CN1395617A - A humanized antibody to surface antigen S of hepatitis B virus and preparing method thereof - Google Patents

Abstract

The present invention relates to the humanized antibodies to surface antigen S of hepatitis B virus and a preparing method thereof. Particularly, it relates to the humanized antibodies which comprise heavy and light chains having amino acid sequences originated from human antibodies at the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 of their variable regions, expression vectors containing each of the heavy and light chain genes of the humanized antibody and transformant which can produce humanized antibody by transfection with heavy and light chain expression vectors and a preparing method thereof. A humanized antibody of the present invention is more humanized than that of the previous arts. So, it minimizes the probability of immune response in humans and has good antigen binding capacity, making it a excellent candidate for prevention and treatment of the hepatitis B virus infection.

Description

Humanized antibody of surface antigen S of hepatitis B virus and preparation method thereof

Invention field

The present invention relates to humanized antibody of surface antigen S of hepatitis B virus and preparation method thereof.Specifically, it relates to a kind of like this humanized antibody, it is included in the HCDR1 of its variable region, HCDR2, HCDR3 and LCDR1, LCDR2, the heavy chain and the light chain that have the aminoacid sequence that derives from people's antibody on the LCDR3, by from the deletion of the heavy chain of above-mentioned humanized antibody in conjunction with the peptide sequence of MHC II quasi-molecule make the people anti--replying of mouse antibodies (HAMA) minimize, it also relates to each heavy chain that contains above-mentioned humanized antibody and the expression vector of light chain gene, by the transformant that can produce humanized antibody with heavy chain and light chain expression vector transfection, and by cultivating the method for above-mentioned transformant scale operation humanized antibody.

Background of invention

Hepatitis B virus (HBV) is invaded human body, causes chronic and acute hepatitis.If worse off, it may be a kind of cause of disease that causes liver cirrhosis and liver cancer.The whole world is up to 300,000,000 patient infection HBV (Tiollais and Buendia, Sci.Am., 264,48,1991) according to estimates.The coating of HBV is made up of three kinds of protein.Specifically, it is by comprising the antigenic main protein of S, comprise the middle protein of S antigen and preS 2 antigen and comprising that the larger protein of S antigen, preS 2 antigen and pre S 1 antigen forms (Neurath, A.R. and Kent, S.B.H., Adv.Vir.Res., 34:65-142,1988).During above-mentioned all HBV surface antigen protein matter all can be induced and the antibody of HBV.Wherein, S antigen accounts for the about 80% of total envelope protein, and has the neutralizing epitope that is referred to as " having a " that all exists in all HBV hypotypes usually.

When HBV infects, for example, the office worker of the Institute for Medical Research of newborn infant that the positive mother of HBV produces or contact HBV or accept under recipient's situation of liver transplantation of the relevant liver problem sufferer of HBV, used HB immunoglobulin (Ig) (HBIG) prevention HBV to infect (Beasley etc., Lancet, 2,1099,1983; Todo etc., Hepatol., 13,619,1991).Yet the HBIG that uses is prepared by human plasma at present, therefore has the low relative higher shortcoming with contamination of heavy of anti-antigenic specificity.And, human blood must be provided continuously.

In order to overcome these shortcomings, developed the method for the mouse monoclonal antibody of using hepatitis B vaccine surface antigen.Usually, mouse monoclonal antibody has advantage affine to the antigen height and can scale operation, but, it also has such shortcoming, and what can produce the human anti-mouse antibody in the time of promptly in being administered to human body in human body replys (being referred to as hereinafter, " HAMA replys ") (Shawler etc., J.Immunol., 135,1530,1985).

In order to overcome these shortcomings, developed humanized antibody, wherein the high-affinity of mouse monoclonal antibody and specificity are kept, and HAMA reply be reduced to minimum.The framework region of the complementary determinant (hereinafter being referred to as " CDRs ") of mouse monoclonal antibody being transplanted to people's antibody by gene engineering method makes up humanized antibody.Reported that this humanized antibody shows much smaller immunogenicity (Riechmann etc., Nature, 332,323,1988 in human body; Nakatani etc., Protein Engineering, 7,435,1994).Yet, it is reported that the humanized antibody that this method produces also has shortcoming, promptly when using to human body repeatedly, the CDRs that derives from mouse in the antibody can cause immunne response (Stephens etc., Immunology, 85,668-674,1995; Sharkey etc., CancerResearch, 55,5935s-5945s, 1995).Therefore, when expectation was substituted residue into people's antibody when the amino-acid residue of the CDRs that derives from mouse antibodies in the humanized antibody, HAMA replied and will be lowered.

3 heavy chain CDR ring-HCDR1 are arranged, HCDR2 and HCDR3-and 3 light chain CDR ring-LCDR1, LCDR2 and LCDR3 in the variable region of antibody molecule.In order to produce humanized antibody, whole 6 CDR rings have been transplanted with this area aforesaid method.Yet, because all direct conjugated antigen (Padlan etc. of not all residue among the CDR, FASEB J., 9,133,1995), so be transplanted to humanized antibody that the framework region of people's antibody makes up by the specificity decision residue (hereinafter being referred to as " SDRs ") that will directly relate to antigen bonded mouse monoclonal antibody and will produce the HAMA lower and reply than known humanized antibody.

On the other hand, be essential in the early stage reactivation process of helper cell in immunne response, the necessary effect of performance in selection and activating T cell of MHC (main histocompatibility complex) II quasi-molecule.MHC II proteinoid is in conjunction with the peptide with 9 amino-acid residues that obtains from proteantigen digestion, and it is at the surface expression of antigen presenting cell.When forming activated T cell by mixture with the TXi Baoshouti (TCR) of the above-mentioned MHC II quasi-molecule of identification, immunne response initial (Germain, R.N.Cell 76,287-299,1994) then.Therefore, there is not possibility to incite somebody to action not induce immune response in human body in conjunction with the humanized antibody of the peptide sequence of MHC molecule.

In the past, the humanized antibody that the inventor provides the CDR-transfer methods of the gene of a kind of mouse monoclonal antibody H67 by using hepatitis B vaccine surface antigen to make up, Korean Patent Application No. 163163 (on September 3rd, 1998), and on this antibody basis, worked out the humanized antibody HZII-H67 that compares the immunogenicity reduction that has with above-mentioned humanized antibody, and also apply for Korean Patent, application number is 98-32644.

Therefore, minimize for HAMA being replied by further humanization to above-mentioned humanized antibody HZII-H67, the inventor works out such humanized antibody, they are included in the HCDR1 of its variable region, HCDR2, HCDR3 and LCDR1, LCDR2, have heavy chain and light chain on the LCDR3 from the aminoacid sequence of people's antibody, by deleted peptide sequence from the heavy chain of above-mentioned humanized antibody in conjunction with MHC II quasi-molecule, and make the people anti--mouse antibodies (HAMA) replys and minimizes, and by prove it in human body the possibility that has minimized immunne response and have good antigen binding capacity, make it become the outstanding material standed for of prevention and treatment hepatitis B virus infection, thereby finish the present invention.

Summary of the invention

The object of the present invention is to provide the humanized antibody of hepatitis B vaccine surface antigen S, it makes the immunogenicity in the human body reduce to minimum, and having outstanding antigen binding capacity simultaneously, the present invention also provides its preparation method, so that effectively prevent and treat hepatitis B virus infection.

The accompanying drawing summary

Fig. 1 shows the Nucleotide between the humanization variable region of heavy chain mutant of the variable region of heavy chain of humanized antibody HZII-H67 and hepatitis B vaccine surface antigen S of the present invention and the comparative result of aminoacid sequence.

Fig. 2 provides schematic view illustrating humanization heavy chain mutant I34V the preparation method of (wherein Xie Ansuan replaces the 34th the Isoleucine of mouse HCDR1).

Fig. 3 demonstration comprises the restriction map by the expression vector pcDdA-HzSIIIh-I34V of the I34V mutant of the method preparation of Fig. 2.

The ELISA result of the antigen binding capacity of the humanized antibody with humanization heavy chain mutant of the present invention of the anti-surface antigen S of Fig. 4 explicit declaration,

A; The HCDR1 mutant, B; The HCDR2 mutant,

C; The HCDR3 mutant,

H/K; The humanized antibody HZII-H67 that forms by heavy chain and the light chain of HZII-H67.

Fig. 5 explicit declaration comprises the ELISA result of antigen binding capacity of anti-surface antigen S of the humanized antibody of two or more humanization heavy chain sudden change of the present invention.

Fig. 6 shows the Nucleotide between the humanization variable region of light chain mutant of the variable region of light chain of humanized antibody HZII-H67 and hepatitis B vaccine surface antigen S of the present invention and the comparative result of aminoacid sequence.

Fig. 7 shows synoptic diagram, and the humanization heavy chain mutant Q89L preparation method of (wherein leucine replaces the 89th glutamine of mouse LCDR1) is described.

Fig. 8 demonstration comprises the restriction map by the expression vector pcDdA-HzSIIIh-Q89L of the Q89L mutant of the method preparation of Fig. 7.

Fig. 9 shows ELISA result, and the antigen binding capacity of the humanized antibody of the anti-surface antigen S with humanization light chain mutant of the present invention is described.

H/K; The humanized antibody HZII-H67 that forms by heavy chain and the light chain of HZII-H67

Figure 10 shows Western engram analysis result, and the existence of the humanized antibody HzS-III with humanization reorganization heavy chain mutant of the present invention and light chain mutant is described.

A；HzS-III，????????????????????????B；HZII-H67

Figure 11 shows ELISA result, and the antigen binding capacity of the humanized antibody HzS-III of the hepatitis B vaccine surface antigen S with humanization reorganization heavy chain mutant of the present invention and light chain mutant is described.

Figure 12 shows the result of competitive ELISA, and the humanized antibody HzS-III of the hepatitis B vaccine surface antigen S with humanization reorganization heavy chain mutant of the present invention and light chain mutant and the antigen binding affinity of HzS-IV are described.

Figure 13 shows the aminoacid sequence comparative result between the variable region of heavy chain of the variable region of heavy chain of humanized antibody HzS-III of the present invention and HzS-IV and wild-type mice monoclonal antibody H67 and humanized antibody HZII-H67.

Figure 14 shows the aminoacid sequence comparative result between the variable region of light chain of humanized antibody HzS-III of the present invention and wild-type mice monoclonal antibody H67 and humanized antibody HZII-H67.

                         Detailed Description Of The Invention

The invention provides the humanized antibody of hepatitis B vaccine surface antigen S, it comprises that employment source amino acid residue replaces heavy chain and the light chain of the amino acid residue of CDRs on the heavy chain in mouse source and variable region of light chain; Comprise the heavy chain of each humanized antibody and the expression vector of light chain gene; And with the transformant of above-mentioned expression vector transfection.

Below, the present invention will be described in more detail.

The invention provides the humanization heavy chain mutant, wherein choose amino-acid residue on the heavy chain antibody of source replaces the HCDR1 of corresponding variable region of heavy chain of the humanized antibody of hepatitis B vaccine surface antigen S, HCDR2, mouse source amino-acid residue in the HCDR3 district, and the expression vector that comprises above-mentioned humanization heavy chain mutant.

HCDR1 in the variable region of heavy chain of comparison mouse source humanized antibody HZII-H67 antibody, the corresponding HCDR1 of HCDR2 and HCDR3 and human antibody, the aminoacid sequence in HCDR2 and the HCDR3 district screens the most similar CDR sequence.Then, the amino-acid residue in personnel selection antibody source replaces does not influence the mouse source amino-acid residue of antigen-binding activity, and makes up humanized antibody of the present invention.

For the mouse source amino-acid residue in HCDR1 district among the source aminoacid replacement humanized antibody HZII-H67 that chooses, the Asp-Tyr-Asn-Ile-Gln of SEQ.ID.NO:1 representative, the inventor is with the aminoacid sequence in mouse HCDR1 district and comparing of people's antibody.Found that the amino-acid residue Asp-Tyr-Asn-Val-Asn height homology of the HCDR1 of the people HCDR1 antibody sequence that mouse HCDR1 and SEQ.ID.NO:2 from Kabat database ID numbers 35920 represent.Therefore, the present invention has made up the HCDR1 mutant, shows that wherein the 4th and the 5th different amino acids residues is substituted.

In order so to do, made up mutant I34V or Q35N, wherein Xie Ansuan or l-asparagine replace the Isoleucine or the glutamine of the 34th or the 35th amino acids residue, and this two amino acids residue is different between mouse and people's antibody HCDR1 district.

By PCR and recombinant PCR, the heavy chain expression carrier pRc/CMV-HC-HZIIS (KCTC 0489BP) that uses humanized antibody makes up described mutant (referring to Fig. 1 and Fig. 2) as template.

The result, use respectively from the HL of SEQ.ID.No:3 and SEQ.ID.No:4 representative and P1 primer to and the P2 of SEQ.ID.No:5 and SEQ.ID.No:6 representative and HC primer to the dna fragmentation that obtains as template, it is right as primer to use HL and HC, has made up the variable region of heavy chain mutant I34V of humanized antibody with the recombinant PCR reaction.Afterwards, above-mentioned I34V mutant clone in the expression vector of the cDNA that has cloned the humanization heavy chain antibody, and is made up recombinant expression vector, it is named as pcDdA-HzSIIIh-I34V (referring to Fig. 3).Behind the nucleotide sequence analysis of the variable region of heavy chain I34V mutant that has carried out humanized antibody, the inventor finds that the variable region of heavy chain I34V mutant of humanized antibody has correctly inserted above-mentioned recombinant expression vector.

Use SEQ.ID.No:3, SEQ.ID.No:7, the primer of SEQ.ID.No:8 and SEQ.ID.No.6 representative is right, by having made up the Q35N mutant with PCR and recombinant PCR with quadrat method, wherein on the HCDR1 sequence of mouse and people's antibody, show in the different amino-acid residues, use l-asparagine from people's heavy chain to replace glutamine on the 5th amino acids residue.Afterwards, Q35N is cloned into generation recombinant expression vector pcDdA-HzSIIIh-Q35N in the expression vector.

In addition, in humanized antibody of the present invention, wherein replace amino-acid residue in the mouse HCDR2 district of humanized antibody HZII-H67 from the amino-acid residue of people's antibody, preferred people source HCDR2 aminoacid sequence replaces all mouse source HCDR2 aminoacid sequences, except directly relating to antigen bonded amino-acid residue.For the amino-acid residue that derives from mouse HCDR2 among the humanized antibody HZII-H67 is changed to people source amino-acid residue, the aminoacid sequence in mouse and the HCDR2 district, people source is made comparisons.After finding the homology between the HCDR2 district on mouse HCDR2 and the people's antibody DP-15 heavy chain, use above-mentioned same procedure, directly do not relate to and antigen bonded amino-acid residue by replacing, and make up the HCDR2 mutant.

The result, made up on the 51st amino acids residue of HCDR2 with methionine(Met) and replaced the I51M mutant primer of SEQ.ID.No:9 and SEQ.ID.No:10 representative (use to) of Isoleucine and comprise the recombinant expression vector pcDdA-HzSIIIh-I51M of said mutation body, and on the 54th amino acids residue, replace the T54S mutant primer of SEQ.ID.No:11 and SEQ.ID.No:12 representative (use to) of Threonine and comprise this mutant recombinant expression vector pcDdA-HzSIIIh-T54S, and on the 65th amino acids residue, replace the S65G mutant (use primer that SEQ.ID.No:13 and SEQ.ID.No:14 represent to) of Serine and comprise the recombinant expression vector pcDdA-HzSIIIh-S65G of this mutant with glycine with Serine.

In addition, in humanized antibody of the present invention, wherein replaced amino-acid residue in the mouse HCDR3 district of humanized antibody HZII-H67 from the amino-acid residue of people's antibody, preferred people source HCDR3 aminoacid sequence replaces all mouse source HCDR3 aminoacid sequences, unless amino-acid residue directly relates to the antigen combination.For the amino-acid residue that derives from mouse HCDR3 among the humanized antibody HZII-H67 is changed to people source amino-acid residue, at first carry out the alanine scanning mutagenesis of mouse HCDR3, directly relate to the antigen combination so that verify those amino-acid residues.

The result, made up on the 95th amino acids residue of HCDR3 with L-Ala and replaced the N95A mutant primer of SEQ.ID.No:15 and ISEQ.ID.No:16 representative (use to) of aspartic acid and comprise the recombinant expression vector pcDdA-HzSIIIh-N95A of said mutation body, on the 96th amino acids residue, replace the Y96A mutant primer of SEQ.ID.No:17 and SEQ.ID.No:18 representative (use to) of tyrosine and comprise this mutant recombinant expression vector pcDdA-HzSIIIh-Y96A with L-Ala, on the 97th amino acids residue with the G97A mutant of the L-Ala substituted glycinic acid primer of SEQ.ID.No:19 and SEQ.ID.No:20 representative (use to) with comprise the recombinant expression vector pcDdA-HzSIIIh-G97A of this mutant, on the 98th amino acids residue, replace the Y98A mutant primer of SEQ.ID.No:21 and SEQ.ID.No:22 representative (use to) of tyrosine and comprise the recombinant expression vector pcDdA-HzSIIIh-Y98A of said mutation body with L-Ala, on the 99th amino acids residue, replace the D99A mutant primer of SEQ.ID.No:23 and SEQ.ID.No:24 representative (use to) of l-asparagine and comprise this mutant recombinant expression vector pcDdA-HzSIIIh-D99A with L-Ala, on the 100th amino acids residue, replace the E100A mutant primer of SEQ.ID.No:25 and SEQ.ID.No:26 representative (use to) of L-glutamic acid and comprise the recombinant expression vector pcDdA-HzSIIIh-E100A of this mutant, and on the 100th amino acids residue, replace the S100aA mutant (use primer that SEQ.ID.No:27 and SEQ.ID.No:28 represent to) of Serine and comprise the recombinant expression vector pcDdA-HzSIIIh-S100aA of this mutant with L-Ala with L-Ala.

As mentioned above, by directly not relating to antigen bonded mouse source amino acid with the people source aminoacid replacement in the humanized antibody CDR district, and make the more humanization of humanized antibody of the present invention, and therefore expectation does not influence antigen-binding affinity than prior art.Specifically, use the SDR-transfer methods in the present invention, reply so that humanized antibody has less HAMA, and improve therapeutic action, the SDR district that wherein directly relates in conjunction with the HBV surface antigen S is substituted the antibody into the people.This with the CDR district of mouse antibodies in all amino acid of performance homology all be substituted into the method for the prior art of people's antibody different.

In order to exist the humanized antibody of sudden change to express on the heavy chain that proves humanized antibody of the present invention, the inventor uses the every kind of recombinant expression vector transfecting animal cells that comprises various mutator genes on the heavy chain.After measuring the antibody concentration that reaches from above-mentioned transfection body surface by sandwich ELISA, measure the antigen binding capacity of the humanized antibody of the humanization heavy chain mutant that comprises anti-surface antigen S again with indirect ELISA.Found that in all HCDR1 mutant, the binding ability of the I34V of the hepatitis B vaccine surface antigen S almost binding ability with the heavy chain of wild-type humanized antibody HZS-H67 is identical.For the HCDR2 mutant, T54S and the almost identical antigen binding capacity (referring to Fig. 4) of S65G performance with wild-type antibody.For the HCDR3 mutant, the antigen binding capacity that E100A performance is lower slightly than wild-type antibody, the glutaminic acid residue (E100) that the HCDR3 mutant is described is with inessential during the HBV surface antigen S combines.

Therefore, the aminoacid sequence of the mouse HCDR3 among the aminoacid sequence of the people's antibody HCDR3 in the database and the humanized antibody HZII-H67 is compared, be the amino-acid residue of people's antibody so that the glutaminic acid residue in the aminoacid sequence of mouse HCDR3 replaced.Found that, with the human antibody sequence of mouse HCDR3 sequence performance high homology in (Kabat database ID No 24562,39669,44760), Serine on the 100th and 101 amino acids residues of HCDR3 and glycine are total amino-acid residues.Therefore, the inventor has made up such mutant, wherein replaces L-glutamic acid or L-Ala on the 100th and 101 amino acid positions with Serine or glycine respectively, measures their antigen binding capacity then.

With above-mentioned same method, made up the E100S mutant, wherein Serine has replaced L-glutamic acid on the 100th amino acids residue primer of SEQ.ID.No:29 and SEQ.ID.No:30 representative (use to) and has comprised the recombinant expression vector pcDdA-HzSIIIh-E100S of said mutation body, the A101G mutant, wherein glycine has replaced L-Ala on the 101st amino acids residue primer of SEQ.ID.No:31 and SEQ.ID.No:32 representative (use to) and has comprised the recombinant expression vector pcDdA-HzSIIIh-A101G of said mutation body.After having measured the antigen binding capacity of humanized antibody of anti-surface antigen S, find that the antigen binding capacity of E100S is similar to the antigen binding capacity of wild-type with humanization heavy chain mutant, and the lower slightly antigen binding capacity of A101G performance.

From The above results, the inventor determines in all humanization heavy chain mutants, I34V, and T54S, the antigen binding capacity of S65G and E100S is similar to the antigen binding capacity of wild-type.Therefore, we have made up the recombinant mutant that comprises above-mentioned whole 4 kinds of sudden changes simultaneously.

At first, the recombinant mutant that comprises T54S and I34V sudden change for member, with pcDdA-HzSIIIh-T54S as template by previously described same quadrat method, carry out PCR and subclone, the expression vector of Gou Jianing is named as pcDdA-HzSIIIh-T54S+I34V like this.

Simultaneously, comprise T54S, the recombinant mutant of I34V and E100S sudden change in order to make up, with pcDdA-HzSIIIh-I34V as template, by previously described same quadrat method, carry out PCR and subclone, the expression vector of Gou Jianing is named as pcDdA-HzSIIIh-T54S+I34V+E100S like this.

In addition, comprise T54S in order to make up, I34V, the recombinant mutant of E100S and S65G sudden change as template, by previously described same quadrat method, carries out PCR and subclone with pcDdA-HzSIIIh-E100s.Therefrom made up and comprised T54S, I34V, the recombinant expression vector pcDdA-HzSIIIh-T54S+I34V+E100S+S65G of E100S and all sudden changes of S65G, it is named as pcDdA-HzSIIIh.

In order to investigate the antigen binding capacity that on the heavy chain of humanized antibody, has the humanized antibody of sudden change of the present invention, the inventor uses and comprises the recombinant expression vector transfecting animal cells of said mutation, and measures the antigen binding capacity of the humanized antibody of anti-surface antigen S with indirect ELISA.

As a result, contain I34V+T54S respectively, every kind of humanization heavy chain of I34V+T54S+E100S and I34V+T54S+E100S+S65G sudden change and wild-type humanized antibody HZS-H67 almost show the binding ability (referring to Fig. 5) of identical anti-surface antigen S.The expression vector pcDdA-HzSIIIh that expresses the heavy chain recombinant mutant of the antigen binding capacity that shows hepatitis B vaccine surface antigen S has been preserved in Korea S typical case's culture collecting center of Korea S's life science and biotechnology research institute (KRIBB) on September 22nd, 2000, preserving number is KCTC 10083BP.After having analyzed the aminoacid sequence of the humanized antibody HzS-III that gives expression to from expression vector pcDdA-HzSIIIh, the inventor finds that it is made up of the humanization heavy chain HzS-III-VH of the variable region amino acid sequence that comprises the SEQ.ID.No:43 representative.

Further, the invention provides the humanization light chain mutant of producing by the SDR-transfer methods, wherein use the LCDR1 of corresponding variable region of light chain that replaces the humanized antibody of hepatitis B vaccine surface antigen S from the amino-acid residue on people's the light chain antibody, LCDR2, amino-acid residue in the LCDR3 district, and the expression vector that comprises mutant.

Humanized antibody of the present invention is to make up like this, and the endogenous light chain amino-acid residue of promptly choosing replaces the LCDR1 of the variable region of light chain of humanized antibody HZII-H67, the amino-acid residue on LCDR2 and the LCDR3.For the humanization light chain, the LCDR1 of humanized antibody HZII-H67 and LCDR2 have had that (for LCDR1 is D27cS, S31N, M33I from the amino-acid residue of people's antibody; For LCDR2 is Q54K, S56T).Therefore, the inventor attempts to change the amino-acid residue on the LCDR3.Specifically, for the mouse source amino-acid residue in the LCDR3 district of humanized antibody HZII-H67 is changed be the amino-acid residue of people's antibody, the inventor compared the humanization light chain aminoacid sequence and with the LCDR3 aminoacid sequence (referring to Fig. 6) of the humanization B1 light chain of antibody of its apparent altitude homology.Infer that from this inventor glutamine and Threonine on the 89th and 91 amino acids residues of mouse source LCDR3 directly do not relate to the antigen combination.Therefore, we are amino-acid residue leucine and Serine in people's antibody with their replacements.

The result has made up the Q89L mutant, wherein leucine has replaced glutamyl on the 89th amino acids residue (use primer that SEQ.ID.No:33 represents to SEQ.ID.No:36 to) and has comprised the recombinant expression vector pcDdA-HzSIIIk-Q89L (referring to Fig. 7 and Fig. 8) of said mutation body, and T91S mutant, wherein Serine has replaced Threonine (the application SEQ.ID.No:33 on the 91st amino acids residue, SEQ.ID.No:36, the primer of SEQ.ID.No:37 and SEQ.ID.No:38 representative to) and comprise the recombinant expression vector pcDdA-HzSIIIk-T91S of said mutation body.

Antigen binding capacity for the humanized antibody of investigating the hepatitis B vaccine surface antigen S that has sudden change on the humanized antibody light chain of the present invention, the inventor is with the recombinant expression vector transfecting animal cells that comprises each mutant, with the antigen binding capacity of indirect ELISA mensuration humanized antibody.

The result, contain the humanization light chain performance of T91S mutant (wherein Serine has replaced the Threonine on the 91st amino acids residue) and the binding ability of the anti-surface antigen S of the heavy chain same degree of wild-type humanized antibody HZS-H67, the expression vector pCMV-dhfr-HzSIIIk-T91S of expression T91S mutant of the present invention is named as pCMV-dhfr-HzSIIIk.The expression vector pcDdA-HzSIIIk of mutant T91S that expression has the antigen binding capacity of hepatitis B vaccine surface antigen S has been preserved in the Korea S typical case culture collecting center of Korea S's life science and biotechnology research institute (KRIBB) on September 22nd, 2000, preserving number is KCTC 10084BP.After having analyzed the aminoacid sequence of the humanized antibody HzS-III that gives expression to from expression vector pcDdA-HzSIIIk, the inventor finds that it is made up of the humanization heavy chain HzS-III-VL of the variable region amino acid sequence that comprises the SEQ.ID.No:42 representative.

The present invention also provides such humanized antibody, and it comprises using from people's amino-acid residue and replaces the heavy chain of mouse monoclonal antibody H67 of hepatitis B vaccine surface antigen S and the heavy chain and the light chain of the mouse source amino-acid residue on the variable region of light chain.

In order to make up the humanized antibody that comprises reorganization heavy chain and light chain mutant, the inventor uses humanization heavy chain expression carrier pcDdA-HzSIIIh and humanization light chain expression vector pCMV-dhfr-HzSIIIk transfecting animal cells.Afterwards, the transformant that incubation chooses 48 hours is expressed the humanized antibody HzS-III that has humanization heavy chain and light chain recombination mutation simultaneously.Measure the concentration of the humanized antibody HzS-III that exists in the medium supernatant by the sandwich ELISA method after, using indirect ELISA mensuration antigen binding capacity.

Found that humanized antibody HzS-III performance and wild-type humanized antibody HZS-H67 (H/K) be the binding ability of the anti-surface antigen S of degree (referring to Figure 11) much at one.

In addition, the inventor has measured the antigen binding affinity of humanized antibody HzS-III with competitive ELISA (competitive ELISA, Ryu etc., J.Med.Virol., 52,226,1997).Found that, although personnel selection source amino-acid residue has replaced mouse source propylhomoserin acid residue, (referring to Figure 12) much at one of the antigen binding affinity of humanized antibody HzS-III of the present invention and wild-type humanized antibody HZII-H67.

Screened in the humanized antibody HzS-III heavy chain behind the peptide sequence in conjunction with MHC II quasi-molecule, the inventor has made up the humanization heavy chain of not expecting in conjunction with MHC II quasi-molecule that has amino-acid residue to replace, and has measured their antigen binding capacity.In order to produce described humanization heavy chain, made up with Threonine and replace the S44T mutant primers of SEQ.ID.No:46 and 47 representatives (use to) of the Serine on the 44th and replace the T68D mutant primers of SEQ.ID.No:48 and 49 representatives (use to) of the Threonine on the 68th and the recombinant expression vector that comprises them with aspartic acid.This expression vector has been preserved in Korea S typical case's culture collecting center of Korea S's life science and biotechnology research institute (KRIBB) September 26 calendar year 2001, preserving number is KCTC 10080BP.After humanization heavy chain expression carrier pcDdA-HzSIVh and humanization light chain expression vector pCMV-dhfr-HzSIIIk transfection are in the animal cell line,, make it express humanized antibody HzS-IV with above-mentioned transformant insulation 48 hours.The antigen binding affinity of the humanized antibody HzS-IV that exists in the supernatant liquor that mensuration obtains is found humanized antibody HzS-IV performance of the present invention and humanized antibody HzS-III affinity (referring to Figure 12) much at one.

With comparing with the aminoacid sequence of wild-type mice monoclonal antibody H67 and humanized antibody HZII-H67 of exhibits excellent at the binding ability of HBV surface antigen S and the humanized antibody HzS-III of antigen binding affinity and the heavy chain of HzS-IV and the aminoacid sequence of variable region of light chain.Found that, humanized antibody HzS-III of the present invention and HzS-IV contain less mouse source amino acid than mouse monoclonal antibody H67 and humanized antibody HZII-H67, because heavy chain HCDR1, HCDR2, HCDR3 or light chain LCDR1, LCDR2, some the mouse source amino-acid residue on the LCDR3 have changed and have been people source amino-acid residue.And, in the aminoacid sequence on humanized antibody HzS-IV variable region of heavy chain of the present invention, framework region have two may be by the amino-acid residue of induce immune response be changed in conjunction with people MHC II quasi-molecule, thereby make immunne response minimize (referring to Figure 13 and Figure 14).

Therefore, humanized antibody HzS-III of the present invention and HzS-IV can become the outstanding candidate of prevention hepatitis B virus infection because their performances than the more outstanding HBV of the humanized antibody of prior art in conjunction with active and induce lower HAMA to reply.

Below, we will be by the present invention of embodiment more detailed description.

Yet the following examples purpose is to illustrate the present invention, rather than in order to limit the present invention. Embodiment Embodiment 1: the sudden change of the gene of coding humanization heavy chain antibody The structure of 1-1:HCDR1 mutant

Mouse source amino-acid residue for the HCDR1 district of the source aminoacid replacement humanized antibody HZII-H67 that chooses, the Asp-Tyr-Asn-Ile-Gln (Korea Korean Patent Application No. 98-32644) of SEQ.ID.NO:1 representative, the inventor has compared the aminoacid sequence in mouse HCDR1 district and people's antibody HCDR1 district.Found that the HCDR1 amino-acid residue Asp-Tyr-Asn-Val-Asn height homology of mouse CDR1 and the people HCDR1 antibody sequence that is numbered 35920 SEQ.ID.NO:2 representative from Kabat database ID.Therefore, the inventor has made up and has shown the different substituted HCDR1 mutant of the 4th and 5 amino acids residues. 1-1-1: Xie Ansuan replaces the structure of the mutant of Isoleucine on the 34th amino acids residue

In order to make up the mutant of Xie Ansuan replacement Isoleucine on the 34th amino acids residue, the HCDR1 district of mouse and people's antibody shows different and this position is thought and is not antigenic directly in conjunction with epi-position on this position, and the sequence of representing to SEQ.ID.No:6 with SEQ.ID.No:3 is carried out PCR and recombinant PCR (Fig. 1 and Fig. 2) as the heavy chain expression carrier of primer, humanized antibody HZII-H67pRc/CMV-HC-HZIIS (KCTC 0489BP) as template.Specifically, (Takara Co.) carries out the PCR circulating reaction 30 times with the Taq archaeal dna polymerase, 94 ℃ 30 seconds, 55 ℃ of 30 seconds and 72 ℃ 1 minute.

Found that, HL and P1 primer with SEQ.ID.No:3 and SEQ.ID.No:4 representative obtain the dna fragmentation that length is 181bp from PCR reaction amplification, and P2 and the HC primer represented with SEQ.ID.No:5 and SEQ.ID.No:6 obtain the dna fragmentation that length is 284bp from PCR reaction amplification.With above-mentioned two kinds of dna fragmentations as template and HL and HC primer to after being connected these two segmental recombinant PCRs reactions, obtain the variable region of heavy chain mutant I34V of size for the humanized antibody of 448bp.Behind two ends with Restriction Enzyme EcoRI and ApaI digestion I34V mutant variable region, the inventor has been by having made up recombinant expression vector with above-mentioned fragment cloning to the EcoRI-ApaI restriction enzyme site of pcDdA-HC expression vector (it is that the heavy chain gene of the humanized antibody of anti-HBV pre S 1 antigen (Korea Korean Patent Application No. 1998-49663) is cloned into the expression vector that obtains on the EcoRI-NotI site of pcDNA2 of Invitrogen company), and with its called after pcDdA-HzSIIIh-I34V (Fig. 3).After the nucleotide sequence of the variable region of heavy chain I34V mutant of humanized antibody analyzed, the inventor found that the variable region of heavy chain I34V mutant of humanized antibody is correct and inserts above-mentioned recombinant expression vector. 1-1-2: l-asparagine replaces the structure of the mutant of the glutamine on the 35th amino acids residue

In order to make up the mutant Q35N that l-asparagine replaces the glutamine on the 35th amino acids residue, different and this position of amino-acid residue between mouse on this position and people HCDR1 antibody is not thought antigenic directly in conjunction with epi-position, as embodiment＜1-1-1〉the same terms under carry out PCR.

Specifically, HL and the P3 primer amplification represented with SEQ.ID.No:3 and SEQ.ID.No:7 obtain the dna fragmentation that size is 187bp, and HC and the P4 primer amplification represented with SEQ.ID.No:6 and SEQ.ID.No:8 obtain the dna fragmentation that size is 278bp.By being coupled together, these two fragments obtain the big or small humanized antibody variable region of heavy chain mutant Q35N of 448bp that is.Behind two ends with Restriction Enzyme EcoRI and ApaI digestion Q35N mutant variable region, the present invention has been by having made up recombinant expression vector with above-mentioned fragment cloning to the EcoRI-ApaI restriction enzyme site of pcDdA-HC expression vector, and with its called after pcDdA-HzSIIIh-Q35N.After the nucleotide sequence of humanized antibody variable region of heavy chain Q35N mutant analyzed, the inventor found that humanization antibody heavy chain variable region Q35N mutant is correct and inserts above-mentioned recombinant expression vector. The structure of 1-2:HCDR2 mutant

For the mouse source amino-acid residue in the HCDR2 district of the humanized antibody HZII-H67 of SEQ.ID.NO:39 representative is substituted by humanization amino acid, the inventor compares the aminoacid sequence of mouse HCDR1 antibody and the aminoacid sequence in people's antibody HCDR1 district.Found that the kind of mouse CDR2 and SEQ.ID.NO:40 representative is the aminoacid sequence height homology in the HCDR2 district on the heavy chain DP-15 fragment of people's antibody.Therefore, the inventor seems directly not relate to antigen bonded amino-acid residue by replacement and has made up the HCDR2 mutant. 1-2-1: methionine(Met) replaces the structure of the mutant of the Isoleucine on the 51st amino acids residue

In order to make up the HCDR2 mutant, with P5 and the P6 primer rather than the embodiment＜1-1-1 of SEQ.ID.No:9 and SEQ.ID.No:10 representative〉P1 and P2 primer in the same procedure carry out PCR.Obtain the dna fragmentation that size is 236bp with HL and P5 primer amplification, obtain the dna fragmentation that size is 230bp with HC and P4 primer.React being connected these two segmental recombinant PCRs with the HC primer as template and HL with above-mentioned two kinds of dna fragmentations, obtain size and be the humanized antibody variable region of heavy chain mutant I51M of 448bp.Behind two ends with Restriction Enzyme EcoRI and ApaI digestion said mutation body I51M variable region, the inventor has been by having made up recombinant expression vector with above-mentioned fragment cloning to the EcoRI-ApaI restriction enzyme site of pcDdA-HC expression vector, and with its called after pcDdA-HzSIIIh-I51M.After the nucleotide sequence of the I51M mutant of the variable region of heavy chain of humanized antibody analyzed, the inventor found that the variable region of heavy chain I51M mutant of humanized antibody is correct and inserts above-mentioned recombinant expression vector. 1-2-2: Serine replaces the structure of the mutant of the Threonine on the 54th amino acids residue

P7 and P8 primer rather than enforcement＜1-1-1 with SEQ.ID.No:11 and SEQ.ID.No:12 representative〉P1 and the P2 primer that use in the same procedure carry out PCR.Obtain the dna fragmentation that size is 249bp with HL and P7 primer amplification, obtain the dna fragmentation that size is 218bp with HC and P8 primer amplification.React being connected these two segmental recombinant PCRs with the HC primer as template and HL with above-mentioned two kinds of dna fragmentations, obtain size and be the humanized antibody variable region of heavy chain mutant T54S of 448bp.Behind two ends with Restriction Enzyme EcoRI and ApaI digestion said mutation body T54S variable region, the inventor has been by having made up recombinant expression vector with above-mentioned fragment cloning to the EcoRI-ApaI restriction enzyme site of pcDdA-HC expression vector, and with its called after pcDdA-HzSIIIh-T54S.After the nucleotide sequence of the mutant T54S of the variable region of heavy chain of humanized antibody analyzed, the inventor found that the variable region of heavy chain T54S mutant of humanized antibody is correct and inserts above-mentioned recombinant expression vector. 1-2-: Serine replaces the structure of the mutant of the glycine on the 65th amino acids residue

P9 and P10 primer rather than enforcement＜1-1-1 with SEQ.ID.No:13 and SEQ.ID.No:14 representative〉P1 and the P2 primer that use in the same procedure carry out PCR.Obtain the dna fragmentation that size is 278bp with HL and P9 primer amplification, obtain the dna fragmentation that size is 185bp with HC and P10 primer amplification.React being connected these two segmental recombinant PCRs with the HC primer as template and HL with above-mentioned two kinds of dna fragmentations, obtain the variable region of heavy chain mutant S65G of size for the humanized antibody of 448bp.Behind two ends with Restriction Enzyme EcoRI and ApaI digestion said mutation body S65G variable region, the inventor has been by having made up recombinant expression vector with above-mentioned fragment cloning to the EcoRI-ApaI restriction enzyme site of pcDdA-HC expression vector, and with its called after pcDdA-HzSIIIh-S65G.After the nucleotide sequence of the mutant S65G of the variable region of heavy chain of humanized antibody analyzed, the inventor found that the variable region of heavy chain S65G mutant of humanized antibody is correct and inserts above-mentioned recombinant expression vector. The structure of 1-3:HCDR3 mutant

For the mouse source amino-acid residue on the HCDR3 of the humanized antibody HZII-H67 of SEQ.ID.NO:41 representative is substituted by humanization amino acid, the inventor compares the aminoacid sequence of mouse HCDR3 antibody and the aminoacid sequence in people's antibody HCDR3 district.Afterwards, the inventor has made up the HCDR3 mutant by seeming directly not relate to antigen bonded amino-acid residue in the replacement mouse HCDR3 sequence. 1-3-1: L-Ala replaces the structure of the mutant of the l-asparagine on the 95th amino acids residue

P11 and P12 primer rather than enforcement＜1-1-1 with SEQ.ID.No:15 and SEQ.ID.No:16 representative〉P1 and the P2 primer that use in the same procedure carry out PCR.Obtain the dna fragmentation that size is 381bp with HL and P11 primer amplification, obtain the dna fragmentation that size is 88bp with HC and P12 primer amplification.React being connected these two segmental recombinant PCRs with the HC primer as template and HL with above-mentioned two kinds of dna fragmentations, obtain the variable region of heavy chain mutant N95A of size for the humanized antibody of 448bp.Behind two ends with Restriction Enzyme EcoRI and ApaI digestion said mutation body N95A variable region, the inventor has been by having made up recombinant expression vector with above-mentioned fragment cloning to the EcoRI-ApaI restriction enzyme site of pcDdA-HC expression vector, and with its called after pcDdA-HzSIIIh-N95A.After the nucleotide sequence of the mutant N95A of the variable region of heavy chain of humanized antibody analyzed, the inventor found that the variable region of heavy chain N95A mutant of humanized antibody is correct and inserts above-mentioned recombinant expression vector. 1-3-2: L-Ala replaces the structure of the mutant of the tyrosine on the 96th amino acids residue

P13 and P14 primer rather than enforcement＜1-1-1 with SEQ.ID.No:17 and SEQ.ID.No:18 representative〉P1 and the P2 primer that use in the same procedure carry out PCR.Obtain the dna fragmentation that size is 380bp with HL and P13 primer amplification, obtain the dna fragmentation that size is 85bp with HC and P14 primer amplification.React being connected these two segmental recombinant PCRs with the HC primer as template and HL with above-mentioned two kinds of dna fragmentations, obtain the variable region of heavy chain mutant Y96A of size for the humanized antibody of 448bp.Behind two ends with Restriction Enzyme EcoRI and ApaI digestion said mutation body Y96A variable region, the inventor has been by having made up recombinant expression vector with above-mentioned fragment cloning to the EcoRI-ApaI restriction enzyme site of pcDdA-HC expression vector, and with its called after pcDdA-HzSIIIh-Y96A.After the nucleotide sequence of the mutant Y96A of the variable region of heavy chain of humanized antibody analyzed, the inventor found that the variable region of heavy chain Y96A mutant of humanized antibody is correct and inserts above-mentioned recombinant expression vector. 1-3-3: L-Ala replaces the structure of the mutant of the glycine on the 97th amino acids residue

P15 and P16 primer rather than enforcement＜1-1-1 with SEQ.ID.No:19 and SEQ.ID.No:20 representative〉P1 and the P2 primer that use in the same procedure carry out PCR.Obtain the dna fragmentation that size is 383bp with HL and P15 primer amplification, obtain the dna fragmentation that size is 82bp with HC and P14 primer amplification.React being connected these two segmental recombinant PCRs with the HC primer as template and HL with above-mentioned two kinds of dna fragmentations, obtain the variable region of heavy chain mutant G97A of size for the humanized antibody of 448bp.Behind two ends with Restriction Enzyme EcoRI and ApaI digestion said mutation body G97A variable region, the inventor has been by having made up recombinant expression vector with above-mentioned fragment cloning to the EcoRI-ApaI restriction enzyme site of pcDdA-HC expression vector, and with its called after pcDdA-HzSIIIh-G97A.After the nucleotide sequence of the mutant G97A of the variable region of heavy chain of humanized antibody analyzed, the inventor found that the variable region of heavy chain G97A mutant of humanized antibody is correct and inserts above-mentioned recombinant expression vector. 1-3-4: L-Ala replaces the structure of the mutant of the tyrosine on the 98th amino acids residue

P17 and P18 primer rather than enforcement＜1-1-1 with SEQ.ID.No:21 and SEQ.ID.No:22 representative〉P1 and the P2 primer that use in the same procedure carry out PCR.Obtain the dna fragmentation that size is 386bp with HL and P17 primer amplification, obtain the dna fragmentation that size is 78bp with HC and P18 primer amplification.React being connected these two segmental recombinant PCRs with the HC primer as template and HL with above-mentioned two kinds of dna fragmentations, obtain the variable region of heavy chain mutant Y98A of size for the humanized antibody of 448bp.Behind two ends with Restriction Enzyme EcoRI and ApaI digestion said mutation body Y98A variable region, the inventor has been by having made up recombinant expression vector with above-mentioned fragment cloning to the EcoRI-ApaI restriction enzyme site of pcDdA-HC expression vector, and with its called after pcDdA-HzSIIIh-Y98A.After the nucleotide sequence of the mutant Y98A of the variable region of heavy chain of humanized antibody analyzed, the inventor found that the variable region of heavy chain Y98A mutant of humanized antibody is correct and inserts above-mentioned recombinant expression vector. 1-3-5: L-Ala replaces the structure of the mutant of the aspartic acid on the 99th amino acids residue

P19 and P20 primer rather than enforcement＜1-1-1 with SEQ.ID.No:23 and SEQ.ID.No:24 representative〉P1 and the P2 primer that use in the same procedure carry out PCR.Obtain the dna fragmentation that size is 390bp with HL and P19 primer amplification, obtain the dna fragmentation that size is 73bp with HC and P20 primer amplification.React being connected these two segmental recombinant PCRs with the HC primer as template and HL with above-mentioned two kinds of dna fragmentations, obtain the variable region of heavy chain mutant D99A of size for the humanized antibody of 448bp.Behind two ends with Restriction Enzyme EcoRI and ApaI digestion said mutation body D99A variable region, the inventor has been by having made up recombinant expression vector with above-mentioned fragment cloning to the EcoRI-ApaI restriction enzyme site of pcDdA-HC expression vector, and with its called after pcDdA-HzSIIIh-D99A.After the nucleotide sequence of the mutant D99A of the variable region of heavy chain of humanized antibody analyzed, the inventor found that the variable region of heavy chain D99A mutant of humanized antibody is correct and inserts above-mentioned recombinant expression vector. 1-3-6: L-Ala replaces the structure of the mutant of the L-glutamic acid on the 100th amino acids residue

P21 and P22 primer rather than enforcement＜1-1-1 with SEQ.ID.No:25 and SEQ.ID.No:26 representative〉P1 and the P2 primer that use in the same procedure carry out PCR.Obtain the dna fragmentation that size is 390bp with HL and P21 primer amplification, obtain the dna fragmentation that size is 73bp with HC and P22 primer amplification.React being connected these two segmental recombinant PCRs with the HC primer as template and HL with above-mentioned two kinds of dna fragmentations, obtain the variable region of heavy chain mutant E100A of size for the humanized antibody of 448bp.Behind two ends with Restriction Enzyme EcoRI and ApaI digestion said mutation body E100A variable region, the inventor has been by having made up recombinant expression vector with above-mentioned fragment cloning to the EcoRI-ApaI restriction enzyme site of pcDdA-HC expression vector, and with its called after pcDdA-HzSIIIh-E100A.After the nucleotide sequence of the mutant E100A of the variable region of heavy chain of humanized antibody analyzed, the inventor found that the variable region of heavy chain E100A mutant of humanized antibody is correct and inserts above-mentioned recombinant expression vector. 1-3-7: L-Ala replaces the structure of the mutant of the Serine on the 100a amino acids residue

P23 and P24 primer rather than enforcement＜1-1-1 with SEQ.ID.No:27 and SEQ.ID.No:28 representative〉P1 and the P2 primer that use in the same procedure carry out PCR.Obtain the dna fragmentation that size is 395bp with HL and P23 primer amplification, obtain the dna fragmentation that size is 67bp with HC and P24 primer amplification.React being connected these two segmental recombinant PCRs with the HC primer as template and HL with above-mentioned two kinds of dna fragmentations, obtain the variable region of heavy chain mutant S100aA of size for the humanized antibody of 448bp.Behind two ends with Restriction Enzyme EcoRI and ApaI digestion said mutation body S100aA variable region, the inventor has been by having made up recombinant expression vector with above-mentioned fragment cloning to the EcoRI-ApaI restriction enzyme site of pcDdA-HC expression vector, and with its called after pcDdA-HzSIIIh-S100aA.After the nucleotide sequence of the mutant S100aA of the variable region of heavy chain of humanized antibody analyzed, the inventor found that the variable region of heavy chain S100aA mutant of humanized antibody is correct and inserts above-mentioned recombinant expression vector. Embodiment 2: the expression with humanized antibody of humanization heavy chain mutant

In order in cell, directly to express humanized antibody with humanization heavy chain mutant, with expression vector transfection of the present invention in animal cell line.

Specifically, with the COS7 cell in the DMEM substratum (GIBCO) of additional 10% foetal calf serum at 37 ℃, 5%CO ₂Incubator in the cultivation of going down to posterity.With cell with 1 * 10 ⁶The concentration of individual cell/m is inoculated in the 100mm culture dish, 37 ℃ of overnight incubation.Afterwards, clean 3 times with OPTI-MEMI (GIBCO).What simultaneously, embodiment 1 is made up comprises humanization heavy chain mutant I34V, Q35N, I51M, T54S, S65G, N95A, Y96A, G97A, Y98A, D99A, each 5 μ g of the light chain expression vector pKC-dhfr-HZIIS of the recombinant expression vector of E100A or S100aA and humanized antibody HZII (KCTC 0490BP) dilute with 800 μ l OPTI-MEM I, and 50 μ lLipofectamine (GIBCO) are also diluted with 800 μ l OPTI-MEM I.Above-mentioned diluent mixed obtaining the DNA-lipofectamine mixture in the 15ml test tube, insulation at room temperature is at least 15 minutes then.6.4ml OPTI-MEM I is added each DNA-Lipofectamine mixture, then with they with clean after COS7 cell uniform mixing and rotaring redyeing COS 7 cell.At 37 ℃, 5%CO ₂The cell 48 hours that incubation DNA-Lipofectamine mixture is handled in the incubator and carry out transfection.Then, collect supernatant liquor, measure antibody concentration with the sandwich ELISA method.In order to carry out sandwich ELISA, with Anti-Human IgG (Sigma Co.) as capture antibody, use with horseradish peroxidase (Sigma Co.) blended Anti-Human antibody (Fc specificity) as second antibody. Embodiment 3: the antigen combination of anti-surface antigen S with humanized antibody of humanization heavy chain mutant The mensuration of ability

The surface antigen S (Greencross Co.) of 250ng HBV is added in each hole of microplate, applies the hole.Each substratum of the DNA-lipofectamine mixture transfection that will obtain with the foregoing description 2 adds dull and stereotyped, and each antibody concentration that makes the antibody concentration of measuring based on embodiment 2 is for being respectively 0,0.2,0.4,0.8,1.8,3.5 and 7ng.Carry out measuring optical density(OD) at 492nm after the indirect ELISA as second antibody by in the hole of above-mentioned flat board, adding with Anti-Human's antibody of horseradish peroxidase combination.In contrast, test equally as heavy chain and light chain expression vector respectively with HZII-H67 pRc/CMV-HC-HZIIS (KCTC 0489BP) and pKC-dhfr-HZIIS.The result shows in Fig. 4.

As shown in Figure 4, in the HCDR1 mutant, the binding ability of the hepatitis B vaccine surface antigen S of I34V is identical with the heavy chain of wild-type humanized antibody HZS-H67 almost.For the HCDR2 mutant, T54S and S65G performance almost with the antigen binding capacity of wild-type antibody same degree.On the other hand, for the HCDR3 mutant, the antigen binding capacity that E100A performance is lower slightly than wild-type antibody, the glutaminic acid residue (E100) that the HCDR3 mutant is described is with inessential during the HBV surface antigen S combines. The structure of embodiment 4:HCDR2 mutant

The inventor confirms mouse HCDR3 sequence in the foregoing description 3 the 100th amino acids L-glutamic acid is with inessential during the HBV surface antigen S combines.Therefore, we compare it with the aminoacid sequence of people's antibody CDR3 in the database, are the amino-acid residue of people's antibody so that replace above-mentioned amino-acid residue replaced.

Found that, showing in the human antibody sequence of high homology that with mouse HCDR3 sequence Serine on the 100th and 101 amino acids residues of HCDR3 and glycine are total amino-acid residues.Therefore the inventor has made up respectively with Serine or glycine and has replaced the L-glutamic acid on the 100th and 101 amino acids residues or the mutant of L-Ala, and has measured their antigen binding capacity. 4-1: Serine replaces the structure of the mutant of the L-glutamic acid on the 100th amino acids residue

P25 and P26 primer rather than enforcement＜1-1-1 with SEQ.ID.No:29 and SEQ.ID.No:30 representative〉P1 and the P2 primer that use in the same procedure carry out PCR.Obtain the dna fragmentation that size is 390bp with HL and P25 primer amplification, obtain the dna fragmentation that size is 73bp with HC and P26 primer amplification.React being connected these two segmental recombinant PCRs with the HC primer as template and HL with above-mentioned two kinds of dna fragmentations, obtain the variable region of heavy chain mutant E100A of size for the humanized antibody of 448bp.Behind two ends with Restriction Enzyme EcoRI and ApaI digestion said mutation body E100A variable region, the inventor has been by having made up recombinant expression vector with above-mentioned fragment cloning to the EcoRI-ApaI restriction enzyme site of pcDdA-HC expression vector, and with its called after pcDdA-HzSIIIh-E100A.After the nucleotide sequence of the mutant E100A of the variable region of heavy chain of humanized antibody analyzed, the inventor found that the variable region of heavy chain E100A mutant of humanized antibody is correct and inserts above-mentioned recombinant expression vector. 4-2: glycine replaces the structure of the mutant of the L-Ala on the 101st amino acids residue

P27 and P28 primer rather than enforcement＜1-1-1 with SEQ.ID.No:31 and SEQ.ID.No:32 representative〉P1 and the P2 primer that use in the same procedure carry out PCR.Obtain the dna fragmentation that size is 396bp with HL and P27 primer amplification, obtain the dna fragmentation that size is 68bp with HC and P28 primer amplification.React being connected these two segmental recombinant PCRs with the HC primer as template and HL with above-mentioned two kinds of dna fragmentations, obtain the variable region of heavy chain mutant A101G S100aA of size for the humanized antibody of 448bp.Behind two ends with Restriction Enzyme EcoRI and ApaI digestion said mutation body A101G variable region, the inventor has been by having made up recombinant expression vector with above-mentioned fragment cloning to the EcoRI-ApaI restriction enzyme site of pcDdA-HC expression vector, and with its called after pcDdA-HzSIIIh-A101G.After the nucleotide sequence of the mutant A101G of the variable region of heavy chain of humanized antibody analyzed, the inventor found that the variable region of heavy chain A101G mutant of humanized antibody is correct and inserts above-mentioned recombinant expression vector. 4-3: the mensuration of the antigen binding capacity of anti-surface antigen S

With the foregoing description＜4-1〉and＜4-2〉2 recombinant expression vectors making up are with after the same procedure transfection of embodiment 2 and 3 is in the COS cell, and the inventor has measured the antigen binding capacity of selecting transformant with indirect elisa method.As a result, the antigen binding capacity of E100S is similar to the antigen binding capacity of wild-type, and the antigen binding capacity that the A101G performance reduces slightly.

From embodiment 3 and＜4-3 the result, the present invention be sure of in the humanization heavy chain mutant, I34V, T54S, the antigen binding capacity of S65G and E100S is similar to wild-type.Therefore, we have made up the recombinant mutant that contains all these four kinds of sudden changes simultaneously. Embodiment 5: the recombinant mutant of humanization heavy chain gene 5-1: the structure of recombinant mutant T54S+I34V

In order to make up the recombinant mutant that contains T54S and I34V sudden change, with front embodiment＜1-2-1〉heavy chain gene expression vector pcDdA-HzSIIIh-T54S as template, with embodiment＜1-1-1 same procedure carry out PCR and subclone.Therefrom the expression vector of Gou Jianing is named as pcDdA-HzSIIIh-T54S+I34V. 5-2: the structure of recombinant mutant T54S+I34V+E100S

Contain T54S in order to make up, the recombinant mutant of I34V and E100S sudden change is with front embodiment＜5-1〉heavy chain gene expression vector p pcDdA-HzSIIIh-I34V as template, with embodiment＜1-1-1 same procedure carry out PCR and subclone.Therefrom the expression vector of Gou Jianing is named as pcDdA-HzSIIIh-T54S+I34V+E100S. 5-3: the structure of recombinant mutant T54S+I34V+E100S+S65G

Contain T54S in order to make up, I34V, the recombinant mutant of E100S and S65G sudden change is with front embodiment＜5-2〉heavy chain gene expression vector pcDdA-HzSIIIh-E100s as template, with embodiment＜1-1-1 same procedure carry out PCR and subclone.The result has made up and has comprised T54S, I34V, and the recombinant expression vector pcDdA-HzSIIIh-T54S+I34V+E100S+S65G of E100S and all sudden changes of S65G is with its called after pcDdA-HzSIIIh. Embodiment 6: the mensuration of antigen binding capacity with humanized antibody of humanization heavy chain recombinant mutant

The surface antigen S (Greencross Co.) of 250ng HBV is joined in each hole of microplate, after applying, with each recombinant expression vector or the foregoing description＜4-1〉to＜4-3〉the pKC-dhfr-HZIIS carrier that makes up is with the same procedure rotaring redyeing COS 7 cell among the embodiment 2.Each substratum that will contain above-mentioned transfectional cell adds dull and stereotyped, makes the concentration of each antibody be respectively 0.625,1.25, and 2.5,5,10 and 20ng.Then, measure optical density(OD) with the same procedure of embodiment 2.In contrast, carry out same test as heavy chain and the light chain expression vector of HZII-H67 respectively with pRc/CMV-HC-HZIIS and pKC-dhfr-HZIIS.The result as shown in Figure 5.

As shown in Figure 5, contain I34V+T54S respectively, each humanization heavy chain of the recombinant mutant of I34V+T54S+E100S and I34V+T54S+E100S+S65G, wherein there are several sudden changes simultaneously, show heavy chain binding ability much at one at surface antigen S with wild-type humanized antibody HZS-H67.The expression vector pcDdA-HzSIIIh that shows the expression heavy chain recombinant mutant of antigen binding capacity at the HBV surface antigen S has been preserved in Korea S typical case's culture collecting center of Korea S's life science and biotechnology research institute (KRIBB) on September 22nd, 2000, preserving number is: KCTC 10083BP.After the amino acid of the humanized antibody HzS-III that gives expression to from expression vector pcDdA-HzSIIIh analyzed, the inventor found that it is made up of the humanization heavy chain HzS-III-VH of the variable region amino acid sequence that comprises the SEQ.ID.No:43 representative. Embodiment 7: the sudden change of humanization light chain gene

For the humanization light chain, the LCDR1 of humanized antibody HZII-H67 and LCDR2 have had the amino-acid residue from people's antibody.Therefore, the inventor attempts to change the amino-acid residue among the LCDR3.Specifically, be the amino-acid residue of people's antibody for the mouse source amino-acid residue in the LCDR3 district of humanized antibody HZII-H67 is changed, the inventor compares the aminoacid sequence of humanization light chain and the LCDR3 aminoacid sequence of light chain that has the humanization B1 antibody of high homology with it.Therefrom, the inventor infers that glutamine and Threonine on the 89th and 91 amino acids residues of mouse source LCDR3 directly do not relate to the antigen combination.Therefore, our the choose leucine and the Serine of the corresponding light chain residue on the B1 antibody replaces them. 7-1: leucine replaces the structure of the mutant of the glutamine on the 89th amino acids residue

The mutant that replaces the glutamine on the 89th amino acids residue of mouse source LCDR3 for the leucine residue that makes up people's antibody, carries out PCR and recombinant PCR (Fig. 6 and Fig. 7) with the light chain expression vector pKC-dhkr-HzIIS of humanized antibody HZII-H67 as template at the primer of representing with SEQ.ID.No:33 and SEQ.ID.No:36.

Specifically, carry out 25 PCR circulation with the Taq archaeal dna polymerase, 94 ℃ 30 seconds, 55 ℃ of 30 seconds and 72 ℃ 1 minute.LL and P29 primer with SEQ.ID.No:33 and SEQ.ID.No:36 representative are the dna fragmentation of 336bp by the pcr amplification size, and P30 and the LC primer represented with SEQ.ID.No:35 and SEQ.ID.No:36 are the dna fragmentation of 273bp by the pcr amplification size.Right as primer with above-mentioned two dna fragmentations as template, LL and P29, connect these two fragments and carry out recombinant PCR reaction, obtain the variable region of light chain mutant Q89L of size for the humanized antibody of 743bp.Behind two ends with Restriction Enzyme HindIII and SalI digestion said mutation body Q89L variable region, the inventor has made up recombinant plasmid pBlue-HzSIIIk-Q89L by they being cloned into pBluescript KS+ (StrataGen).Behind the nucleotide sequence analysis to humanized antibody variable region of light chain G89L mutant, the inventor finds the above-mentioned expression vector of the correct insertion of Q89L.After digesting above-mentioned pBlue-HzSIIIk-Q89L DNA, obtain humanization light chain mutant fragment with Restriction Enzyme HindIII and ApaI, the inventor is by being cloned into above-mentioned fragment again the HindIII-ApaI site of pCMV-dhfr expression vector, and made up recombinant expression vector (Fig. 7), and with its called after pCMV-dhfr-HzSIIIk-Q89L (Fig. 8). 7-2: Serine replaces the structure of the mutant of the Threonine on the 91st amino acids residue

Use SEQ.ID.No:33, SEQ.ID.No:36, the primer of SEQ.ID.No:37 and SEQ.ID.No:38 representative is at embodiment＜7-1〉carry out PCR under the identical reaction conditions.Is the dna fragmentation of 369bp with LL and P31 primer by pCR amplification size, is the dna fragmentation of 391bp by the pcr amplification size with P32 and LC primer.Right as primer with above-mentioned two dna fragmentations as template, LL and LC, connect these two fragments and carry out recombinant PCR reaction, obtain the variable region of light chain mutant T91S of size for the humanized antibody of 743bp.Behind two ends with Restriction Enzyme HindIII and SalI digestion said mutation body T91S variable region, the inventor has made up recombinant plasmid by they are cloned into pBluescriptKS+.Behind the nucleotide sequence analysis to humanized antibody variable region of light chain T91S mutant, the inventor finds the above-mentioned recombinant expression vector of the correct insertion of T91S.

After digesting above-mentioned pBlue-HzSIIIk-T89S DNA, obtain humanization light chain mutant fragment with Restriction Enzyme HindIII and ApaI, the inventor is by being cloned into above-mentioned fragment again the HindIII-ApaI site of pCMV-dhfr expression vector, and made up recombinant expression vector pCMV-dhfr-HzSIIIk-T91S, and with its called after pCMV-dhfr-HzSIIIk-T91S. Embodiment 8: the combination of hepatitis B vaccine surface antigen S with humanized antibody of humanization light chain mutant The mensuration of ability

With embodiment＜7-1〉and＜7-2〉the expression vector pCMV-dhfr-HzSIIIk-Q89L or the pCMV-dhfr-HzSIIIk-T91S that obtain, with the heavy chain expression carrier of pRc/CMV-HC-HZIIS carrier as humanized antibody HZII-H67, behind the same procedure rotaring redyeing COS 7 cell according to embodiment 2, to the transformant incubation selected 48 hours.Then, by the antibody concentration in the sandwich ELISA method mensuration supernatant liquor.Same procedure according to embodiment 3 is added to antibody in the flat board, makes the concentration of each antibody be respectively 0,0.2, and 0.4,0.8,1.8,3.5 and 7ng.In contrast, use pRc/CMV-HC-HZIIS and pKC-dhfr-HZIIS, carry out identical test respectively as heavy chain and the light chain expression vector of HZII-H67.The result as shown in Figure 9.

As shown in Figure 9, contain the binding ability of humanization light chain performance and the anti-surface antigen S of the heavy chain same degree of wild-type humanized antibody HZS-H67 that Serine replaces the T91S mutant of the Threonine on the 91st amino acids residue.The expression vector pcDdA-HzSIIIk that expresses the mutant T91S of the antigen binding capacity that shows hepatitis B vaccine surface antigen S has been preserved in Korea S typical case's culture collecting center of Korea S's life science and biotechnology research institute (KRIBB) on September 22nd, 2000, preserving number is: KCTC 10084BP.Behind the amino acid sequence analysis to the humanized antibody HzS-IIId that expresses from expression vector pcDdA-HzSIIIk, the inventor finds that it is made up of the humanization heavy chain HzS-III-VL of the variable region amino acid sequence that comprises the SEQ.ID.No:42 representative. Embodiment 9: the antigen with humanized antibody (HzS-III) of humanization heavy chain and light chain recombinant mutant The mensuration of binding ability

With embodiment＜5-2〉the humanization heavy chain expression carrier pCDdA-HzSIIIh and the embodiment＜7-2 that obtain〉the humanization light chain expression vector pCMV-dhfr-HzSIIIk that obtains, behind the same quadrat method rotaring redyeing COS 7 cell according to embodiment 2, the transformant of selecting to obtain by insulation gave expression to the humanized antibody HzS-III that has humanization heavy chain and light chain recombination mutation simultaneously in 48 hours.In order to determine from the supernatant liquor that substratum obtains, there is humanized antibody HzS-III, combine with Anti-Human's antibody (Fc specificity) and the second antibody that obtains is carried out the Western engram analysis with horseradish peroxidase (Sigma Co.).The result detects the humanization heavy chain of the about 55kDa of size and the humanization hydrogen chain of the about 27kDa of size as shown in figure 10.Therefore, the inventor finds that humanized antibody of the present invention is correctly expressed and secretes from transformant.

Measured the concentration of the secretory antibody in the supernatant liquor by the sandwich ELISA method after, with the same quadrat method of embodiment 3 antibody is added and to be coated with in the flat board of HBV surface antigen S, make the concentration of each antibody be respectively 0.625,1.25,2.5,5,10 and 20ng.In contrast, carry out identical test with pRc/CMV-HC-HZIIS and pKC-dhfr-HZIIS as heavy chain and the light chain expression vector of HZII-H67 respectively.The result as shown in figure 11.

As shown in figure 11, the humanized antibody HzS-III performance that has humanization heavy chain and light chain recombination mutation simultaneously almost with the binding ability of the anti-surface antigen S of wild-type humanized antibody HZS-H67 (H/K) same degree. Embodiment 10: the mensuration of the antigen binding affinity of humanized antibody HzS-III

Measure the antigen binding affinity (competitive ELISA, Ryu etc., J.Med.Virol., 52,226,1997) of humanized antibody HzS-III with comparing the ELISA method.(from 1 * 10 for humanized antibody HzS-III that 5ng is produced from the COS7 cell of embodiment 8 and surface antigen S ^-7M to 1 * 10 ^-12M) 37 ℃ be incubated 3 hours in advance after, mixture added be coated with in advance in the 96 hole flat boards of surface antigen S.Make the hole flat board after 3 hours, measure optical density(OD) (Figure 12) by ELISA with the same procedure of embodiment 3 37 ℃ of reactions.As contrast, use humanized antibody HZII-H67.

The antigen binding affinity that found that humanized antibody HzS-III of the present invention is about 4 * 10 ⁹M ^-1, it and contrast HZII-H67 (3.2 * 10 ⁹M ^-1) big-difference very not. Embodiment 11: the people source of removing from humanized antibody HzS-III in conjunction with the peptide sequence of mhc class ii molecule Change the structure of antibody HzS-IV

The inventor uses Stumiolo ' s method (TEPITOPE) (Sturniolo etc., NatureBiotechnology, 17,555-561,1999) to investigate the peptide sequence that whether exists among the above-mentioned humanized antibody HzS-III in conjunction with MHC II quasi-molecule.In addition, the inventor is a retrieve sequence the DP-25 albumen from the ethnic group with above-mentioned humanized antibody apparent altitude homology, it is got rid of consider then.

Found that has 2 sections peptide sequences in conjunction with MHC II quasi-molecule on the humanization heavy chain.First section peptide has the aminoacid sequence of SEQ.ID.No:44 representative, finds that it is in conjunction with 22 in 51 MHC II quasi-molecules.Second peptide has the aminoacid sequence of SEQ.ID.No:45 representative, and finds that it is in conjunction with 14 MHC II quasi-molecules (table 1).

＜table 1〉the TEPITOPE analytical results of humanization heavy chain HzSIIIh

Peptide	Before the replacement	After the replacement	Before the replacement	After the replacement
					WVRQAPGK S (SEQ.ID.No.4 4)	??WVKQAPGKT ??(SEQ.ID.N ??o.46)	??FQGRVTLTV ??(SEQ.ID.No.4 ??5)	??FQGRVDLTV ??(SEQ.ID.N ??o.47
	The MHC II quasi-molecule of binding peptide	??DRB1_0101 ??DRB1_0301 ??DRB1_0305 ??DRB1_0309 ??DRB1_0401 ??DRB1-0405 ??DRB1_0408 ??DRB1_0421 ??DRB1_0426 ??DRB1_0801 ??DRB1_0802 ??DRB1_1101 ??DRB1_1102 ??DRB1_1104 ??DBR1_1106 ??DRB1_1107 ??DRB1_1114 ??DRB1_1120 ??DRB1_1121 ??DRB1_1128 ??DRB1_1302 ??DRB1_1305 ??DRB1_1307 ??DRB1_1311 ??DRB1_1321 ??DRB1_1322 ??DRB1_1323 ??DRB5_0101 ??DRB5_0105	??DRB1_0101 ??DRB1_0305 ??DRB1_0309 ??DRB1_1101 ??DRB1_1307 ??DRB1_1321 ??DRB5_0101 ??DRB1_0105	??DRB1_0402 ??DRB1_0421 ??DRB1_0701 ??DRB1_0703 ??DRB1_0801 ??DRB1_0802 ??DRB1_0804 ??DRB1_0806 ??DRB1_0813 ??DRB1_0817 ??DRB1_1101 ??DRB1_1114 ??DRB1_1120 ??DRB1_1128 ??DRB1_1302 ??DRB1_1305 ??DRB1_1323 ??DRB1_1502					??DRB1 *1502
Sum					????29	????8	????18	????1

In order to weaken the binding affinity of peptide and MHC II molecule, the inventor is with the Serine on the aminoacid sequence P9 position of Threonine replacement SEQ.ID.No:44 representative, with the Threonine on the aminoacid sequence P6 position of aspartic acid replacement SEQ.ID.No:45 representative.As a result, for SEQ.ID.No:44, the number of MHC II quasi-molecule that can binding peptide is reduced to 11 (55%).For SEQ.ID.No:45, in 51 MHC II quasi-molecules, only there is 1 molecule can binding peptide.

Therefore, in order to produce the humanization heavy chain that weakens at MHC II quasi-molecule binding affinity, made up S44T/T68D mutant with S44T and T68D sudden change, wherein Threonine replace Serine on the 44th (with the primers of SEQ.ID.No:46 and 47 representatives to) and aspartic acid replace Threonine on the 68th (with SEQ.ID.No:48 and 49 primers of representing to).

Behind two ends with the variable region of Restriction Enzyme EcoRI and ApaI digestion said mutation body S44T/T68D, the inventor is the EcoRI-ApaI restriction enzyme site of above-mentioned fragment cloning to the pcDdA-HC expression vector, and made up recombinant expression vector.This expression vector has been preserved in Korea S typical case's culture collecting center of Korea S's life science and biotechnology research institute (KRIBB) on September 26th, 200, preserving number is: KCTC 10080BP.

Humanization heavy chain expression carrier pcDdA-HzSIVh and humanization light chain expression vector pCMV-dhfr-HzSIIIk transfection behind animal cell line, are therefrom expressed humanized antibody HzS-IV with above-mentioned transformant incubation 48 hours.The antigen binding affinity of the humanized antibody HzS-IV that exists in the supernatant liquor that mensuration obtains is found humanized antibody HzS-IV demonstration of the present invention and the humanized antibody HzS-III binding affinity of degree (referring to Figure 12) much at one. Embodiment 12: the aminoacid sequence of humanized antibody HzS-III and HzS-IV relatively

The aminoacid sequence of heavy chain/variable region of light chain of humanized antibody HzS-III and HzS-IV and the aminoacid sequence of wild-type mice monoclonal antibody H67 and humanized antibody HZII-H67 are made comparisons.

Result such as Figure 13 and shown in Figure 14, humanized antibody HzS-III of the present invention and HzS-IV expection may produce lower HAMA and reply, because the aminoacid sequence of heavy chain/variable region of light chain of humanized antibody HzS-III and HzS-IV is at the HCDR1 of heavy chain, HCDR2, the LCDR1 of HCDR3 or light chain, LCDR2, the last corresponding site than mouse monoclonal antibody H67 and humanized antibody HZII-H67 of LCDR3 contains less mouse source amino-acid residue, and HzS-IV than HzS-IH contain less can be in conjunction with the aminoacid sequence of MHC II quasi-molecule.Therefore, humanized antibody HzS-III of the present invention and HzS-IV can become the outstanding candidate of prevention hepatitis B virus, because they induce lower HAMA to reply than the humanized antibody of prior art.

                       Industrial applicibility

As mentioned above, by using heavy chain HCDR1, HCDR2, HCDR3 and light chain LCDR1, L CDR2, humanization amino acid residue on LCDR3 replace mouse source amino acid residue and the humanized antibody that makes hepatitis B vaccine surface antigen S of the present invention than the humanized antibody of prior art humanization more. And, by reducing in the FR district immunogenicity possibility that may significantly be reduced in conjunction with the amino acid sequence of MHC II quasi-molecule in human body, and the antigen binding affinity of exhibits excellent. Therefore, humanized antibody of the present invention can be used in the prevention hepatitis B virus infection.

sequence table, sequence table,＜110 〉, Korea Institute of Bioengineering institute,＜120 〉, humanized antibody of surface antigen S of hepatitis B virus and preparation method thereof,＜130 〉, lfpo-09-05,＜150 〉, KR10-2000-0057891,＜151 〉, 2000-10-02,＜150 〉, KR10-2001-0060966,＜151 〉, 2001-09-29,＜160 〉, 49,＜170 〉, KopatentIn, 1.55,＜210 〉, 1,＜211 〉, 5,＜212 〉, PRT,＜213 〉, mouse,＜400 〉, 1, Asp, Tyr, Asn, Ile, Gln, 1, 5,＜210 〉, 2,＜211 〉, 5,＜212 〉, PRT,＜213 〉, the people,＜400 〉, 2, Asp, Tyr, Asn, Val, Asp, 1, 5,＜210 〉, 3,＜211 〉, 26,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, HL,＜400 〉, 3, gagaattcac, attcacgatg, tacttg,＜210 〉, 4,＜211 〉, 23,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P1,＜400 〉, 4, cccactgaac, gttgtagtca, gtg, 23,＜210 〉, 5,＜211 〉, 25,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P2,＜400 〉, 5, ctacaacgtt, cagtgggtgc, gcaag, 25,＜210 〉, 6,＜211 〉, 20,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, HC,＜400 〉, 6, gatgggccct, tggtggaggc, 20,＜210 〉, 7,＜211 〉, 27,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P3,＜400 〉, 7, cgcacccagt, taatgttgta, gtcagtg, 27,＜210 〉, 8,＜211 〉, 27,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P4,＜400 〉, 8, caacattaac, tgggtgcgcc, aggcccc, 27,＜210 〉, 9,＜211 〉, 30,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P5,＜400 〉, 9, gtaaggatac, atatatccca, tccactcaag, 30,＜210 〉, 10,＜211 〉, 27,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P6,＜400 〉, 10, gggatatatg, tatccttaca, ctggtgg, 27,＜210 〉, 11,＜211 〉, 30,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P7,＜400 〉, 11, cagtaccacc, actgtaagga, taaatatatc, 30,＜210 〉, 12,＜211 〉, 26,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P8,＜400 〉, 12, ccttacagtg, gtggtactgg, ctacag, 26,＜210 〉, 13,＜211 〉, 24,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P9,＜400 〉, 13, gtgactctgc, cctggaactt, ctgg, 24,＜210 〉, 14,＜211 〉, 23,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P10,＜400 〉, 14, ccagggcaga, gtcacattga, ctg, 23,＜210 〉, 15,＜211 〉, 30,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P11,＜400 〉, 15, gtaaccatag, gctcttgcac, agtaatagac, 30,＜210 〉, 16,＜211 〉, 28,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P12,＜400 〉, 16, gtgcaagagc, ctatggttac, gacgagtc, 28,＜210 〉, 17,＜211 〉, 28,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P13,＜400 〉, 17, gtaaccggcg, tttcttgcac, agtaatag, 28,＜210 〉, 18,＜211 〉, 27,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P14,＜400 〉, 18, caagaaacgc, cggttacgac, gagtctg, 27,＜210 〉, 19,＜211 〉, 25,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P15,＜400 〉, 19, gtcgtaggca, tagtttcttg, cacag, 25,＜210 〉, 20,＜211 〉, 29,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P16,＜400 〉, 20, gaaactatgc, ctacgacgag, tctgcctac, 29,＜210 〉, 21,＜211 〉, 26,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P17,＜400 〉, 21, ctcgtcggca, ccatagtttc, ttgcac, 26,＜210 〉, 22,＜211 〉, 24,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P18,＜400 〉, 22, ctatggtgcc, gagtctgctt, actg, 24,＜210 〉, 23,＜211 〉, 27,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P19,＜400 〉, 23, cagactcggc, gtaaccatag, tttcttg, 27,＜210 〉, 24,＜211 〉, 26,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P20,＜400 〉, 24, ggttacggcg, agtctgctta, ctgggg, 26,＜210 〉, 25,＜211 〉, 24,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P21,＜400 〉, 25, cagaggcgtc, gtaaccatag, tttc, 24,＜210 〉, 26,＜211 〉, 27,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P22,＜400 〉, 26, ggttacgacg, cctctgctta, ctggggc, 27,＜210 〉, 27,＜211 〉, 25,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P23,＜400 〉, 27, gtaagcggcc, tcgtcgtaac, catgg, 25,＜210 〉, 28,＜211 〉, 26,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P24,＜400 〉, 28, gacgaggccg, cttactgggg, ccaagg, 26,＜210 〉, 29,＜211 〉, 25,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P25,＜400 〉, 29, gtaagcagac, gagtcgtaac, catag, 25,＜210 〉, 30,＜211 〉, 26,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P26,＜400 〉, 30, cgactcgtct, gcttactggg, gccaag, 26,＜210 〉, 31,＜211 〉, 24,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P27,＜400 〉, 31, ccagtaacca, gactcgtcgt, aacc, 24,＜210 〉, 32,＜211 〉, 25,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P28,＜400 〉, 32, cgagtctggt, tactggggcc, aaggg, 25,＜210 〉, 33,＜211 〉, 26,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, LL,＜400 〉, 33, caaagcttgg, aagcaagatg, gaatca, 26,＜210 〉, 34,＜211 〉, 30,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P29,＜400 〉, 34, ccttagtttg, cagacagaaa, taatttgcag, 30,＜210 〉, 35,＜211 〉, 25,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P30,＜400 〉, 35, ctgtctgcaa, actaaggcgg, ttccg, 25,＜210 〉, 36,＜211 〉, 27,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, LC,＜400 〉, 36, gaagtcgacc, taacactctc, ccctgtt, 27,＜210 〉, 37,＜211 〉, 30,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P31,＜400 〉, 37, cctccttact, ttgctgacag, aaataatttg, 30,＜210 〉, 38,＜211 〉, 28,＜212 〉, DNA,＜213 〉, artificial sequence,＜220 〉,＜223 〉, P32,＜400 〉, 38, gtcagcaaag, taaggaggtt, ccgtacac, 28,＜210 〉, 39,＜211 〉, 17,＜212 〉, PRT,＜213 〉, mouse,＜400 〉, 39, Tyr, Ile, Tyr, Pro, Tyr, Thr, Gly, Gly, Thr, Gly, Tyr, Ser, Gln, Lys, Phe, Lys, 1, 5, 10, 15, Ser,＜210 〉, 40,＜211 〉, 17,＜212 〉, PRT,＜213 〉, the people,＜400 〉, 40, Trp, Met, Asn, Pro, Asn, Ser, Gly, Asn, Thr, Gly, Tyr, Ala, Gln, Lys, Phe, Gln, 1, 5, 10, 15, Gly,＜210 〉, 41,＜211 〉, 8,＜212 〉, PRT,＜213 〉, mouse,＜400 〉, 41, Asn, Tyr, Gly, Tyr, Asp, Glu, Ser, Ala, 1, 5,＜210 〉, 42,＜211 〉, 112,＜212 〉, PRT,＜213 〉, people HzS-III-VL,＜400 〉, 42, Asp, Ile, Val, Leu, Thr, Gln, Ser, Pro, Ala, Ser, Leu, Ala, Val, Ser, Pro, Gly, 1, 5, 10, 15, Gln, Arg, Ala, Thr, Ile, Thr, Cys, Arg, Ala, Ser, Glu, Ser, Val, Ser, Asn, Tyr

20??????????????????????25??????????????????????30Gly?Ile?Asn?Phe?Ile?Asn?Trp?Phe?Gln?Gln?Lys?Pro?Gly?Gln?Pro?Pro

35?????????????????????40??????????????????????45Lys?Leu?Leu?Ile?Tyr?Thr?Ala?Ser?Asn?Lys?Gly?Thr?Gly?Val?Pro?Ala

50??????????????????????55?????????????????????60Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Asn?65??????????????????????70?????????????????????75??????????????????????80Pro?Val?Glu?Ala?Glu?Asp?Thr?Ala?Asn?Tyr?Phe?Cys?Gln?Gln?Ser?Lys

85??????????????????????90??????????????????????95Glu?Val?Pro?Tyr?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg

100 105 110＜210〉43＜211〉118＜212〉PRT＜213〉people HzS-III-VH＜400〉43Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 15 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20?????????????????????25??????????????????????30Asn?Val?Gln?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Ser?Leu?Glu?Trp?Met

35?????????????????????40??????????????????????45Gly?Tyr?Ile?Tyr?Pro?Tyr?Ser?Gly?Gly?Thr?Gly?Tyr?Ser?Gln?Lys?Phe

50?????????????????????55??????????????????????60Gln?Gly?Arg?Val?Thr?Leu?Thr?Val?Asp?Asn?Ser?Ala?Ser?Thr?Ala?Tyr?65??????????????????????70?????????????????????75??????????????????????80Met?Glu?Leu?Ser?Ser?Leu?Arg?Ser?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys

85??????????????????????90??????????????????????95Ala?Arg?Asn?Tyr?Gly?Tyr?Asp?Ser?Ser?Ala?Tyr?Trp?Gly?Gln?Gly?Thr

100?????????????????????105????????????????????110Leu?Val?Thr?Val?Ser?Ser

115＜210〉44＜211〉9＜212〉PRT＜213〉artificial sequence＜220〉＜223〉be attached to the peptide sequence of mhc class ii molecule＜400〉44Trp Val Arg Gln Ala Pro Gly Lys Ser 15＜210〉45＜211〉9＜212〉PRT＜213〉artificial sequence＜220〉＜223 be attached to the peptide sequence of mhc class ii molecule＜400〉45Phe Gln Gly Arg Val Thr Leu Thr Val

(A) Bio: Plasmodium falciparum (XI) SEQUENCE DESCRIPTION: SEQ ID NO: 11: AAAAATGGGG CCCAAGGAGG CTGCAGGTGG GGATGATATT GAGGATGAAA GTGCCAAACA 60 TATGTTTGAT AGGATAGGAA AAGATGTGTA CGATAAAGTA AAAGAGGAAG CTAAAGAACG 120 TGGTAAAGGC TTGCAAGGAC GTTTGTCAGA AGCAAAATTT GAGAAAAATG AAAGCGATCC 180 ACAAACACCA GAAGATCCAT GCGATCTTGA TCATAAATAT CATACAAATG TAACTACTAA 240 TGTAATTAAT CCGTGCGCTG ATAGATCTGA CGTGCGTTTT TCCGATGAAT ATGGAGGTCA 300 ATGTACACAT AATAGAATAA AAGATAGTCA ACAGGGTGAT AATAAAGGTG CATGTGCTCC 360 ATATAGGCGA TTGCATGTAT GCGATCAAAA TTTAGAACAG ATAGAGCCTA TAAAAATAAC 420 AAATACTCAT AATTTATTGG TAGATGTGTG TATGGCAGCA AAATTTGAAG GACAATCAAT 480 AACACAAGAT TATCCAAAAT ATCAAGCAAC ATATGGTGAT TCTCCTTCTC AAATATGTAC 540 TATGCTGGCA CGAAGTTTTG CGGACATAGG GGACATTGTC AGAGGAAGAG ATTTGTATTT 600 AGGTAATCCA CAAGAAATAA AACAAAGACA ACAATTAGAA AATAATTTGA AAACAATTTT 660 CGGGAAAATA TATGAAAAAT TGAATGGCGC AGAAGCACGC TACGGAAATG ATCCGGAATT 720 TTTTAAATTA CGAGAAGATT GGTGGACTGC TAATCGAGAA ACAGTATGGA AAGCCATCAC 780 ATGTAACGCT TGGGGTAATA CATATTTTCA TGCAACGTGC AATAGAGGAG AACGAACTAA 840 AGGTTACTGC CGGTGTAACG ACGACCAAGT TCCCACATAT TTTGATTATG TGCCGCAGTA 900 TCTTCGCTGG TTCGAGGAAT GGGCAGAAGA TTTTTGTAGG AAAAAAAATA AAAAAATAAA 960 AGATGTTAAA AGAAATTGTC GTGGAAAAGA TAAAGAGGAT AAGGATCGAT ATTGTAGCCG 1020 TAATGGCTAC GATTGCGAAA AAACTAAACG AGCGATTGGT AAGTTGCGTT ATGGTAAGCA 1080 ATGCATTAGC TGTTTGTATG CATGTAATCC TTACGTTGAT TGGATAAATA ACCAAAAAGA 1140 ACAATTTGAC AAACAGAAAA AAAAATATGA TGAAGAAATA AAAAAATATG AAAATGGAGC 1200 ATCAGGTGGT AGTAGGCAAA AACGGGATGC AGGTGGTACA ACTACTACTA ATTATGATGG 1260 ATATGAAAAA AAATTTTATG ACGAACTTAA TAAAAGTGAA TATAGAACCG TTGATAAATT 1320 TTTGGAAAAA TTAAGTAATG AAGAAATATG CACAAAAGTT AAAGACGAAG AAGGAGGAAC 1380 AATTGATTTT AAAAACGTTA ATAGTGATAG TACTAGTGGT GCTAGTGGCA CTAATGTTGA 1440 AAGTCAAGGA ACATTTTATC GTTCAAAATA TTGCCAACCC TGCCCTTATT GTGGAGTGAA 1500 AAAGGTAAAT AATGGTGGTA GTAGTAATGA ATGGGAAGAG AAAAATAATG GCAAGTGCAA 1560 GAGTGGAAAA CTTTATGAGC CTAAACCCGA CAAAGAAGGT ACTACTATTA CAATCCTTAA 1620 AAGTGGTAAA GGACATGATG ATATTGAAGA AAAATTAAAC AAATTTTGTG ATGAAAAAAA 1680 TGGTGATACA ATAAATAGTG GTGGTAGTGG TACGGGTGGT AGTGGTGGTG GTAACAGTGG 1740 TAGACAGGAA TTGTATGAAG AATGGAAATG TTATAAAGGT GAAGATGTAG TGAAAGTTGG 1800 ACACGATGAG GATGACGAGG AGGATTATGA AAATGTAAAA AATGCAGGCG GATTATGTAT 1860 ATTAAAAAAC CAAAAAAAGA ATAAAGAAGA AGGTGGAAAT ACGTCTGAAA AGGAGCCTGA 1920 TGAAATCCAA AAGACATTCA ATCCTTTTTT TTACTATTGG GTTGCACATA TGTTAAAAGA 1980 TTCCATACAT TGGAAAAAAA AACTTCAGAG ATGTTTACAA AATGGTAACA GAATAAAATG 2040 TGGAAACAAT AAATGTAATA ATGATTGTGA ATGTTTTAAA AGATGGATTA CACAAAAAAA 2100 AGACGAATGG GGGAAAATAG TACAACATTT TAAAACGCAA AATATTAAAG GTAGAGGAGG 2160 TAGTGACAAT ACGGCAGAAT TAATCCCATT TGATCACGAT TATGTTCTTC AATACAATTT 2220 GCAAGAAGAA TTTTTGAAAG GCGATTCCGA AGACGCTTCC GAAGAAAAAT CCGAAAATAG 2280 TCTGGATGCA GAGGAGGCAG AGGAACTAAA ACACCTTCGC GAAATCATTG AAAGTGAAGA 2340 CAATAATCAA GAAGCATCTG TTGGTGGTGG CGTCACTGAA CAAAAAAATA TAATGGATAA 2400 ATTGCTCAAC TACGAAAAAG ACGAAGCCGA TTTATGCCTA GAAATTCACG AAGATGAGGA 2460 AGAGGAAAAA GAAAAAGGAG ACGGAAACGA ATGTATCGAA GAGGGCGAAA ATTTTCGTTA 2520 TAATCCATGT AGTGGCGAAA GTGGTAACAA ACGATACCCC GTTCTTGCGA ACAAAGTAGC 2580 GTATCAAATG CATCACAAGG CAAAGACACA ATTGGCTAGT CGTGCTGGTA GAAGTGCGTT 2640 GAGAGGTGAT ATATCCTTAG CGCAATTTAA AAATGGTCGT AACGGAAGTA CATTGAAAGG 2700 ACAAATTTGC AAAATTAACG AAAACTATTC CAATGATAGT CGTGGTAATA GTGGTGGACC 2760 ATGTACAGGC AAAGATGGAG ATCACGGAGG TGTGCGCATG AGAATAGGAA CGGAATGGTC 2820 AAATATTGAA GGAAAAAAAC AAACGTCATA CAAAAACGTC TTTTTACCTC CCCGACGAGA 2880 ACACATGTGT ACATCCAATT TAGAAAATTT AGATGTTGGT AGTGTCACTA AAAATGATAA 2940 GGCTAGCCAC TCATTATTGG GAGATGTTCA GCTCGCAGCA AAAACTGATG CAGCTGAGAT 3000 AATAAAACGC TATAAAGATC AAAATAATAT ACAACTAACT GATCCAATAC AACAAAAAGA 3060 CCAGGAGGCT ATGTGTCGAG CTGTACGTTA TAGTTTTGCC GATTTAGGAG ACATTATTCG 3120 AGGAAGAGAT ATGTGGGATG AGGATAAGAG CTCAACAGAC ATGGAAACAC GTTTGATAAC 3180 CGTATTTAAA AACATTAAAG AAAAACATGA TGGAATCAAA GACAACCCTA AATATACCGG 3240 TGATGAAAGC AAAAAGCCCG CATATAAAAA ATTACGAGCA GATTGGTGGG AAGCAAATAG 3300 ACATCAAGTG TGGAGAGCCA TGAAATGCGC AACAAAAGGC ATCATATGTC CTGGTATGCC 3360 AGTTGACGAT TATATCCCCC AACGTTTACG CTGGATGACT GAATGGGCTG AATGGTATTG 3420 TAAAGCGCAA TCACAGGAGT ATGACAAGTT AAAAAAAATC TGTGCAGATT GTATGAGTAA 3480 GGGTGATGGA AAATGTACGC AAGGTGATGT CGATTGTGGA AAGTGCAAAG CAGCATGTGA 3540 TAAATATAAA GAGGAAATAG AAAAATGGAA TGAACAATGG AGAAAAATAT CAGATAAATA 3600 CAATCTATTA TACCTACAAG CAAAAACTAC TTCTACTAAT CCTGGCCGTA CTGTTCTTGG 3660 TGATGACGAT CCCGACTATC AACAAATGGT AGATTTTTTG ACCCCAATAC ACAAAGCAAG 3720 TATTGCCGCA CGTGTTCTTG TTAAACGTGC TGCTGGTAGT CCCACTGAGA TCGCCGCCGC 3780 CGCCCCGATC ACCCCCTACA GTACTGCTGC CGGATATATA CACCAGGAAA TAGGATATGG 3840 GGGGTGCCAG GAACAAACAC AATTTTGTGA AAAAAAACAT GGTGCAACAT CAACTAGTAC 3900 CACGAAAGAA AACAAAGAAT ACACCTTTAA ACAACCTCCG CCGGAGTATG CTACAGCGTG 3960 TGATTGCATA AATAGGTCGC AAACAGAGGA GCCGAAGAAA AAGGAAGAAA ATGTAGAGAG 4020 TGCCTGCAAA ATAGTGGAGA AAATACTTGA GGGTAAGAAT GGAAGGACTA CAGTAGGTGA 4080 ATGTAATCCA AAAGAGAGTT ATCCTGATTG GGATTGCAAA AACAATATTG ACATTAGTCA 4140 TGATGGTGCT TGTATGCCTC CAAGGAGACA AAAACTATGT TTATATTATA TAGCACATGA 4200 GAGTCAAACA GAAAATATAA AAACAGACGA TAATTTGAAA GATGCTTTTA TTAAAACTGC 4260 AGCAGCAGAA ACTTTTCTTT CATGGCAATA TTATAAGAGT AAGAATGATA GTGAAGCTAA 4320 AATATTAGAT AGAGGCCTTA TTCCATCCCA ATTTTTAAGA TCCATGATGT ACACGTTTGG 4380 AGATTATAGA GATATATGTT TGAACACAGA TATATCTAAA AAACAAAATG ATGTAGCTAA 4440 GGCAAAAGAT AAAATAGGTA AATTTTTCTC AAAAGATGGC AGCAAATCTC CTAGTGGCTT 4500 ATCACGCCAA GAATGGTGGA AAACAAATGG TCCAGAGATT TGGAAAGGAA TGTTATGTGC 4560 CTTAACAAAA TACGTCACAG ATACCGATAA CAAAAGAAAA ATCAAAAACG ACTACTCATA 4620 CGATAAAGTC AACCAATCCC AAAATGGCAA CCCTTCCCTT GAAGAGTTTG CTGCTAAACC 4680 TCAATTTCTA CGTTGGATGA TCGAATGGGG AGAAGAGTTT TGTGCTGAAC GTCAGAAGAA 4740 GGAAAATATC ATAAAAGATG CATGTAATGA AATAAATTCT ACACAACAGT GTAATGATGC 4800 GAAACATCGT TGTAATCAAG CATGTAGAGC ATATCAAGAA TATGTTGAAA ATAAAAAAAA 4860 AGAATTTTCG GGACAAACAA ATAACTTTGT TCTAAAGGCA AATGTTCAGC CCCAAGATCC 4920 AGAATATAAA GGATATGAAT ATAAAGACGG CGTACAACCG ATACAGGGGA ATGAGTATTT 4980 ACTGCAAAAA TGTGATAATA ATAAATGTTC TTGCATGGAT GGAAATGTAC TTTCCGTCTC 5040 TCCAAAAGAA AAACCTTTTG GAAAATATGC CCATAAATAT CCTGAGAAAT GTGATTGTTA 5100 TCAAGGAAAA CATGTACCTA GCATACCACC TCCCCCCCCA CCTGTACAAC CACAACCGGA 5160 AGCACCAACA GTAACAGTAG ACGTTTGCAG CATAGTAAAA ACACTATTTA AAGACACAAA 5220 CAATTTTTCC GACGCTTGTG GTCTAAAATA CGGCAAAACC GCACCATCCA GTTGGAAATG 5280 TATACCAAGT GACACAAAAA GTGGTGCTGG TGCCACCACC GGCAAAAGTG GTAGTGATAG 5340 TGGTAGTATT TGTATCCCAC CCAGGAGGCG ACGATTATAT GTGGGGAAAC TACAGGAGTG 5400 GGCTACCGCG CTCCCACAAG GTGAGGGCGC CGCGCCGTCC CACTCACGCG CCGACGACTT 5460 GCGCAATGCG TTCATCCAAT CTGCTGCAAT AGAGACTTTT TTCTTATGGG ATAGATATAA 5520 AGAAGAGAAA AAACCACAGG GTGATGGGTC ACAACAAGCA CTATCACAAC TAACCAGTAC 5580 ATACAGTGAT GACGAGGAGG ACCCCCCCGA CAAACTGTTA CAAAATGGTA AGATACCCCC 5640 CGATTTTTTG AGATTAATGT TCTATACATT AGGAGATTAT AGGGATATTT TAGTACACGG 5700 TGGTAACACA AGTGACAGTG GTAACACAAA TGGTAGTAAC AACAACAATA TTGTGCTTGA 5760 AGCGAGTGGT AACAAGGAGG ACATGCAAAA AATACAAGAG AAAATAGAAC AAATTCTCCC 5820 AAAAAATGGT GGCACACCTC TTGTCCCAAA ATCTAGTGCC CAAACACCTG ATAAATGGTG 5880 GAATGAACAC GCCGAATCTA TCTGGAAAGG TATGATATGT GCATTGACAT ATACAGAAAA 5940 GAACCCTGAC ACCAGTGCAA GAGGCGACGA AAACAAAATA GAAAAGGATG ATGAAGTGTA 6000 CGAGAAATTT TTTGGCAGCA CAGCCGACAA ACATGGCACA GCCTCAACCC CAACCGGCAC 6060 ATACAAAACC CAATACGACT ACGAAAAAGT CAAACTTGAG GATACAAGTG GTGCCAAAAC 6120 CCCCTCAGCC TCTAGTGATA CACCCCTTCT CTCCGATTTC GTGTTACGCC CCCCCTACTT 6180 CCGTTACCTT GAAGAATGGG GTCAAAATTT TTGTAAAAAA AGAAAGCATA AATTGGCACA 6240 AATAAAACAT GAGTGTAAAG TAGAAGAAAA TGGTGGTGGT AGTCGTCGTG GTGGTATAAC 6300 AAGACAATAT AGTGGGGATG GCGAAGCGTG TAATGAGATG CTTCCAAAAA ACGATGGAAC 6360 TGTTCCGGAT TTAGAAAAGC CGAGTTGTGC CAAACCTTGT AGTTCTTATA GAAAATGGAT 6420 AGAAAGCAAG GGAAAAGAGT TTGAGAAACA AGAAAAGGCA TATGAACAAC AAAAAGACAA 6480 ATGTGTAAAT GGAAGTAATA AGCATGATAA TGGATTTTGT GAAACACTAA CAACGTCCTC 6540 TAAAGCTAAA GACTTTTTAA AAACGTTAGG ACCATGTAAA CCTAATAATG TAGAGGGTAA 6600 AACAATTTTT GATGATGATA AAACCTTTAA ACATACAAAA GATTGTGATC CATGTCTTAA 6660 ATTTAGTGTT AATTGTAAAA AAGATGAATG TGATAATTCT AAAGGAACCG ATTGCCGAAA 6720 TAAAAATAGT ATTGATGCAA CAGATATTGA AAATGGAGTG GATTCTACTG TACTAGAAAT 6780 GCGTGTCAGT GCTGATAGTA AAAGTGGATT TAATGGTGAT GGTTTAGAGA ATGCTTGTAG 6840 AGGTGCTGGT ATCTTTGAAG GTATTAGAAA AGATGAATGG AAATGTCGTA ATGTATGTGG 6900 TTATGTTGTA TGTAAACCGG AAAACGTTAA TGGGGAAGCA AAGGGAAAAC ACATTATACA 6960 AATTAGAGCA CTGGTTAAAC GTTGGGTAGA ATATTTTTTT GAAGATTATA ATAAAATAAA 7020 ACATAAAATT TCACATCGCA TAAAAAATGG TGAAATATCT CCATGTATAA AAAATTGTGT 7080 AGAAAAATGG GTAGATCAGA AAAGAAAAGA ATGGAAGGAA ATTACTGAAC GTTTCAAAGA 7140 TCAATATAAA AATGACAATT CAGATGATGA CAATGTGAGA AGTTTTTTGG AGACCTTGAT 7200 ACCTCAAATT ACTGATGCAA ACGCTAAAAA TAAGGTTATA AAATTAAGTA AGTTCGGTAA 7260 TTCTTGTGGA TGTAGTGCCA GTGCGAACGA ACAAAACAAA AATGGTGAAT ACAAGGACGC 7320 TATAGATTGT ATGCTTAAAA AGCTTAAAGA TAAAATTGGC GAGTGCGAAA AGAAACACCA 7380 TCAAACTAGT GATACCGAGT GTTCCGACAC ACCACAACCG CAAACCCTTG AAGACGAAAC 7440 TTTGGATGAT GATATAGAAA CAGAGGAGGC GAAGAAGAAC ATGATGCCGA AAATTTGTGA 7500 AAATGTGTTA AAAACAGCAC AACAAGAGGA TGAAGGCGGT TGTGTCCCAG CAGAAAATAG 7560 TGAAGAACCG GCAGCAACAG ATAGTGGTAA GGAAACCCCC GAACAAACCC CCGTTCTCAA 7620 ACCCGAAGAA GAAGCAGTAC CGGAACCACC ACCTCCACCC CCACAGGAAA AAGCCCCGGC 7680 ACCAATACCC CAACCACAAC CACCAACCCC CCCCACACAA CTCTTGGATA ATCCCCACGT 7740 TCTAACCGCC CTGGTGACCT CCACCCTCGC CTGGAGCGTT GGCATCGGTT TTGCTACATT 7800 CACTTATTTT TATCTAAAGG TAAATGGAAG TATATATATG GGGATGTGGA TGTATGTGGA 7860 TGTATGTGAA TGTATGTGGA TGTATGTGGA TGTATGTGGA TGTGTTTTAT GGATATGTAT 7920 TTGTGATTAT GTTTGGATAT ATATATATAT ATATATATGT TTATGTATAT GTGTTTTTGG 7980 ATATATATAT GTGTATGTAT ATGATTTTCT GTATATGTAT TTGTGGGTTA AGGATATATA 8040 TATATGGATG TACTTGTATG TGTTTTATAT ATATATTTTA TATATATGTA TTTATATTAA 8100 AAAAGAAATA TAAAAACAAA TTTATTAAAA TGAAAAAAAG AAAAATGAAA TATAAAAAAA 8160 AATTTATTAA AATAAAAAAA AAAAAAAAAA AAAAGGAGAA AAATTTTTTA AAAAATAATA 8220 (2) SEQ ID NO: 12 items: (I) SEQUENCE CHARACTERISTICS: ...

Claims (25)

1. specificity is in conjunction with the humanization heavy chain mutant of hepatitis B virus surface antigen S, and wherein all or part amino-acid residue among the people source heavy chain HCDRs has replaced and is selected from mouse source heavy chain HCDR1, all or part amino-acid residue of HCDR2 and HCDR3.

2. according to the humanization heavy chain mutant of claim 1, wherein Xie Ansuan has replaced the Isoleucine on HCDR1 the 34th amino acids residue.

3. according to the humanization heavy chain mutant of claim 1, wherein Serine has replaced the Threonine on HCDR2 the 54th amino acids.

4. according to the humanization heavy chain mutant of claim 1, wherein glycine has replaced the Serine on HCDR2 the 65th amino acids.

5. according to the humanization heavy chain mutant of claim 1, wherein Serine has replaced the L-glutamic acid on HCDR3 the 100th amino acids.

6. according to the humanization heavy chain mutant of claim 1, wherein it contains all sudden changes of claim 2 to 5.

7. the expression vector pcDdA-HzSIIIh (preserving number: KCTC 10083BP) that comprises the humanization heavy chain mutant of claim 6.

8. specificity is in conjunction with the humanization heavy chain mutant of hepatitis B virus surface antigen S, wherein all or part amino-acid residue among the people source heavy chain HCDRs has replaced and has been selected from mouse source heavy chain HCDR1, the all or part amino-acid residue of HCDR2 and HCDR3, and removed peptide sequence in conjunction with MHC II quasi-molecule.

9. according to the humanization heavy chain mutant of claim 8, wherein represent by SEQ.ID.No:44 or SEQ.ID.No:45 in conjunction with the peptide sequence of MHC II quasi-molecule.

10. according to the humanization heavy chain mutant of claim 8, wherein Threonine has replaced the Serine on the sequence P9 position of SEQ.ID.No:44 representative.

11. according to the humanization heavy chain mutant of claim 8, wherein aspartic acid has replaced the Threonine on the sequence P6 position of SEQ.ID.No:45 representative.

12. according to the humanization heavy chain mutant of claim 8, wherein it contains all sudden changes of claim 10 to 11.

13. contain the expression vector pcDdA-HzSIVh (preserving number: KCTC 10080BP) of the humanization heavy chain mutant of claim 12.

14. specificity is in conjunction with the humanization light chain mutant of hepatitis B virus surface antigen S, wherein all or part of amino-acid residue among people's endogenous light chain LCDRs has replaced and has been selected from mouse endogenous light chain LCDR1, all or part of amino-acid residue of LCDR2 and LCDR3.

15. according to the humanization light chain mutant of claim 14, wherein Serine has replaced the aspartic acid on the 27th amino acids of LCDR1.

16. according to the humanization light chain mutant of claim 14, wherein arginine has replaced the Serine on the 31st amino acids of LCDR1.

17. according to the humanization light chain mutant of claim 14, wherein Isoleucine has replaced the methionine(Met) on the 33rd amino acids of LCDR1.

18. according to the humanization light chain mutant of claim 14, wherein Methionin has replaced the glutamine on the 54th amino acids of LCDR2.

19. according to the humanization light chain mutant of claim 14, wherein Threonine has replaced the Serine on the 56th amino acids of LCDR2.

20. according to the humanization light chain mutant of claim 14, wherein Serine has replaced the Threonine on the 91st amino acids of LCDR3.

21. according to the humanization light chain mutant of claim 14, wherein it contains all sudden changes of claim 15 to 20.

22. contain the expression vector pCMV-dhfr-HzSIIIk (preserving number: KCTC 10084BP) of the humanization light chain mutant of claim 21.

23. use the transformant of the humanization light chain expression vector transfection of the humanization heavy chain expression carrier of claim 7 or claim 13 and claim 22 simultaneously.

24. the transformant of Accessory Right requirement 23 is expressed the humanized antibody of the surface antigen S of hepatitis B virus that obtains.

25. prepare the method for humanized antibody, it is the CDR sequence between comparison mouse and the people's antibody, select the highest people CDR sequence of homology, replace amino-acid residue among the mouse antibodies CDR with amino-acid residue among the personnel selection antibody CDR, wherein the amino-acid residue among the mouse CDR does not influence the antigen combination.

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