KR20040058606A - A humanized antibody against s-surface antigen of hepatitis b virus and a method for preparing thereof - Google Patents

Abstract

PURPOSE: A humanized antibody against S-surface antigen of hepatitis B virus and a method for preparing the same are provided, thereby treating a person suffering chronic hepatitis B, preventing infection of hepatitis B to a liver transplanted person, and preventing the direct infection of hepatitis B to a fetus from a mother. CONSTITUTION: A humanized heavy chain gene encoding the humanized antibody against S-surface antigen of hepatitis B virus comprising a variable region of the amino acid sequence set forth in SEQ ID NO:25 has the nucleotide sequence set forth in SEQ ID NO:23. A humanized light chain gene encoding the humanized antibody against S-surface antigen of hepatitis B virus comprising a variable region of the amino acid sequence set forth in SEQ ID NO:26 has the nucleotide sequence set forth in SEQ ID NO:24. A leader sequence gene of humanized heavy chain comprises the nucleotide sequence set forth in SEQ ID NO:1. A leader sequence gene of humanized heavy chain comprises the nucleotide sequence set forth in SEQ ID NO:13. A heavy chain expression vector of the humanized antibody pHAB-A9V7FW42HF(KCTC 10371BP) comprises the humanized heavy chain gene of SEQ ID NO:23. A light chain expression vector of the humanized antibody pHAB-A9V7LF(KCTC 10372BP) comprises the humanized light chain gene of SEQ ID NO:24. A transformant CHO-hA9-11-5 is produced by transforming CHO cell with the heavy chain expression vector pHAB-A9V7FW42HF(KCTC 10371BP) and light chain expression vector pHAB-A9V7LF(KCTC 10372BP). The method for preparing the humanized antibody against S-surface antigen of HBV comprises culturing the transformant CHO-hA9-11-5.

Description

HUMANIZED ANTIBODY AGAINST S-SURFACE ANTIGEN OF HEPATITIS B VIRUS AND A METHOD FOR PREPARING THEREOF}

The present invention relates to a humanized antibody that recognizes the S-surface antigen of Hepatitis B Virus (hereinafter referred to as "HBV"), and more particularly to the S-surface antigen of Hepatitis B virus. By substituting the variable gene A9-11-5 (Korean Patent Application No. 2000-28938) of the mouse antibody against the variable gene of the human antibody, the immune response of the mouse antibody is minimized and the HBV of the original mouse antibody is minimized. Humanized antibody genes, antibody expression vectors comprising the same, transformants transformed with the expression vectors, and the transformants, which can be effectively used for the treatment of hepatitis B patients by maintaining reactivity to S-surface antigens. It relates to a humanized antibody obtained by culturing and a method for producing the same.

Hepatitis B virus is a spherical particle of about 42 nm, known as the Dane particle. The outer envelope contains a large amount of Hepatitis B surface antigens and about 180 hepatitis B cores. It surrounds a nucleocapsid composed of proteins (Hepatitis B core proteins). This nucleocapsid contains the HBV gene, polymerase and the like (Summers et al., Proc. Nat. Acad. Sci, 72, 4579, 1975; Pierre Tiollais et al., Science, 213, 406-411, 1981 ).

Within the HBV gene, the HBV gene region encoding hepatitis B surface antigen contains three in-frame initiation sites, all of which share the end codon of the S-domain end and have. Thus, HBV surface antigens are known as (1) small Hepatitis B Virus Surface Antigen (hereinafter referred to as "S-Surface Antigen") containing only S-domain, (2) S-domain and Pre-S2. Middle Hepatitis B Virus Surface Antigen (hereinafter referred to as “M-Surface Antigen”) comprising 55 amino acids, and (3) large HBV surface comprising S-domain, Pre-S2 and Pre-S1 Antigen (Large Hepatitis B Virus Surface Antigen, hereinafter referred to as "L-surface antigen"). Of these surface antigens, S-surface antigens make up about 80% or more of the total surface antigens expressed.

Subtypes of S-surface antigens are classified by antibody recognition. That is, the antigenic sites present in all surface antigens are classified as "a" determinants and the other four subtype antigenic sites as "d" / "y" and "w" / "r" determinants. The "d" determinant is lysine, whereas the "y" determinant is arginine, the "w" determinant is lysine, whereas the "r" determinant is arginine (Kennedy RC et. al., J. Immunol. 130, 385, 1983). Thus, serotypes are classified into subtypes such as adr, adw, ayr and ayw (Peterson et al., J. Biol. Chem. 257, 10414, 1982; Lars O. Marnius et al., Intervirology, 38, 24-34, 1995), mainly "adr" type is detected in Korea.

The S-surface antigen can specifically bind to liver cells (Leenders et al., Hepatology, 12, 141, 1990; Irina Ionescu-Matiu et al., J. Med. Virology, 6, 175-178, 1980; Swan NT et al., Gastroenterology, 80, 260-264, 1981; Swan NT et al., Gastroenterology, 85, 466-468, 1993; Marie, LM et al., Proc. Nat. Acad. Sci. 81 , 7708-7712, 1982), Apolipoprotein H and endonexin II as S-surface antigen-specific binding proteins have been identified on human liver plasma membranes (Mehdi H. et al. , J. Virol. 68, 2415, 1994; Hertogs K. et al., Virology, 197, 265, 1993).

Currently, interferon alpha and lamivudine are widely used as treatment agents for chronic hepatitis (CL Lai, PC Wu, HKMJ, 3, 289-96, 1997), but interferon alpha is an immune response to viruses in vivo. Because of its strong toxicity, many side effects of use have been suggested as problems. Lamivudine, on the other hand, acts as a nucleic acid derivative by inhibiting viral DNA synthase, which acts as an antiviral. It is developed for oral use and has a high therapeutic effect, but it is highly likely that mutations resistant to lamivudine may occur after long-term use. T.-Y. Lau et al, Hepatology 32, 828-34, 2000). Antibody therapeutics can act directly on the virus to neutralize the antibody or effectively remove the virus through the function of secondary immune cells. In addition, anti-HBV polyantibodies isolated from human plasma which are currently used for liver transplantation and prevention of vertical infection have various disadvantages such as low specificity for antigen and separation from human blood.

Antibody therapeutics are therapeutic methods that use humoral immune responses, one of the two immune responses in the human body. Serum therapy, the origin of antibody therapeutics, was first attempted in the early 20th century (Casadevall A, Scharff MD. Antimicrob. Agents Chemother. 38, 1695-1702, 1994; Casadevall A, Scharff MD, Cli. Infect. Dis. 21, 150-161, 1995), and then monoclonal antibodies using hybridoma technology (Kohler G, Milstein C., Nature 256, 495-497, 1975) that were not used for antibiotics and were developed in the 1970s. Because of its development, OKT3, the first monoclonal antibody therapeutic, has been used for kidney transplant suppression (Morrison SL, Johnson, MJ, et al., Proc. Natn. Acad. Sci. USA 81, 6851-6855, 1984). However, long-term use of mouse monoclonal antibodies, such as OKT3, produces human antibodies against mouse antibodies called the HAMA (Human Anti-Mouse Antibody, HAMA) reaction, which has been reported to cause problems when used as a long-term therapeutic agent (Dimaggio JJ, Scheinberg DA et al., Cancer Chemother. Biol.Response Modif. 11, 177-203, 1990).

The development of genetic recombination technology in the 1980s has been made possible to replace the mouse antibody shortcomings by substituting the remaining portions of the antibody except for the portion that binds the antigen of the antibody to the human antibody, and the humanized antibody was thus developed. Humanized antibodies have been actively researched by PDL, Genentech, etc. of the United States, and several humanized antibodies are currently being developed and sold as products. Heceptin is a product developed for the treatment of terminal metastatic breast cancer and acts as a mechanism for attacking cancer cells by binding to the Her2 receptor, a specific antigen expressed on the surface of breast cancer cells (Paul Carter and H. Michael Shepard., Proc. Natn Acad.Sci. USA 89, 4285-4289, 1992; G. Schaller. Et al., J. Cancer Res. Clin.Oncol. 125, 520-524, 1999). In addition, Herceptin When administered in combination with the existing Taxol (Taxol) has been shown to inhibit the progression of cancer cells for more than six months. Rituxan, which has been developed as an antibody for the treatment of non-Hodgkin's lymphoma, is also effective in treating cancer patients. Remicade, developed and sold by Centocor, is currently being developed and sold for the treatment of rheumatoid arthritis and inflammatory bowel disease. Is evaluated as a high therapeutic agent (Ravinder Maini., The Lancet 354, 1932-1939, 1999).

Antibodies in the field of infectious diseases were first developed as blood-treatment drugs against pneumonia, and the first anti-Synagis drug, Synazis, was developed and sold by Novartis. Michael Boeckh et al., The Journal of Infectious Disease 184, 350-354, 2001. Synagis is a drug used to prevent respiratory infections caused by a virus called RSV, which is fatal to children. In addition, as the field of development for the treatment of infectious diseases, prevention and treatment for viruses such as HBV and HCV, and treatment for microorganisms such as bacteria and fungi are being developed.

Currently, the mouse antibody is replaced with a humanized antibody by selecting the most similar human antibody gene for the antibody to be replaced and replacing only the CDR region of the mouse antibody with the human antibody CDR position by a method called CDR transplantation. In the case of PDL's Zenapax, the mouse antibody gene was developed using the mouse antibody gene as it is based on the human antibody gene, whereas Herceptin, the Genentech breast cancer drug Herceptin, was CDR2. The humanized antibody was developed to further reduce immunogenicity by changing the terminal region with the human antibody gene.

Currently, the development of monoclonal antibodies as a treatment for chronic hepatitis B has been studied by various research institutes and pharmaceutical companies in the world, but has not been developed as a product. Therefore, there is an urgent need for the development of antibodies that can neutralize viruses more efficiently for the development of innovative hepatitis B treatments.

Accordingly, the inventors of the present invention have repeatedly studied to solve the above problems. As a result, the antibody specificity of the monoclonal antibody against the S-surface antigen of the hepatitis B virus is maintained, while the variable region of the mouse antibody is substituted with a human antibody gene to replace the mouse antibody. Humanized antibodies have been developed that recognize the S-surface antigens of hepatitis B virus with minimally reduced immune response to.

Accordingly, an object of the present invention is to provide a humanized antibody that recognizes the S-surface antigen of hepatitis B virus and a method for producing the same.

It is also an object of the present invention to provide humanized antibody heavy and light chain genes encoding humanized antibody heavy and light chains that recognize S-surface antigens of hepatitis B virus.

It is also an object of the present invention to provide humanized antibody heavy and light chain expression vectors encoding humanized antibody heavy and light chains that recognize S-surface antigens of hepatitis B virus.

It is also an object of the present invention to provide a transformant transformed with an expression vector comprising humanized antibody heavy and light chain genes that recognize S-surface antigens of hepatitis B virus.

1 shows a comparison of the amino acid sequence of the mouse monoclonal antibody and the humanized antibody heavy chain with the human antibody template against HBV S-surface antigen,

FIG. 2 shows a comparison of the amino acid sequence of the mouse monoclonal antibody and the humanized antibody light chain with the human antibody template against HBV S-surface antigen,

Figure 3 shows the process for producing a humanized antibody heavy chain expression vector pHAB-A9V7FW42HF,

Figure 4 shows the process for preparing the humanized antibody light chain expression vector pHAB-A9V7LF,

Figure 5 shows the polyacylamide gel electrophoresis (SDS-PAGE) of humanized antibodies produced and purified in animal cells.

In order to achieve the above object, in the present invention, the variable gene A9-11-5 (Korean Patent Application No. 2000-28938) of the mouse antibody against the S-surface antigen of hepatitis B virus is used. By substituting with a gene, the humanized antibody and its light or heavy chains can be efficiently used for the treatment of hepatitis B patients by minimizing the immune response of the mouse antibody and maintaining the reactivity of the original mouse antibody to the HBV S-surface antigen. Provide an amino acid sequence.

In addition, the present invention recognizes the S- surface antigen of hepatitis B virus that can reduce the immune response to the mouse antibody to a minimum while maintaining the antibody specificity of the monoclonal antibody to the S- surface antigen of the hepatitis B virus Humanized antibody light or heavy chain genes are provided.

In addition, the present invention is to culture the transformants transformed with an expression vector containing a humanized antibody heavy and light chain gene that recognizes the S- surface antigen of hepatitis B virus to recognize the S- surface antigen of hepatitis B virus Provided are methods for making humanized antibodies.

Hereinafter, the present invention will be described in detail.

In the present invention, the humanized antibody against the S-surface antigen of the humanized hepatitis B virus comprising the variable region of the amino acid sequence of SEQ ID NO: 25 and the S of the hepatitis B virus comprising the humanized light chain comprising the amino acid sequence of SEQ ID NO: 26 Provide humanized antibodies against surface antigens.

Furthermore, in the present invention, the humanized heavy chain gene A9V7FW42 for the S-surface antigen of hepatitis B virus comprising the nucleotide sequence of SEQ ID NO: 23 encoding the variable region of the amino acid sequence of SEQ ID NO: 25. And a humanized light chain gene A9V7 for the S-surface antigen of hepatitis B virus comprising the nucleotide sequence of SEQ ID NO: 24 encoding the variable region of the amino acid sequence of SEQ ID NO: 26.

The present invention also provides a leader sequence gene of the humanized antibody heavy chain comprising the nucleotide sequence of SEQ ID NO: 1 and a leader sequence gene of the humanized antibody light chain comprising the nucleotide sequence of SEQ ID NO: 13.

In addition, in the present invention, the heavy chain expression vector pHAB-A9V7FW42HF (human accession number: KCTC 10371BP) of the humanized antibody against the S-surface antigen of the hepatitis B virus containing the gene A9V7FW42 (accession number: KCTC 10371BP) and the hepatitis B virus containing the gene A9V7 The light chain expression vector pHAB-A9V7LF (Accession No .: KCTC 10372BP) of the humanized antibody against S-surface antigen is provided.

The present invention also provides a transformant CHO-hA9-11-5 obtained by transforming the expression vectors pHAB-A9V7FW42HF and pHAB-A9V7LF into CHO cells.

The present invention also provides a method for producing a humanized antibody against HBV S-surface antigen, wherein the transformant CHO-hA9-11-5 is cultured to obtain a humanized antibody hA9-11-5.

To prepare a humanized antibody against the gene A9-11-5 (Korean Patent Application No. 2000-28938) of a mouse antibody against hepatitis B virus S-surface antigen previously filed by the inventors of the present invention, The amino acid sequences most similar to the light and heavy chain genes can be retrieved from the Gene bank database. As a result of the search, the heavy chain 2 human heavy chain 2 sequence according to the definition of Kabat, the light chain human light chain 7 sequence or germline 3 sequence was confirmed that the human gene most similar to the variable region gene of the mouse antibody Humanized heavy and light chain genes can be prepared based on the human genes selected above.

Substitution of amino acids in the production of humanized heavy and light chain genes affects binding to antigens based on known genetic analysis data and antibody structure analysis data, or amino acids important for antibody structure are preferably used as the original residues of mouse antibodies. (Kabat EA et al., DHHS 91-3242, 1991; Chothia C. et al., Nature 342, 877-883, 1989; Gary M. Studnicka. Et al., Protein Engineering 7, 805-814, 1994; Linda Harris, and Bajorath, J. Protein Science 4, 306-310, 1995).

Specifically, the humanized heavy chain variable gene is excluding the portion of the heavy chain variable region gene of the mouse monoclonal antibody A9-11-5 (Korean Patent Application No. 2000-28938) against hepatitis B virus S-surface antigen. It can be prepared by substituting the human antibody gene sequence. That is, the humanized heavy chain variable gene selects primers to humanize the heavy chain gene of the mouse antibody A9-11-5 as a template, performs PCR using each primer, and then, each PCR product is subjected to restriction enzyme and DNA ligase ( by using ligase) A9V7FW42HV, which may be prepared (see FIG. 1).

To prepare a humanized heavy chain gene, first, the humanized heavy chain variable gene A9V7FW42HV prepared above was inserted into a PCR vector to prepare pCR-A9V7FW42HV, and the heavy chain variable gene was isolated from the pCR-A9V7FW42HV, and then the isolated heavy chain variable gene was isolated. Humanized heavy chain gene can be prepared by inserting the antibody heavy chain expression vector pHAB-HC (Korean Patent Application No. 2000-0045765) into which the human antibody heavy chain constant gene previously filed by the present inventors was inserted, and named it A9V7FW42. . In addition, the humanized heavy chain expression vector inserted with the gene was pHAB-A9V7FW42HF (Accession No .: KCTC 10371BP).

In addition, the humanized light chain variable gene is the remainder of the light chain variable region gene of the mouse monoclonal antibody A9-11-5 (Korean Patent Application No. 2000-28938) against hepatitis B virus S-surface antigen except for the portion that binds the antigen. Can be prepared by substituting for a human antibody gene sequence. That is, the humanized light chain variable gene selects primers to humanize the light chain gene of the mouse antibody A9-11-5 as a template, performs PCR using each primer, and then, each PCR product is subjected to restriction enzyme and DNA ligase ( by using ligase) It can be prepared and named A9V7LV (see FIG. 2).

In order to prepare a humanized light chain gene, first, the humanized light chain variable gene prepared above is inserted into a PCR vector to prepare pCR-A9V7LV, and the light chain variable gene is separated from the pCR-A9V7LV, and then the separated light chain variable gene is viewed. The humanized light chain gene can be prepared by inserting the antibody light chain expression vector pHAB-LC (Korean Patent Application No. 2000-0045767) into which the human antibody light chain constant gene previously filed by the inventors was inserted, which was named A9V7. In addition, the humanized light chain expression vector pHAB-A9V7LF inserted with the gene (Accession Number: KCTC 10372BP).

The transformant CHO-hA9-11-5 was prepared by transforming the antibody expression vectors pHAB-A9V7FW42HF and pHAB-A9V7LF containing the humanized heavy and light chain genes prepared above into CHO cells, respectively. In this case, a suitable transformation solution such as Gene POTER may be used. The humanized antibody of the present invention is isolated by purifying the culture medium obtained by culturing the transformant CHO-hA9-11-5 with protein-A and goat anti-human immunoglobulin G. It was named hA9-11-5.

The purified humanized antibody hA9-11-5 was identified by SDS-PAGE electrophoresis, and the protein band was composed of an antibody heavy chain band of 50,000 daltons and an antibody light chain band of 25,000 daltons. It was confirmed that it was lost. In addition, the affinity of the humanized antibody hA9-11-5 for hepatitis B virus S-surface antigen can be measured using the Sandwich Eliza method. As a result, the antigen-binding affinity of the humanized antibody hA9-11-5 was 2.2x10 ⁹ M ^-1 , indicating that there was no difference from the affinity of the mouse antibody for antigen.

Therefore, the humanized antibody against the hepatitis B virus S-surface antigen of the present invention can significantly prevent the hepatitis B virus while maintaining an antigen-binding ability similar to that of a conventional mouse antibody, thereby significantly reducing immunogenicity.

Hereinafter, the present invention will be described in detail by examples. However, this does not limit the present invention.

Example

Example 1 Preparation of Humanized Heavy Chain Variable Genes

The amino acid sequence most similar to the heavy chain variable region gene (Korean Patent Application No. 2000-28938) of mouse antibody A9-11-5 against anti-hepatitis B S-surface antigen was searched through a blast program of the Gene Bank. . As a result of selecting the template of several candidate groups based on the search results, it was confirmed that the antibody heavy chain belongs to No. 2 (Hh2) of the antibody heavy chain variable subgroup (Kabat, 1991). Therefore, the main sequence of the subgroup 2 was used as a template of the human antibody heavy chain, and the remainder except for the CDR region of the mouse antibody heavy chain variable region was replaced with the amino acid sequence of the human antibody heavy chain subgroup 2. However, in the case of amino acids important for the activity of the antibody, a humanized heavy chain variable gene was prepared using the mouse antibody sequence as it was, named A9V7FW42HV (see FIG. 1). Primers prepared for humanizing mouse antibody heavy chain variable genes are described in Table 1 below.

PCR Conditions for Humanized Antibody Heavy Chain Development Primers order primer Result of amplification substitution 2-step PCR conditions Denature Annealing Extension One SEQ ID NO: 1 / SEQ ID NO: 12 A 94 ° C, 1 minute 30 seconds 55 ℃, 2 minutes 72 ° C., 3 minutes 2 SEQ ID NO: 1 / SEQ ID NO: 2 B 94 ° C, 1 minute 30 seconds 50 ℃, 2 minutes 72 ° C., 2 minutes 30 seconds 3 SEQ ID NO: 3 / SEQ ID NO: 12 94 ° C, 1 minute 30 seconds 50 ℃, 2 minutes 72 ° C., 2 minutes 30 seconds 4 SEQ ID NO: 4 / SEQ ID NO: 12 C 94 ° C, 1 minute 30 seconds 55 ℃, 2 minutes 72 ° C., 2 minutes 30 seconds 5 SEQ ID NO: 1 / SEQ ID NO: 5 94 ° C, 1 minute 30 seconds 55 ℃, 2 minutes 72 ° C., 2 minutes 30 seconds 6 SEQ ID NO: 6 / SEQ ID NO: 12 D 94 ° C, 1 minute 30 seconds 55 ℃, 2 minutes 72 ° C., 2 minutes 30 seconds 7 SEQ ID NO: 1 / SEQ ID NO: 7 94 ° C, 1 minute 30 seconds 55 ℃, 2 minutes 72 ° C., 2 minutes 30 seconds 8 SEQ ID NO: 8 / SEQ ID NO: 12 E 94 ° C, 1 minute 30 seconds 55 ℃, 2 minutes 72 ° C., 2 minutes 30 seconds 9 SEQ ID NO: 1 / SEQ ID NO: 9 94 ° C, 1 minute 30 seconds 55 ℃, 2 minutes 72 ° C., 2 minutes 30 seconds 10 SEQ ID NO: 10 / SEQ ID NO: 12 A9V7FW42HV 94 ° C, 1 minute 30 seconds 55 ℃, 2 minutes 72 ° C., 2 minutes 30 seconds 11 SEQ ID NO: 1 / SEQ ID NO: 11 94 ° C, 1 minute 30 seconds 55 ℃, 2 minutes 72 ° C., 2 minutes 30 seconds

Specifically, in order to prepare a humanized heavy chain variable gene, first, the mouse antibody variable gene was amplified by the heavy chain variable gene (A) including the leader sequence using SEQ ID NO: 1 and SEQ ID NO: 12. Gene fragments amplified by PCR were recovered using agarose electrophoresis and etiquette (QIAgel extraction kit, Qiagen, USA). Next, based on the separated and purified heavy chain variable gene (A), amplification was performed in SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 12, and the amplified genes were linked to each other to prepare a heavy chain variable gene (B). Next, the heavy chain variable gene (C) was prepared by linking genes amplified with SEQ ID NOs: 4 and 12 and SEQ ID NOs: 1 and 5 based on the heavy chain variable gene (B). PCR was performed in the same manner as described above in Table 1 PCR was largely divided into three stages, and the first stage consisted only of decomposition, and the degradation conditions were performed once at 95 ° C. for 5 minutes. Step 2 was repeated 30 times as a time according to the conditions of Table 1 above. Step 3 consisted only of synthesis and performed the same at 72 ° C. for 10 minutes. PCR was carried out in the same manner as described above to finally produce a humanized heavy chain variable gene, which was named A9V7FW42HV.

Example 2. Preparation of humanized heavy chain gene and expression vector

In order to prepare a humanized heavy chain gene, the antibody heavy chain expression vector pHAB-HC (Korean Patent Application No. 2002-0045765) into which the human antibody heavy chain constant gene inserted by the inventors of the present invention was inserted was used. For high expression of the humanized heavy chain, the Kozak sequence (Kozak, M. Nuc. Acids Res. 15, 8125-8148, 1987) was inserted in front of the humanized heavy chain leader and leucine, the fourth amino acid of the heavy chain leader, was inserted. A primer with high expression efficiency was prepared by substituting (Leucin) nucleotide codon with CTG, a codon frequently used in animal cells in the conventional TTA. The substituted humanized heavy chain leader sequences are described in Table 2 below.

Heavy Chain Leader Sequence (SEQ ID NO: 1) primer order Mouse heavy chain leader sequence CGAC ATG GCT GTC TTA TTC CTG CTC CTC TGC CTGM AV L F LLLCL Substituted heavy chain leader sequence C ACC ATGGCT GTC CTG TTC CTG CTC CTC TGC CTGM A VLF L L L C L

PCR amplification was performed using the primers to increase the expression efficiency of the humanized heavy chain gene. As a result, the amplified humanized heavy chain variable gene was inserted into a PCR vector, which was named pCR-A9V7FW42HV.

In order to insert the humanized heavy chain variable gene into the antibody heavy chain expression vector pHAB-HC (Korean Patent Application No. 2000-0045765) into which a human antibody constant gene was inserted, pCR-A9V7FW42HV was restriction enzyme HindIII / ApaI (Bioreb, USA). After cutting for 2 hours at 37 ° C., the fragments were identified by 1.5% agarose gel electrophoresis and ethidium bromide staining. Similarly, the antibody heavy chain expression vector pHAB-HC was digested with restriction enzyme HindIII / ApaI to confirm a cleaved expression vector of about 6.5 Kb. Each gene fragment was then recovered using a QIAgel extraction kit (QIAgen, USA) and then overnight at 16 ° C. using T4 DNA ligase (DNA ligase, BioLab, USA). After the reaction, E. coli XL1-Blue was transformed to obtain a transformant. The resulting transformants were incubated overnight in LB medium containing 100 μg / ml of ampicillin, and then the plasmids were recovered, and then digested with restriction enzyme HindIII / ApaI to undergo electrophoresis of the entire antibody heavy chain variable and constant genes of about 1.4 Kb. It was confirmed through (see Figure 3).

According to the method described above, a humanized heavy chain gene in which a humanized heavy chain variable gene and a heavy chain constant gene were connected was prepared, which was named A9V7FW42. In addition, the humanized heavy chain expression vector pHAB-A9V7FW42HF into which the gene A9V7FW42 was inserted was deposited on November 8, 2002 to the Biotechnology Research Institute Gene Bank (KCTC) (Accession No .: KCTC 10371BP).

Example 3 Preparation of Humanized Light Chain Variable Genes

The amino acid sequence most similar to the light chain variable region gene (Korean Patent Application No. 2000-28938) of mouse antibody A9-11-5 against anti-hepatitis B S-surface antigen was searched through a blast program of the Gene Bank. . As a result of selecting the template of several candidate groups based on the search results, the antibody light chain was identified in V3 of antibody light chain lambda variable subgroup 7 and gene bank antibody lambda germline V genes of Genebank Ig blast. It was confirmed to belong (Kawasaki K. et al, 1997). In particular, it was found very similar to 4A (M. L. Anderson et al., Nuc. Acids Res. 12, 6647-6661, 1984) in subgroup 7 above. Therefore, the variable gene of the 4A antibody light chain of subgroup 7 was used as a template of human antibody light chain. In addition, the remainder except the CDR region of the mouse antibody light chain variable region was substituted with the amino acid sequence of human antibody light chain lambda variable subgroup 7. However, in the case of amino acids important for the activity of the antibody, a humanized antibody light chain variable gene was prepared using the mouse antibody sequence as it is, and named A9V7LV (see FIG. 2). Primers prepared to humanize the mouse antibody light chain variable genes are described in Table 3 below.

PCR Conditions for Humanized Antibody Light Chain Development Primers order primer Result of amplification substitution 2-step PCR conditions Denature Annealing Extension One SEQ ID NO: 13 / SEQ ID NO: 22 A 94 ° C, 1 minute 30 seconds 55 ℃, 2 minutes 72 ° C., 3 minutes 2 SEQ ID NO: 14 / SEQ ID NO: 22 B 94 ° C, 1 minute 30 seconds 55 ℃, 2 minutes 72 ° C., 3 minutes 3 SEQ ID NO: 13 / SEQ ID NO: 15 94 ° C, 1 minute 30 seconds 55 ℃, 2 minutes 72 ° C., 3 minutes 4 SEQ ID NO: 16 / SEQ ID NO: 22 C 94 ° C, 1 minute 30 seconds 49 ℃, 2 minutes 72 ° C., 3 minutes 5 SEQ ID NO: 13 / SEQ ID NO: 17 94 ° C, 1 minute 30 seconds 50 ℃, 2 minutes 72 ° C., 3 minutes 6 SEQ ID NO: 18 / SEQ ID NO: 22 D 94 ° C, 1 minute 30 seconds 55 ℃, 2 minutes 72 ° C., 3 minutes 7 SEQ ID NO: 13 / SEQ ID NO: 19 94 ° C, 1 minute 30 seconds 55 ℃, 2 minutes 72 ° C., 3 minutes 8 SEQ ID NO: 20 / SEQ ID NO: 22 A9V7LV 94 ° C, 1 minute 30 seconds 55 ℃, 2 minutes 72 ° C., 3 minutes 9 SEQ ID NO: 13 / SEQ ID NO: 21 94 ° C, 1 minute 30 seconds 50 ℃, 2 minutes 72 ° C., 3 minutes

Specifically, in order to prepare a humanized light chain variable gene, first, the light chain variable gene (A) including the leader sequence was amplified using the mouse antibody variable genes (SEQ ID NO: 13 and SEQ ID NO: 22). Gene fragments amplified by PCR were confirmed by agarose electrophoresis and ethidium bromide (EtBr) staining, and recovered using a QIAgel extraction kit. Next, the light chain variable gene (B) was prepared by amplifying the sequences 14 and 22 and the sequences 13 and 15 based on the separated and purified light chain variable genes (A). Next, light chain variable genes (C) were prepared by linking genes amplified with SEQ ID NOs: 16 and 22 and SEQ ID NOs: 13 and 17 based on the light chain variable genes (B). PCR was performed in the same manner as described above in the order of Table 3. PCR was largely divided into three stages, and the first stage consisted only of decomposition, and the degradation conditions were performed once at 95 ° C. for 5 minutes. Step 2 was performed according to the conditions of Table 3 above. Step 3 consisted only of synthesis and performed the same at 72 ° C. for 10 minutes. PCR was performed in the same manner as above to finally prepare a humanized light chain variable gene A9V7LV.

Example 4 Preparation of Humanized Light Chain Gene and Expression Vector

In order to prepare a humanized light chain gene, an antibody light chain expression vector pHAB-LC (Korean Patent Application No. 2002-0045767) into which a human antibody light chain constant gene inserted by a inventor of the present invention was inserted was used. For high expression of the humanized light chain, a Kozak sequence (Kozak, M. Nuc. Acids Res. 15, 8125-8148, 1987) was inserted in front of the humanized light chain leader sequence, and the eighth amino acid of the light chain leader sequence gene was inserted. In the existing phenylalanine (Phenylalanine) was substituted with leucine with high expression efficiency in animal cells, CTT encoding the 6th leucine to CTG and ATA encoding the 7th isoleucine to ATC to replace the high efficiency of the light chain Primer was prepared. The humanized light chain leader sequence substituted as described above is described in Table 4 below.

Light Chain Leader Sequence (SEQ ID NO: 13) primer order Mouse Light Chain Leader Sequence TTC ATG GCC TGG ATT TCA CTT ATA TTC TCT CTC CTGM AWISLI F S LL Substituted Light Chain Leader Sequences C ACC ATG GCC TGG ATT TCA CTG ATC CTC TCT CTC CTGM AWISLI L S LL

In order to increase the expression efficiency of the humanized light chain gene, PCR amplification was performed using the primers. The amplified humanized antibody light chain variable gene was inserted into a PCR vector, which was named pCR-A9V7LV.

In addition, in order to insert the humanized light chain variable gene into the antibody light chain expression vector pHAB-LC (Korean Patent Application No. 2002-0045767) into which the human antibody constant gene was inserted, pCR-A9V7LV was added at 37 ° C by restriction enzyme HindIII / AvrII. After cleavage by reaction for a period of time, the gene fragment of about 400bp was confirmed by 1.5% agarose gel electrophoresis and ethidium bromide staining. Similarly, the light chain expression vector pHAB-LC was also digested with restriction enzyme HindIII / AvrII to confirm the cleaved expression vector of about 5.6 Kb. Then, each gene fragment was recovered using a Qiagen extraction kit (Qiagen, USA), and then reacted overnight at 16 ° C. using T4 DNA ligase (DNA ligase, BioLab, USA). Transformants were obtained by transforming XL1-Blue. The resulting transformants were incubated overnight in LB medium containing 100 μg / ml of ampicillin, and then the plasmids were recovered, and then digested with restriction enzyme HindIII / NotI (Biorepsa, USA) to cut the entire gene of about 0.7 Kb of the antibody light chain. It was confirmed by electrophoresis (see Fig. 4).

According to the method described above, a humanized light chain gene having a humanized light chain variable gene and a light chain constant gene connected thereto was prepared and named as A9V7. In addition, the humanized light chain expression vector into which the gene A9V7 was inserted was deposited with pHAB-A9V7LF to the Biotechnology Research Institute Gene Bank (KCTC) on November 8, 2002 (Accession Number: KCTC 10372BP).

Example 5. Transformation of Humanized Antibodies in CHO Cells

In order to measure the activity of humanized antibodies in animal cells, antibody genes were transformed into CHO cells with the heavy and light chain humanized antibody expression vectors pHAB-A9V7FW42HF and pHAB-A9V7LF prepared in Examples 2 and 4, respectively. First, to culture CHO cells, a culture medium in which 1.5 g / L of sodium bicarbonate and 10% dialyzed FBS was added to DMEM / F12 medium (JRH, USA) was used. 5% after the carbon dioxide is supplied to culture the cells for 2 days to 3 days at 37 ℃ incubator (humidified CO ₂ incubator) which the cells were recovered and separated for 5 minutes centrifugation at 1200 rpm at room temperature (15 ℃). Next, the recovered CHO cells were stained with 0.4% trypan blue solution, and the number of cells was measured using a hematocytometer, and about 6 × 10 ⁶ cells were placed in a T-225 flask ( Or T-75 flask) and subcultured again. The passaged CHO cells were sufficiently grown to about 60-90% of the surface of the flask, and then 10 μg of the gene to be transformed and 40 μl of GenePoter solution, a transformation solution, were added to DMEM / F12 (serum-free medium). 5 ml of medium was mixed and mixed. The mixture was left at room temperature for 10 to 45 minutes, and then cultured with the cells from which the culture solution was removed in a 37 ° C. incubator for about 3 to 5 hours to obtain a transformant CHO-hA9-11-5.

Example 6. Purification of Humanized Antibodies

The transformant CHO-hA9-11-5 obtained in Example 5 was cultured to express the antibody gene. Specifically, the cell culture was inoculated with 2 × 10 ⁶ cells in a T-75 flask and cultured in DMEM / F12 medium containing 10% calf serum. The cell culture solution was recovered, centrifuged and filtered to purify only the culture solution containing the antibody. In addition, in order to purify only antibodies in the culture medium, Sepharose 4B (protein A) and goat anti-human immunoglobulin (goat-antihuman Immunoglobulin G) were used. To bind the antibody to Protein-A, the cell culture medium in which the antibody is expressed is passed through a protein-A bound column, and then the protein-A coupled antibody is passed through a glycine buffer of pH 2.5. To elute the humanized antibody. Thereafter, 1 molar Tris-Cl (1M Tris-Cl, pH 8.0) buffer was added to 1/10 volume of the antibody recovery solution to neutralize the pH of the antibody eluate. The eluted humanized antibody was passed again through Sepharose 4B to which goat anti-human immunoglobulin was bound, so that only antibody protein was specifically bound. The antibody was eluted purely using the same method as the elution method in Protein-A, which was named hA9-11-5. Purified antibody protein was confirmed by SDS-PAGE electrophoresis to identify the antibody heavy chain at 50,000 dalton molecular weight position and the antibody light chain protein band at 25,000 Dalton molecular weight position (see FIG. 5).

Example 7. Determination of affinity of humanized antibodies for HBV S-surface antigens

The humanized antibody purified in Example 6 was quantified using a sandwich ELISA method, and its activity was analyzed using S-surface antigen (International Enzyme, USA) AD type. To quantify the antibody, 100 ng of goat anti-human immunoglobulin GAM was added to each well of a microplate (Dynatek, USA), reacted overnight at 4 ° C., and then coated. The amount of the antibody was measured using an antibody known as a control. Next, in order to measure the affinity of the antibody for HBV S-surface antigen, the antigen was coated by reacting overnight at 4 ° C. with 1 μg or 30ng of HBV S-surface antigen in each well of the microplate. Then, 200 μl of 0.5% casein-phosphate buffered saline (PBS) solution was added to each well, and the mixture was left at 37 ° C. for about 1 hour. Then, each well was purified with 1 mg / ml of the antibody purified in Example 6 above. Diluted two times in sequence in the concentration was added. After that, 100-fold dilutions of goat anti-human polyclonal antibodies (Goat anti-mouse polyclonalantibody, Biorad, USA) combined with horseradish peroxidase were added to each well at 100 캜. After reacting for 1 hour, an optical density was measured at a wavelength of 410 nm using an ELISA reader (Dynatek, USA).

From the absorbance measured above, the affinity of the antibody for antigen was determined by converting the concentration of the antibody that did not bind to the antigen (Friguet, et al. 1985, J. Immunol. Meth. 77, 305-319). The results are shown in Table 5. To as described in Table 5, and the mouse antibody antigen-binding affinity is 2.4 x 10 ⁹ M ^-1, for chimeric antibody appears to 2.1x 10 ⁹ M ^-1 having a substantially similar activity when using the same variable genes It turned out. In addition, the antigen-binding affinity of the humanized antibody hA9-11-5 of this Example was 2.2x10 ⁹ M ^-1 , and it was confirmed that there is no difference in antigen-binding affinity compared to the mouse antibody.

Comparison of affinity for antigens of mouse, chimeric and humanized antibodies antigen Mouse antibody mA9-11-5 Chimeric Antibody cA9-11-5 Humanized Antibody hA9-11-5 Affinity for antigen (M ^-1 ) 2.4 x 10 ⁹ 2.1 x 10 ⁹ 2.2 x 10 ⁹

The humanized antibody of the present invention is a humanized antibody which shows an antigen-binding ability similar to that of a mouse antibody or chimeric antibody, but has a markedly reduced immunogenic ability in the human body, and is used for treating chronic hepatitis B patients, preventing infection in liver transplant patients, and B. It can be used to prevent vertical infection from mother to fetus infected with hepatitis virus.

<110> YUHAN CORPORATION <120> HUMANIZED ANTIBODY AGAINST A S-SURFACE ANTIGEN OF HEPATITIS B          VIRUS AND METHOD FOR PREPARING THEREOF <130> YU200209 <160> 26 <170> KopatentIn 1.71 <210> 1 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 caccatggct gtcctgttcc tgctcctctg cctg 34 <210> 2 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 caggcctagt gaagccctca cagaccctgt ccctcacctg cacag 45 <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 taggcctggt ccgctctctt gcagctgcac ctg 33 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gctgcagaca cagccgtgta ttattgtgcc ag 32 <210> 5 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 tctgcagcag tcacactgga cagtttcagg aaaacttggc 40 <210> 6 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 cgtcgacacc tccaagaacc aattctcact gaaactgtc 39 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 tgtcgacgct gatggtcact ctggatatg 29 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ttctctttaa aactgtccag tg 22 <210> 9 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 ttttaaagag aattggttct tggaggtgtc cttgctgatg 40 <210> 10 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 atctagagtg accatcagca aggacacctc caagaaccaa gtttcactga aa 52 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ctctagatat gaaagctgca tt 22 <210> 12 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 agagctcacg gtgaccgtgg tcccagcgcc cc 32 <210> 13 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 caccatggcc tggatttcac tgatcctctc tctcctg 37 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 ggtggtaccg tcacactcac ttgtc 25 <210> 15 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 cggtaccgcc aggtgagacg gtgagtgaag gttcctg 37 <210> 16 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 ctggccaggc tcctagaact ctaatatatg atacc 35 <210> 17 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 ctggccaggt ttctgttgga accagttggc 30 <210> 18 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 ggtgcacagc ctgaagatgc agaatattat tgtgc 35 <210> 19 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 tgtgcaccga gtagggtgag ggcagccttg tttccgagca ggg 43 <210> 20 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 tcccgggtgt tcctgacaga ttctc 25 <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 acccgggact cggttgttgg tatc 24 <210> 22 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 cccaagcttc caccaggggc caggggatag acggatgg 38 <210> 23 <211> 342 <212> DNA <213> Artificial Sequence <220> <223> variable region of humanized heavy chain (A9V7FW42) <400> 23 caggtgcagc tgcaagagag cggaccaggc ctagtgaagc cctcacagac cctgtccctc 60 acctgcacag tctctggttt ctcattaagt acctatggtg tacagtgggt tcgccagcct 120 ccaggaaagg gtctggagtg gctgggagtg atatggagtg gtggaaacac agactataat 180 gcagctttca tatccagagt gaccatcagc aaggacacct ccaagaacca agtttcactg 240 aaactgtcca gtgtgactgc tgcagacaca gccgtgtatt attgtgccag agcacggtac 300 ttcgatgtct ggggcgctgg gaccacggtc accgtgagct ct 342 <210> 24 <211> 327 <212> DNA <213> Artificial Sequence <220> <223> variable region of humanized light chain (A9V7) <400> 24 caggctgttg tgactcagga accttcactc accgtctcac ctggtggtac cgtcacactc 60 acttgtcgct caagtactgg ggctattaca actaataact ttgccaactg gttccaacag 120 aaacctggcc aggctcctag aactctaata tatgatacca acaaccgagt tccgggtgtt 180 cctgacagat tctcaggctc cctgctcgga aacaaggctg ccctcaccat cacaggtgca 240 cagcctgaag atgaggcaga atattattgt gctctatggt acaacaactg ggtgttcggt 300 ggaggaacca aactgactgt cctaggc 327 <210> 25 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> variable region of humanized heavy chain (A9V7FW42) <400> 25 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln   1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Thr Tyr              20 25 30 Gly Val Gln Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu          35 40 45 Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Ala Ala Phe Ile      50 55 60 Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu  65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                  85 90 95 Arg Ala Arg Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val             100 105 110 Ser Ser <210> 26 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> variable region of humanized light chain (A9V7) <400> 26 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly   1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Ile Thr Thr Asn              20 25 30 Asn Phe Ala Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Thr          35 40 45 Leu Ile Tyr Asp Thr Asn Asn Arg Val Pro Gly Val Pro Asp Arg Phe      50 55 60 Ser Gly Ser Leu Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala  65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Asn Asn                  85 90 95 Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly             100 105

Claims (10)

Humanized antibody against the S-surface antigen of the hepatitis B virus of the humanized heavy chain which contains the variable region of the amino acid sequence of SEQ ID NO: 25. Humanized antibody against the S-surface antigen of the hepatitis B virus of the humanized light chain which contains the variable region of the amino acid sequence of SEQ ID NO: 26. A humanized heavy chain gene for the S-surface antigen of hepatitis B virus comprising the nucleotide sequence of SEQ ID NO: 23 encoding the variable region of the amino acid sequence of SEQ ID NO: 25. A humanized light chain gene for the S-surface antigen of hepatitis B virus comprising the nucleotide sequence of SEQ ID NO: 24 encoding the variable region of the amino acid sequence of SEQ ID NO: 26. Leader sequence gene of the humanized antibody heavy chain comprising the nucleotide sequence of SEQ ID NO: 1. Leader sequence gene of the humanized antibody light chain comprising the nucleotide sequence of SEQ ID NO: 13. Heavy chain expression vector pHAB-A9V7FW42HF of humanized antibody against the S-surface antigen of hepatitis B virus containing the gene of Claim 3 (Accession No .: KCTC 10371BP). Light chain expression vector pHAB-A9V7LF of humanized antibody against S-surface antigen of hepatitis B virus containing the gene of claim 4 (Accession No .: KCTC 10372BP). Transformant CHO-hA9-11-5 obtained by transforming heavy chain expression vector pHAB-A9V7FW42HF of humanized antibody and light chain expression vector pHAB-A9V7LF of humanized antibody into CHO cells. A method for producing a humanized antibody against S-surface antigen of hepatitis B virus, wherein the transformant CHO-hA9-11-5 of claim 9 is cultured to produce a humanized antibody.