KR100345467B1 - Humanized antibody against surface antigen S of hepatitis B virus and preparation method thereof - Google Patents

Abstract

The present invention relates to a humanized antibody against hepatitis B virus (hereinafter abbreviated as "HBV"), specifically the variable region of the mouse monoclonal antibody against the surface antigen S of HBV The heavy chain and light chain genes of humanized antibodies prepared by targeting similar human genes and having much lower amino acid residues derived from mice than conventional humanized antibodies against surface antigen S of HBV, including the expressions comprising the same By producing a humanized antibody against the surface antigen S of HBV by culturing the vector, the transformant transformed with the expression vector, and the transformant, the humanized antibody of the present invention maintains antigen-binding ability similar to that of the existing humanized antibody. At the same time, the immunogenic ability is significantly reduced compared to the existing humanized antibodies, effectively preventing the hepatitis B virus.

Description

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

The present invention relates to a humanized antibody against hepatitis B virus (hereinafter abbreviated as "HBV"), specifically the variable region of the mouse monoclonal antibody against the surface antigen S of HBV Heavy chains of humanized antibodies prepared by targeting similar human genes, which have significantly reduced human-derived amino acid residues compared to conventional humanized antibodies against surface antigen S of HBV, which can significantly improve the human immune response compared to conventional humanized antibodies. ) A gene and a light chain gene, an expression vector comprising the same, a transformant transformed with the expression vector, and a method for producing a humanized antibody against surface antigen S of HBV by culturing the transformant. will be.

HBV is a pathogen that invades the human body and causes chronic and acute hepatitis and, if worse, causes cirrhosis and liver cancer, has an estimated 300 million patients worldwide (Tiollais and Buendia,Sci. Am.,264, 48, 1991). The coat of HBV is composed 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. It consists of a large protein containing. (Neurath, A.R. and Kent, S.B.H.,Adv. Virus Res., 34: 65-142, 1988). All of these surface antigen proteins induce antibodies that neutralize and neutralize HBV, of which S antigens make up 80% of the total coat protein and are common to all subtypes of HBV. in It has a neutralizing epitope.

HBV infections include, but are not limited to, HB immunoglobulins (hereinafter referred to as "to prevent HBV infections", for example, newborns born to HBV-positive mothers, health care workers exposed to HBV, or liver transplant patients with HBV-related liver disease). HBIG "(Beasley et al. , Lancet , 2 , 1099, 1983; Todo et al. , Hepatol. , 13 , 619, 1991). However, since HBIG currently used is extracted from plasma, there is a problem of low specificity to antigen, exposure to pollutants during the manufacturing process, and continuous supply of human blood.

In order to solve the above problem, a method of using a monoclonal antibody against HBV surface antigen of a mouse has been developed. Mouse monoclonal antibodies generally have high antigen affinity and are capable of mass production, but have the disadvantage of inducing an immune response in humans when injected into humans (Shawler et al. , J. Immunol., 135 , 1530, 1985).

In order to solve the above problems, a technique using a human monoclonal antibody has been reported, but a technique for producing a large amount of human monoclonal antibody having high affinity for HBV surface antigen S has not been put to practical use.

Recently, "humanized antibodies" have been developed in which only the antigen-binding sites of mice are implanted into human antibodies in such a way as to reduce the immunogenic ability in the human body while maintaining the high affinity and specificity of mouse-derived monoclonal antibodies. Such humanized antibodies have the advantage that they can be mass-cultured genetically and humanized most of the genes, which greatly reduces the immune response in the human body (Riechmann et al. , Nature , 332 , 323, 1988; Nakatani). et al. , Protein Engineering , 7 , 435, 1994). The challenge remains to reduce the adverse effects of human immune responses that still exist in humanized antibodies.

Therefore, the present inventors have reduced the amino acid residues derived from the mouse much more than the humanized antibody against the surface antigen S of the conventional HBV to produce a humanized antibody with improved human immunogenic ability, the variable region of the mouse antibody against the surface antigen S of HBV After searching for the human genes most similar to the sequences, the light and heavy chain genes of the humanized antibody were prepared, cloned into the expression vectors, the host cells were transformed, and then cultured to obtain the humanized antibodies. Although several amino acid residues were more humanized than humanized antibodies of, the present invention was completed by confirming that the antigen-binding ability similar to the existing humanized antibodies was maintained.

It is an object of the present invention to provide a humanized antibody against HBV surface antigen S, wherein the amino acid residues derived from the mouse are more humanized, resulting in a significantly reduced immunogenic capacity than conventional humanized antibodies against HBV surface antigen S.

1 shows a comparison of amino acid sequences and nucleotide sequences of mouse monoclonal and humanized antibody heavy chains against HBV surface antigen S,

Figure 2 schematically shows a process for obtaining a heavy chain gene pHHC-HZIIS of the humanized antibody against the surface antigen S of HBV by performing recombinant PCR with the humanized antibody heavy chain gene expression vector pRc / CMV-HC-huS as a template,

3 shows a comparison of amino acid sequences and nucleotide sequences of mouse monoclonal and humanized antibody light chains against HBV surface antigen S,

4 schematically shows a process of obtaining recombinant light chain gene pHKC-HZIIS of humanized antibody against surface antigen S of HBV by performing recombinant PCR with humanized antibody light chain gene expression vector pKC-dhfr-huS as a template.

5 shows the heavy chain expression vector pRc / CMV-HC-HZIIS of a humanized antibody against surface antigen S of HBV of the present invention,

Figure 6 shows the light chain expression vector pKC-dhfr-HZIIS of the humanized antibody against the surface antigen S of the HBV of the present invention,

Figure 7 shows the antigen binding affinity of the humanized antibody HZII-H67 of the present invention in comparison with CS131A.

---- ◇ ---- CS131A

---- □ ---- HZⅡ-H67

In order to achieve the above object, the present invention provides an amino acid sequence of a humanized antibody and light or heavy chain thereof reduced in amino acid residues derived from mice more than the existing humanized antibody against the surface antigen S of HBV.

The present invention also provides humanized antibody light and heavy chain genes encoding humanized antibody light and heavy chains with much fewer amino acid residues derived from mice than conventional humanized antibodies against surface antigen S of HBV.

The present invention provides a method of producing a humanized antibody against the surface antigen S of HBV by culturing a transformant transformed with an expression vector comprising the light and heavy chain genes of the humanized antibody against the surface antigen S of HBV.

Hereinafter, the present invention will be described in detail.

In the present invention, the humanized antibody against the surface antigen S of the human antigen HBV of the humanized heavy chain comprising the variable region of the amino acid sequence of SEQ ID NO: 19 and the humanized antibody against the surface antigen S of HBV of the humanized light chain comprising the amino acid sequence of SEQ ID NO: 20 to provide.

The present invention also provides humanized heavy chain gene pHHC-HZIIS for surface antigen S of HBV encoding the amino acid sequence of SEQ ID NO: 19 and humanized light chain gene pHKC-HZIIS for surface antigen S of HBV encoding the amino acid sequence of SEQ ID NO: 20. do.

In the present invention, the expression vector pRc / CMV-HC-HZIIS containing the gene pHHC-HZIIS (Accession No .: KCTCO489BP) and the expression vector pKC-dhfr-HZIIS containing the gene pHKC-HZIIS (Accession No .: KCTCO490BP) To provide.

The present invention also provides a transformant HZII-H67 obtained by transforming COS7 cells with the expression vector pRc / CMV-HC-HZIIS and the expression vector pKC-dhfr-HZIIS.

The present invention also provides a method for producing a humanized antibody against HBV surface antigen S, wherein the transformant HZII-H67 is cultured to obtain a humanized antibody against HBV surface antigen S.

In the present invention, in order to prepare a humanized antibody against the surface antigen S of HBV, first, a human gene is searched most similar to the variable region sequence of the mouse monoclonal antibody H67 against the surface antigen S of HBV.

Gene Bank data base (GenBank data base) After a screening the gene of the person from a human _{V H 21/28 (J. Mol. Biol} ., 227, 776, 1992) is the most similar genes and the mouse antibody variable region gene of the H67 heavy chain Human B1 ( Eur. J. Immunol., 24 , 827, 1994) was confirmed to be the gene most similar to the variable region gene of the mouse antibody H67 light chain.

Therefore, light and heavy chain genes of humanized antibodies are prepared for the genes.

Specifically, to prepare the heavy chain gene of the humanized antibody, all or all of the antigen-binding sites in the heavy chain variable region of mouse monoclonal antibody H67 (Ryu, et al. , Gene ., 144 , 313, 1994) against HBV surface antigen S SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 using an expression vector pRc / CMV-HC-huS (Korean Patent Application No. 96-17625) containing a humanized heavy chain prepared by transplanting a portion into a heavy chain (γ1) of a human antibody PCR is performed using SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and primers of SEQ ID NO: 7 and SEQ ID NO: 8, respectively. The PCR product of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 was conjugated to form a template, and the recombinant PCR was carried out using the primers of SEQ ID NO: 1 and SEQ ID NO: 6, thereby performing a recombinant PCR. Gets a fragment of PCR was carried out using PCR products using the primers of SEQ ID NO: 7 and SEQ ID NO: 8, respectively, to obtain 1428 bp of humanized heavy chain gene, cloned into pBluescript SK (+), and cloned into pHHC-HZIIS. It was named.

The primers of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 used in the process of synthesizing the humanized heavy chain gene are shown in FIG. 2 . Specifically, the primer of SEQ ID NO: 1 has a restriction enzyme Eco RI sequence at its terminal and the primer of SEQ ID NO: 8 has a restriction enzyme Sal I sequence at its terminal.

The variable region of the pHHC-HZIIS humanized heavy chain prepared as above was obtained by substituting the variable region amino acid residues of the seven mouse antibodies of the existing humanized antibody heavy chain with those of the human antibody VH 21/28 heavy chain (see FIG. 1 ). The humanized heavy chain of the present invention comprises a variable region consisting of the amino acid sequence of SEQ ID NO: 19.

The substitution of amino acids is expected to affect the structure of the antibody in existing humanized antibodies, and antigen-antibody binding with several residues that are expected to have a minor effect on antigen binding affinity among the mouse framework amino acid residues used as mice. In the protein database analysis, the amino acid residues at the antigen-binding site that are not considered to participate in the binding to the antigens are used.

In addition, in order to prepare the light chain gene of the humanized antibody, the humanized light chain prepared by transplanting all or part of the antigen-binding site in the light chain variable region of the mouse monoclonal antibody H67 against HBV surface antigen S into the light chain (κ) of the human antibody. SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 17 containing the expression vector pKC-dhfr-huS (Korean Patent Application No. 96-17625) as a template. PCR is performed using each primer of SEQ ID NO: 18. The PCR product of SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 was conjugated to form a template, and recombinant PCR was performed using the primers of SEQ ID NO: 9 and SEQ ID NO: 12 to obtain a fragment of about 274 bp. A 407 bp fragment was synthesized by PCR using the primers of SEQ ID NO: 9 and SEQ ID NO: 16 together with the PCR products using the primers of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively. Using the PCR product using the primers of SEQ ID NO: 17 and SEQ ID NO: 18, respectively, as a template, PCR was performed using the primers of SEQ ID NO: 9 and SEQ ID NO: 18 to obtain a humanized light chain gene of 736 bp, cloned into pBluescript SK (+), and then cloned into pHKC-. Named HZIIS.

The primers of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18 used in the synthesis of the humanized light chain gene are shown in FIG. 4 . Specifically, the primer of SEQ ID NO: 9 has a restriction enzyme Hind III sequence at its terminal and the primer of SEQ ID NO: 18 has a restriction enzyme Sal I sequence at its terminal.

The variable region of the pHKC-HZIIS humanized light chain prepared as described above is a substitution of the variable region amino acid residues of the nine mouse antibodies of the existing humanized antibody heavy chain with those of the human antibody VH 21/28 heavy chain (see FIG. 3 ). The humanized light chain of the present invention comprises a variable region consisting of the amino acid sequence of SEQ ID NO: 20.

The substitution of amino acids, like humanized heavy chains, is expected to affect the structure of antibodies in existing humanized antibodies, with several residues of mouse framework amino acids that are expected to have a minor effect on antigen binding affinity. Protein database analysis of antigen-antibody binding is directed to using residues of human antibodies against amino acid residues at antigen-binding sites that are not thought to participate in binding to antigen.

The present invention provides a method of producing a humanized antibody against surface antigen S of HBV by culturing a transformant transformed with an expression vector comprising the light and heavy chain genes of the humanized antibody against surface antigen S of HBV.

In order to prepare a humanized antibody against the surface antigen S of HBV, first of all, in the present invention, an expression vector including light and heavy chain genes of the humanized antibody is prepared.

Specifically, the humanized heavy chain of the present invention was isolated from the pHHC-HZIIS containing a humanized heavy chain using a restriction enzyme and then inserted into pRc / CMV to prepare the humanized heavy chain expression plasmid pRc / CMV-HC-HZIIS of the present invention. . In addition, the humanized light chain of the present invention was isolated from the above pHKC-HZIIS containing a humanized light chain using a restriction enzyme and then inserted into pCMV-dhfr to prepare the humanized light chain expression plasmid pKC-dhfr-HZIIS of the present invention.

E. coli DH5α strains were transformed with the expression vectors pRc / CMV-HC-HZIIS or pKC-dhfr-HZIIS, respectively, to prepare E. coli transformants. Deposited on June 5, 1989 (Accession No .: KCTC0489BP, KCTC 0490BP).

In the present invention, in order to prepare a humanized antibody against the surface antigen S of HBV, expression vectors pRc / CMV-HC-HZIIS and pKC-dhfr-HZIIS containing the humanized heavy and humanized light chain genes of the present invention are respectively transfected into COS7 cells. Conversion to obtain transformant HZII-H67 and then cultured to isolate the humanized antibody of the present invention.

As a result of investigating the binding capacity and antigen binding affinity of HBV surface antigen S of the humanized antibody obtained as described above, the humanized antibody to HBV surface antigen S of the present invention is similar to the existing humanized antibody, while maintaining the antigen-binding ability. It was confirmed that the immunogenic ability is significantly reduced than the humanized antibody.

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

However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

Example 1 Preparation of Humanized Heavy Chain Gene

To prepare a humanized heavy chain variable region, first, human V _H 21/28 ( J. Mol. Biol. 227 , 776, 1992) having the amino acid sequence most similar to the variable region gene of H67 (Korean Patent Registration No. 102173) was first obtained . Selection was made from the GenBank database. Comparing the amino acid sequence and the nucleotide sequence of the heavy chain variable region of the H67 and the heavy chain variable region of the V _H 21/28 antibody and devised the first humanized antibody Hz I based on this, and then improved the first humanized antibody designed to the second humanized Antibody HzII was designed (see FIG. 1 ).

In addition, recombinant PCR was performed using the previously applied humanized antibody heavy chain gene expression vector pRc / CMV-HC-huS (Korean Patent Application No. 96-17625) as a template to make the heavy chain gene of the second humanized antibody (see FIG. 2 ). ). In the second improved humanized antibody, several residues of the mouse framework amino acid residues used as mice that are expected to affect the antibody's structure in the first humanized antibody are expected to have a minor effect on antigen binding affinity. In the protein database analysis of the antigen-antibody binding, the residue of the human antibody was used as it is for the antigen-binding amino acid residues that are not considered to participate in the binding with the antigen. The primers of recombinant PCR inducing amino acid changes are described in SEQ ID NOs: 1-8.

PCR reaction conditions were performed 30 times using Taq DNA polymerase (1 minute at 94 ℃, 1 minute at 55 ℃, 1 minute at 72 ℃). First, among the DNA fragments obtained using the primers of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 111 bp), the DNA fragments obtained using the primers of SEQ ID NO: 5 and SEQ ID NO: 6 (96 bp) were annealed and linked by recombinant PCR with the primers of SEQ ID NO: 1 and SEQ ID NO: 6, and the synthesized fragment (281 bp) was linked to SEQ ID NO: 7 and SEQ ID NO: 8 DNA fragment (1165bp) synthesized using the primers of SEQ ID NO. 1 and the primers of SEQ ID NO: 1 and SEQ ID NO: 8 were linked. Finally, the synthesized humanized antibody heavy chain gene (about 1428 bp) was cut at both ends by restriction enzymes Eco RI and restriction enzyme Sal I, cloned into pBluescript SK (+), and named pHHC-HZIIS. The base sequence of the prepared humanized antibody heavy chain gene was confirmed by DNA sequencing.

Example 2 Preparation of Humanized Light Chain Gene

In order to prepare a humanized light chain variable region, first, a human B1 ( Eur. J. Immunol. 24 , 827, 1994) having the most similar amino acid sequence to the variable region gene of H67 was selected from the Genbank database, and the light chain variable region of H67 was selected. After comparing the amino acid sequence and the nucleotide sequence of the light chain variable region of the B1 antibody, the first humanized antibody HzI was devised and the second humanized antibody HzII was designed by improving the designed first humanized antibody.

In addition, recombinant PCR was performed using the previously applied humanized antibody light chain gene expression vector pKC-dhfr-huS (Korean Patent Application No. 96-17625) as a template to generate the light chain gene of the second humanized antibody (see FIG. 4 ).

In the second improved humanized antibody, several residues of the mouse framework amino acid residues used as mice that are expected to affect the antibody's structure in the first humanized antibody are expected to have a minor effect on antigen binding affinity. In the protein database analysis of antigen-antibody binding, the residues of human antibodies were used as they were for amino acid residues at positions not believed to participate in binding to the antigen. The primers of recombinant PCR inducing amino acid changes are described in SEQ ID NOs: 9-18.

PCR reaction conditions were performed 30 times in 1 minute at 94 ℃, 1 minute at 55 ℃, 1 minute at 72 ℃. First, among the DNA fragments obtained using the primers of SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 9, SEQ ID NO: 10 (188 bp), SEQ ID NO: DNA fragments obtained using the primers of SEQ ID NO: 11 and SEQ ID NO: 12 (96 bp) were annealed and linked by recombinant PCR with the primers of SEQ ID NO: 9 and SEQ ID NO: 12, and the synthesized fragment (274 bp) was again sequenced using SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 And annealed with 99 bp and 81 bp of DNA fragments synthesized with the primer of SEQ ID NO: 16, followed by recombinant PCR with the primers of SEQ ID NO: 9 and SEQ ID NO: 16. The DNA fragment (407 bp) thus prepared and the DNA fragment (350 bp) synthesized with the primers of SEQ ID NO: 17 and SEQ ID NO: 18 were linked to the primers of SEQ ID NO: 9 and SEQ ID NO: 18 to finally synthesize a humanized antibody light chain gene (736 bp). The synthesized humanized antibody light chain gene was cut at both ends by restriction enzyme Hind III and restriction enzyme Sal I, cloned into pBluescript SK (+), and named as pHKC-HZIIS. The base sequence of the synthesized humanized antibody light chain gene was confirmed by DNA sequencing.

Example 3 Preparation of Humanized Heavy Chain Expression Plasmids

The plasmid pHHC-HZIIS was cut with restriction enzyme Sal I and blunted with Klenow enzyme, and then cut again with restriction enzyme Not I to isolate the humanized heavy chain gene. Meanwhile, pRc / CMV (Invitrogen) was cut with restriction enzyme Xba I, blunted with Klenau enzyme, and cut again with restriction enzyme Not I to separate the vector. The humanized heavy chain gene was linked to the isolated vector to prepare a humanized heavy chain expression vector pRc / CMV-HC-HZIIS. The prepared humanized heavy chain expression vector pRc / CMV-HC-HZIIS was deposited in KCTC (Accession No .: KCTC0489BP), and the completed vector is shown in FIG. 5 .

Example 4 Preparation of Humanized Light Chain Expression Plasmids

The plasmid pHKC-HZIIS was cut with restriction enzymes Hind III and Apa I to separate humanized light chain genes and inserted at the Hind III and Apa I positions of pCMV-dhfr (KCTC8671P) to prepare a humanized light chain expression vector pKC-dhfr-HZIIS. The prepared humanized light chain expression vector pKC-dhfr-HZIIS was deposited in KCTC (Accession No .: KCTC0490BP), and the completed vector is shown in FIG. 6 .

Example 5 Expression of COS7 Cells of Humanized Antibodies

COS7 cells were passaged while maintaining 37 ° C. and 5% carbon dioxide in DMEM culture medium (GIBCO Co., Ltd.) to which calf serum was added 10%. The cells were seeded 1 × 10 ⁶ in a 100 mm dish and incubated at 37 ° C. overnight. Meanwhile, 5 μg of the expression vectors pRc / CMV-HC-HZIIS and pKC-dhfr-HZIIS obtained in Examples 3 and 4 were diluted with 800 μl of OPTI MEM I and 50 μl of lipofectamine (Lipofectamine, GIBCO, respectively). Company) was also diluted with 800 μl OPTI MEM I. The mixtures were mixed in a 15 ml tube and then left at room temperature for at least 15 minutes. COS7 cells inoculated the day before incubating the mixture were washed twice with OPTI MEM I (manufactured by GIBCO). 6.4 ml of OPTI MEM I was added to the DNA-lipopeptamine mixture, mixed and poured uniformly on the washed COS7 cells. After incubation for 72 hours in a carbon dioxide gas incubator, the supernatant of the culture was collected and concentrated by ultrafiltration kit (Ultrafiltration Kit), and then the concentration of the antibody was measured using Sandwich ELISA.

Example 6 Binding Ability of Humanized Antibody to HBV Surface Antigen S

Antibody culture solution obtained in Example 5 was coated with 1 μg of surface antigen S of HBV in each well of a microplate, and sandwiched eliza using the entirety of an anti-human IgG as a capture antibody. The antibody concentration of was quantified. Add 0, 0.25, 0.5, 1, 2, 3, 4, 5, 7.5, 10, 20, 50 ng of each antibody based on the concentration of the quantified antibody, and then add horseradish hydrogen peroxide to the anti-human antibody (Fc specific). Absorbance was measured at 492 nm by an indirect eliza that binds a horseradish peroxidase enzyme conjugated antibody to a secondary antibody. As a control, purified CS131A (Ryu et al. , J. Med. Virol. , 52 , 226, 1997) was used, and the results are shown in Table 1 .

Binding Capability of CS13 and HZII-H67 to HBV Surface Antigen S (Absorbance, 492nm) Amount (ng) 0 0.25 0.5 One 2 3 4 5 7.5 10 20 50 CS13 0.04 0.26 0.43 0.79 1.23 1.67 1.98 2.23 2.64 2.90 3.34 3.40 HZⅡ-H67 0.04 0.15 0.22 0.42 0.67 0.94 1.16 1.37 1.73 2.02 2.69 3.24

Example 7 Antigen Binding Affinity of Humanized Antibodies

Antigen binding affinity was measured using a competitive ELISA method (Competitive ELISA; Ryu et al. , J. Med. Virol. , 52 , 226, 1997) to determine the affinity of the humanized antibody for HBV antigen. First, to the HBV antigen of 1 × 10 ^-7 ~ 1 × 10 ^-12 M concentration, the antibody produced in COS7 and 5 ng of CS131A, a control group, were respectively bound at 37 ° C. for 2 hours. Binding to antigen coated in 96 wells. From the results, the amount of antigen bound to HBV was finally measured, and the results are shown in FIG. 7 . Comparison of antigen binding affinity between CS131A and HZII-H67 showed that CS131A was about 6 × 10 ⁸ M ^-1 and HZII-H67 was 5 × 10 ⁸ M ^-1 , which showed a large difference in antigen binding affinity between the two antibodies. It was confirmed that no.

As described above, the humanized antibody against the HBV surface antigen S of the present invention exhibits similar antigen binding ability as the conventional humanized antibody, whereas the amino acid residues are more humanized than the existing humanized antibody, thereby significantly reducing the immunogenic ability in the human body. The antibody may be useful for the prevention of hepatitis B virus.

[ List of Sequences]

SEQ ID NO: 1

Sequence length: 26

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO: 1]

[ List of Sequences]

SEQ ID NO: 2

Sequence length: 39

Type of sequence: Nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO 2]

[ List of Sequences]

SEQ ID NO: 3

Sequence length: 39

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO 3]

[ List of Sequences]

SEQ ID NO: 4

Sequence length: 30

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO 4]

[ List of Sequences]

SEQ ID NO: 5

Sequence length: 36

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO: 5]

[ List of Sequences]

SEQ ID NO: 6

Sequence length: 27

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO: 6]

[ List of Sequences]

SEQ ID NO: 7

Sequence length: 27

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO: 7]

[ List of Sequences]

SEQ ID NO: 8

Sequence length: 26

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO: 8]

[ List of Sequences]

SEQ ID NO: 9

Sequence length: 26

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO: 9]

[ List of Sequences]

SEQ ID NO: 10

Sequence length: 36

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO: 10]

[ List of Sequences]

SEQ ID NO: 11

Sequence length: 36

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO: 11]

[ List of Sequences]

SEQ ID NO: 12

Sequence length: 24

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO: 12]

[ List of Sequences]

SEQ ID NO: 13

Sequence length: 24

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO: 13]

[ List of Sequences]

SEQ ID NO: 14

Sequence length: 36

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO: 14]

[ List of Sequences]

SEQ ID NO: 15

Sequence length: 36

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO: 15]

[ List of Sequences]

SEQ ID NO: 16

Sequence length: 21

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO: 16]

[ List of Sequences]

SEQ ID NO: 17

Sequence length: 21

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO: 17]

[ List of Sequences]

SEQ ID NO: 18

Sequence length: 27

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO: 18]

[ List of Sequences]

SEQ ID NO: 19

Sequence length: 119

Type of sequence: amino acid

Number of chains: 1 chain

Form: Straight

Type of sequence: protein

[SEQ ID NO: 19]

[ List of Sequences]

SEQ ID NO: 20

Sequence length: 112

Type of sequence: amino acid

Number of chains: 1 chain

Form: Straight

Type of sequence: protein

[SEQ ID NO: 20]

Claims (8)

A humanized antibody against surface antigen S of HBV, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 19. A humanized antibody against surface antigen S of HBV, wherein the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 20. Humanized heavy chain gene for surface antigen S of HBV encoding a humanized heavy chain, wherein the amino acid sequence of the variable region is set forth in SEQ ID NO: 19. Humanized light chain gene for surface antigen S of HBV encoding a humanized light chain, wherein the amino acid sequence of the variable region is set forth in SEQ ID NO: 20. Heavy chain expression vector pRc / CMV-HC-HZIIS of humanized antibody against the surface antigen S of HBV containing the humanized heavy chain gene of Claim 3 (Accession No .: KCTC 0489BP). Light chain expression vector pKC-dhfr-HZIIS of a humanized antibody against the surface antigen S of HBV comprising the humanized light chain gene of claim 4 (Accession No .: KCTC 0490BP). Transformant HZII-H67 obtained by transforming COS7 cells with expression vector pRc / CMV-HC-HZIIS and expression vector pKC-dhfr-HZIIS. A method for producing a humanized antibody against HBV surface antigen S, wherein the transformant of claim 7 is cultured to obtain a humanized antibody against HBV surface antigen S.