KR100484054B1 - A humanized antibody to surface antigen S of hepatitis B virus and a preparing method thereof - Google Patents

Abstract

The present invention relates to a humanized antibody specific for surface antigen S of hepatitis B virus (HBV) and a method for preparing the same, and specifically to a CDR-grafting method. Amino acid residues of each of the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 moieties in the heavy and light chain variable regions of the humanized antibody against HBV surface antigen S. Humanized antibody against HBV surface antigen S consisting of a humanized heavy chain and a humanized light chain substituted with a humanized antibody, and a humanized antibody further minimizing a human anti-mouse antibody (HAMA) reaction by removing a peptide sequence that binds MHC class II from the humanized heavy chain Transformed by an expression vector comprising each of the heavy or light chain genes of the humanized antibody, the heavy chain expression vector and the light chain expression vector; Is a method for producing a humanized antibody that mass production of the transformant, and a humanized antibody by culturing the transformant which can produce the humanized antibody. The humanized antibody of the present invention is more humanized than the existing humanized antibody, so that the immunogenic reactivity in the human body is reduced, while showing excellent antigen binding ability, it can be usefully used for the prevention and treatment of hepatitis B virus infection.

Description

A humanized antibody to surface antigen S of hepatitis B virus and a preparing method

The present invention relates to a humanized antibody specific for surface antigen S of hepatitis B virus (HBV) and a method for preparing the same, and specifically to a CDR-grafting method. Amino acid residues of each of the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 moieties in the heavy and light chain variable regions of the humanized antibody against HBV surface antigen S. Humanized antibody against HBV surface antigen S consisting of a humanized heavy chain and a humanized light chain substituted with a humanized antibody, and a humanized antibody further minimizing a human anti-mouse antibody (HAMA) reaction by removing a peptide sequence that binds MHC class II from the humanized heavy chain Transformed by an expression vector comprising each of the heavy or light chain genes of the humanized antibody, the heavy chain expression vector and the light chain expression vector; Is a method for producing a humanized antibody that mass production of the transformant, and a humanized antibody by culturing the transformant which can produce the humanized antibody.

Hepatitis B virus (abbreviated as "HBV") is a pathogen that invades humans and causes chronic and acute hepatitis, and when it worsens, causes cirrhosis and liver cancer. It is estimated that patients are infected with HBV (Tiollais & Buendia, Sci. Am. , 264, 48, 1991). The coat of HBV consists of three proteins, specifically, a major protein containing S antigen, a middle protein including S antigen and free S2 antigen, and a S antigen, free S2 antigen and free S1 antigen. Consisting of large proteins (Neurath, AR and Kent, SBH, Adv. Vir. Res ., 34: 65-142, 1988). All of the above-described HBV surface antigen proteins induce antibodies that neutralize and neutralize HBV, of which the S antigen accounts for about 80% of all encapsulated proteins and is called "Common a ", which is common to all subtypes of HBV. It has a neutralizing epitope.

If infected with HBV, HB immunoglobulins may be used to prevent beneficiary HBV infection during liver transplantation from, for example, newborns born from HBV-positive mothers, healthcare workers exposed to HBV, or patients with HBV-related liver disease. immunoglobulin, IG) has been used (Beasley et al., Lancet , 2, 1099, 1983; Todo et al., Hepatol. , 13, 619, 1991). However, HBIG, which is currently used, is extracted from human plasma, and thus has a disadvantage of low antigen specificity, exposure to contaminants, and continuous supply of human blood.

To solve this problem, a method of using monoclonal antibodies against HBV surface antigens of mice has been developed. In general, mouse monoclonal antibodies have the advantage of high affinity for the antigen and mass production, but when administered to humans, the human anti-mouse antibody response in the human body is " Has a disadvantage of causing an "HAMA response" (Shawler et al., J. Immunol. , 135, 1530, 1985).

As a method for overcoming this problem, a method for minimizing the HAMA response while maintaining high affinity and specificity of mouse-derived monoclonal antibodies, abbreviated as complementarity determining regions (CDRs) of mouse monoclonal antibodies. "Humanized antibodies" have been developed in which only human antibodies have been transplanted into human antibodies, and these humanized antibodies are genetically engineered for mass cultivation and humanized most of the genes, thereby significantly reducing the immune response in the human body. Has the advantage that it can be achieved (Riechmann et al., Nature , 332, 323, 1988; Nakatani et al., Protein Engineering, 7,435, 1994).

As a conventional method for producing humanized antibodies, CDR-grafting methods are generally used. In the CDR-grafting method, humans having homology with the genes of CDRs, which are sites that bind antigens in mouse monoclonal antibody variable regions, are used. Humanized antibodies are prepared by replacing some amino acid residues in framework regions that affect the conformation of CDRs with those of the original mouse monoclonal antibody from those of the human antibody after transplantation into the genetic region of the antibody. Method (Riechmann et al., Nature , 332, 323, 1988; Queen et al., Proc. Natl. Acad. Sci. , USA 86, 10029, 1989; Nakatani et al., Protein Engineerin g, 7,435, 1994). . However, it has been reported that humanized antibodies prepared as described above also have a problem that CDRs derived from mice present in the antibody may induce HAMA response when administered repeatedly to the human body (Stephens et al., Immunology, 85, 668-674). , 1995; Sharkey et al., Cancer Research , 55, 5935s-5945s, 1995). Therefore, it is expected that the substitution of CDRs amino acid residues derived from mouse antibodies in the humanized antibodies with residues of human antibodies may reduce the HAMA response.

In the variable region of the antibody molecule, there are three heavy chain CDR loops HCDR1, HCDR2 and HCDR3 and three light chain CDR loops LCDR1, LCDR2 and LCDR3, using conventional CDR-transplantation methods to prepare humanized antibodies. To transplant all six CDR loops. However, in view of the fact that only some of the amino acid residues in the CDRs bind directly to the antigen (Padlan et al., FASEB J. , 9, 133, 1995), specificity determining residues directly involved in antigen binding, The SDR-transplantation method of implanting only "SDRs" as human acupuncture) into human antibodies sought to develop humanized antibodies with improved therapeutic effect while inducing less HAMA response than conventional humanized antibodies.

As a result, the present inventors have disclosed a humanized antibody developed by CDR-transplantation method using the genes of mouse monoclonal antibody H67 against surface antigen S of HBV in Korean Patent Registration No. 163163 (September 3, 1998). From this, the humanized antibody HZII-H67 with reduced immunogenicity than the humanized antibody has been developed and applied for a patent (Korean Patent Application No. 98-32644).

On the other hand, Helper T cells play an essential role early in the immune response, and Major Histocompatibility Complex (MHC) class II plays a central role in the selection and activation of T cells. MHC class II binds to a peptide consisting of nine amino acids cleaved from protein antigens, is displayed on the surface of antigen-presenting cells, and forms a complex with T cell receptors (TCRs) that recognize them. When T cells are activated, an immune response begins (Germain, RN Cell 76, 287-299, 1994). Thus, initial removal of peptide sequences that specifically bind to MHC molecules in a protein antigen may prevent the human immune response to the protein.

Accordingly, the inventors of the present invention have described each HCDR1, HCDR2, HCDR3 moiety in the heavy chain variable region of the humanized antibody against HBV surface antigen S prepared by the CDR-transplantation method in order to further humanize the humanized antibody HZII-H67. HBV surface consisting of a humanized heavy chain in which the amino acid residues of the human antibody are substituted with amino acids derived from the heavy chain of the human antibody, and a humanized light chain in which the amino acid residues of the LCDR1, LCDR2, and LCDR3 portions in the light chain variable region of the humanized antibody are substituted with the light chain derived amino acids of the human antibody. Humanized antibodies against antigen S and peptide sequences that bind to MHC class II were removed from the humanized heavy chains to produce humanized antibodies that further minimized the HAMA response. Immunogenic reactivity is reduced while showing good antigen binding capacity, type B By identifying that can be useful in the prevention and treatment of salt infection and completed the present invention.

SUMMARY OF THE INVENTION An object of the present invention is to provide a humanized antibody against HBV surface antigen S having a superior antigen binding ability while minimizing the induction of immune response in the human body, and a method for preparing the same, in order to effectively prevent and treat infection of hepatitis B virus. .

In order to achieve the above object, the present invention provides a humanized antibody comprising heavy and light chain genes in which mouse-derived CDRs amino acid residues in the heavy and light chain variable region genes of humanized antibodies against surface antigen S of HBV are substituted with human antibody-derived amino acid residues. ; An expression vector comprising each of the heavy and light chain genes of the humanized antibody; It provides a transformant transformed with the expression vector.

In addition, the present invention provides a method for producing a humanized antibody that produces a humanized antibody in a large amount by culturing the transformant. As used herein, the term "humanized heavy chain gene" or "humanized light chain gene" refers to a "humanized antibody". Heavy chain gene "or" light chain gene of a humanized antibody. "

Hereinafter, the present invention will be described in detail.

The present invention provides humanized heavy chain gene variants in which the amino acid residues of the HCDR1, HCDR2, and HCDR3 portions of the heavy chain variable region of the humanized antibody against the surface antigen S of HBV are replaced with the heavy chain-derived amino acids of a human antibody, and an expression vector comprising the same. .

The humanized antibody of the present invention is most compared with the amino acid sequences of the HCDR1, HCDR2 and HCDR3 moieties derived from the mouse antibody present in the heavy chain variable region of the humanized antibody HZII-H67 compared with the amino acid sequences of the heavy chain HCDR1, HCDR2 and HCDR3 derived from the human antibody, respectively. It is prepared by selecting similar CDR sequences and substituting amino acid residues from human antibodies which do not affect antigen binding within each CDR.

We compared mouse HCDR1 with the amino acid sequence of human antibody HCDR1 to replace the mouse HCDR1 derived amino acid residue Asp-Tyr-Asn-Ile-Gln as set forth in SEQ ID NO: 1 present in humanized antibody HZII-H67 with that of a human antibody. As a result, it was confirmed that mouse HCDR1 has high homology with the HCDR1-derived amino acid residue Asp-Tyr-Asn-Val-Asp of the human antibody set forth in SEQ ID NO: 2 of Kabat Database ID Number 35920, and shows a difference between the two sequences. HCDR1 gene variants are prepared by replacing the fourth and fifth residues.

To this end, first of all, the inventors of the present invention valine the isoleucine No. 34, which is thought not to bind directly to the epitope of the antigen among the residues showing differences between the HCDR1 amino acid sequences of the mouse HCDR1 and the human antibody. Gene variant Q35N was prepared by substituting asparagine for gene variant I34V and amino acid residue No. 35 glutamine.

These gene variants are prepared by performing PCR and recombinant PCR using the heavy chain gene expression vector pRc / CMV-HC-HZIIS (KCTC 0489BP) of the humanized antibody HZII-H67 as a template (see FIGS . 1 and 2 ).

As a result, DNA fragments obtained from PCR using HL and P1 primer pairs described in SEQ ID NO: 3 and SEQ ID NO: 4 and DNA obtained from PCR using P2 and HC primer pairs described in SEQ ID NO: 5 and SEQ ID NO: 6 Humanized antibody heavy chain variable region gene variant I34V is obtained by fragments as a template and recombinant PCR with HL and HC primer pairs. The gene variant I34V was cloned into an expression vector in which the cDNA gene encoding the humanized heavy chain was cloned as a humanized heavy chain expression vector to prepare a recombinant expression vector, which was named pcDdA-HzSIIIh-I34V (see FIG. 3 ). The base sequence of the humanized antibody heavy chain variable region gene variant I34V was analyzed by DNA sequencer to confirm that I34V, a humanized antibody heavy chain variable region gene variant, was inserted into the recombinant expression vector.

Among the residues showing differences between the mouse HCDR1 and the HCDR1 sequences of the human antibody, the fifth variant, glutamine (Gln), was substituted with asparagine (Asn) of DP-15 derived from human antibody heavy chain, SEQ ID NO: 3 , SEQ ID NO: 7 , SEQ ID NO: 8 and using a primer pair described in SEQ ID NO: 6 by PCR and recombinant PCR obtained in the same manner as described above and cloned into an expression vector to produce a recombinant expression vector pcDdA-HzSIIIh-Q35N.

In the humanized antibody of the present invention wherein the mouse HCDR2 derived amino acid residue present in the humanized antibody HZII-H67 is substituted with the amino acid residue of the human antibody, all the HCDR2 derived amino acid sequences derived from the mouse are replaced with the HCDR2 amino acid sequence derived from the human antibody. Preferably, amino acid residues directly involved in antigen binding are not substituted. In order to replace the mouse HCDR2-derived amino acid residues present in humanized antibody HZII-H67 with those of human antibodies, we compared mouse HCDR2 with the amino acid sequence of human antibody HCDR2 to HCDR2 of DP-15 where the mouse HCDR2 is a fragment of the human antibody heavy chain. Confirming high homology with the sequence of and the residues that do not seem to be directly involved in antigen binding among the residues showing the difference between the two sequences by replacing the HCDR2 gene variants are prepared in the same manner as above.

As a result, the gene variant I51M (using the primer pairs of SEQ ID NO: 9 and SEQ ID NO: 10 and SEQ ID NO: 10 ) in which HCDR2-derived amino acid residue 51 isoleucine was substituted with methionine, and the amino acid residue of recombinant expression vector pcDdA-HzSIIIh-I51M Gene variant T54S with threonine 54 substituted with serine (using primer pairs of SEQ ID NO: 11 and SEQ ID NO: 12 ) and recombinant expression vector pcDdA-HzSIIIh-T54S comprising the same, amino acid residue 65 serine glycine Gene variant S65G (using primer pairs of SEQ ID NO: 13 and SEQ ID NO: 14 ) substituted with (glycine) and recombinant expression vector pcDdA-HzSIIIh-S65G comprising the same were prepared.

In addition, in the humanized antibody of the present invention in which the mouse HCDR3 derived amino acid residue present in the humanized antibody HZII-H67 is substituted with the amino acid residue of the human antibody, all the HCDR3 derived amino acid sequences derived from the mouse are replaced with the HCDR3 amino acid sequence derived from the human antibody. Preferably, amino acid residues directly involved in antigen binding are not substituted. In order to replace the amino acid residues derived from the mouse HCDR3 sequence present in the humanized antibody HZII-H67 with those of human antibodies, firstly, to determine whether each amino acid residue of mouse HCDR3 is directly involved in antigen binding, each amino acid residue is alanine. HCDR3 gene variants were prepared.

As a result, the gene variant N95A (using the primer pairs of SEQ ID NO: 15 and SEQ ID NO: 15 and SEQ ID NO: 15 ) and amino acid residue 96 of tyrosine, including the gene variant N95A having the amino acid residue 95 aspartazine substituted with alanine gene variant Y96A (tyrosine) substituted with alanine (using primer pairs of SEQ ID NO: 17 and SEQ ID NO: 18 ) and recombinant expression vector pcDdA-HzSIIIh-Y96A comprising the same, gene variant G97A substituted with alanine for amino acid residue 97 (Using the primer pairs of SEQ ID NO: 19 and SEQ ID NO: 20 ) and the recombinant expression vector pcDdA-HzSIIIh-G97A comprising the same, the gene variant Y98A in which the amino acid residue No. 98 tyrosine was substituted with alanine (the primer pair of SEQ ID NO: 21 and SEQ ID NO: 22) And the recombinant expression vector pcDdA-HzSIIIh-Y98A and amino acid residue No. 99 aspartic acid (aspartate) containing alanine. Substituted gene mutant D99A (SEQ ID NO: 23 and SEQ ID NO primer pair 24) and ● recombinant expression comprising the same vector pcDdA-HzSIIIh-D99A, 100 times the amino acid residue glutamic acid (glutamate) is E100A gene variants replaced with an alanine (SEQ ID NO: 25 and the primer pair of SEQ ID NO: 26 ) and the recombinant expression vector pcDdA-HzSIIIh-E100A comprising the same, the gene variant S100aA wherein serine of amino acid residue 100a is substituted with alanine (using the primer pair of SEQ ID NO: 27 and SEQ ID NO: 28 ), and A recombinant expression vector pcDdA-HzSIIIh-S100aA comprising the same was prepared.

As described above, in the present invention, the amino acid residues of the mouse-derived CDR regions present in the existing humanized antibodies do not directly participate in the binding with the antigen, so that some amino acid residues are not expected to affect the antigen binding affinity. The humanization antibodies were further humanized by replacing them with amino acid residues of the human antibody. In particular, in the present invention, unlike conventional CDR-transplantation methods in which the entire amino acid residues of the CDRs of a mouse antibody are implanted into a human antibody having homology with them, SDRs sites directly involved in binding to HBV S surface antigens, particularly within CDRs Using human SDR-transplantation methods to transplant only human antibodies, humanized antibodies with improved therapeutic effect while inducing less HAMA response than conventional humanized antibodies were developed.

The present inventors transformed each recombinant expression vector containing each humanized heavy chain gene variant into an animal cell line to confirm the expression level of the humanized antibody having the humanized heavy chain gene variant of the present invention prepared as described above. The concentration of the expressed humanized antibody was measured by a sandwich ELISA method, and the antigen binding ability of the humanized antibody having humanized heavy chain gene variants to surface antigen S was measured by performing an indirect ELISA. As a result, among the HCDR1 gene variants, the binding capacity of I34V to HBV surface antigen S was almost the same as the heavy chain of wild-type humanized antibody HZS-H67, and in the HCDR2 gene variant, T54S and S65G had almost the same level of antigen binding capacity as wild type. (See FIG. 4 ). On the other hand, in the case of HCDR3 gene variants, E100A showed slightly less antigen-binding ability than the wild type, suggesting that glutamic acid residues (E100) of the HCDR3 gene variants do not play a significant role in binding to HBV surface antigen S.

As described above, the present inventors confirmed that glutamic acid (E100), which is the 100th amino acid residue derived from the mouse HCDR3 sequence, did not play an important role in binding to HBV surface antigen S and to replace the amino acid residue with that of another human antibody. As compared with the amino acid sequence database of the human antibody sequences (Kabat Data Base ID No 24562, 39669, 44760) showing high homology to the mouse HCDR3 sequence, the serine of residue 100 and glycine of residue 101 After confirming the commonness, gene variants in which the 100th glutamic acid of mouse-derived HCDR3 was substituted with serine or 101st alanine with glycine were prepared to measure their antigen binding ability.

Gene variant E100S (using primer pairs of SEQ ID NO: 29 and SEQ ID NO: 29 and SEQ ID NO: 29) and amino acid residue 100 of glutamic acid substituted by serine in the same manner as described above, pcDdA-HzSIIIh-E100S, amino acid 101 alanine Gene variant A101G substituted with this glycine (using a primer pair of SEQ ID NO: 31 and SEQ ID NO: 32 ) and a recombinant expression vector pcDdA-HzSIIIh-A101G comprising the same were prepared.

As a result of measuring the antigen binding ability to the surface antigen S of the humanized antibody having the humanized heavy chain gene variants thus prepared, it was confirmed that the antigen binding ability of E100S was similar to that of the wild type, while A101G slightly decreased the antigen binding ability.

Based on the above results, the present inventors confirmed that the antigen-binding ability of I34V, T54S, S65G, and E100S is similar to that of the wild type among the variants of the humanized heavy chains prepared in the present invention, and the recombinant variants including the four gene variants simultaneously. To prepare.

First, in order to prepare recombinant gene variants including T54S and I34V gene variants, PCR and subcloning were carried out in the same manner as above using the heavy chain gene expression vector pcDdA-HzSIIIh-T54S of T54S as a template. The resulting recombinant expression vector was named pcDdA-HzSIIIh-T54S + I34V.

In addition, PCR and subcloning were performed in the same manner as above, using the heavy chain gene expression vector pcDdA-HzSIIIh-I34V of I34V as a template to prepare recombinant gene variants including T54S, I34V, and E100S gene variants. The resulting recombinant expression vector was named pcDdA-HzSIIIh-T54S + I34V + E100S.

In addition, PCR and subcloning were performed in the same manner as described above using the heavy chain gene expression vector pcDdA-HzSIIIh-E100S as a template to prepare recombinant gene variants including T54S, I34V, E100S and S65G gene variants. From this, a recombinant expression vector pcDdA-HzSIIIh-T54S + I34V + E100S + S65G containing all of the T54S, I34V, E100S and S65G gene variants was prepared and named pcDdA-HzSIIIh.

In order to measure the antigen binding capacity of the humanized antibody having the humanized heavy chain recombinant gene variant thus prepared, a recombinant expression vector comprising the same is transformed into an animal cell line and indirectly the antigen binding ability to the surface antigen S of the humanized antibody expressed therefrom. Confirmation was performed by ELISA.

As a result, each humanized heavy chain having a recombinant gene variant of I34V + T54S, I34V + T54S + E100S and I34V + T54S + E100S + S65G, which contains several gene variants of the present invention, had a heavy chain of wild-type humanized antibody HZS-H67. Binding capacity was shown for approximately the same level of surface antigen S (see FIG. 5 ). As such, the expression vector pcDdA-HzSIIIh expressing the heavy-chain recombinant gene variant of which the antigen-binding ability to HBV surface antigen S was confirmed was deposited on September 22, 2000 to the Korea Biotechnology Research Institute Gene Bank (Accession Number: KCTC 10083BP). As a result of analyzing the heavy chain amino acid sequence of the humanized antibody HzS-III expressed from the expression vector pcDdA-HzSIIIh, it was confirmed that the humanized heavy chain HzS-III-VH including the variable region of the amino acid sequence set forth in SEQ ID NO: 43 . .

In addition, the present invention provides a humanized light chain gene variant in which the amino acid residues of the LCDR1, LCDR2, and LCDR3 portions in the light chain variable region of the humanized antibody against the surface antigen S of HBV prepared by the CDR-transplantation method are substituted with light chain-derived amino acids of a human antibody, and An expression vector comprising the same is provided.

Humanized antibodies of the invention are prepared by substituting the amino acid residues of the LCDR1, LCDR2 and LCDR3 moieties present in the light chain variable region of humanized antibody HZII-H67 with light chain amino acid sequences derived from human antibodies. In the case of humanized light chains, since LCDR1 and LCDR2 of humanized antibody HZII-H67 already contain amino acid residues of human antibodies (D27cS, S31N, M33I for LCDR1; Q54K, S56T for LCDR2), we found amino acid residues of LCDR3. I wanted to change them. Specifically, the present inventors compared the amino acid residues of mouse-derived LCDR3 present in humanized antibody HZII-H67 with those of human antibodies, compared with the LCDR3 amino acid sequence of human light chain B1 having high homology with LCDR3 (see FIG. 6 ). From this, it was determined that glutamine No. 89 and threonine residue No. 91 of the mouse-derived LCDR3 sequence would not be directly involved in antigen binding and were substituted with leucine and serine, residues of human antibodies.

As a result, the gene variant Q89L (using the primer pair of SEQ ID NOS: 33 to 36 ) in which amino acid residue 89 glutamine was substituted with leucine and the recombinant expression vector pCMV-dhfr-HzSIIIk-Q89L containing the same ( FIGS. 7 and 8) Gene variant T91S (using SEQ ID NO: 33 and SEQ ID NO: 36 to SEQ ID NO: 38 primer pair) and a recombinant expression vector pCMV-dhfr-HzSIIIk-T91S comprising the same were prepared.

In order to measure the binding capacity of the humanized antibody having the humanized light chain gene variant prepared as described above to HBV surface antigen S, a recombinant expression vector including each of the gene variants was transformed into an animal cell line and then the humanized antibody expressed therefrom. Indirect ELISA method was used.

As a result, it was confirmed that the humanized light chain having a mutation of T91S in which the threonine 91 of the gene variants was substituted with serine showed the same level of binding to surface antigen S as the light chain of the wild-type humanized antibody HZS-H67, and the T91S gene variant was identified. The expression vector pCMV-dhfr-HzSIIIk-T91S which expresses was named pCMV-dhfr-HzSIIIk (refer FIG. 9 ). Thus, the recombinant expression vector pCMV-dhfr-HzSIIIk expressing the gene variant T91S expressing the binding ability to HBV surface antigen S was deposited on September 22, 2000 to the Biotechnology Research Institute Gene Bank (Accession Number: KCTC 10084BP). As a result of analyzing the light chain amino acid sequence of the humanized antibody HzS-III expressed from the expression vector pCMV-dhfr-HzSIIIk, the humanized light chain HzS-III-VL comprising the variable region of the amino acid sequence represented by SEQ ID NO: 42 was obtained. Confirmed.

In addition, the present invention provides a humanized antibody comprising heavy and light chain genes in which mouse-derived amino acid residues in the heavy and light chain variable region genes of the mouse monoclonal antibody H67 against the surface antigen S of HBV are substituted with human-derived amino acid residues.

In order to prepare humanized antibodies having humanized heavy chain recombinant gene variants and humanized light chain gene variants prepared as described above, the present inventors transformed the humanized heavy chain expression vector pcDdA-HzSIIIh and humanized light chain expression vector pCMV-dhfr-HzSIIIk into animal cell lines. And cultured for 48 hours to express humanized antibody HzS-III with humanized heavy chain recombinant gene variant / humanized light chain gene variant from the transformants. The concentration of the humanized antibody HzS-III present in the culture supernatant obtained therefrom was measured by the sandwich ELISA method, and antigen binding capacity was measured using the indirect ELISA method in the same manner as described above.

As a result, the humanized antibody HzS-III having the humanized heavy chain recombinant gene variant and the humanized light chain gene variant of the present invention showed the ability to bind surface antigen S to the same level as the wild type humanized antibody HZS-H67 (H / K) ( FIG. 11 ).

In addition, the antigen-binding affinity of the humanized antibody HzS-III was measured using a competitive ELISA method (competitive ELISA, Ryu et al., J. Med. Virol ., 52, 226, 1997). HzS-III confirmed that some amino acid residues derived from the mouse were replaced with human ones but did not significantly affect antigen binding affinity, thus showing no significant difference with the affinity of HZII-H67 used as a control (see FIG. 12 ).

In the present invention, whether a peptide sequence that binds to MHC class II is present in the heavy chain sequence of the humanized antibody HzS-III by Sturniolo et al. (Sturniolo et al., Nature Biotechnology , 17, 555-561, 1999). By searching, a humanized heavy chain substituted with residues suspected of not binding to MHC II was prepared and its antigen binding capacity was measured. In order to prepare such a humanized heavy chain, a gene mutation S44T in which amino acid residue serine was substituted with threonine (using a primer pair of SEQ ID NO: 46 and SEQ ID NO: 47 ) and a gene mutation in which amino acid residue 68 threonine was substituted with aspartic acid T68D (using a primer pair of SEQ ID NO: 48 and SEQ ID NO: 49 ) and a recombinant expression vector pcDdA-HzSIVh comprising the same were prepared and deposited on September 26, 2001 to the Korea Biotechnology Research Institute Gene Bank (Accession Number; KCTC). 10080BP). The humanized heavy chain expression vector pcDdA-HzSIVh and the humanized light chain expression vector pCMV-dhfr-HzSIIIk were transformed into animal cell lines and cultured for 48 hours to express the humanized antibody HzS-IV from the transformants. As a result of measuring the antigen binding ability of the humanized antibody HzS-IV present in the culture supernatant obtained therefrom, the humanized antibody HzS-IV of the present invention showed an antigen binding affinity almost similar to that of the humanized antibody HzS-III (see FIG. 12 ). ).

Thus, the amino acid sequences of the heavy and light chain variable regions of the humanized antibodies HzS-III and HzS-IV of the present invention showing excellent binding ability and antigen binding affinity for HBV surface antigen S were wild-type mouse monoclonal antibody H67 and humanized antibody HZII. As compared with the amino acid sequence of -H67, the amino acid sequences of the heavy and light chain variable regions of the humanized antibody HzS-III of the present invention were compared with the HCDR1, HCDR2 and HCDR3 of the heavy chain genes compared to the mouse monoclonal antibody H67 and the humanized antibody HZII-H67. Some mouse-derived amino acid residues in LCDR1, LCDR2 and LCDR3 of the light chain gene are substituted with human-derived amino acid residues to contain fewer amino acid residues from mouse, and the amino acid sequence of the heavy chain variable region of the humanized antibody HzS-IV of the present invention Substitution of two amino acid residues in the FR (framework region), which are believed to bind human MHC II and trigger an immune response It can be seen that by minimizing the control immune responses (see Fig. 13 and 14).

Therefore, the humanized antibodies HzS-III and HzS-IV of the present invention exhibit excellent therapeutic effects while inducing less HAMA response than conventional humanized antibodies, and thus may be useful for the treatment of hepatitis B virus.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Mutation of Humanized Heavy Chain Genes

<1-1> Preparation of HCDR1 Gene Variants

In order to replace the mouse HCDR1-derived amino acid residue Asp-Tyr-Asn-Ile-Gln as described in SEQ ID NO: 1 present in the humanized antibody HZII-H67 (Korean Patent No. 98-32644) to the human antibody, The amino acid sequence of HCDR1 was compared. As a result, it was confirmed that mouse HCDR1 has high homology with the HCDR1-derived amino acid residue Asp-Tyr-Asn-Val-Asp of the human antibody described by SEQ ID NO: 2 of Kabat Database ID Number 35920. HCDR1 gene variants were prepared by replacing the visible fourth and fifth residues.

<1-1-1> Preparation of Gene Variants Substituted with Valine for Isoleucine Amino Acid Residue No. 34

Among the residues showing differences between the HCDR1 amino acid sequences of the mouse HCDR1 and the human antibody, a genetic variant in which the fourth 34 isoleucine (Ile) was substituted with valine, which is not thought to bind directly to the epitope of the antigen. To prepare I34V, PCR (polymerase chain) was carried out using the primers described in SEQ ID NOS: 3 to SEQ ID NO: 6 with the heavy chain gene expression vector pRc / CMV-HC-HZIIS (KCTC 0489BP) of humanized antibody HZII-H67. Reaction, polymerase chain reaction, hereinafter abbreviated as "PCR" and recombinant PCR were performed ( FIGS. 1 and 2 ). Specifically, PCR was performed using Taq DNA polymerase (polymerase, manufactured by Takara) for 30 seconds at 94 ° C, 30 seconds at 55 ° C, and 1 minute at 72 ° C.

As a result, DNA fragments of 181 bp are amplified from PCR using HL and P1 primer pairs described in SEQ ID NO: 3 and SEQ ID NO: 4 , and PCR using P2 and HC primer pairs described in SEQ ID NO: 5 and SEQ ID NO: 6 , respectively. It was confirmed that the DNA fragment of 284 bp size was amplified. The two DNA fragments thus obtained were used as templates and recombinant PCR was performed with HL and HC primer pairs to connect them to each other to obtain a 448 bp humanized antibody heavy chain variable region gene variant I34V. Both ends of the variable region of the gene variant I34V were digested with restriction enzymes EcoR I and Apa I, and the expression vector pcDdA-, which is a cDNA gene encoding the humanized heavy chain at the positions of restriction enzymes EcoR I and Not I, was cloned as a humanized heavy chain expression vector. Restriction enzyme EcoR I- Apa I of HC ( expression vector in which heavy chain gene of humanized antibody against preS1 antigen of HBV is cloned between EcoR I- Not I positions of pcDNA3 of Invitrogen) (Korean Patent Application No. 1998-49663) Recombinant expression vectors were prepared by cloning in position and named pcDdA-HzSIIIh-I34V ( FIG. 3 ). The base sequence of the humanized antibody heavy chain variable region gene variant I34V was analyzed by DNA sequencer to confirm that I34V, a humanized antibody heavy chain variable region gene variant, was inserted into the recombinant expression vector.

<1-1-2> Preparation of Gene Variant with Glutamin Amino Acid Residue 35 Substituted with Asparagine

The above Example to prepare gene variant Q35N in which the fifth residue, glutamine (Gln), was substituted with asparagine (Asn) of DP-15 derived from human antibody heavy chain, among residues showing differences between the HCDR1 sequence of mouse HCDR1 and human antibody. PCR was performed under the same conditions as in <1-1-1>.

Specifically, amplify a DNA fragment of 187 bp using the HL described in SEQ ID NO: 3 and the P3 primer pair described in SEQ ID NO: 7 , and the HC described in SEQ ID NO: 6 and the P4 primer pair described in SEQ ID NO: 8 448 bp humanized antibody heavy chain variable region gene variant Q35N was obtained by amplifying DNA fragments of 278 bp size, using them as templates, and performing recombinant PCR with HL and HC primer pairs. Both ends of the variable region of the gene variant Q35N were digested with restriction enzymes EcoR I and Apa I and cloned into the restriction enzyme EcoR I -Apa I position of the expression vector pcDdA-HC to prepare a recombinant expression vector, which was called pcDdA-HzSIIIh-Q35N. Named it. The nucleotide sequence of the humanized antibody heavy chain variable region gene variant Q35N was analyzed by DNA sequencing to confirm that the humanized antibody heavy chain variable region gene variant Q35N was inserted into the recombinant expression vector.

<1-2> Preparation of HCDR2 Gene Variants

In order to replace the mouse HCDR2 derived amino acid residue represented by SEQ ID NO: 39 present in humanized antibody HZII-H67 with that of a human antibody, mouse HCDR2 was compared with the amino acid sequence of human antibody HCDR2. As a result, mouse HCDR2 exhibited high homology with the sequence of HCDR2 described in SEQ ID NO: 40 of DP-15, a fragment of the germline human antibody heavy chain, and was not directly involved in antigen binding among the residues showing differences between the two sequences. HCDR2 gene variants were prepared by substituting residues that were not thought to be.

<1-2-1> Preparation of Gene Variant with Amino Acid Residue 51 Isoleucine Substituted with Methionine

To prepare the HCDR2 gene variant, PCR was performed using the P5 and P6 primer pairs set forth in SEQ ID NO: 9 and SEQ ID NO: 10 instead of P1 and P2 primer pairs in the same manner as in Example <1-1-1>. It was. Amplify a 236 bp DNA fragment using HL and P5 primer pairs, amplify a 230 bp DNA fragment using HC and P4 primer pairs, and then use the two DNA fragments obtained as a template. Recombinant PCR was performed with a primer pair to connect them to each other to obtain a humanized antibody heavy chain variable region gene variant I51M of 448 bp in size. Both ends of the variable region of the gene variant I51M were digested with restriction enzymes EcoR I and Apa I and cloned into the restriction enzyme EcoR I -Apa I positions of the expression vector pcDdA-HC to prepare a recombinant expression vector, which was pcDdA-HzSIIIh-I51M. Named it. The nucleotide sequence of the humanized antibody heavy chain variable region gene variant I51M was analyzed by DNA sequencing to confirm that I51M, the humanized antibody heavy chain variable region gene variant, was inserted into the recombinant expression vector.

<1-2-2> Preparation of Gene Variants Substituted with Serine at Threonine Amino Acid Residue 54

PCR was performed using the P7 and P8 primer pairs described by SEQ ID NO: 11 and SEQ ID NO: 12 instead of P1 and P2 primer pairs in the same manner as in Example <1-1-1>. Amplify a 249 bp DNA fragment using HL and P7 primer pairs, amplify a 218 bp DNA fragment using HC and P8 primer pairs, and then use the two DNA fragments obtained as a template. Recombinant PCR was performed with the primer pairs to connect them to each other to obtain a humanized antibody heavy chain variable region gene variant T54S having a size of 448 bp. Both ends of the variable region of the gene variant T54S were digested with restriction enzymes EcoR I and Apa I and cloned into the restriction enzyme EcoR I -Apa I position of the expression vector pcDdA-HC to prepare a recombinant expression vector, which was called pcDdA-HzSIIIh-T54S. Named it. The base sequence of the humanized antibody heavy chain variable region gene variant T54S was analyzed by DNA sequencing to confirm that T54S, a humanized antibody heavy chain variable region gene variant, was inserted into the recombinant expression vector.

<1-2-3> Preparation of Gene Variant with Serine of Amino Acid Residue 65 Substituted with Glycine

PCR was performed using the P9 and P10 primer pairs described in SEQ ID NO: 13 and SEQ ID NO: 14 instead of P1 and P2 primer pairs in the same manner as in Example <1-1-1>. Amplify a 278 bp DNA fragment using HL and P9 primer pairs, amplify a 185 bp DNA fragment using HC and P10 primer pairs, and then use the two DNA fragments obtained as a template. Recombinant PCR was performed with a primer pair to connect them to each other to obtain a humanized antibody heavy chain variable region gene variant S65G having a size of 448 bp. Both ends of the variable region of the gene variant S65G were digested with restriction enzymes EcoR I and Apa I and cloned into the restriction enzyme EcoR I -Apa I positions of the expression vector pcDdA-HC to prepare a recombinant expression vector, which was called pcDdA-HzSIIIh-S65G. Named it. The base sequence of the humanized antibody heavy chain variable region gene variant S65G was analyzed by DNA sequencing to confirm that S65G, the humanized antibody heavy chain variable region gene variant, was inserted into the recombinant expression vector.

<1-3> Preparation of HCDR3 Gene Variants

In order to change the amino acid residue from the mouse HCDR3 sequence represented by SEQ ID NO: 41 present in humanized antibody HZII-H67 to that of a human antibody, mouse HCDR3 is compared with the amino acid sequence of human antibody HCDR3 to antigen-binding among amino acid residues of mouse HCDR3 sequence HCDR3 gene variants were prepared by substituting residues that do not directly affect.

<1-3-1> Preparation of Gene Variants Substituted with Alanine for Asparagine at Amino Acid Residue 95

PCR was performed using the P11 and P12 primer pairs described in SEQ ID NO: 15 and SEQ ID NO: 16 instead of P1 and P2 primer pairs in the same manner as in Example <1-1-1>. Amplify a 381 bp DNA fragment using HL and P11 primer pairs, amplify a 88 bp DNA fragment using HC and P12 primer pairs, and then use these two DNA fragments as templates. Recombinant PCR was performed with the primer pairs to connect them to each other to obtain a humanized antibody heavy chain variable region gene variant N95A of 448 bp in size. Both ends of the variable region of the gene variant N95A were digested with restriction enzymes EcoR I and Apa I and cloned into the restriction enzyme EcoR I -Apa I position of the expression vector pcDdA-HC to prepare a recombinant expression vector, which was referred to as pcDdA-HzSIIIh-N95A. Named it. The nucleotide sequence of the humanized antibody heavy chain variable region gene variant N95A was analyzed by DNA sequencing to confirm that N95A, a humanized antibody heavy chain variable region gene variant, was inserted into the recombinant expression vector.

<1-3-2> Preparation of Gene Variants with Tyrosine Amino Acid Residue 96 Substituted with Alanine

PCR was performed using the P13 and P14 primer pairs described by SEQ ID NO: 17 and SEQ ID NO: 18 instead of P1 and P2 primer pairs in the same manner as in Example <1-1-1>. Amplify DNA fragments of 380 bp using HL and P13 primer pairs, amplify DNA fragments of 85 bp using HC and P14 primer pairs, and then use these two DNA fragments as templates. Recombinant PCR was performed with the primer pairs to connect them to each other to obtain a 448 bp humanized antibody heavy chain variable region gene variant Y96A. Both ends of the variable region of the gene variant Y96A were digested with restriction enzymes EcoR I and Apa I and cloned into the restriction enzyme EcoR I -Apa I positions of the expression vector pcDdA-HC to prepare a recombinant expression vector, which was referred to as pcDdA-HzSIIIh-Y96A. Named it. The base sequence of the humanized antibody heavy chain variable region gene variant Y96A was analyzed by DNA sequencer to confirm that the humanized antibody heavy chain variable region gene variant Y96A was inserted into the recombinant expression vector.

<1-3-3> Preparation of Gene Variants Substituted with Alanine for Glycine Amino Acid Residue 97

PCR was performed using the P15 and P16 primer pairs described by SEQ ID NO: 19 and SEQ ID NO: 20 instead of P1 and P2 primer pairs in the same manner as in Example <1-1-1>. Amplify a 383 bp DNA fragment using HL and P15 primer pairs, amplify a 82 bp DNA fragment using HC and P14 primer pairs, and then use the two DNA fragments obtained as a template. Recombinant PCR was performed with the primer pairs to connect them to each other to obtain a humanized antibody heavy chain variable region gene variant G97A of 448 bp in size. Both ends of the variable region of the gene variant G97A were digested with restriction enzymes EcoR I and Apa I and cloned into the restriction enzyme EcoR I -Apa I position of the expression vector pcDdA-HC to prepare a recombinant expression vector, which was referred to as pcDdA-HzSIIIh-G97A. Named it. The nucleotide sequence of the humanized antibody heavy chain variable region gene variant Y96A was analyzed by DNA sequencing to confirm that G97A, a humanized antibody heavy chain variable region gene variant, was inserted into the recombinant expression vector.

<1-3-4> Preparation of Gene Variant Substituted with Alanine for Tyrosine Amino Acid Residue No. 98

PCR was performed using P17 and P18 primer pairs described by SEQ ID NO: 21 and SEQ ID NO: 22 instead of P1 and P2 primer pairs in the same manner as in Example <1-1-1>. HL and P17 primer pairs were used to amplify DNA fragments of 386 bp, HC and P18 primer pairs were used to amplify 78 bp DNA fragments, and the two DNA fragments obtained therefrom as templates. Recombinant PCR was performed with the primer pairs to connect them to each other to obtain a humanized antibody heavy chain variable region gene variant Y98A of 448 bp in size. Both ends of the variable region of the gene variant Y98A were digested with restriction enzymes EcoR I and Apa I and cloned into the restriction enzyme EcoR I -Apa I positions of the expression vector pcDdA-HC to prepare a recombinant expression vector, which was referred to as pcDdA-HzSIIIh-Y98A. Named it. The nucleotide sequence of the humanized antibody heavy chain variable region gene variant Y98A was analyzed by DNA sequencing to confirm that Y98A, a humanized antibody heavy chain variable region gene variant, was inserted into the recombinant expression vector.

<1-3-5> Preparation of Gene Variants in which Aspartate Amino Acid Residue 99 is Substituted with Alanine

PCR was performed using P19 and P20 primer pairs described by SEQ ID NO: 23 and SEQ ID NO: 24 instead of P1 and P2 primer pairs in the same manner as in Example <1-1-1>. Amplify a 390 bp DNA fragment using HL and P19 primer pairs, amplify a 73 bp DNA fragment using HC and P20 primer pairs, and then use these two DNA fragments as templates. Recombinant PCR was performed with the primer pairs to connect them to each other to obtain a humanized antibody heavy chain variable region gene variant D99A having a size of 448 bp. Both ends of the variable region of the gene variant D99A were digested with restriction enzymes EcoR I and Apa I and cloned into the restriction enzyme EcoR I -Apa I position of the expression vector pcDdA-HC to prepare a recombinant expression vector, which was referred to as pcDdA-HzSIIIh-D99A. Named it. The base sequence of the humanized antibody heavy chain variable region gene variant Y98A was analyzed by DNA sequencing to confirm that D99A, a humanized antibody heavy chain variable region gene variant, was inserted into the recombinant expression vector.

<1-3-6> Preparation of Gene Variants Substituted with Alanine for Glutamate Amino Acid Residue No. 100

PCR was performed using the P21 and P22 primer pairs described in SEQ ID NO: 25 and SEQ ID NO: 26 instead of P1 and P2 primer pairs in the same manner as in Example <1-1-1>. Amplify a 390 bp DNA fragment using HL and P21 primer pairs, amplify a 73 bp DNA fragment using HC and P22 primer pairs, and then use these two DNA fragments as templates. Recombinant PCR was performed with a primer pair to connect them to each other to obtain a humanized antibody heavy chain variable region gene variant E100A having a size of 448 bp. Both ends of the variable region of the gene variant E100A were digested with restriction enzymes EcoR I and Apa I and cloned into the restriction enzyme EcoR I -Apa I positions of the expression vector pcDdA-HC to prepare a recombinant expression vector, which was referred to as pcDdA-HzSIIIh-E100A. Named it. The base sequence of the humanized antibody heavy chain variable region gene variant Y98A was analyzed by DNA sequencer to confirm that the humanized antibody heavy chain variable region gene variant E100A was inserted into the recombinant expression vector.

<1-3-7> Preparation of a Gene Variant of Serine of Amino Acid Residue 100a Substituted with Alanine

PCR was performed using P23 and P24 primer pairs as described in SEQ ID NO: 27 and SEQ ID NO: 28 instead of P1 and P2 primer pairs in the same manner as in Example <1-1-1>. Amplify a 395 bp DNA fragment using HL and P23 primer pairs, amplify a 67 bp DNA fragment using HC and P24 primer pairs, and then use these two DNA fragments as templates. Recombinant PCR was performed with a primer pair to connect them to each other to obtain a humanized antibody heavy chain variable region gene variant S100aA having a size of 448 bp. Both ends of the variable region of the gene variant S100aA were digested with restriction enzymes EcoR I and Apa I and cloned into the restriction enzyme EcoR I -Apa I positions of the expression vector pcDdA-HC to prepare a recombinant expression vector, which was referred to as pcDdA-HzSIIIh-S100aA. Named it. The nucleotide sequence of the humanized antibody heavy chain variable region gene variant Y98A was analyzed by DNA sequencer to confirm that S100aA, a humanized antibody heavy chain variable region gene variant, was inserted into the recombinant expression vector.

Example 2 Expression of Humanized Antibodies with Humanized Heavy Chain Gene Variants

The expression vectors were transformed into animal cell lines for direct expression of humanized antibodies with humanized heavy chain gene variants in cells.

Specifically, COS7 cells were seeded in DMEM culture medium (GIBCO) to which 10% fetal bovine serum was added and passaged at 37 ° C., 5% CO ₂ incubator. The cells were seeded at a concentration of 1 × 10 ⁶ cells / ml in a 100 mm culture dish, incubated overnight at 37 ° C., and washed three times with OPTI-MEM I (GIBCO). Meanwhile, each recombinant expression vector and humanized antibody HZII- comprising a humanized heavy chain gene variant of I34V, Q35N, I51M, T54S, S65G, N95A, Y96A, G97A, Y98A, D99A, E100A or S100aA prepared in Example 1 above. 5 μg of light chain expression vector pKC-dhfr-HZIIS (KCTC 0490BP) of H67 was taken and diluted with 800 μl of OPTI-MEM I, and 50 μl of lipofectamine (GIBCO) was also diluted with 800 μl of OPTI-MEM I. . The dilutions were mixed in a 15 ml tube to prepare a DNA-lipopeptamine mixture, which was left at room temperature for at least 15 minutes. 6.4 ml of OPTI-MEM I was added to each of the DNA-lipopeptamine mixtures, and the cells were evenly mixed with the washed COS7 cells and transformed into cells. Transformation was performed by incubating the cell culture medium to which the DNA-lipopeptamine mixture was added for 48 hours at 37 ° C. and 5% CO ₂ incubator, and recovering the culture supernatant from the antibody by sandwich ELISA. The concentration was measured. In order to perform sandwich ELISA, an anti-human antibody (anti-human IgG, manufactured by Sigma) was used as a capture antibody, and horseradish peroxidase (Hrad) was used for anti-human antibody (Fc specific). Sigma) bound antibody was used as a secondary antibody.

Example 3 Determination of Antigen Binding Ability to Surface Antigen S of Humanized Antibodies with Humanized Heavy Chain Gene Variants

The surface antigen S (green cross) of HBV was added to each well of the microplate by coating 250 ng, and then the cell culture medium transformed with the respective DNA-lipopeptamine mixture obtained from Example 2 was prepared in Example 2. Based on the measured antibody concentrations, the concentrations of the antibodies were added to be 0, 0.2, 0.4, 0.8, 1.8, 3.5 and 7 ng, respectively. The absorbance was measured at 492 nm after an indirect ELISA using an antibody conjugated with horseradish peroxidase to an anti-human antibody in the well plate as a secondary antibody. As a control, the same experiment was performed using pRc / CMV-HC-HZIIS (KCTC 0489BP) and pKC-dhfr-HZIIS, which are heavy and light chain expression vectors of HZII-H67, and the results are shown in FIG. 4 .

As shown in FIG . 4 , among the HCDR1 gene variants, the binding capacity of I34V to HBV surface antigen S was almost the same as that of the wild-type humanized antibody HZS-H67, and in the HCDR2 gene variant, T54S and S65G were almost the same as the wild type. Levels of antigen binding capacity. On the other hand, in the case of HCDR3 gene variants, E100A showed slightly less antigen-binding ability than the wild type, suggesting that glutamic acid residues (E100) of the HCDR3 gene variants do not play a significant role in binding to HBV surface antigen S.

Example 4 Preparation of HCDR3 Gene Variants

The present inventors confirmed that glutamic acid (E100), which is the 100th amino acid residue derived from the mouse HCDR3 sequence, did not play an important role in binding to HBV surface antigen S from the results of Example 3, and replaced the amino acid residue with that of another human antibody. For comparison, the amino acid sequence database of human antibody HCDR3 was compared.

As a result, it was confirmed that serine, which is residue 100 of HCDR3, and glycine, which was residue 101, were common in human antibody sequences showing high homology to the mouse HCDR3 sequence, and the 100th glutamic acid of mouse-derived HCDR3 was serine or residue 101. Gene variants in which phosphorus alanine was substituted with glycine were prepared to measure their antigen binding ability.

<4-1> Preparation of Gene Variant Substituted with Serine for Glutamic Acid at Amino Acid Residue No. 100

PCR was performed using the P25 and P26 primer pairs described by SEQ ID NO: 29 and SEQ ID NO: 30 instead of P1 and P2 primer pairs in the same manner as in Example <1-1-1>. Amplify a 390 bp DNA fragment using HL and P25 primer pairs, amplify a 73 bp DNA fragment using HC and P26 primer pairs, and then use these two DNA fragments as templates. Recombinant PCR was performed with a primer pair to connect them to each other to obtain a humanized antibody heavy chain variable region gene variant E100S having a size of 448 bp. Both ends of the variable region of the gene variant E100S were digested with restriction enzymes EcoR I and Apa I and cloned into the restriction enzyme EcoR I -Apa I position of the expression vector pcDdA-HC to prepare a recombinant expression vector, which was called pcDdA-HzSIIIh-E100S. Named it. The nucleotide sequence of the humanized antibody heavy chain variable region gene variant E100S was analyzed by DNA sequencer to confirm that the humanized antibody heavy chain variable region gene variant E100S was inserted into the recombinant expression vector.

<4-2> Preparation of Gene Variant with Alanine Amino Acid No. 101 Replaced with Glycine

PCR was performed using P27 and P28 primer pairs described by SEQ ID NO: 31 and SEQ ID NO: 32 instead of P1 and P2 primer pairs in the same manner as in Example <1-1-1>. Amplify a 396 bp DNA fragment using HL and P27 primer pairs, amplify a 68 bp DNA fragment using HC and P28 primer pairs, and then use the two DNA fragments obtained as a template. Recombinant PCR was performed with a primer pair to connect them to each other to obtain a humanized antibody heavy chain variable region gene variant A101G having a size of 448 bp. Both ends of the variable region of the gene variant A101G were digested with restriction enzymes EcoR I and Apa I and cloned into the restriction enzyme EcoR I -Apa I position of the expression vector pcDdA-HC to prepare a recombinant expression vector, which was called pcDdA-HzSIIIh-A101G. Named it. The nucleotide sequence of the humanized antibody heavy chain variable region gene variant A101G was analyzed by DNA sequencer to confirm that A101G, a humanized antibody heavy chain variable region gene variant, was inserted into the recombinant expression vector.

<4-3> Measurement of antigen binding capacity to surface antigen S

Two recombinant expression vectors prepared in Examples <4-1> and <4-2> were transformed into COS cells in the same manner as in Examples 2 and 3, and then the antigen binding ability of the transfectants selected therefrom. Indirect ELISA method showed that the antigen-binding ability of E100S was similar to that of wild type, while A101G showed a slight decrease in antigen-binding ability.

The inventors confirmed that the antigen binding ability of I34V, T54S, S65G and E100S is similar to that of the wild type among the variants of the humanized heavy chain through the results of Examples 3 to <4-3>, and includes the four gene variants simultaneously. To prepare a recombinant variant.

Example 5 Recombinant Variant of Humanized Heavy Chain Gene

<5-1> Preparation of T54S + I34V Recombinant Gene Variants

In order to prepare recombinant gene variants comprising T54S and I34V gene variants, the heavy chain gene expression vector pcDdA-HzSIIIh-T54S of Example <1-2-1> was used as a template. PCR and subcloning were performed in the same manner, and the recombinant expression vector obtained therefrom was named pcDdA-HzSIIIh-T54S + I34V.

<5-2> Preparation of T54S + I34V + E100S Recombinant Gene Variant

In order to prepare recombinant gene variants comprising T54S, I34V and E100S gene variants, the heavy chain gene expression vector pcDdA-HzSIIIh-I34V of Example <5-1> was used as a template and PCR and subcloning were performed in the same manner, and the recombinant expression vector obtained therefrom was named pcDdA-HzSIIIh-T54S + I34V + E100S.

<5-3> Preparation of T54S + I34V + E100S + S65G recombinant gene variants

To prepare a recombinant gene variant comprising the T54S, I34V, E100S and S65G gene variants, the heavy chain gene expression vector pcDdA-HzSIIIh-E100S of Example <5-2> was used as a template. PCR and subcloning were performed in the same manner as> to prepare a recombinant expression vector pcDdA-HzSIIIh-T54S + I34V + E100S + S65G containing all of the T54S, I34V, E100S and S65G gene variants, which were named pcDdA-HzSIIIh.

Example 6 Determination of antigen-binding ability of a humanized antibody having a humanized heavy chain recombinant gene variant

The surface antigen S (green cross) of HBV was added to each well of the microplate by coating 250 ng, and then coated with each recombinant expression vector and HZII- prepared from Examples <4-1> to <4-3>. PKC-dhfr-HZIIS, the light chain expression vector of H67, was transformed into COS7 cells in the same manner as in Example 2, and the cell culture medium of the transformants obtained therefrom was found to have an antibody concentration of 0.625, 1.25, 2.5, 5, 10 and 20 ng were added to the well plates. The absorbance was measured by ELISA in the same manner as in Example 2. In this case, the same experiment was performed using pRc / CMV-HC-HZIIS and pKC-dhfr-HZIIS, which are heavy and light chain expression vectors of HZII-H67, as shown in FIG. 5 .

As shown in FIG . 5 , each humanized heavy chain having recombinant genetic variants of I34V + T54S, I34V + T54S + E100S and I34V + T54S + E100S + S65G comprising several gene variants of the present invention is a wild-type humanized antibody HZS- It showed binding capacity to surface antigen S at about the same level as the heavy chain of H67. Thus, the expression vector pcDdA-HzSIIIh expressing the heavy chain recombinant gene variant confirmed the antigen-binding ability to HBV surface antigen S was deposited on September 22, 2000 to the Biotechnology Research Institute Gene Bank (Accession Number: KCTC 10083BP). As a result of analyzing the heavy chain amino acid sequence of the humanized antibody HzS-III expressed from the expression vector pcDdA-HzSIIIh, it was confirmed that the humanized heavy chain HzS-III-VH including the variable region of the amino acid sequence set forth in SEQ ID NO: 43 . .

Example 7 Mutation of Humanized Light Chain Gene

In the case of the humanized light chain, since the LCDR1 and LCDR2 of the humanized antibody HZII-H67 have amino acid residues derived from human antibodies, the present inventors sought to alter the amino acid residues of LCDR3. Specifically, the inventors compared the amino acid residues of mouse-derived LCDR3 present in humanized antibody HZII-H67 with those of human antibodies, and compared them with the LCDR3 amino acid sequence of human light chain B1 having high homology with the amino acid sequence of the humanized light chain. From this, it was determined that the glutamine No. 89 and the threonine residue of the mouse-derived LCDR3 sequence would not be directly involved in antigen binding and were replaced with leucine and serine, which are residues of the human B1 light chain.

<7-1> Preparation of Gene Variant of Glutamin Substituted with Amino Acid Residue No. 89

89 one glutamine residue in the mouse-derived LCDR3 is for the production of a gene mutant substituted with leucine of a human antibody, to the conventional humanized antibodies HZII-H67 light chain expression vector pKC-dhfr-HzIIS as a template and SEQ ID NO: 33 to SEQ ID NO: PCR and recombinant PCR were performed using primers described as number 36 ( FIGS. 6 and 7 ).

Specifically, PCR was performed 25 times of reaction for 30 minutes at 94 ℃, 30 seconds at 55 ℃, 1 minute at 72 ℃ using Taq DNA polymerase. DNA fragments of 336 bp are amplified from PCR using LL and P29 primer pairs as depicted in SEQ ID NO: 33 and SEQ ID NO: 34 , and 273 bp from PCR using P30 and LC primer pairs as depicted in SEQ ID NO: 35 and SEQ ID NO: 36 It was confirmed that the DNA fragment of size was amplified. The two DNA fragments thus obtained were used as templates, and recombinant PCR was performed using LL and P29 primer pairs to connect them to each other to obtain 743 bp humanized antibody light chain variable region gene variant Q89L. Both ends of the variable region of the gene variant Q89L were cut with restriction enzymes Hind III and Sal I and cloned into pBluescript KS ⁺ (StrataGen) to prepare a recombinant plasmid pBlue-HzSIIIk-Q89L. The nucleotide sequence of the humanized antibody light chain variable region gene variant G89L was analyzed to confirm that Q89L was correctly inserted into the expression vector. The pBlue-HzSIIIk-Q89L DNA was digested with restriction enzymes Hind III and Apa I to cut humanized light chain gene variant fragments, and then cloned back into the Hind III -Apa I position of pCMV-dhfr as an expression vector to prepare a recombinant expression vector. ( Fig. 7 ) It was named pCMV-dhfr-HzSIIIk-Q89L ( Fig. 8 ).

<7-2> Preparation of Gene Variant Substituted with Serine of Amino Acid 91 Threonine

PCR was performed using the primers of SEQ ID NO: 33 and SEQ ID NO: 36 to SEQ ID NO: 38 under the same reaction conditions as in Example <7-1>. Amplify a 369 bp DNA fragment using the LL and P31 primer pairs, amplify a 391 bp DNA fragment using the P32 and LC primer pairs, and then use the two DNA fragments obtained as a template. Recombinant PCR was performed with a primer pair to connect them to each other to obtain 743 bp humanized antibody light chain variable region gene variant T91S. Both ends of the variable region of the gene variant T91S were digested with restriction enzymes Hind III and Sal I and cloned into pBluescript KS ⁺ to prepare a recombinant plasmid pBlue-HzSIIIk-T91S. The nucleotide sequence of the humanized antibody light chain variable region gene variant T91S was analyzed to confirm that T91S was correctly inserted into the expression vector. The pBlue-HzSIIIk-T91S DNA was digested with restriction enzymes Hind III and Apa I to cut humanized light chain gene fragment fragments, and then cloned back into the Hind III -Apa I position of the expression vector pCMV-dhfr and the recombinant expression vector pCMV-dhfr- HzSIIIk-T91S was prepared and named pCMV-dhfr-HzSIIIk.

Example 8 Determination of Binding Ability to HBV Surface Antigen S of Humanized Antibodies with Humanized Light Chain Gene Variants

PRc / CMV-HC- which is a heavy chain expression vector of pCMV-dhfr-HzSIIIk-Q89L or pCMV-dhfr-HzSIIIk-T91S and the humanized antibody HZII-H67 obtained from Examples <7-1> and <7-2> After transforming HZIIS into COS7 cells in the same manner as in Example 2, the selected transformants were incubated for 48 hours. The concentration of the humanized antibody present in the culture supernatant obtained therefrom was measured by the sandwich ELISA method, and the HBV concentration was 0, 0.2, 0.4, 0.8, 1.8, 3.5 and 7 ng in the same manner as in Example 3, respectively. Antigen binding ability was measured by indirect ELISA method by adding to surface antigen S-coated well plate. In this case, the same experiment was performed using pRc / CMV-HC-HZIIS and pKC-dhfr-HZIIS, which are heavy and light chain expression vectors of HZII-H67, and the results are shown in FIG. 9 .

As shown in FIG . 9 , the humanized light chain having a mutation of T91S in which the threonine 91 of the gene variants was substituted with serine showed the same level of surface antigen S binding as that of the wild-type humanized antibody HZS-H67. Thus, the recombinant expression vector pCMV-dhfr-HzSIIIk expressing the gene variant T91S expressing the binding ability to HBV surface antigen S was deposited on September 22, 2000 to the Biotechnology Research Institute Gene Bank (Accession Number: KCTC 10084BP). As a result of analyzing the light chain amino acid sequence of the humanized antibody HzS-III expressed from the expression vector pCMV-dhfr-HzSIIIk, the humanized light chain HzS-III-VL comprising the variable region of the amino acid sequence represented by SEQ ID NO: 42 was obtained. Confirmed.

Example 9 Determination of Antigen Binding Ability of Humanized Antibody (HzS-III) with Humanized Heavy Chain Recombinant Gene Variant / Humanized Light Chain Gene Variant

The humanized heavy chain expression vector pcDdA-HzSIIIh obtained in Example <5-2> and the humanized light chain expression vector pCMV-dhfr-HzSIIIk obtained in Example <7-2> were transformed into COS7 cells in the same manner as in Example 2. It was then cultured for 48 hours to express humanized antibody HzS-III with humanized heavy chain recombinant gene variant / humanized light chain gene variant from the transformants. In order to confirm the humanized antibody HzS-III existing in the culture supernatant obtained therefrom, a secondary antibody was prepared by binding horseradish peroxidase (Sigma) to an anti-human antibody (Fc specific). Western blot analysis was performed. As a result, as shown in FIG . 10 , a humanized heavy chain of about 55 kDa and a humanized light chain of about 27 kDa were detected, confirming that the humanized antibody of the present invention was correctly expressed and secreted from the transformant.

The concentration of the humanized antibody HzS-III secreted into the culture medium was measured by the sandwich ELISA method, and the surface of the HBV was adjusted to 0.625, 1.25, 2.5, 5, 10, and 20 ng in the same manner as in Example 3, respectively. Antigen S was added to the coated well plate and antigen binding capacity was measured by indirect ELISA method. At this time, the same experiment was performed using pRc / CMV-HC-HZIIS and pKC-dhfr-HZIIS, which are heavy and light chain expression vectors of HZII-H67, and the results are shown in FIG. 11 .

As shown in FIG . 11 , the humanized antibody HzS-III having a humanized heavy chain recombinant gene variant and a humanized light chain gene variant of the present invention exhibited the ability to bind surface antigen S to the same level as the wild type humanized antibody HZS-H67 (H / K). Indicated.

Example 10 Determination of antigen binding affinity of humanized antibody HzS-III

To determine the antigen binding affinity of the humanized antibody HzS-III, a competitive ELISA method (competitive ELISA, Ryu et al., J. Med. Virol ., 52, 226, 1997) was used. In Example 8, 5 ng of humanized antibody HzS-III produced from COS7 cells and surface antigen S (1 × 10 ⁻⁷ M to 1 × 10 ⁻¹² M) were previously reacted at 37 ° C. for 3 hours, and then the reaction mixture was reacted. It was added to a 96 well plate previously coated with surface antigen S. After reacting the well plate at 37 ° C. for 30 minutes, ELISA was performed in the same manner as in Example 3 to measure absorbance ( FIG. 12 ). At this time, humanized antibody HZII-H67 was used as a control.

As a result, the antigen-binding affinity of the humanized antibody HzS-III of the present invention was about 4 × 10 ⁹ M ⁻¹ , showing no significant difference from the affinity (3.2 × 10 ⁹ M ⁻¹ ) of HZII-H67 used as a control group. Was confirmed.

Example 11 Preparation of Humanized Antibody HzS-IV Removed Peptide Sequence Binding to MHC Class II from Humanized Antibody HzS-III

The present inventors analyzed the presence of peptide sequence binding to MHC class II in the humanized antibody HzS-III using Sturniolo et al. (TEPITOPE) (Sturniolo et al., Nature Biotechnology , 17, 555-561, 1999). . In addition, the same peptide sequence was excluded from consideration by analyzing the sequence of human germline DP-25 having high similarity with the humanized antibody.

As a result, it was analyzed that two peptides bind to MHC class II in the humanized heavy chain. The first peptide had an amino acid sequence as set out in SEQ ID NO: 44 and was analyzed to bind 22 MHC class II molecules out of 51 MHC class II molecules, and the second peptide had an amino acid sequence as set out in SEQ ID NO: 45 It was analyzed to bind to 14 MHC class II molecules ( Table 1 ).

TEPITOPE Assay of Humanized Heavy Chain HzSIIIh Peptide Before substitution After substitution Before substitution After substitution WVKQAPGK S ( SEQ ID NO: 44 ) WVKQAPGK T ( SEQ ID NO: 46 ) FQGRV T LTV ( SEQ ID NO 45 ) FQGRV D LTV ( SEQ ID NO 47 ) MHC class II molecules that bind to peptides DRB1_0101DRB1_0301DRB1_0305DRB1_0309DRB1_0401DRB1_0405DRB1_0408DRB1_0421DRB1_0426DRB1_0801DRB1_0802DRB1_1101DRB1_1102DRB1_1104DRB1_1106DRB1_1DRDR1_1BDR_1_1DR1_1DR1_11221 DRB1_0101DRB1_0305DRB1_0309DRB1_1101DRB1_1307DRB1_1321DRB5_0101DRB5_0105 DRB1_0402DRB1_0421DRB1_0701DRB1_0703DRB1_0801DRB1_0802DRB1_0804DRB1_0806DRB1_0813DRB1_0817DRB1_1101DRB1_1114DRB1_1120DRB1_1128DRB1_1302DRB1_1305DRB1_1323DRB1_1502 DRB1 * 1502 Total 29 14 18 One

In order to weaken the binding capacity of the peptides to MHC II, the present inventors replaced serine at the P9 position of the amino acid sequence of SEQ ID NO: 44 with threonine, resulting in eleven (50%) of MHC class II molecules. When the threonine residue present in P6 of the amino acid sequence set forth in SEQ ID NO: 45 was replaced with aspartic acid, only one of the 51 MHC II molecules was analyzed.

Accordingly, the present inventors have proposed that the genetic modification S44T ( SEQ ID NO: 46 and SEQ ID NO: 47) in which the serine of the heavy chain sequence 44 serine of the humanized antibody HzS-III is substituted with threonine to prepare a humanized heavy chain having weakened binding to MHC class II Gene variants S44T / T68D were obtained, including the gene variant T68D (using the primer pairs of SEQ ID NO: 48 and SEQ ID NO: 49 ) in which the amino acid residue 68 threonine was substituted with aspartic acid. Both ends of the variable region of the gene variant S44T / T68D were digested with restriction enzymes EcoR I and Apa I and cloned into the restriction enzyme EcoR I -Apa I positions of the expression vector pcDdA-HC to prepare a recombinant expression vector, which was referred to as pcDdA-HzSIVh. It was deposited on September 26, 2001 to the Korea Institute of Biotechnology Gene Bank (Accession Number; KCTC 10080BP).

The humanized heavy chain expression vector pcDdA-HzSIVh and the humanized light chain expression vector pCMV-dhfr-HzSIIIk were transformed into an animal cell line and cultured for 48 hours to express the humanized antibody HzS-IV from the transformant, and the cultured upper layer obtained therefrom. As a result of measuring the antigen binding ability of the humanized antibody HzS-IV present in the solution, the humanized antibody HzS-IV of the present invention showed an antigen binding affinity almost similar to that of the humanized antibody HzS-III (see FIG. 12 ).

Example 12 Comparison of Amino Acid Sequences of Humanized Antibodies HzS-III and HzS-IV

The amino acid sequences of the heavy and light chain variable regions of humanized antibodies HzS-III and HzS-IV of the present invention were compared with the amino acid sequences of wild type mouse monoclonal antibody H67 and humanized antibody HZII-H67.

As a result, as shown in Figs . 13 and 14 , the amino acid sequences of the heavy and light chain variable regions of the humanized antibodies HzS-III and HzS-IV of the present invention were compared to those of the heavy chain genes compared to mouse monoclonal antibody H67 and humanized antibody HZII-H67. LCDR1, LCDR2 and LCDR3 of the HCDR1, HCDR2 and HCDR3 or light chain genes contain fewer amino acid residues from the mouse, and HzS-IV has a lower MHC class binding sequence than the heavy chain of HzS-III, resulting in a lower HAMA response. It is expected. Therefore, the humanized antibodies HzS-III and HzS-IV of the present invention exhibit excellent therapeutic effects while inducing less HAMA response than conventional humanized antibodies, and thus may be useful for the treatment of hepatitis B virus.

As described above, the humanized antibody against HBV surface antigen S of the present invention is a human-derived amino acid residues in the HCDR1, HCDR2, HCDR3 of the heavy chain gene and the LCDR1, LCDR2, LCDR3 of the light chain gene are replaced with human-derived amino acids, and thus, humanized antibodies. By including more humanized amino acid residues than antibodies and also reducing the sequence of binding to MHC class II in FR, immunogenic responsiveness in humans is markedly reduced, while exhibiting excellent antigen binding ability to treat hepatitis B virus. It can be usefully used.

1 compares the nucleotide and amino acid sequences of the heavy chain variable region of the humanized antibody HZII-H67 against the surface antigen S of HBV and the humanized heavy chain variable region gene variants of the present invention,

Figure 2 is a schematic diagram showing the manufacturing process of humanized heavy chain gene variant I34V substituted with valine no. 34 isoleucine of mouse HCDR1,

Figure 3 shows a cleavage map of the expression vector pcDdA-HzSIIIh-I34V comprising the gene variant I34V prepared as in Figure 2 ,

4 is a result of measuring the antigen binding capacity of the humanized antibody having a humanized heavy chain gene variant of the present invention to the surface antigen S by ELISA,

A; HCDR1 gene variant B; HCDR2 gene variants

C; HCDR3 gene variants

H / K; Humanized antibody HZII-H67 consisting of heavy and light chains of HZII-H67

5 is a result of measuring the antigen binding ability to the surface antigen S of the humanized antibody having a recombinant gene variant comprising two or more humanized heavy chain gene variants of the present invention by ELISA,

Figure 6 compares the nucleotide and amino acid sequences of the light chain variable region of humanized antibody HZII-H67 against the surface antigen S of HBV and the humanized light chain variable region gene variants of the present invention,

Figure 7 is a schematic diagram showing the manufacturing process of the humanized light chain gene variant Q89L in which glutamine No. 89 of mouse LCDR1 is substituted with leucine,

Figure 8 shows a cleavage map of the expression vector will pcDdA-HzSIIIh-Q89L comprising a gene mutant Q89L produced as shown in Figure 7,

9 is a result of measuring the antigen binding capacity to the surface antigen S of the humanized antibody having a humanized light chain gene variant of the present invention by ELISA,

H / K; Humanized antibody HZII-H67 consisting of heavy and light chains of HZII-H67

10 shows the results obtained by Western blot analysis of humanized antibody HzS-III having a humanized heavy chain recombinant gene variant and a humanized light chain gene variant of the present invention,

A; HzS-III B; HZⅡ-H67

11 is a result of measuring the antigen binding capacity of the humanized antibody HzS-III having the humanized heavy chain recombinant gene variant and humanized light chain gene variant of the present invention to HBV surface antigen S by ELISA method,

12 is a result of measuring the antigen binding affinity of humanized antibodies HzS-III and HzS-IV to HBV surface antigen S having humanized heavy chain recombinant gene variant and humanized light chain gene variant of the present invention by a competitive ELISA method,

FIG. 13 shows the results of comparing the amino acid sequences of the heavy chain variable regions of the humanized antibodies HzS-III and HzS-IV of the present invention with those of the wild-type mouse monoclonal antibody H67 and the humanized antibody HZII-H67.

Figure 14 shows the result of comparing the amino acid sequence of the light chain variable region of the humanized antibody HzS-III of the present invention with the amino acid sequences of the wild type mouse monoclonal antibody H67 and humanized antibody HZII-H67.

<110> Korea Research Institute of Bioscience and Biotechnology <120> A humanized antibody to surface antigen S of hepatitis B virus and a preparing method <130> 1p-07-07B <150> KR10-2000-0057891 <151> 2000-10-02 <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> Homo sapiens <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 26 <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> Homo sapiens <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> Homo sapiens 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 30 Gly Ile Asn Phe Ile Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Thr Ala Ser Asn Lys Gly Thr Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Glu Asp Thr Ala Asn Tyr Phe Cys Gln Gln Ser Lys 85 90 95 Glu 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> Homo sapiens HzS-III-VH <400> 43 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Val Gln Trp Val Arg Gln Ala Pro Gly Lys Ser Leu Glu Trp Met 35 40 45 Gly Tyr Ile Tyr Pro Tyr Ser Gly Gly Thr Gly Tyr Ser Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Val Asp Asn Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Tyr Gly Tyr Asp Ser Ser Gly Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 44 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide sequence which binds to MHC class II molecule <400> 44 Trp Val Arg Gln Ala Pro Gly Lys Ser 1 5 <210> 45 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide sequence which binds to MHC class II molecule <400> 45 Phe Gln Gly Arg Val Thr Leu Thr Val 1 5 <210> 46 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> P33 <400> 46 ggaaagaccc ttgagtggat ggg 23 <210> 47 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> P34 <400> 47 ctcaagggtc tttccggggg cc 22 <210> 48 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> P35 <400> 48 gcagagtcga tttgactgta gacaattc 28 <210> 49 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> P36 <400> 49 cagtcaaatc gactctgccc tggaac 26

Claims (25)

Heavy chain gene variant of a humanized antibody that specifically binds to surface antigen S of hepatitis B virus, comprising amino acid substitutions of one or more of (a) to (d): (a) the amino acid residue 34 isoleucine of HCDR1 of a mouse-derived humanized antibody is substituted with valine; (b) the threonine at amino acid residue 54 of HCDR2 of a mouse-derived humanized antibody is substituted with a serine; (c) the serine of amino acid residue 65 of HCDR2 of the mouse-derived humanized antibody is substituted with glycine; (d) glutamate amino acid residue No. 100 of HCDR3 of a mouse-derived humanized antibody is substituted with serine. The heavy chain gene variant of the humanized antibody of Claim 1 in which the amino acid residue 34 isoleucine of HCDR1 was substituted by valine. The heavy chain gene variant of the humanized antibody of Claim 1 in which the amino acid residue No. 54 threonine of HCDR2 was substituted with serine. The heavy chain gene variant of humanized antibody of Claim 1 whose serine amino acid residue 65 of HCDR2 is substituted by glycine. The heavy chain gene variant of the humanized antibody of Claim 1 in which the amino acid residue 100 glutamate of HCDR3 is substituted with serine. The heavy chain gene variant of the humanized antibody according to claim 1, which comprises all of the gene variants of claims 2 to 5. A pcDdA-HzSIIIh expression vector (Accession Number: KCTC 10083BP) comprising the heavy chain gene variant of the humanized antibody of claim 6. The surface antigen S of claim 1, wherein some amino acids in the peptide sequence that bind to human MHC class II described in SEQ ID NO: 44 or SEQ ID NO: 45 are substituted. Heavy chain gene variants of the binding humanized antibody. delete 9. The heavy chain gene variant of the humanized antibody of claim 8, wherein the P9 position serine of the sequence set forth in SEQ ID NO: 44 is substituted with threonine. The heavy chain gene variant of the humanized antibody of Claim 8 in which the P6 position threonine of the sequence shown by SEQ ID NO: 45 was substituted with aspartic acid. The heavy chain gene variant of humanized antibody of Claim 8 containing all the gene variants of Claims 10-11. A pcDdA-HzSIVh expression vector (Accession Number: KCTC 10080BP) comprising the heavy chain gene variant of the humanized antibody of claim 12. Light chain gene variants of humanized antibodies that specifically bind to surface antigen S of hepatitis B virus, comprising amino acid substitutions of one or more of (a) to (f): (a) amino acid residue 27c of aspartic acid of LCDR1 of a mouse-derived humanized antibody substituted with serine; (b) the amino acid residue 31 serine of LCDR1 of a mouse-derived humanized antibody is substituted with asparagine; (c) the amino acid residue No. 33 methionine of LCDR1 of the mouse-derived humanized antibody is substituted with isoleucine; (d) the amino acid residue 54 glutamine of LCDR2 of the mouse-derived humanized antibody is substituted with lysine; (e) the serine amino acid residue of amino acid residue 56 of LCDR2 of the mouse-derived humanized antibody; (f) Substituted with serine for amino acid residue 91 of the LCDR3 amino acid of a mouse-derived humanized antibody. The light chain gene variant of the humanized antibody of Claim 14 in which the aspartic acid amino acid residue 27c of LCDR1 is substituted with serine. 15. The light chain gene variant of humanized antibody according to claim 14, wherein the serine amino acid residue No. 31 of LCDR1 is substituted with asparagine. 15. The light chain gene variant of humanized antibody according to claim 14, wherein the amino acid residue No. 33 methionine of LCDR1 is substituted with isoleucine. 15. The light chain gene variant of the humanized antibody according to claim 14, wherein the amino acid residue No. 54 glutamine of LCDR2 is substituted with lysine. 15. The light chain gene variant of humanized antibody of claim 14, wherein serine amino acid residue No. 56 of LCDR2 is substituted with threonine. 15. The light chain gene variant of the humanized antibody according to claim 14, wherein the mouse-derived light chain LCDR3 amino acid residue 91 threonine is substituted with serine. 15. The light chain gene variant of humanized antibody according to claim 14, comprising all of the gene variants of claims 15-20. A pCMV-dhfr-HzSIIIk expression vector (Accession Number: KCTC 10084BP) comprising the light chain gene variant of the humanized antibody of claim 21. A transformant in which the heavy chain expression vector of the humanized antibody of claim 7 or 13 and the light chain expression vector of the humanized antibody of claim 22 are simultaneously transformed. A humanized antibody against surface antigen S of hepatitis B virus (HBV) expressed from the transformant of claim 23. 1) searching for a variable region of a heavy or light chain of a humanized antibody composed of mouse-derived CDRs, each searching for a human CDR sequence having the highest sequence homology with each mouse-derived CDR; 2) comparing the mouse-derived CDR sequences with the human CDR sequences having the highest sequence homology found in step 1) and replacing the amino acids of the mouse-derived CDR sequences with amino acids of the human CDR sequences; 3) testing the activity of the genetic variants of the humanized antibody obtained in step 2) to the antigen; 4) mouse-derived CDRs comprising the step of determining the heavy or light chain variable regions of the genetic variants of the humanized antibody by selecting only those substituents that do not degrade the antigen activity based on the results obtained in step 3) A method of making a genetic variant of a humanized antibody that encodes an antibody that is more humanized than a humanized antibody.