KR100467706B1 - Human antibodies against the surface antigen of HBV - Google Patents

Abstract

The present invention relates to a nucleotide sequence of the human antibody gene (DNA) against the surface antigen (HBsAg) of hepatitis B virus (HBV), an animal cell expression vector, a cell line transformed with the expression vector, and a human antibody therefrom. It relates to a method of manufacturing.

Description

Human antibodies against the surface antigen of HBV

The present invention relates to a human antibody against the surface antigen of Hepatitis B virus (HBV), and more particularly to the hepatitis B virus surface antigen. The present invention relates to a nucleotide sequence of a human antibody gene (DNA), an animal cell expression vector, a cell line transformed with the expression vector, and a method for producing a human antibody from the same.

In general, the hepatitis B virus is a major pathogen of hepatitis and liver cancer, which infects about 300 million people worldwide and about 4 million people in Korea. It is an important infectious disease that threatens human health. Progresses to liver cancer. Although vaccines are now preventable, there are still a large number of patients with chronic hepatitis caused by the hepatitis B virus.

Hepatitis B antibody preparations are administered to prevent vertical infection of newborns born to mothers of HBV carriers when exposed to blood containing HBV, and have recently been used to prevent HBV infection during liver transplantation. In addition, administration of antibodies has been attempted for the treatment of chronic hepatitis patients.

Hepatitis B antibody preparations that have been used so far are collected by the blood of people who carry them and are produced using advanced purification technology and virus inactivation technology, but the purchase of raw plasma is difficult and costly. Compared to this, there is a problem that it is not possible to flexibly cope with the increasing demand. There is also a disadvantage that it takes a lot of time and money to remove contamination from viruses derived from human plasma.

Since the monoclonal antibody (MAb) manufacturing method was established by Kohler and Milstein in 1975, the main antibody used is the MAb made in the mouse for diagnosis and some treatment. However, when a mouse antibody is applied to a human body for therapeutic purposes, there is a problem in that a human antibody to a mouse antibody (human anti-mouse Ab: HAMA) is generated in the human body, thereby causing a problem of continuous administration for therapeutic purposes. In order to overcome this problem, chimeric and humanized antibodies have been developed by reducing the amino acid residues of mouse antibodies and replacing them with amino acid residues of human antibodies. However, the HAMA problem has not been solved. Chimeric antibodies have about 70% human nucleotide sequences and humanized antibodies have 90-95% human nucleotide sequences. However, chronic hepatitis treatment and liver transplantation are required to repeatedly administer the antibody over a long period of time, in which case the use of human antibodies rather than the use of chimeric or humanized antibody is absolutely required to reduce the side effects of repeated administration.

The present invention solves the above problems, and the object of the present invention is to provide a human antibody for the prevention and treatment of HBV infection.

1 is an illustration of the phage-display vector pFAB73 into which the F chain and the L chain of an H chain of an antibody used in the present invention are inserted.

Figure 2 is a picture of the electrophoresis of the antibody Fd and L chain DNA synthesized by PCR in 1.2% agarose gel.

FIG. 3 shows a panning of antibodies from an antibody library. FIG.

4 is a graph measuring the affinity of HBsAg of antibodies (Fab) selected from antibody libraries.

Figure 5 shows the H chain animal cell expression vector pRC13-Hpa of the antibody.

Fig. 6 shows the L chain animal cell expression vector pKC12-BH of an antibody.

Figure 7 is a photograph of the SDS-PAGE analysis of purified antibody expressed in animal cell line.

8 is a graph measuring the affinity for HBsAg of antibodies expressed in animal cell lines.

In order to achieve the above object, the present invention provides an amino acid sequence of SEQ ID NO: 1 encoding the variable region of the human antibody H chain to the surface antigen of hepatitis B virus (HBAb-H4); And

Provided is a human antibody protein against the surface antigen of hepatitis B virus comprising the amino acid sequence of SEQ ID NO: 2 (HBAb-L9) encoding the variable region of the human antibody L chain against the surface antigen of hepatitis B virus.

In another aspect, the present invention is a sequence encoding a variable region of human antibody DNA and L chain to the surface antigen of hepatitis B virus comprising the base sequence (baseHBAb-H4) of SEQ ID NO: 3 encoding the variable region of the H chain Human antibody DNA to the surface antigen of hepatitis B virus containing the base sequence of information 4 (baseHBAb-L9) is provided.

In addition, the present invention is an animal that can easily insert the antibody H chain expression plasmid pRC13-Hpa and the L chain variable region of the human antibody in the animal cell that can easily insert the variable region of the H chain of the human antibody Antibody L chain expression plasmid pKC12-BH in cells is provided.

The present invention also encodes the variable region of the plasmid pRC13-Hpa-HB-H4 and the L chain of a human antibody, including the nucleotide sequence of base information 3 (baseHBAb-H4) encoding the variable region of the H chain of the human antibody. It provides a plasmid pKC12-BH-HB-L9 comprising the nucleotide sequence of SEQ ID NO: 4 (baseHBAb-L9).

In addition, the present invention is the above plasmid pRC13-Hpa-HB-H4; And the Chinese hamster ovary (CHO) cell line HBAb-49 (KCLRF-BP-00054) transformed with the plasmid pKC12-BH-HB-L9.

The cell line of the present invention was deposited with the strain accession number KCLRF-BP-00054 on November 30, 2001 to the Korea Cell Line Research Foundation located at Seoul National University College of Medicine, Jongno-gu, Seoul, Korea.

The present invention also comprises the steps of culturing the cells of claim 8; Expressing a human antibody protein against the surface antigen of hepatitis B virus using the cultured cells; And it provides a human antibody method for the surface antigen of hepatitis B virus comprising the step of purifying the expressed protein and human antibody protein for HBV surface antigen prepared by the above method.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

In the present invention, human antibody genes for HBsAg were selected and animal cell lines expressing human antibodies were established to develop human antibodies with increased safety, which can be used for the prevention of HBV infection, treatment of chronic hepatitis, and liver transplantation aids. .

In particular, in the present invention, phage-display technology was used to select human antibodies against HBsAg. This phage-display technology (Smith, Science, 228, 1315-1317, 1985; Hoogenboom & Chames, Immunol Today, 21, 371-378, 2000) is a filamentous phage (M13, Fd, F1) gene that expresses epidermal protein. The gene expressing the antibody of interest is fused to (gene III) to generate antibody-phage virus particles exposed to the surface of the bacteriophage particle, thereby exposing the high specificity and affinity and phage of the exposed antibody. It is a state-of-the-art molecular biology technology widely used in the study of antibody engineering that allows high infectivity to select desired antibodies from phage libraries using biopanning techniques (Burton & Barbas, Adv Immunol., 57, 191-280). , 1994; Winter et al., Annu Rev Immunol., 12, 433-455, 1994); Hoogenboom et al., Immunotechnology, 4, 1-20, 1998).

The phage-display vector pFAB73 used in the present invention is Dr. Used from Jan Engberg (Engberg et al., Mol Biotechnol., 6, 287-310, 1996) (see FIG. 1).

In addition, the primers used for cloning the human antibody DNA used in the present invention are shown in Table 1.

TABLE 1 Primers used for cloning and sequencing of human antibody DNA

Ab chain Prime name Primer sequences (5 '→ 3') Fd VH1 (38 bp) VH2 (38 bp) VH3 (38 bp) VH4 (38 bp) CAG CCA GCA ATG GCA CAG GTG CAG CTG CTC GAG TCT GGCAG CCA GCA ATG GCA CAG GTG CAG CTG CTC GAG TCG GGCAG CCA GCA ATG GCA CAG GTG CAA CTG CTC GAG TCT GGCAG CCA GCA ATG GCA CAG GTG GA CTG CTC CH1 (30 bp) GGT GCA TGC GGC CGC ACA AGA TTT GGG CTC VH.EXT (39 bp) GCT GGA TTG TTA TTG CTA GCA GCA CAG CCA GCA ATG GCA Kappa VLK (51 bp) CGC GAA TTG GCC CAG CCG GCC ATG GCC GAG CTC GTG ATG ACC CAG TCT CCA CLK (48 bp) GTC TCC TTC TCG A GG CGC GCC TCA TTA ACA CTC TCC CCT GTT GAA GCT Lambda VLL (47 bp) CGC GAA TTG GCC CAG CCG GCC ATG GCC GAG CTC GTG GTG ACT CAG CC CLL (45 bp) GTC TCC TTC TCG AGG CGC GCC TCA TTA TGA ACA TTC TGT AGG GGC Sequencing Fd GTG CCC CCA GAG GTG CTC TTG G Kappa GGC AGT TCC AGA TTT CAA CTG Lambda GCG GAT AAC AAT TTC ACA CAG

Hereinafter, the present invention will be described in more detail with reference to non-limiting examples.

Example 1: Isolation of RNA

In the present invention, hepatitis B vaccine (Hepavax-Gene; Green Cross vaccine, Korea) was injected into 10 persons twice at 4 week intervals and blood was collected by 10 ml after 7 days to secure the gene of the hepatitis B antibody. The collected blood was collected and diluted with 100 ml of Hank's balanced salt solution (HBSS; Sigma, USA), 10 ml of Histoprep (Sigma) was added to a 50 ml tube, and 20 ml of HBSS and diluted blood were superimposed thereon. The leukocyte layer was separated by centrifugation at 3000 rpm. The white blood cells thus collected were collected, and 6 ml of HBSS and 2 ml of white blood cells were mixed and centrifuged at 1000 rpm to collect white blood cells. RNA was isolated by mixing 100 μl of white blood cells with 1 ml of TRIZOL (Life Technology, USA). The RNA thus separated was diluted with distilled water and the absorbance was measured at 260 nm to calculate the amount thereof (1.8 μg / μl). The detailed process is as follows.

1 ml of trizol per 100 μl of leukocytes were added to the cells, followed by pipetting, followed by waiting at room temperature (15 ° C. to 30 ° C.) for 5 minutes. Next, 200 μl of chloroform per 1 ml of Trizol was mixed well, the lid was closed and shaken vigorously for 15 seconds by hand, and then waited at room temperature for 3 minutes. Thereafter, the mixture was centrifuged at 15,000 rpm for 15 minutes at 2 to 8 ° C, and only the supernatant was transferred to a new tube. 500 μl of isopropyl alcohol was added per 1 ml of Trizol, mixed well, and then waited at room temperature for 10 minutes. Thereafter, the mixture was centrifuged at 15,000 rpm for 10 minutes at 2 to 8 ° C, and the supernatant was removed. 1 ml of 75% ethanol was added per 1 ml of Trizol, and centrifuged at 15,000 rpm for 5 minutes at 2 ° C. to 8 ° C., the ethanol was discarded, and the RNA pellet was dried briefly. 150 μl of distilled water was dissolved in the RNA, and then the absorbance was measured and measured at 260 nm. It was then stored at -20 ° C.

Example 2: Antibody Gene Amplification

5 μl of RNA isolated in Example 1, 1 μl of pd (T) _12-18 (0.5 μg / μl), and 34 μl of distilled water were added and mixed so that the total volume was 40 μl. The mixture was reacted at 65 ° C. for 5 minutes and then cooled slowly to 45 ° C. for 25 minutes. Ready-To-Go RT-PCR Bead (Amersham Pharmacia Biotech, USA) was added thereto and reacted at 42 ° C. for 30 minutes to synthesize cDNA. It was then left at 95 ° C for 5 minutes and immediately cooled on ice. The beads include Taq DNA polymerase, dNTP, Moloney Murine Leukemia Virus Reverse Transcriptase, RNAguard Ribonuclease inhibitors and stabilizers. In the cooled mixture, 2 μl (25 pmol / μl) of 5 'primer, 2 μl (25 pmol / μl) of 3' primer and 6 μl of distilled water were added to the Fd, kappa, and lambda chains. The PCR reaction was carried out for 5 minutes at 95 ° C., for 1 minute at 95 ° C., 2 minutes at 56 ° C., and 2 times at 72 ° C., and finally at 72 ° C. for 15 minutes.

Primers VH1 to VH4 were mixed in the same ratio, and PCR was performed with primer CH1. PCR was performed using primers VH.EXT and primer CH1 again to clone Fd DNA using 2 μl of the PCR DNA obtained here.

The primers VLK and VLL were mixed in the same amount, and the primers CLK and CLL were also mixed in the same amount and PCR was performed to clone the light chain DNA.

The antibody DNA thus synthesized was confirmed by electrophoresis on 1.2% agarose gel (see FIG. 2), and each DNA was purified by a Qiagen kit (Qiagen, Germany). As shown in Figure 2, PCR obtained about 700 base pairs of DNA bands specific for the Fd and L chain. In FIG. 2, M is a DNA size label, H is a PCR result of Fd, and L is a PCR result of light chain.

Example 3: Restriction Enzyme Cleavage of Antibody DNA

In the DNA prepared in Example 2, Fd was cut using restriction enzymes NheI / NotI, kappa and lambda using restriction enzyme SfiI / AscI, and each chain cut with restriction enzyme was separated by electrophoresis using 1.2% agarose gel. The isolated DNA was purified by Qiagen kit.

Example 4: ligation and library preparation of antibody DNA

Phage-display vector pFAB73 was digested with restriction enzyme NheI / NotI and purified by 1.2% agarose gel electrophoresis and Qiagen kit. 180 ng of Fd prepared in Example 3 and 600 ng of pFAB73 (all cut with NheI / NotI) were mixed, and T4 DNA ligase (New England BioLabs, USA) was added and reacted at room temperature overnight. The ligation mixture was purified by Qiagen kit, transformed into E. coli XL1-blue cells by electroporation, grown in 100 ml of medium overnight for isolation of plasmids, and named HB-ΔL-Fd-pFAB73.

The HB-ΔL-Fd-pFAB73 plasmid was digested with restriction enzyme SfiI / AscI and purified as above, and 1.4 μg of HB-ΔL-Fd-pFAB73 plasmid and 300 ng of L chain PCR DNA (all were cut with SfiI / AscI). ) And T4 DNA ligase (New England BioLabs, USA) was added and allowed to react overnight at room temperature. The ligation mixture was purified by Qiagenkit and transformed into E. coli XL1-blue cells by electroporation. Carbenicillin and tetracyclin were added to 100 ml of medium and grown at 37 ° C for 2 hours, followed by M13 helper phage (Stratagene, USA). Phage libraries were made by inoculation for 16 hours and reported (Engberg et al., Mol Biotechnol., 6, 287-310, 1996). Plasmids were isolated from cultured E. coli and named HB-Fab-pFAB73.

Example 5: Antibody Screening for HBsAg

Antibodies that bind HBsAg were selected using panning techniques (Engberg et al., Mol Biotechnol., 6, 287-310, 1996; Kim et al., Gene, 241, 19-25, 2000). The method is diluted HBsAg (provided with green cross vaccine) to 5μg / ml with PBS buffer and coated in 1 ml each time on Immunotube (NUNC, Denmark), 1ml of the phage library prepared above was reacted for 2 hours at 37 ?? After washing, remove the phages attached to HBsAg with 0.1M Glycine Buffer (pH 2.0) containing 1% BSA, neutralize them with 1M Tris solution, and then infect E. coli XL1-blue cells and add helper phages. Incubate overnight in 100 ml of medium, add 1/5 volume of polyethylen glycol (PEG) solution (solution containing 20% PEG 8,000 and 15% NaCl) and store on ice for 30 minutes to precipitate phage particles to collect phage. The above process was repeated five times by reacting the HBsAg coated Immunotube. The process of antibody screening using phage-display library is shown in FIG. 3.

Each colony of five panning libraries was grown in 2 ml of medium according to known methods (Kim et al., Gene, 241, 19-25, 2000) to induce antibody expression by treatment with IPTG.

Antibody expression was measured by ELISA using a 96 well plate coated with HBsAg.

Example 6: Sequencing of Selected Antibodies

The antibody secreting colony selected in Example 5 was incubated overnight in 10 ml of LB medium containing 50 μg / ml carbenicillin, followed by recombinant plasmid using a Qiagen plasmid mini kit (Qiagen, Valencia, CA, USA). Was isolated and cleaved with each restriction enzyme (Fd: NheI / NotI; L: SfiI / AscI) and subjected to electrophoresis on an agarose gel to confirm that the fragments of each antibody were correctly inserted into the Fd and L chains of the plasmid. DNA sequencing was confirmed.

The sequencing primer exemplified in Table 1 was used as the primer for sequencing, and the sequencing primer was analyzed by Macrogen (Seoul, Korea) according to a known method.

Example 7: Affinity Measurement of Selected Antibodies

To determine the affinity of the selected antibodies for HBsAg, affinity was measured using a competetion ELISA method (Kim et al., Hybridoma, 20, 265-272, 2001). The results of measuring the affinity of the selected antibodies are shown in FIG. 4.

The detailed process is as follows.

(1) Determination of Proper Antibody Concentration

A. Preparation of Plates

HBsAg (provided with green cross vaccine) was diluted to 10 μg / ml in PBS buffer, aliquoted by μl and incubated at 4 ° C. overnight. The plate was washed once with PBST, then 200 μl of 1% BSA-PBS solution was stored and stored at room temperature for 1 hour.

B. First Reaction

The E. coli culture containing the expressed antibody was diluted with PBS in several steps, 100 μl of each well of the plate was reacted at room temperature for 2 hours, and washed 5 times with PBST.

C. Secondary Reaction

Goat anti-human IgG (Fab specific) -peroxidase conjugate (Sigma) was diluted 5,000-fold with 1% BSA-PBS solution, added to 100 μl of each well, and then reacted at room temperature for 1 hour and washed 5 times with PBST.

D. Substrate Reaction

100 μl of ABTS (KPL, MD, USA) solution was added to each well, and O.D. was measured at 405 nm. A half concentration of the antibody concentration showing the maximum binding value was determined as an appropriate antibody concentration.

(2) competitive ELISA

A. Preparation of Plates

HBsAg (provided with green cross vaccine) was diluted to 10 μg / ml with PBS buffer, then 100 μl was dispensed into each well and incubated at 4 ° C. overnight. The plates were washed once with PBST, then 200 μl of 1% BSA-PBS solution was dispensed into each well and stored at room temperature for 1 hour.

B. First Reaction

Dilute HBsAg to 500 μg / ml-1 ng / ml and place it in the plate prepared in 10μl, and then dilute the antibody to the appropriate antibody concentration determined in (1), add 90μl each, react for 2 hours at room temperature, and wash 5 times with PBST. It was.

C. Secondary Reaction

Goat anti-human IgG (Fab specific) -peroxidase conjugate (Sigma) diluted 5,000-fold with 1% BSA-PBS solution was added to each well 100μl, reacted for 1 hour at room temperature, and then washed 5 times with PBST.

D. Substrate Reaction

100 μl of ABTS (KPL, MD, USA) solution was added to each well, and O.D. was measured at 405 nm. The HBsAg concentration that inhibits 50% of the maximum binding value (ELISA O.D value without competing HBsAg) was calculated as affinity (Kd).

Example 8 Preparation of Animal Cell Expression Vector

In order to convert the selected antibody fragments into a form for producing a complete form of immunoglobulin, the antibody expression vectors of Green Cross [(pRC / CMV-HC-huS and pKC-dhfr-huS: "humanized antibodies against hepatitis B S" patent (Korean Patent No. 191900)] However, in order to use these vectors as expression vectors of other antibodies, the method of splicing by overlap extension PCR (Horton et al., Gene, 15, 77, 61-68, 1989) and There are inconveniences in using the PCR, primers, etc. for each antibody, such as several steps, etc. Therefore, in the present invention, the variable region of the antibody can be easily inserted into a well-known expression vector. -huS and pKC-dhfr-huS were modified.

That is, as illustrated in Figure 5, the Npa site using primer-Nhe (CAG GTC GAC TAG AGC TAG CTA TTC TAT AGT G) for the ApaI site present in the C terminal of the H chain expression vector pRC / CMV-HC-huS The HpaI site, which was not converted to the N terminal of the vector, was newly created using primer-Hpa (CAG AGT GAG GTT AAC CTG GTG CAA TCT GG), and the resulting vector was named pRC13-Hpa. These site-directed mutagenesis were performed according to the method presented using the GeneEditor in vitro Site-Directed Mutagenesis System (Promega, USA). Therefore, by using the ApaI site present in the middle portion, the antibody fragments produced by PCR can be easily inserted into the HpaI / ApaI site. The experimental method is as follows. Vector pRC / CMV-HC-huS 0.05 pmol, phosphorylated selection oligonucleotide 0.25 pmol, phosphorylated primer-Nhe 1.25 pmol, phosphorylated primer-Hpa 1.25 pmol, annealing 10 x buffer 2 μl, and distilled water to a total volume of 20 μl gave. The mixture was reacted with boiling water for 5 minutes, ice for 5 minutes and then at room temperature for 30 minutes. 5 μl of distilled water, 3 μl of systhesis 10 x buffer, 1 μl of T4 DNA polymerase, and 1 μl of T4 DNA ligase were added to the mixture, followed by reaction at 37 ° C. for 90 minutes. The reaction mixture was transformed into XLmuts Kan ^r competent cells (Stratagene, USA) by heat-shock method. Plasmids were isolated from the transformed cells, and the plasmids were transformed into JM 109 competent cells by heat-shock method. Plasmids were isolated from transformed JM 109 competent cells, cut with restriction enzymes HpaI / ApaI and electrophoresed on agarose gel. The plasmid with the appropriate size fragment was named pRC13-Hpa. In the present invention, pRC13-Hpa is prepared by PCR using a primer of DNA (GCC ATG GCC CAG GTT AAC CTG CTC GAG TCG) and primer (GGG AAG ACC GAT GGG CCC TTG GTG GAG GCT G). And inserted into the pRC13-Hpa-HB-H4.

As illustrated in FIG. 6, for the L chain expression vector pKC-dhfr-huS, a primer-reHind (CTC ACT ATA GGG AGA CCC GCT AGC GGA AGC AAG ATG G) was used as the NheI site for the HindIII site present in the N terminal. Substitute the BstEII site [primer-BstE (GGG GAC ATT GTG GTG ACC CAA TCT CCA GC)] on the N terminal of the vector and the HindIII site [primer-Hind (GGA GGG GGG ACC AAG CTT GAA ATA AAA CGG) Used, and the resulting vector was named pKC12-BH. Therefore, the antibody fragment produced by PCR can be easily inserted into the BstEII / HindIII site. pKC12-BH was prepared in the same manner as pRC13-Hpa, but instead of 1.25 pmol phosphorylated primer-Nhe and 1.25 pmol phosphorylated primer-Hpa, 1.25 pmol, phosphorlyated primer-BstE 1.25 pmol, phosphorlyated primer-Hind 1.25 pmol The difference is that I wrote. In the present invention, the DNA of the HBAb-L9 by PCR using a primer (GAG CTC GTG GTG ACC CAG TCT CCA) and a primer (TCG TTT GAT TTC AAG CTT GGT CCC TTG) is inserted into pKC12-HB. It was named -BH-HB-L9.

Example 9: Preparation of Antibody-releasing Animal Cell Line

24 hours prior to the introduction of pRC13-Hpa-HB-H4 and pKC12-BH-HB-L9 prepared in Example 8, 2 * 10 ⁵ chinese hamster overy (CHO) cells were added with 10% FBS (Life Technologies, USA). Culture in a T-25 (NUNC, Denmark) flask containing Alpha-MEM (Life Technologies, USA). CHO cells are cultured in 5% CO ₂ and 37 ° C incubators. The next day, the cell confluency is about 50%, and 30 μg of lipofectin (Life Technologies, USA) is placed in 1.5 ml Opti-MEM (Life Technologies, USA) medium and left at room temperature for 90 minutes. At the same time, 8 μg of pRC13-Hpa-HB-H4 and 7 μg of pKC12-BH-HB-L9 are mixed and placed in 1.5 ml of Opti-MEM medium and left at room temperature for 90 minutes. After 90 minutes, the medium containing lipofectin and the medium containing pRC13-Hpa-HB-H4 and pKC12-BH-HB-L9 are mixed and allowed to react at room temperature for 15 minutes. During the reaction, the medium in the flask in which the cells to be introduced are grown is removed and washed three times with PBS. Put the mixed solution reacted for 15 minutes to the washed cells and incubate. After 6 hours, remove the mixed solution, add Alpha-MEM medium and incubate for 48 hours. After 48 hours of incubation, the cells were treated with trypsin (Life Technologies, USA), removed from the flask, diluted with Alpha-MEM medium, and passaged to a 96 well plate (NUNC, Denmark). Alpha-MEM medium does not contain ribonucleosides and deoxyribonucleosides and contains 10% dialyzed FBS (Life Technologies, USA) and 550 μg / ml G418 (Sigma, USA). The medium is changed every 2 days, the culture supernatant of the colony-formed wells is recovered and subjected to ELISA, and the selected cells are transferred to 12 wells. If you sleep well in 12 wells, transfer to 6 wells. If you sleep well in 6 wells, treat MTX (Sino-Pharma). Start at 20 nM and treat 80 nM if you sleep well, then gradually increase the MTX concentration to 320 nM, 1 μM. Cell lines with high antibody secretion are selected and survived at 1 μM and cultured in large quantities. Mass cultivation uses spinner flasks and serum-free media and conditions are performed at 65 rpm, 5% CO ₂ , 37 ° C. incubator. In a 250 ml spinner flask, put 10 ⁸ cells in 100 ml of serum-free medium and grow. When the number of cells is doubled, the culture solution is centrifuged at 1000 rpm for 5 minutes to separate the culture supernatant and the cells separately. The isolated cells are incubated in a 500 ml spinner flask containing 200 ml of medium. When the number of cells doubled, centrifuge to separate the culture supernatant and cells, and incubate the separated cells in a 1 liter spinner flask containing 400 ml medium. When doubled, the cells are also separated and transferred to a 3 liter spinner flask containing 1 liter of medium. Sodium butyrate (Aldrich, USA) is added to a final concentration of 2 mM and incubated for 5 days and the culture supernatant is recovered. Antibodies were purified using protein A-agarose column (Amersham Pharmacia Biotech, USA) from all culture supernatants recovered by incubation in spinner flasks, band identification on SDS-PAGE (FIG. 7), affinity measurement (8 degrees), etc. Was implemented.

As shown in Figure 7, SDS-PAGE results in the heavy chain of about 50 kd and light chain band of about 25 kd was observed under reducing conditions, it can be seen that the antibody is synthesized correctly produced. As compared with FIG. 8, the affinity was measured to confirm that the affinity was about 9 times higher than that of the humanized antibody of Green Cross [(“humanized antibody against hepatitis B” patent ”(Korean Patent No. 191900)). .

The present invention has established a method for producing human antibodies against surface antigens of hepatitis B virus. It can overcome the difficulty of supplying raw materials of antibodies isolated from blood and the fear of human-derived virus infection, and can be developed as a treatment for chronic hepatitis caused by HBV.

The human antibodies produced in this way can reduce side effects caused by human anti-mouse antibody (HAMA) reactions of chimeric and humanized antibodies from mouse antibodies. It is possible.

In addition, the phage library production and antibody screening technology, antibody expression vector production, animal cell line construction and screening technology and the like established through the present invention can be applied to the development of other therapeutic antibodies and protein medicines.

<110> KOREA GREEN CROSS CORPORATION <120> Human antibodies against the surface antigen of HBV <160> 26 <170> KopatentIn 1.71 <210> 1 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Variable region of human antibody H chain <400> 1 Gln Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Ser Leu Thr Lys Tyr 20 25 30 Lys Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Thr Ser Arg Asp Ile Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Val Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Asp Gly Trp Leu Trp Gly Trp Asp Val Arg Ser Asn Tyr 100 105 110 Tyr Asn Ala Leu Asp Val Trp Gly Gln Gly Thr Thr Val Val Val Val Ser 115 120 125 Ser <210> 2 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Variable region of human antibody L chain <400> 2 Glu Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Tyr Asn Ser 20 25 30 Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu 35 40 45 Tyr Ser Thr Ser Thr Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Thr Asn Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Th r Tyr Tyr Cys Gln Gln Tyr Phe Val Thr Pro Glu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 3 <211> 387 <212> DNA <213> Artificial Sequence <220> < 223> Base sequence encoding variable region of H chain <400> 3 caggtgaaac tgctcgagtc ggggggaggc ctggtcaagc cgggggggtc cctgagactc 60 tcctgttcag cctctggatt cagcctcact aaatataaga tgacctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcatcc attagtagta caagtagaga catcgactac 180 gcagactcag tgaagggccg attcaccatc tccagagaca acgccaagaa ctcgctcttt 240 ctccaaatga gcagcctgag agtcgacgac acggctgttt attactgtac cagagacggg 300 tggctctggg gctgggacgt caggtccaac tactattaca acgctttgga cgtctggggc 360 caagggacca cggtcaccgt ctcctca 387 <210> 4 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Base sequence encoding variable reg ion of L chain <400> 4 gagctcgtga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcgagtca gggcatttac aactctatag cctggtatca gcagaaacca 120 gggaaagccc ccaaactcct gctctattct acatccacat tgcttagtgg ggtcccatcc 180 aggttcagtg gcagtggctc tgggacggat tacactctca ccatcaccaa cctgcagcct 240 gaagattttg caacttatta ttgtcaacag tactttgtta ccccggagac gttcggccaa 300 gggaccaagg tggaaatcaa acga 324 <210> 5 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> VH1 primer of Fd chain <400> 5 cagccagcaa tggcacaggt gcagctgctc gagtctgg 38 <210> 6 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> VH2 primer of Fd chain <400> 6 cagccagcaa tggcacaggt gcagctgctc gagtcggg 38 <210> 7 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> VH3 primer of Fd chain < 400> 7 cagccagcaa tggca caggt gcaactgctc gagtctgg 38 <210> 8 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> VH4 primer of Fd chain <400> 8 cagccagcaa tggcacaggt gcaactgctc gagtcggg 38 <210> 9 <211> 30 < 212> DNA <213> Artificial Sequence <220> <223> CH1 primer of Fd chain <400> 9 ggtgcatgcg gccgcacaag atttgggctc 30 <210> 10 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> VH.EXT primer of Fd chain <400> 10 gctggattgt tattgctagc agcacagcca gcaatggca 39 <210> 11 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> VLK primer of Kappa chain <400> 11 cgcgaattgg cccagccggc catggccgag ctcgtgatga cccagtctcc a 51 <210> 12 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> CLK primer of Kappa chain <400> 12 gtctccttct cgaggcgcgc ctcattaaca ctctcccctg tt gaagct 48 <210> 13 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> VLL primer of Lambda chain <400> 13 cgcgaattgg cccagccggc catggccgag ctcgtggtga ctcagcc 47 <210> 14 <211> 45 <212 > DNA <213> Artificial Sequence <220> <223> CLL primer of Lambda chain <400> 14 gtctccttct cgaggcgcgc ctcattatga acattctgta ggggc 45 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223 > Fd primer for sequencing <400> 15 gtgcccccag aggtgctctt gg 22 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Kappa primer for sequencing <400> 16 ggcagttcca gatttcaact g 21 <210 > 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Lambda primer for sequencing <400> 17 gcggataaca atttcacaca g 21 <210> 18 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Primer-Nhe <400> 18 caggtcgact agagctagct attctatagt g 31 <210> 19 <211> 29 <212> DNA <213 Artificial Sequence <220> <223> Primer-Hpa <400> 19 cagagtgagg ttaacctggt gcaatctgg 29 <210> 20 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> PCR primer of HBAb-H4 < 400> 20 gccatggccc aggttaacct gctcgagtcg 30 <210> 21 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> PCR primer of HBAb-H4 <400> 21 gggaagaccg atgggccctt ggtggaggct g 31 <210> 22 < 211> 37 <212> DNA <213> Artificial Sequence <220> <223> Primer-reHind <400> 22 ctcactatag ggagacccgc tagcggaagc aagatgg 37 <210> 23 <211> 29 <212> DNA <213> Artific ial Sequence <220> <223> Primer-BstE <400> 23 ggggacattg tggtgaccca atctccagc 29 <210> 24 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer-Hind <400> 24 ggagggggga ccaagcttga aataaaacgg 30 <210> 25 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR primer of HBAb-L9 <400> 25 gagctcgtgg tgacccagtc tcca 24 <210> 26 <211> 27 <212 > DNA <213> Artificial Sequence <220> <223> PCR primer of HBAb-L9 <400> 26 tcgtttgatt tcaagcttgg tcccttg 27

Claims (10)

a) the amino acid sequence of SEQ ID NO: 1 encoding the variable region of the human antibody H chain against the surface antigen of hepatitis B virus (HBAb-H4); And b) Human antibody protein against the surface antigen of hepatitis B virus comprising the amino acid sequence of SEQ ID NO: 2 (HBAb-L9) encoding the variable region of the human antibody L chain against the surface antigen of hepatitis B virus. Human antibody DNA against the surface antigen of hepatitis B virus comprising the nucleotide sequence of SEQ ID NO: 3 (baseHBAb-H4) encoding the variable region of the H chain of claim 1. Human antibody DNA to the surface antigen of hepatitis B virus comprising the nucleotide sequence of SEQ ID NO: 4 (baseHBAb-L9) encoding the variable region of the L chain of claim 1. delete delete The plasmid pRC13-Hpa-HB-H4 comprising the nucleotide sequence of SEQ ID NO: 3 encoding the variable region of the H chain of the human antibody of claim 2 (baseHBAb-H4). The plasmid pKC12-BH-HB-L9 comprising the nucleotide sequence of SEQ ID NO: 4 encoding the variable region of the L chain of the human antibody of claim 3 (baseHBAb-L9). a) the plasmid pRC13-Hpa-HB-H4 of claim 6; And b) Chinese hamster ovary (CHO) cell line HBAb-49 (KCLRF-BP-00054) transformed with plasmid pKC12-BH-HB-L9 of claim 7 a) culturing the cells of claim 8; b) expressing a human antibody protein against the surface antigen of hepatitis B virus using the cultured cells; And c) A method for producing a human antibody against the surface antigen of hepatitis B virus comprising the step of purifying the expressed protein. A human antibody protein against HBV surface antigen prepared by the method of claim 9.