

325 330 335

Gly Lys Asn Tyr Met Gly Asn Leu Ser Lys Thr Arg Ser Gly Leu Thr

340 345 350

Cys Ser Met Trp Asp Lys Asn Met Glu Asp Leu His Arg His Ile Phe

355 360 365

Trp Glu Pro Asp Ala Ser Lys Leu Asn Lys Asn Tyr Cys Arg Asn Pro

370 375 380

Asp Asp Asp Ala His Gly Pro Trp Cys Tyr Thr Gly Asn Pro Leu Ile

385 390 395 400

Pro Trp Asp Tyr Cys Pro Ile Ser Arg Cys Glu Gly Asp Thr Thr Pro

405 410 415

Thr Ile Val Asn Leu Asp His Pro Val Ile Ser Cys Ala Lys Thr Lys

420 425 430

Gln Leu Arg Val Val Asn Gly Ile Pro Thr Gln Thr Thr Val Gly Trp

435 440 445

Met Val Ser Leu Arg Tyr Arg Asn Lys His Ile Cys Gly Gly Ser Leu

450 455 460

Ile Lys Glu Ser Trp Val Leu Thr Ala Arg Gln Cys Phe Pro Ser Arg

465 470 475 480

Asp Leu Lys Asp Tyr Glu Ala Trp Leu Gly Ile His Asp Val His Gly

485 490 495

Arg Gly Asp Glu Lys Cys Lys Gln Val Leu Asn Val Ser Gln Leu Val

500 505 510

Tyr Gly Pro Glu Gly Ser Asp Leu Val Leu Met Lys Leu Ala Arg Pro

515 520 525

Ala Ile Leu Asp Asn Phe Val Ser Thr Ile Asp Leu Pro Ser Tyr Gly

530 535 540

Cys Thr Ile Pro Glu Lys Thr Thr Cys Ser Ile Tyr Gly Trp Gly Tyr

545 550 555 560

Thr Gly Leu Ile Asn Ala Asp Gly Leu Leu Arg Val Ala His Leu Tyr

565 570 575

Ile Met Gly Asn Glu Lys Cys Ser Gln His His Gln Gly Lys Val Thr

580 585 590

Leu Asn Glu Ser Glu Leu Cys Ala Gly Ala Glu Lys Ile Gly Ser Gly

595 600 605

Pro Cys Glu Gly Asp Tyr Gly Gly Pro Leu Ile Cys Glu Gln His Lys

610 615 620

Met Arg Met Val Leu Gly Val Ile Val Pro Gly Arg Gly Cys Ala Ile

625 630 635 640

Pro Asn Arg Pro Gly Ile Phe Val Arg Val Ala Tyr Tyr Ala Lys Trp

645 650 655

Ile His Lys Val Ile Leu Thr Tyr Lys Leu Cys Gly Arg His His His

660 665 670

His His His

675

Claims (16)

1. isolating conjugated protein that combines human hepatocyte growth factor (HGF), it comprises:

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

(i) CDR _H1Form by aminoacid sequence SEQ ID NO.15,

(ii) CDR _H2Form by aminoacid sequence SEQ ID NO.204 or SEQ ID NO.205, and

(iii) CDR _H3Form by aminoacid sequence SEQ ID NO.17; And

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

(i) CDR _L1Form by aminoacid sequence SEQ ID NO.18,

(ii) CDR _L2Form by aminoacid sequence SEQ ID NO.19 or SEQ ID NO.206, and

(iii) CDR _L3Form by aminoacid sequence SEQ ID NO.20;

Wherein said immunoglobulin heavy chain variable region and said immunoglobulin light chain variable region define the single binding site that is used to combine people HGF together.

2. the described conjugated protein of claim 1, wherein immunoglobulin heavy chain variable region comprises structure C DR _H1-CDR _H2-CDR _H3, wherein

(i) CDR _H1Form by aminoacid sequence SEQ ID NO.15,

(ii) CDR _H2Form by aminoacid sequence SEQ ID NO.205, and

(iii) CDR _H3Form by aminoacid sequence SEQ ID NO.17;

And immunoglobulin light chain variable region comprises structure C DR _L1-CDR _L2-CDR _L3, wherein

(i) CDR _L1Form by aminoacid sequence SEQ ID NO.18,

(ii) CDR _L2Form by aminoacid sequence SEQ ID NO.206, and

(iii) CDR _L3Form by aminoacid sequence SEQ ID NO.20.

3. claim 1 or 2 described conjugated proteins, wherein said CDR is inserted between people or the humanized Tegeline framework region.

4. claim 1 or 2 described conjugated proteins, wherein said conjugated protein is monoclonal antibody or its Fab.

5. claim 1 or 2 described isolating conjugated proteins, wherein said conjugated protein and human hepatocyte growth factor bonded k _dBe 4.0x10 ^-5s ^-1Or it is lower.

6. claim 1 or 2 described isolating conjugated proteins, wherein said conjugated protein and human hepatocyte growth factor bonded K _DBe 20pM or lower.

7. the conjugated protein of the described combination human hepatocyte growth factor of claim 1 (HGF), it comprises and is selected from following immunoglobulin light chain variable region and immunoglobulin heavy chain variable region:

(a) comprise the immunoglobulin light chain variable region of aminoacid sequence SEQ ID NO.193 and comprise the immunoglobulin heavy chain variable region of aminoacid sequence SEQ ID NO.183;

(b) comprise the immunoglobulin light chain variable region of aminoacid sequence SEQ ID NO.193 and comprise the immunoglobulin heavy chain variable region of aminoacid sequence SEQ ID NO.189;

(c) comprise the immunoglobulin light chain variable region of aminoacid sequence SEQ ID NO.199 and comprise the immunoglobulin heavy chain variable region of aminoacid sequence SEQ ID NO.183;

(d) comprise the immunoglobulin light chain variable region of aminoacid sequence SEQ ID NO.199 and comprise the immunoglobulin heavy chain variable region of aminoacid sequence SEQ ID NO.189.

8. the described conjugated protein of claim 7, wherein said immunoglobulin light chain variable region comprises aminoacid sequence SEQ ID NO.199 and said immunoglobulin heavy chain variable region comprises aminoacid sequence SEQ ID NO.189.

9. the conjugated protein of the described combination human hepatocyte growth factor of claim 1 (HGF), it comprises and is selected from following light chain immunoglobulin sequence and heavy chain immunoglobulin sequence:

(a) comprise the light chain immunoglobulin sequence of aminoacid sequence SEQ ID NO.197 and comprise the heavy chain immunoglobulin sequence of aminoacid sequence SEQ ID NO.187;

(b) comprise the light chain immunoglobulin sequence of aminoacid sequence SEQ ID NO.197 and comprise the heavy chain immunoglobulin sequence of aminoacid sequence SEQ ID NO.191;

(c) comprise the light chain immunoglobulin sequence of aminoacid sequence SEQ ID NO.201 and comprise the heavy chain immunoglobulin sequence of aminoacid sequence SEQ ID NO.187;

(d) comprise the light chain immunoglobulin sequence of aminoacid sequence SEQ ID NO.201 and comprise the heavy chain immunoglobulin sequence of aminoacid sequence SEQ ID NO.191.

10. the described conjugated protein of claim 9, wherein said light chain immunoglobulin comprises aminoacid sequence SEQ ID NO.201 and said heavy chain immunoglobulin comprises aminoacid sequence SEQ ID NO.191.

11. claim 1,2 or 7-10 in each described conjugated protein in the purposes of preparation in the medicine, said medicine is used for suppressing or reducing the tumor growth of Mammals.

12. an isolating nucleic acid, it comprises nucleotide sequence and the nucleotide sequence of the immunoglobulin heavy chain variable region of the described conjugated protein of coding claim 2 of the immunoglobulin light chain variable region of coding claim 2 described conjugated protein.

13. an expression vector, it comprises the described nucleic acid of claim 12.

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

15. host cell; It comprises first expression vector and second expression vector; Said first expression vector comprises the nucleotide sequence of the immunoglobulin light chain variable region of the described conjugated protein of coding claim 2, and said second expression vector comprises the nucleotide sequence of the immunoglobulin heavy chain variable region of the described conjugated protein of coding claim 2.

16. a method for preparing the conjugated protein that combines human hepatocyte growth factor (HGF), said conjugated protein is a monoclonal antibody, or its Fab, and this method comprises:

(i) cultivate claim 14 or 15 described host cells under certain condition, make this host cell expression comprise the polypeptide and the polypeptide that comprises immunoglobulin light chain variable region of immunoglobulin heavy chain variable region; And

(ii) antibody purification or its Fab.

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