JP2007014267A - Monoclonal antibody against HBs antigen of hepatitis B virus, genes and peptides related thereto, hepatitis B virus assay method, hepatitis B diagnosis method, treatment method - Google Patents

Abstract

An HBs antigen-specific monoclonal antibody capable of having neutralizing activity against all types of HBV is provided.
An HBs antigen-specific monoclonal antibody. Single chain antibody (scFv) against HBs antigen of hepatitis B virus, heavy chain variable region constituting HBs antigen-specific monoclonal antibody and protein that is light chain variable region, heavy chain constituting HBs antigen-specific monoclonal antibody Peptides that are complementarity determining regions 1 to 3 and light chain complementarity determining regions 1 to 3, DNAs (genes) encoding the heavy chain variable region and the light chain variable region constituting the HBs antigen-specific monoclonal antibody, and the above A DNA (gene) encoding heavy chain complementarity determining regions 1 to 3 and light chain complementarity determining regions 1 to 3 constituting an HBs antigen-specific monoclonal antibody. A method for assaying hepatitis B virus using these monoclonal antibodies, a method for diagnosing hepatitis B, and a method for treating hepatitis B.
[Selection figure] None

Description

  The present invention relates to a gene encoding the variable region or complementarity determining region of the heavy or light chain of a monoclonal antibody against the HBs antigen of hepatitis B virus. Furthermore, the present invention relates to a peptide that is a variable region or complementarity determining region of a heavy or light chain of a monoclonal antibody against HBs antigen of hepatitis B virus. Furthermore, the present invention relates to a monoclonal antibody against a hepatitis B virus HBs antigen, a single chain antibody (scFv), a dimer and a trimer thereof, and a monoclonal antibody, a single chain antibody (scFv), a dimer and a trimer thereof. The present invention relates to a method for assaying hepatitis B virus using a monomer, a method for diagnosing hepatitis B, and a method for treating hepatitis B.

  Hepatitis B is a disease caused by hepatitis B virus infecting hepatocytes and causing death by inducing liver failure and liver cancer. Hepatitis B virus is often infected in hospitals and the like when a patient's blood is accidentally injected into the body due to a needlestick accident or the like. In such a case, hepatitis B by urgently injecting antiserum from the blood of a person who has a large amount of antibody against hepatitis B virus by HB vaccination etc. Generally, infection is prevented by neutralizing (blocking) infection of hepatocytes with a virus with an antibody. However, antiserum prepared from humans in this way is difficult to eliminate the possibility of contamination with unknown viruses, and is not preferable as a therapeutic agent used for humans.

  Therefore, it is considered that an HBs antigen-specific antibody is prepared and used for neutralization (blocking).

[Chimeric antibody, humanized antibody]
HBs antigen-specific antibody production method (screening method) is to immunize mice with HBs antigen, fuse spleen or lymph node B lymphocytes with myeloma cells and secrete HBs antigen-specific antibody. The hybridoma cells are screened by ELISA or the like. The mouse antibody gene is recovered from the established hybridoma and the mouse constant region is replaced (chimerized) with the human constant region, or the antigen binding site (complementarity determining region) of the mouse antibody (CDR1, 2, 3 ) Is substituted (humanized) with that of a human antibody, and a method for bringing it close to a human antibody while retaining the binding property to the HBs antigen is known. [J Waters, M Pignatelli, S Galpin, K Ishihara, HC Thomas, Virus-neutralizing antibodies to hepatitis B virus: The nature of an immunogenic epitope on the S gene peptide. J gen Virol, 67: 2767-2473, 1986 1), S Iwarson, E Tabor, HC Thomas, A Goodall, J Waters, P Snoy, JW-K Shih, RJ Gerety, Neutralization of hepatitis B virus infectivity by a murine monoclonal antibody: and experimental study in the Chimpanzee. J Med Virol, 16: 89-96, 1985 (Non-patent Document 2), CJ Ryu, P Gripon, HR Park, SS Park, YK Kim, C Guguen-Guillouzo, OJ Yoo, HJ Hong, In vitro neutralization of hepatitis B J Med Virol, 52: 226-233, 1997 (Non-Patent Document 5), HJ Hong, CJ Ryu, H Hur, S Kim, HK Oh, MS Oh, SY Park. In vivo neutralization of hepatitis B virus infection by an anti-preS1 humanized antibody in chimpanzees. Virology, 318: 134-141, 2004 (Non-patent Document 6), CJ Ryu, EA Pallan, BR Jin, OJ Yoo, HJ Hong. A humanized a ntibody with specificity for hepatitis B surface antigen. Hum Antibo Hybridomas, 7: 113-122, 1996 (non-patent document 7)]

  As a specific monoclonal antibody, a human monoclonal antibody capable of binding to a hepatitis B virus surface antigen (HBs antigen) is known. Japanese Patent Publication No. 2000-513215 (Patent Document 1) discloses a method for obtaining a hybridoma cell line that produces a human antibody capable of binding to a hepatitis B virus surface antigen (HBs antigen), and a hybridoma cell line and cell line thereof. The antibodies produced are disclosed. Also disclosed are various uses of the above antibodies in the prevention and treatment of HBV infection. In this invention, peripheral blood lymphocytes obtained from a human donor having a high titer of anti-HBs antigen antibody are transplanted into a normal mouse that is lethally irradiated and protected with SCID bone marrow. After immunizing such chimeric mice with HBs antigen, human cells are obtained from the mouse spleen and fused in vitro with heterologous myeloma cells to secrete human antibodies with high affinity and specificity for HBs antigen Generate hybridoma cells.

In addition to the above, HBs antigen-specific antibodies have been reported and described in the following documents.
(1) Maeda F et al. J Med Virol. 58: 338-345, 1999 (non-patent document 8)
(2) Yano A et al. J Med Virol. 73: 208-215, 2004 (Non-patent document 9)
(3) Nihon University Medical Journal 41: 1163-1171, 1982 (Non-Patent Document 10)
(4) Hybridoma 17 (6), 535-540 (1998) (Non-patent Document 11)
(5) Zhonghua Gan Zang Bing Za Zhi 5 (3), 130-132 (2000) (Non-Patent Document 12)
(6) Japanese Patent Laid-Open No. 9-20798 (Patent Document 2)
(7) Biochim. Biophys. Acta 1380 (2), 151-155 (1998) (Non-patent Document 13)
(8) Proc. Natl. Acad. Sci. USA 89 (8), 3175-3179 (1992) (Non-Patent Document 14)

  The following methods are also known as methods for producing (screening methods) for HBs antigen-specific antibodies.

[Human hybridoma method]
Peripheral blood B lymphocytes and myeloma cells are fused by a cell fusion method, and hybridoma cells secreting HBs antigen-specific antibodies are screened by ELISA or the like. [H Sawada, S Iwasa, O Nishimura, K Kitano, Efficient production of anti- (hepatitis B virus) antibodies and their neutralizing activity in chimpanzees. Appl Microbiol Biotechnol, 43: 445-451, 1995 (Non-Patent Document 3), Gary Witherell, XTL-001 XTL Pharmaceuticals. Current Opinion in Investigational Drugs, 3: 684-692, 2002 (non-patent document 4)]

[EB virus transformation method]
Peripheral blood human B lymphocytes are infected with EB virus and immortalized to establish B cell lines, and B cell lines secreting HBs antigen-specific antibodies are screened by ELISA or the like.
Special Table 2000-513215 Japanese Patent Laid-Open No. 9-20798 J Waters, M Pignatelli, S Galpin, K Ishihara, HC Thomas, Virus-neutralizing antibodies to hepatitis B virus: The nature of an immunogenic epitope on the S gene peptide.J gen Virol, 67: 2767-2473, 1986 S Iwarson, E Tabor, HC Thomas, A Goodall, J Waters, P Snoy, JW-K Shih, RJ Gerety, Neutralization of hepatitis B virus infectivity by a murine monoclonal antibody: and experimental study in the Chimpanzee.J Med Virol, 16 : 89-96, 1985 H Sawada, S Iwasa, O Nishimura, K Kitano, Efficient production of anti- (hepatitis B virus) antibodies and their neutralizing activity in chimpanzees.Appl Microbiol Biotechnol, 43: 445-451, 1995 Gary Witherell, XTL-001 XTL Pharmaceuticals.Current Opinion in Investigational Drugs, 3: 684-692, 2002 CJ Ryu, P Gripon, HR Park, SS Park, YK Kim, C Guguen-Guillouzo, OJ Yoo, HJ Hong, In vitro neutralization of hepatitis B virus by monoclonal antibodies against the viral surface antigen.J Med Virol, 52: 226- 233, 1997 HJ Hong, CJ Ryu, H Hur, S Kim, HK Oh, MS Oh, SY Park.In vivo neutralization of hepatitis B virus infection by an anti-preS1 humanized antibody in chimpanzees.Virology, 318: 134-141, 2004 CJ Ryu, EA Pallan, BR Jin, OJ Yoo, HJ Hong.A humanized antibody with specificity for hepatitis B surface antigen.Hum Antibo Hybridomas, 7: 113-122, 1996 Maeda F et al. J Med Virol. 58: 338-345, 1999 Yano A et al. J Med Virol. 73: 208-215, 2004 Nihon University Medical Journal 41: 1163-1171, 1982 Hybridoma 17 (6), 535-540 (1998) Zhonghua Gan Zang Bing Za Zhi 5 (3), 130-132 (2000) Biochim. Biophys. Acta 1380 (2), 151-155 (1998) Proc. Natl. Acad. Sci. USA 89 (8), 3175-3179 (1992)

  As HBV, 10 or more different HBVs are known depending on the amino acid, but there is a problem that only a part of the antibody described in Patent Document 1 can be recognized. Further, the antibody described in Patent Document 1 has a problem that 100% of HBV has not been neutralized as a result of the neutralization experiment.

  In addition, Non-Patent Documents 8 to 10 describe that the antibodies described in Non-Patent Documents 8 to 10 have neutralization, but no specific data is shown. Furthermore, Non-Patent Documents 11 to 14 and Patent Document 2 do not describe whether the antibodies described therein have neutralizing activity.

  Accordingly, an object of the present invention is to provide an HBs antigen-specific monoclonal antibody that can have neutralizing activity against all types of HBV.

Furthermore, the present invention, in addition to the above-mentioned HBs antigen-specific monoclonal antibody,
(1) Single chain antibody (scFv) against HBs antigen of hepatitis B virus
(2) a protein that is a heavy chain variable region and a light chain variable region constituting the HBs antigen-specific monoclonal antibody,
(3) Peptides that are the heavy chain complementarity determining regions 1 to 3 and the light chain complementarity determining regions 1 to 3 constituting the HBs antigen-specific monoclonal antibody,
(4) DNA (gene) encoding the heavy chain variable region and the light chain variable region constituting the HBs antigen-specific monoclonal antibody, and
(5) DNA (gene) encoding heavy chain complementarity determining regions 1 to 3 and light chain complementarity determining regions 1 to 3 constituting the HBs antigen-specific monoclonal antibody
It is also intended to provide.

  The present inventors considered that an antibody against hepatitis B virus was produced using cell chip technology and recombinant DNA technology and used as a therapeutic agent. That is, B lymphocytes are prepared from the blood of a vaccinated person, seeded on a microwell array chip developed by the parties concerned of the present inventors, stimulated with hepatitis B virus surface antigen (HBs antigen), B lymphocytes reacting with HBs antigen were detected using a scanner. The detected B lymphocytes were recovered from the chip, the antibody gene was recovered, and incorporated into an expression vector into an animal cell to produce a recombinant antibody. The produced recombinant antibody was confirmed to bind to the HBs antigen, and further to block (neutralize) virus infection of human hepatocytes, thereby completing the present invention.

The present invention is as follows.
[1] A gene comprising any of the following DNAs.
(1) DNA having the base sequence of SEQ ID NO: 1,
(2) It has a base sequence having a deletion, substitution and / or addition of one to several bases in the base sequence of SEQ ID NO: 1, and encodes the heavy chain variable region of a monoclonal antibody against HBs antigen of hepatitis B virus DNA to perform, and
(3) The heavy chain variable region of a monoclonal antibody against the HBs antigen of hepatitis B virus, which has the base sequence of SEQ ID NO: 1 or a base sequence that hybridizes with a base sequence complementary to this base sequence under stringent conditions DNA encoding
[2] A gene encoding any of the following proteins.
(1) a protein having the amino acid sequence of SEQ ID NO: 2,
(2) A heavy chain variable region of a monoclonal antibody against hepatitis B virus HBs antigen having an amino acid sequence having a deletion, substitution and / or addition of 1 to several amino acids in the amino acid sequence of SEQ ID NO: 2. Protein, and
(3) A protein which has an amino acid sequence having 90% or more homology with the amino acid sequence of SEQ ID NO: 2 and is a heavy chain variable region of a monoclonal antibody against hepatitis B virus HBs antigen.
[3] A gene comprising any of the following DNA.
(1) DNA having the sequence of SEQ ID NO: 3,
(2) It has a base sequence having a deletion, substitution and / or addition of 1 to several bases in the base sequence of SEQ ID NO: 3 and encodes the light chain variable region of a monoclonal antibody against HBs antigen of hepatitis B virus DNA to perform, and
(3) The light chain variable region of a monoclonal antibody against the HBs antigen of hepatitis B virus, which has a base sequence of SEQ ID NO: 3 or a base sequence that hybridizes with a base sequence complementary to this base sequence under stringent conditions DNA encoding
[4] A gene encoding any of the following proteins.
(1) a protein having the amino acid sequence of SEQ ID NO: 4,
(2) A light chain variable region of a monoclonal antibody against hepatitis B virus HBs antigen having an amino acid sequence having a deletion, substitution and / or addition of 1 to several amino acids in the amino acid sequence of SEQ ID NO: 4 Protein, and
(3) A protein which has an amino acid sequence having 90% or more homology with the amino acid sequence of SEQ ID NO: 4 and is a light chain variable region of a monoclonal antibody against hepatitis B virus HBs antigen.
[5] Any of the following proteins.
(1) a protein having the amino acid sequence of SEQ ID NO: 2,
(2) A heavy chain variable region of a monoclonal antibody against hepatitis B virus HBs antigen having an amino acid sequence having a deletion, substitution and / or addition of 1 to several amino acids in the amino acid sequence of SEQ ID NO: 2. Protein, and
(3) A protein which has an amino acid sequence having 90% or more homology with the amino acid sequence of SEQ ID NO: 2 and is a heavy chain variable region of a monoclonal antibody against hepatitis B virus HBs antigen.
[6] Any of the following proteins.
(1) a protein having the amino acid sequence of SEQ ID NO: 4,
(2) A light chain variable region of a monoclonal antibody against hepatitis B virus HBs antigen having an amino acid sequence having a deletion, substitution and / or addition of 1 to several amino acids in the amino acid sequence of SEQ ID NO: 4 Protein, and
(3) A protein which has an amino acid sequence having 90% or more homology with the amino acid sequence of SEQ ID NO: 4 and is a light chain variable region of a monoclonal antibody against hepatitis B virus HBs antigen.
[7] Any of the following genes.
(1) A monoclonal antibody against the hepatitis B virus HBs antigen having the base sequence encoding the 76th to 105th base sequence in the base sequence of SEQ ID NO: 1 or the 26th to 35th amino acid sequence of the amino acid sequence of SEQ ID NO: 3 A gene encoding heavy chain complementarity determining region 1 (CDR-H1) of an antibody.
(2) Monoclonal antibody against hepatitis B virus HBs antigen having a nucleotide sequence of 148 to 198 in the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence encoding the 50th to 66th amino acid sequence of the amino acid sequence of SEQ ID NO: 3 A gene encoding the heavy chain complementarity determining region 2 (CDR-H2).
(3) Monoclonal antibody against hepatitis B virus HBs antigen having a nucleotide sequence of 295 to 333 in the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence encoding amino acids 99 to 111 in the amino acid sequence of SEQ ID NO: 3 A gene encoding the heavy chain complementarity determining region 3 (CDR-H3).
[8] Any of the following genes.
(1) A monoclonal antibody against a hepatitis B virus HBs antigen having a base sequence encoding 69-102 in the base sequence of SEQ ID NO: 3, or a base sequence encoding 24-34 in the amino acid sequence of SEQ ID NO: 4 A gene encoding light chain complementarity determining region 1 (CDR-L1).
(2) A monoclonal antibody against a hepatitis B virus HBs antigen having a base sequence encoding 148 to 168 in the base sequence of SEQ ID NO: 3 or a base sequence encoding 50 to 56 of the amino acid sequence of SEQ ID NO: 4 A gene encoding light chain complementarity determining region 2 (CDR-L2).
(3) A monoclonal antibody against a hepatitis B virus HBs antigen having a nucleotide sequence encoding 265 to 291 in the nucleotide sequence of SEQ ID NO: 3 or an amino acid sequence of 89 to 97 in the amino acid sequence of SEQ ID NO: 4. A gene encoding light chain complementarity determining region 3 (CDR-L3).
[9] Any of the following peptides.
(1) a peptide which is a heavy chain complementarity determining region 1 (CDR-H1) of a monoclonal antibody against HBs antigen of hepatitis B virus having an amino acid sequence of 26 to 35 in the amino acid sequence of SEQ ID NO: 2;
(2) a peptide that is a heavy chain complementarity determining region 2 (CDR-H2) of a monoclonal antibody against HBs antigen of hepatitis B virus, having an amino acid sequence of 50 to 66 in the amino acid sequence of SEQ ID NO: 2;
(3) A peptide that is a heavy chain complementarity determining region 3 (CDR-H3) of a monoclonal antibody against hepatitis B virus HBs antigen, having an amino acid sequence of 99 to 111 in the amino acid sequence of SEQ ID NO: 2.
[10] Any of the following peptides.
(1) A peptide that is a light chain complementarity determining region 1 (CDR-L1) of a monoclonal antibody against hepatitis B virus HBs antigen, having an amino acid sequence of 24-34 in the amino acid sequence of SEQ ID NO: 4,
(2) a peptide that is a light chain complementarity determining region 2 (CDR-L2) of a monoclonal antibody against hepatitis B virus HBs antigen, having an amino acid sequence of 50 to 56 in the amino acid sequence of SEQ ID NO: 4,
(3) A peptide which is a light chain complementarity determining region 3 (CDR-L3) of a monoclonal antibody against hepatitis B virus HBs antigen, having an amino acid sequence of 89 to 97 in the amino acid sequence of SEQ ID NO: 4.
[11] A monoclonal antibody against the hepatitis B virus HBs antigen, comprising a heavy chain comprising a protein having the amino acid sequence of SEQ ID NO: 2 as a variable region and a light chain comprising a protein having the amino acid sequence of SEQ ID NO: 4 as a variable region .
[12] A heavy chain comprising peptides having amino acid sequences of 26 to 35, 50 to 66 and 99 to 111 in the amino acid sequence of SEQ ID NO: 2 as complementarity determining regions 1 to 24 and 24 to 34 in the amino acid sequence of SEQ ID NO: 4 And a light chain comprising peptides having amino acid sequences of 50 to 56 and 89 to 97 as complementarity determining regions 1 to 3, monoclonal antibodies against hepatitis B virus HBs antigen.
[13] A single-chain antibody against hepatitis B virus HBs antigen, wherein a protein having the amino acid sequence of SEQ ID NO: 2 and a protein having the amino acid sequence of SEQ ID NO: 4 are bound by a linker peptide.
[14] a protein comprising peptides having amino acid sequences of 26 to 35, 50 to 66 and 99 to 111 in the amino acid sequence of SEQ ID NO: 2 as complementarity determining regions 1 to 24 and 24 to 34 in the amino acid sequence of SEQ ID NO: 4; A single chain antibody against HBs antigen of hepatitis B virus, wherein a peptide having 50 to 56 and 89 to 97 amino acid sequences as a complementarity determining region 1 to 3 is bound with a linker peptide.
[15] A dimer of the single-chain antibody according to [13] or [14].
[16] A trimer of the single-chain antibody according to [13] or [14].
[17] A type B wherein a protein having the amino acid sequence of SEQ ID NO: 2 and a protein having the amino acid sequence of SEQ ID NO: 4 are linked by a linker peptide, and the CH3 region is bound to the protein having the amino acid sequence of SEQ ID NO: 2 Minibody against hepatitis virus HBs antigen.
[18] a protein comprising peptides having amino acid sequences of 26 to 35, 50 to 66 and 99 to 111 in the amino acid sequence of SEQ ID NO: 2 as complementarity determining regions 1 to 24 and 24 to 34 in the amino acid sequence of SEQ ID NO: 4; A protein comprising peptides having amino acid sequences of 50 to 56 and 89 to 97 as complementarity determining regions 1 to 3 is linked with a linker peptide, and 26 to 35, 50 to 66 and 99 to 99 in the amino acid sequence of SEQ ID NO: 2. A minibody against hepatitis B virus HBs antigen, wherein a CH3 region is bound to the protein comprising a peptide having 111 amino acid sequences as complementarity determining regions 1 to 3.
[19] The protein according to [5] or [6], the peptide according to [9] or [10], the monoclonal antibody according to [11] or [12], or the protein according to [13] or [14] A method for assaying hepatitis B virus using a single chain antibody, a dimer according to [15], a trimer according to [16], or a minibody according to [17] or [18].
[20] The protein according to [5] or [6], the peptide according to [9] or [10], the monoclonal antibody according to [11] or [12], or the protein according to [13] or [14] A method for diagnosing hepatitis B using the single-chain antibody according to [15], the dimer according to [15], the trimer according to [16], or the minibody according to [17] or [18].
[21] The protein according to [5] or [6], the peptide according to [9] or [10], the monoclonal antibody according to [11] or [12], or the protein according to [13] or [14] A method for treating hepatitis B using the single-chain antibody of [15], the dimer according to [15], the trimer according to [16], and the minibody according to [17] or [18].

  According to the present invention, an HBs antigen-specific monoclonal antibody that can have neutralizing activity against all types of HBV can be provided.

  As described above, the recombinant antibody of the present invention stimulates B lymphocytes prepared from the blood of a vaccinated person with hepatitis B virus surface antigen (HBs antigen), and reacts with one B lymph that reacts with HBs antigen. The spheres were collected, antibody genes were collected from the collected B lymphocytes, and prepared using animal cells transformed with an expression vector containing the antibody genes. In this process, the base sequence of the antibody gene and the amino acid sequence of the antibody were determined for the heavy chain and the light chain, respectively, and the complementarity determining regions in the heavy chain and the light chain were determined.

The present invention
(1) Monoclonal antibody against hepatitis B virus HBs antigen and single chain antibody (scFv)
(2) a protein that is a heavy chain variable region and a light chain variable region constituting this monoclonal antibody,
(3) Peptides that are the heavy chain complementarity determining regions 1 to 3 and the light chain complementarity determining regions 1 to 3 constituting this monoclonal antibody,
(4) DNA (gene) encoding heavy chain variable region and light chain variable region constituting this monoclonal antibody, and
(5) It relates to DNA (gene) encoding heavy chain complementarity determining regions 1 to 3 and light chain complementarity determining regions 1 to 3 constituting this monoclonal antibody.

[DNA encoding the variable region (gene)]
The DNA (gene) encoding the heavy chain variable region of the present invention comprises any of the following DNAs.
(1) DNA having the base sequence of SEQ ID NO: 1,
(2) It has a base sequence having a deletion, substitution and / or addition of one to several bases in the base sequence of SEQ ID NO: 1, and encodes the heavy chain variable region of a monoclonal antibody against HBs antigen of hepatitis B virus DNA to perform, and
(3) The heavy chain variable region of a monoclonal antibody against the HBs antigen of hepatitis B virus, which has the base sequence of SEQ ID NO: 1 or a base sequence that hybridizes with a base sequence complementary to this base sequence under stringent conditions DNA encoding

Furthermore, the DNA (gene) encoding the heavy chain variable region of the present invention is a gene encoding any of the following proteins.
(1) a protein having the amino acid sequence of SEQ ID NO: 2,
(2) A heavy chain variable region of a monoclonal antibody against hepatitis B virus HBs antigen having an amino acid sequence having a deletion, substitution and / or addition of 1 to several amino acids in the amino acid sequence of SEQ ID NO: 2. Protein, and
(3) A protein which has an amino acid sequence having 90% or more homology with the amino acid sequence of SEQ ID NO: 2 and is a heavy chain variable region of a monoclonal antibody against hepatitis B virus HBs antigen.

The DNA (gene) encoding the light chain variable region of the present invention comprises any of the following DNAs.
(1) DNA having the sequence of SEQ ID NO: 3,
(2) It has a base sequence having a deletion, substitution and / or addition of 1 to several bases in the base sequence of SEQ ID NO: 3 and encodes the light chain variable region of a monoclonal antibody against HBs antigen of hepatitis B virus DNA to perform, and
(3) The light chain variable region of a monoclonal antibody against the HBs antigen of hepatitis B virus, which has a base sequence of SEQ ID NO: 3 or a base sequence that hybridizes with a base sequence complementary to this base sequence under stringent conditions DNA encoding

Furthermore, the DNA (gene) encoding the light chain variable region of the present invention is a gene encoding any of the following proteins.
(1) a protein having the amino acid sequence of SEQ ID NO: 4,
(2) A light chain variable region of a monoclonal antibody against hepatitis B virus HBs antigen having an amino acid sequence having a deletion, substitution and / or addition of 1 to several amino acids in the amino acid sequence of SEQ ID NO: 4 Protein, and
(3) A protein which has an amino acid sequence having 90% or more homology with the amino acid sequence of SEQ ID NO: 4 and is a light chain variable region of a monoclonal antibody against hepatitis B virus HBs antigen.

  The base sequence of SEQ ID NO: 1 is the base sequence of the H region (heavy chain) V region cDNA, and the amino acid sequence of SEQ ID NO: 2 is the amino acid of the H chain V region. Furthermore, the base sequence of SEQ ID NO: 3 is the base sequence of the cDNA of the V region of the L chain (light chain), and the amino acid sequence of SEQ ID NO: 4 is the amino acid sequence of the V region of the L chain. As described in detail in the Examples, these sequences were obtained by collecting B lymphocytes that reacted with HBs antigen, synthesizing cDNA from the collected B lymphocytes, and using the synthesized cDNA, the antibody H chain and Perform a PCR reaction to amplify the V region of the L chain, prepare cDNA of the H chain and L chain V region of the obtained cell antibody, determine the base sequence of the prepared cDNA, and further, this cDNA It was confirmed after confirming that it was translated into protein. As a result, in the H chain and L chain family (subclass) of the antibody of the present invention, the H chain was VH3, DH, JH2, and the L chain (κ chain) was Vκ3, Jκ4.

  The range of “1 to several” in the “base sequence having deletion, substitution and / or addition of 1 to several bases” as used herein is not particularly limited, but for example, 1 to 40, preferably Means 1 to 30, more preferably 1 to 20, more preferably 1 to 10, further preferably 1 to 5, and particularly preferably about 1 to 3. The same applies to the following.

  The fact that the DNA of the present invention is a DNA encoding the variable region of the heavy chain or light chain of a monoclonal antibody against the HBs antigen of hepatitis B virus indicates that the protein prepared using this DNA as a template is hepatitis B virus. It can confirm by having the reactivity with respect to HBs antigen. The reactivity of hepatitis B virus to HBs antigen is confirmed by, for example, enzyme linked immunosorbent assay (ELISA) using an enzyme-labeled secondary antibody, radio immunoassay (RIA) using a secondary antibody labeled with a radioisotope, etc. can do. In addition, when the protein produced using DNA as a template is a part of an antibody, the reactivity of the antibody to a part of the antigen of the antibody can be similarly measured by ELISA or the like. However, for example, in the case of scFv, since the Fc portion of the antibody is deleted, an enzyme-labeled anti-IgG antibody cannot be used as the secondary antibody. In this case, by adding a tag (a peptide that becomes an epitope of an antibody) to scFv and using an antibody against the tag as a secondary antibody, it can be measured by ELISA or the like.

  ELISA coats the wells of a 96-well plate with HBs antigen. The target antibody can be added to wells not coated with HBs antigen, and binding of the target antibody to HBs antigen can be confirmed by the binding of the secondary antibody labeled with the enzyme using the color development of the enzyme substrate as an indicator. In this case, since the target antibody does not bind to the well not coated with the antigen, the color does not develop and the target antibody binds to the well coated with the antigen and develops color. In RIA, HBs antigen is similarly coated on a 96-well plate. The target antibody can be added to wells coated with and without HBs antigen, and the binding of the target antibody to HBs antigen can be confirmed by using the radioactivity count due to the binding of the secondary antibody labeled with a radioisotope as an indicator. it can.

  The above-mentioned “hybridizes under stringent conditions” means a DNA base sequence obtained by using a DNA as a probe and using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like. For example, after hybridization at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA derived from colonies or plaques or a fragment of the DNA is immobilized, 0. Examples include DNA that can be identified by washing a filter under a condition of 65 ° C. using a 1-2 × SSC solution (1 × SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). Hybridization can be performed according to the method described in Molecular Cloning 2nd edition.

  Examples of DNA that hybridizes under stringent conditions include DNA having a certain degree of homology with the base sequence of DNA used as a probe, for example, 70% or more, preferably 80% or more, more preferably 90%. More preferably, DNA having homology of 93% or more, particularly preferably 95% or more, and most preferably 98% or more is mentioned.

  The range of “1 to several” in the “amino acid sequence having 1 to several amino acid deletions, substitutions and / or additions” as used herein is not particularly limited. For example, 1 to 20, preferably Means 1 to 10, more preferably 1 to 7, further preferably 1 to 5, particularly preferably about 1 to 3.

  The homology in the “amino acid sequence having 90% or more homology to the amino acid sequence described in SEQ ID NO: 2 or 4 of the sequence listing” in the present specification is not particularly limited as long as it is 90% or more. Preferably it is 95% or more, More preferably, it is 97% or more, More preferably, it is 98%, Most preferably, it is 99% or more.

  Several antibodies having reactivity with hepatitis B virus HBs antigen have already been reported. (Patent Documents 1 and 2, Non-Patent Documents 8 to 14) On the other hand, the H chain and L chain families (subclasses) of the antibody of the present invention are as described above, and the antibody of the present invention is combined with the H chain and L chain. Antibodies whose chain family (subclass) is VH3 for H chain and Vκ3 for L chain (κ chain) are shown in the table below. Homology is homology with the amino acid sequence of the corresponding variable region of the antibody of the present invention.

  The method for obtaining the gene of the present invention is not particularly limited. By preparing appropriate probes and primers based on the amino acid sequence and nucleotide sequence information shown in SEQ ID NOs: 1 to 4 in the sequence listing in the present specification, and using them to screen an antibody cDNA library, The gene of the invention can be isolated. Whether the isolated gene is the gene of the present invention can be determined by determining the nucleotide sequence and checking the binding ability of the antibody protein expressed from the gene to the HBs antigen. A cDNA library can be prepared from cells expressing the gene of the present invention by a conventional method.

  The gene of the present invention can also be obtained by PCR. Using the chromosomal DNA library or cDNA library of the antibody as a template, PCR is performed using a pair of primers designed to amplify the base sequence described in SEQ ID NO: 1 or 3. PCR reaction conditions can be set as appropriate. For example, one cycle of a reaction step consisting of 94 ° C. for 30 seconds (denaturation), 55 ° C. for 30 seconds to 1 minute (annealing), and 72 ° C. for 2 minutes (extension) For example, after 30 cycles, a condition of reacting at 72 ° C. for 7 minutes can be exemplified. The amplified DNA fragment can then be cloned into a suitable vector that can be amplified in a host such as E. coli. Whether the isolated gene is the gene of the present invention can be determined by determining the nucleotide sequence and checking the binding ability of the antibody protein expressed from the gene to the HBs antigen.

Operations such as preparation of the probe or primer, construction of a cDNA library, screening of the cDNA library, and cloning of the target gene, determination of the base sequence, expression of the protein from the gene, etc. are known to those skilled in the art, for example, Molecular cloning:.. a laboratory Mannual , 2 nd Ed, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989 ( hereinafter, abbreviated as Molecular cloning 2nd Edition), Current Protocols in Molecular Biology, Supplement 1~38, John Wiley & Sons (1987-1997) (hereinafter abbreviated as “Current Protocols in Molecular Biology”) and the like.

  The above-described method for obtaining a gene of the present invention can be appropriately applied to a method for obtaining a gene encoding a CDR described later.

  In addition, it has an amino acid sequence having a deletion, substitution and / or addition of 1 to several amino acids in the amino acid sequence set forth in SEQ ID NO: 2 or 4 in the sequence listing, and a monoclonal antibody against HBs antigen of hepatitis B virus. A gene encoding a protein that is a heavy chain variable region or a light chain variable region; and a base having a deletion, substitution and / or addition of one to several bases in the base sequence set forth in SEQ ID NO: 1 or 3 in the sequence listing For genes having sequences and encoding heavy chain variable regions or light chain variable regions of monoclonal antibodies against HBs antigen of hepatitis B virus (hereinafter these genes are referred to as mutant genes), SEQ ID NOs: 1-4 Any person known to those skilled in the art, such as chemical synthesis, genetic engineering techniques or mutagenesis, based on the described amino acid sequence and base sequence information In can be prepared.

  For example, it can be carried out using a method of contacting a DNA having the base sequence described in SEQ ID NO: 1 or 3 in the sequence listing with a drug that is a mutagen, a method of irradiating with ultraviolet rays, a genetic engineering method, etc. . Site-directed mutagenesis, which is one of the genetic engineering methods, is useful because it can introduce a specific mutation at a specific position. Molecular cloning 2nd edition, Current Protocols in Molecular. It can be performed according to the method described in biology and the like.

[Variable region protein]
The protein that is the heavy chain variable region constituting the monoclonal antibody against the HBs antigen of the hepatitis B virus of the present invention is any of the following proteins.
(1) a protein having the amino acid sequence of SEQ ID NO: 2,
(2) A heavy chain variable region of a monoclonal antibody against hepatitis B virus HBs antigen having an amino acid sequence having a deletion, substitution and / or addition of 1 to several amino acids in the amino acid sequence of SEQ ID NO: 2. Protein, and
(3) A protein which has an amino acid sequence having 90% or more homology with the amino acid sequence of SEQ ID NO: 2 and is a heavy chain variable region of a monoclonal antibody against hepatitis B virus HBs antigen.

The protein which is the light chain variable region constituting the monoclonal antibody against the HBs antigen of the hepatitis B virus of the present invention is any of the following proteins.
(1) a protein having the amino acid sequence of SEQ ID NO: 4,
(2) A light chain variable region of a monoclonal antibody against hepatitis B virus HBs antigen having an amino acid sequence having a deletion, substitution and / or addition of 1 to several amino acids in the amino acid sequence of SEQ ID NO: 4 Protein, and
(3) A protein which has an amino acid sequence having 90% or more homology with the amino acid sequence of SEQ ID NO: 4 and is a light chain variable region of a monoclonal antibody against hepatitis B virus HBs antigen.

  As described above, the amino acid sequences of SEQ ID NOs: 2 and 4 were determined by confirming that the cDNA of the variable region of the monoclonal antibody against the HBs antigen is translated into a protein.

  The method for obtaining the protein of the present invention is not particularly limited, and may be a protein synthesized by chemical synthesis or a recombinant protein produced by a gene recombination technique. When producing a recombinant protein, first, a gene (DNA) encoding the protein described above is obtained. By introducing this DNA into an appropriate expression system, the protein of the present invention can be produced. Moreover, the CDR peptide of the present invention can be produced in the same manner. The expression of proteins and peptides in the expression system will be described later in this specification.

[Complementarity determining region DNA (gene)]
The DNA (gene) encoding the heavy chain complementarity determining regions 1 to 3 constituting the monoclonal antibody of the present invention is any of the following genes.
(1) having a base sequence encoding the 76th to 105th base sequence (SEQ ID NO: 5) in the base sequence of SEQ ID NO: 1 or the 26th to 35th amino acid sequence (SEQ ID NO: 11) of the amino acid sequence of SEQ ID NO: 3 A gene encoding heavy chain complementarity determining region 1 (CDR-H1) of a monoclonal antibody against HBs antigen of hepatitis B virus.
(2) having a nucleotide sequence encoding the nucleotide sequence of 148 to 198 (SEQ ID NO: 6) in the nucleotide sequence of SEQ ID NO: 1 or the 50th to 66th amino acid sequence (SEQ ID NO: 12) of the amino acid sequence of SEQ ID NO: 3; A gene encoding heavy chain complementarity determining region 2 (CDR-H2) of a monoclonal antibody against hepatitis B virus HBs antigen.
(3) having a base sequence encoding the base sequence of 295 to 333 (SEQ ID NO: 7) in the base sequence of SEQ ID NO: 1 or the 99th to 111th amino acid sequence (SEQ ID NO: 13) of the amino acid sequence of SEQ ID NO: 3, A gene encoding heavy chain complementarity determining region 3 (CDR-H3) of a monoclonal antibody against hepatitis B virus HBs antigen.

Furthermore, the DNA (gene) encoding the light chain complementarity determining regions 1 to 3 constituting the monoclonal antibody of the present invention is any of the following genes.
(1) having a base sequence encoding a base sequence of 69 to 102 (SEQ ID NO: 8) in the base sequence of SEQ ID NO: 3 or an amino acid sequence of 24 to 34 (SEQ ID NO: 14) in the amino acid sequence of SEQ ID NO: 4; A gene encoding the light chain complementarity determining region 1 (CDR-L1) of a monoclonal antibody against HBs antigen of hepatitis B virus.
(2) having a base sequence encoding the base sequence of 148 to 168 (SEQ ID NO: 9) in the base sequence of SEQ ID NO: 3 or the amino acid sequence of 50 to 56 (SEQ ID NO: 15) in the amino acid sequence of SEQ ID NO: 4; A gene encoding the light chain complementarity determining region 2 (CDR-L2) of a monoclonal antibody against HBs antigen of hepatitis B virus.
(3) having a base sequence encoding a base sequence of 265 to 291 (SEQ ID NO: 10) in the base sequence of SEQ ID NO: 3 or an amino acid sequence of 89 to 97 (SEQ ID NO: 16) in the amino acid sequence of SEQ ID NO: 4; A gene encoding the light chain complementarity determining region 3 (CDR-L3) of a monoclonal antibody against HBs antigen of hepatitis B virus.

[Complementarity determining region peptide]
The peptide which is the heavy chain complementarity determining regions 1 to 3 constituting the monoclonal antibody of the present invention is any of the following peptides.
(1) a peptide which is a heavy chain complementarity determining region 1 (CDR-H1) of a monoclonal antibody against HBs antigen of hepatitis B virus having an amino acid sequence of 26 to 35 in the amino acid sequence of SEQ ID NO: 2;
(2) a peptide that is a heavy chain complementarity determining region 2 (CDR-H2) of a monoclonal antibody against HBs antigen of hepatitis B virus, having an amino acid sequence of 50 to 66 in the amino acid sequence of SEQ ID NO: 2;
(3) A peptide that is a heavy chain complementarity determining region 3 (CDR-H3) of a monoclonal antibody against hepatitis B virus HBs antigen, having an amino acid sequence of 99 to 111 in the amino acid sequence of SEQ ID NO: 2.

The peptides that are light chain complementarity determining regions 1 to 3 constituting the monoclonal antibody of the present invention are any of the following peptides.
(1) A peptide that is a light chain complementarity determining region 1 (CDR-L1) of a monoclonal antibody against hepatitis B virus HBs antigen, having an amino acid sequence of 24-34 in the amino acid sequence of SEQ ID NO: 4,
(2) a peptide that is a light chain complementarity determining region 2 (CDR-L2) of a monoclonal antibody against hepatitis B virus HBs antigen, having an amino acid sequence of 50 to 56 in the amino acid sequence of SEQ ID NO: 4,
(3) A peptide which is a light chain complementarity determining region 3 (CDR-L3) of a monoclonal antibody against hepatitis B virus HBs antigen, having an amino acid sequence of 89 to 97 in the amino acid sequence of SEQ ID NO: 4.

  The heavy chain complementarity determining regions 1 to 3 and the light chain complementarity determining regions 1 to 3 of the monoclonal antibody of the present invention were determined according to Kabat's criteria. Furthermore, the heavy chain complementarity determining regions 1 to 3 and the light chain complementarity determining regions 1 to 3 of the monoclonal antibody of the present invention are the same in the H chain and L chain family (subclass) as the monoclonal antibody of the present invention. Tables 2 to 7 show amino acid sequence comparisons and amino acid sequence homologies of antibodies (H chain: VH, L chain (κ chain): Vκ3).

[Monoclonal antibody]
The monoclonal antibody against the HBs antigen of hepatitis B virus of the present invention comprises a heavy chain comprising a protein having the amino acid sequence of SEQ ID NO: 2 as a variable region and a light chain comprising a protein having the amino acid sequence of SEQ ID NO: 4 as a variable region. Is included.

  Furthermore, the monoclonal antibody against HBs antigen of hepatitis B virus of the present invention comprises peptides having amino acid sequences of 26 to 35, 50 to 66 and 99 to 111 in the amino acid sequence of SEQ ID NO: 2 as complementarity determining regions 1 to 3. It comprises a heavy chain and a light chain comprising peptides having amino acid sequences of 24 to 34, 50 to 56 and 89 to 97 in the amino acid sequence of SEQ ID NO: 4 as complementarity determining regions 1 to 3.

  The monoclonal antibody of the present invention can be prepared by using the method described in the following literature [Kanda H, Mori K, Koga H, Taniguchi K, Kobayashi H, Sakahara H, Konishi J, Endo K, Watanabe T Construction and expression of chimeric antibodies by a simple replacement of heavy and light chain V genes into a single cassette vector. Hybridoma. 13: 359-66, 1994]. Specifically, the heavy chain variable region gene and the light chain variable region gene of the antibody gene are amplified by RT-PCR from HBs antigen-specific B lymphocytes. The amplified gene is cloned into pT7Blue-T vector (Novagen, A Brand of EMD Biosciences, Inc, An affiliate of Merck KGaA, Darmstadt, Germany) and the base sequence is determined. For example, the nucleotide sequences are shown in SEQ ID NOs: 1 and 3. PCR is performed using the nucleotide sequence as a template and a primer containing a restriction enzyme site, the amplified gene fragment is cleaved with a restriction enzyme, and introduced into an antibody expression vector using the restriction enzyme site. In addition to the signal sequence necessary for antibody protein secretion, the antibody expression vector has antibody heavy chain constant region DNA or antibody light chain constant region region DNA cloned therein. By introducing the variable region gene, a full-length antibody gene can be constructed.

  The constructed antibody heavy chain gene and antibody light chain gene are simultaneously introduced into animal cells such as human fetal kidney-derived 293T cells and cultured for 2 to 3 days, whereby the desired antibody protein is secreted into the culture supernatant.

  When an antibody is prepared using a combination of CDR genes, it can be performed as follows. The V region of an antibody consists of a framework part (1-4) that forms the basic three-dimensional structure of the antibody and a CDR part (1-3) that binds to the antigen. It is possible to synthesize the V region of any antibody by combining the framework structure with any CDR. As shown in FIG. 12, “overlap PCR” is used to synthesize one V region gene by combining these. For example, when there are Fr1 and CDR1 gene sequences, the Fr1 and CDR3 genes are synthesized by PCR in step 1. At this time, primers (P2, P3) between Fr1 and CDR1 are overlapped by adding the base sequence of CDR1 or Fr1, respectively. The PCR products completed in Step 1 have overlapping base sequences, and when these PCR products are heat-denatured and annealed, they are annealed at the overlapping portions (Step 2). When this is extended with Taq DNA polymerase, Fr1 and CDR1 are connected as shown in Step 3. When this is amplified by PCR using primers P1 and P4, DNA in which Fr1 and CDR1 are connected is amplified. Step 2 and step 3 can be performed simultaneously. Actually, Fr2, CDR2, Fr3, CDR3, and Fr4 are amplified in the same manner. When primers P1 and P5 are used in step 3, the gene in the V region of the antibody is amplified. If the CDR portion is made into a random base sequence, a library of V regions that can bind to any antigen can be created.

[Single-chain antibody]
The single-chain antibody against the HBs antigen of hepatitis B virus of the present invention is a protein in which a protein having the amino acid sequence of SEQ ID NO: 2 and a protein having the amino acid sequence of SEQ ID NO: 4 are bound by a linker peptide. Furthermore, the single-chain antibody against HBs antigen of hepatitis B virus of the present invention comprises peptides having amino acid sequences of 26 to 35, 50 to 66 and 99 to 111 in the amino acid sequence of SEQ ID NO: 2 as complementarity determining regions 1 to 3. And a protein comprising a peptide having amino acid sequences of 24 to 34, 50 to 56, and 89 to 97 in the amino acid sequence of SEQ ID NO: 4 as complementarity determining regions 1 to 3 bound by a linker peptide.

A single chain Fv protein (single chain Fv, scFv) is a protein in which an antibody heavy chain variable region (V _H ) and an antibody light chain variable region (V _L ) are connected in series, and is produced in the same manner as a monoclonal antibody. be able to. That is, using the antibody heavy chain variable region gene and antibody light chain variable region gene cloned in the pT7Blue-T vector described in the above monoclonal antibody production method as a template, these two regions are combined with a linker sequence, and protein synthesis in vitro is performed. A gene containing a T7 promoter, ribosome binding sequence, etc. is prepared using PCR so as to conform to the above. For example, scFv protein can be synthesized by incubating in the cell-free protein synthesis reaction solution PURESYSTEM SS (Post Genome Laboratory, Tokyo) using the gene thus prepared.

  The present invention encompasses dimers and triabodies of the above single chain antibodies. Furthermore, the present invention provides a complementarity determining region comprising a protein having the amino acid sequence of SEQ ID NO: 2 of the above-mentioned single chain antibody or a peptide having the amino acid sequences of 26 to 35, 50 to 66 and 99 to 111 in the amino acid sequence of SEQ ID NO: 2. 1 to 3 include a minibody against hepatitis B virus HBs antigen, in which a CH3 region is bound to the protein contained in 1 to 3.

  The linker constituting the single chain Fv protein is, for example, a series of three (GlyGlyGlyGlySer). As described in the following paper, by changing the length of the linker, a dimer composed of two scFvs and a triabody composed of three scFvs can be prepared (Hudson PJ, Kortt AA, High avidity scFv multimers; diabodies and triabodies.See J Immunol Methods, 231: 177-189 (1999), Edited by Frank Breitling, Sterfan Dubel "Recombinant Antibodies" (1999) A John Willey & Sons, Inc. (New York ) and Spektrum Akademischer Verlag (Heidelberg) Copublication).

  When an antibody is handled as a medicine, its molecular weight is a problem. In order to solve this problem, molecules with reduced molecular weights, such as scFv, dimers, triabodies, and minibodies, that have the ability to bind antigens, have been created and used as antibody drugs. Is being evaluated.

  As shown in FIG. 1, the antibody gene is composed of a VH gene fragment and CH1, (H), CH2, CH3, and (CH4) gene fragments for the H chain, and a VL gene fragment and a CL gene fragment for the L chain. Of these, the VH and VL gene fragments joined by a linker is a single chain Fv (scFv) (FIG. 2). scFv has the structure of 5'-VH-VL-3 'and 5'-VL-VH-3', that is, scFv is formed from the V domain of an antibody and does not have a C region. This is a diabody that forms a dimer, and a triabody that forms a trimer. There are reports that these can be formed by changing the length of the linker (PJ Hudoson and AA Kortt, High avidity scFv multimers; diabodies and triabodies. J Immunol Methods 231: 177-189, 1999). Compared to scFv, diabody and triabody have stronger overall avidity. In addition, the minibody that binds the CH3 domain of IgG to scFv is a product of antibody miniaturization that maintains the antigen-binding ability, and forms a dimer at the CH3 portion (LS Shahied, Y Tang, RK Alpaugh, R Somer, D Greenspon, LM Weiner. Bispecific minibodies targeting HER2 / neu and CD16 exhibit improved tumor lysis when placed in a divalent tumor antigen binding format. J Biol Chem. 279: 53907-53914). There are four variations of IgG from IgG1 to IgG4, and the CH3 portion can be any CH3 of IgG1 to IgG4. However, the CH3 part of IgG1 is preferable.

[Use of monoclonal antibodies]
The present invention includes a method for assaying hepatitis B virus using the monoclonal antibody of the present invention, a method for diagnosing hepatitis B, and a method for treating hepatitis B. In addition to the monoclonal antibody itself, the monoclonal antibody can be used by using a part of the monoclonal antibody of the present invention or a DNA thereof, for example, scFv single chain antibody, single chain dimer (diabody), trimer (triabody). ) Or minibodies, or the use of such genes. Further, as a part of the monoclonal antibody of the present invention, for example, a protein that is a heavy chain variable region and a light chain variable region constituting this monoclonal antibody, and a heavy chain complementarity determining region 1 to 1 constituting this monoclonal antibody. 3 and light chain complementarity determining regions 1-3.

Assay method for hepatitis B virus The assay method for hepatitis B virus using the monoclonal antibody of the present invention can be performed as follows. First, the monoclonal antibody of the present invention is adsorbed to a well of a 96-well plate. After blocking and suppressing nonspecific adsorption, a specimen containing hepatitis B virus is added to the well. If the sample contains a virus, the virus binds to the monoclonal antibody of the present invention adsorbed to the well. Thereafter, the well is washed, and the enzyme-labeled monoclonal antibody of the present invention is added to the well. After washing the well, an enzyme substrate is added to the well, and the presence of virus in the sample is detected by color development of the substrate. In a well to which a specimen containing virus is added, a complex of an antibody adsorbed to the well, a virus bound thereto, and an enzyme-labeled antibody bound to the virus is formed, and the substrate develops color.

The method for assaying hepatitis B virus using the monoclonal antibody of the present invention can also be performed as follows. That is, the monoclonal antibody of the present invention is fixed to a 96-well plate or bead, and a specimen for checking the presence of hepatitis B virus is added thereto. If hepatitis B virus is present, the virus binds to the antibody. Viral DNA is extracted from the virus bound to the antibody, PCR is performed using it as a template, and the viral DNA is amplified and detected. Even in this way, it is possible to detect the presence of the virus in the specimen.
Virus detection by the above method can be carried out by using a part of the monoclonal antibody of the present invention, F (ab) ′ ₂ , F (ab), scFv, a dimer of a single-chain antibody, a trimer or a tribody. A body, a protein that is a heavy chain variable region and a light chain variable region constituting the monoclonal antibody, and heavy chain complementarity determining regions 1 to 3 and light chain complementarity determining regions 1 to 1 constituting the monoclonal antibody. 3 can also be used.

Diagnosis method of hepatitis B Diagnosis of hepatitis B using the monoclonal antibody of the present invention is to detect the hepatitis B virus in the patient serum using the monoclonal antibody of the present invention by examining the patient serum as the sample. Can be done.
It can also be diagnosed by staining the virus in the liver tissue obtained by biopsy from the liver of the patient with the monoclonal antibody of the present invention.
Diagnosis of the virus by the above method can be carried out by using a part of the monoclonal antibody of the present invention, F (ab) ′ ₂ , F (ab), scFv, dimer of single chain antibody, trimer or tribody A body, a protein that is a heavy chain variable region and a light chain variable region constituting the monoclonal antibody, and heavy chain complementarity determining regions 1 to 3 and light chain complementarity determining regions 1 to 1 constituting the monoclonal antibody. 3 can also be used.

Inhibition of Hepatitis B Virus Infection and Treatment Method for Hepatitis B Inhibition of hepatitis B virus infection using the monoclonal antibody of the present invention can be carried out as follows. In other words, in a so-called needlestick accident where the needle with blood, body fluid, culture supernatant, etc. adhering to hepatitis B virus is accidentally stuck, At the same time, a treatment method in which an antiserum having a high antibody titer against hepatitis B virus is injected is generally used. That is, the antibody binds to hepatitis B virus and acts to prevent infection of cells. However, human serum preparations are not desirable because they may be contaminated with unknown viruses because they use serum derived from donated blood. Moreover, it is not always possible to stably supply a preparation having a high titer and an excellent neutralizing action. If the monoclonal antibody of the present invention is produced in a controlled environment, a virus neutralizing antibody free from unknown viruses can be provided in a stable quality and quantity, and can be used in place of the human antiserum.

  In addition, liver transplantation may be an option for treatment in patients who have been infected with hepatitis B virus and suffered from liver cancer, cirrhosis, or liver failure. In this case, if hepatitis B virus remains in the patient, the transplanted liver is reinfected with hepatitis B virus, and the therapeutic effect is reduced by half. In such a liver transplantation field, the monoclonal antibody of the present invention can be used for treatment. That is, when the patient's liver is removed for liver transplantation, the monoclonal antibody of the present invention is administered to the patient alone or in combination with other antibody drugs or antiviral agents, and the remaining hepatitis B virus Neutralizing can prevent virus infection in newly transplanted liver.

Infection prevention and treatment by the above-described methods can be applied to a part of the monoclonal antibody of the present invention, F (ab) ′ ₂ , F (ab), scFv, a dimer of a single chain antibody, a trimer or a trimer. Minibodies, proteins that are the heavy chain variable region and light chain variable region constituting this monoclonal antibody, and heavy chain complementarity determining regions 1 to 3 and light chain complementarity determining region 1 constituting this monoclonal antibody It can also be carried out using a peptide of ~ 3.

Example 1
[Preparation of B lymphocytes]
Recombinant HB vaccine prepared with yeast was immunized to HBs antibody positive volunteers, and lymphocytes were prepared from peripheral blood 9 days after immunization. That is, human lymphocytes from peripheral blood were centrifuged using Lymphosepar I solution (Immuno-Biological Laboratories, Takasaki City, Gunma Prefecture), and further from the lymphocyte fraction using AutoMACS (Miltenyi Biotec, Bergisch Gladbac, Germany). The non-B lymphocyte fraction cells were removed and the B lymphocyte fraction was separated and purified.

[Loading of fluorescent dye to B lymphocytes]
Prepare 2 x 10 ⁶ B lymphocytes at 2μM Fluo-4 / AM (calcium-dependent fluorescent dye, Dojin, Kumamoto) / 1μM CellTracker Orange (Molecular Probe) /0.04% pluronic F-127 (Molecular Probe) / Suspend in loading buffer (137 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl ₂ , 1 mM MgCl ₂ , 1 mg / mL glucose, 1 mg / mL BSA, 20 mM Hepes (pH 7.4)) at room temperature with gentle shaking. Incubated for 30 minutes. The cells were washed with Loading buffer to remove excess Fluo-4 / AM that was not introduced into the cells. Thereafter, the cells were suspended in RPMI1640 / 10% FCS solution. In all the following experiments, RPMI1640 not containing Phenol Red was used. Fluo-4 is a fluorescent dye whose fluorescence intensity increases when calcium binds, and is used to detect activation of B lymphocytes by antigen. CellTracker Orange is a fluorescent dye that emits fluorescence regardless of calcium, and was used for cell recognition using a microarray scanner or for cell recognition when cells were collected under a fluorescence microscope.

[Microwell array chip]
The microwell array chip is manufactured using silicon (manufactured by Toyama Prefectural Industrial Technology Center), and microwells with a diameter of 10 μm and a depth of 14 μm are arranged vertically and horizontally at a pitch of 25 μm (distance between well centers). . The microwells were 30 x 30 (900 wells) and formed one cluster, and 12 clusters were arranged vertically and 22 clusters were arranged horizontally. A sticker having a thickness of 1 mm, a width of about 1 mm, and a length of 2 cm was attached to both sides of the microwell array chip.

[Microarray scanner]
This device basically uses a microarray scanner (CRBIO IIe-FITC) from Hitachi Software Engineering Co., Ltd. (Yokohama), which is equipped with a laser that can emit excitation light with wavelengths of 473 nm and 532 nm. Yes. In addition, analysis is possible with a resolution of 2.5 μm. The depth of focus is set to ± 50 μm.

[Dissemination of lymphocytes into microwell array chip]
In order to fill the RPMI1640 / 10% FCS solution into the microwells of the microwell array chip, the microwell array chip is immersed in the RPMI1640 / 10% FCS solution and the air in the well is removed by applying ultrasonic waves. Filled the solution. Filling the microwells with RPMI1640 / 10% FCS solution was also performed by the following method. Add 100% alcohol solution to the microwell array chip. The alcohol solution penetrates into the microwell. Next, the alcohol solution was replaced with PBS, and further replaced with RPMI1640 / 10% FCS solution. After filling the microwell with RPMI1640 / 10% FCS solution, the excess buffer on the chip was removed, and the cell suspension was added thereto, and allowed to stand for 2 to 5 minutes. Cells that did not enter the microwell were collected, and a cell suspension was further added onto the chip, and allowed to stand for 2 to 5 minutes. Cells that did not enter the microwell were collected, and a cell suspension was further added onto the chip, and allowed to stand for 2 to 5 minutes. Cells that did not enter the microwells were collected, and cells that did not enter the microwells on the chip were washed away using RPMI 1640/10% FCS solution. The diameter of the lymphocyte is about 8 μm, and since the diameter of the microwell used is 10 μm, one lymphocyte is contained in one microwell. A cover glass was placed on the seal and RPMI 1640/10% FCS solution was filled between the chip and the cover glass.

[Detection of HBs antigen-specific B lymphocytes using a microwell array chip]
A microwell array chip seeded with B lymphocytes was inserted into a microarray scanner (CRBIO IIe-FITC), scanned at a resolution of 2.5 μm, measured for fluorescence intensity of Fluo-4 before antigen stimulation, and stored (before antigen stimulation) Fluorescence data A). Next, the chip was taken out of the scanner, the RPMI1640 / 10% FCS solution between the chip and the cover glass was removed, and HBs antigen (100 μg / mL) dissolved in the RPMI1640 / 10% FCS solution was added thereto. Immediately after inserting the microwell array chip into a microarray scanner, the fluorescence intensity of intracellular Fluo-4 was scanned at a resolution of 2.5 μm after adding the antigen, and the data was stored (fluorescence data B after antigen stimulation). ). The ratio of fluorescence intensity before and after stimulation (B / A) was calculated using the analysis software attached to the scanner, and wells with a high ratio were identified. Within these wells are antigen-specific B lymphocytes.

[Recovery of B lymphocytes from microwells]
B lymphocytes with increased intracellular calcium in response to the detected HBs antigen were collected using a micromanipulator under a fluorescence microscope. That is, first, the RPMI 1640/10% FCS solution between the cover glass and the chip was removed, and the cover glass was removed by putting air between the cover glass and the chip. The RPMI1640 / 10% FCS solution was added to the chip so that the chip did not dry, and the target cells were collected with a micromanipulator while observing the fluorescence of CellTracker Orange of the cells under a fluorescence microscope.

[Collecting antibody genes from B lymphocytes]
Transfer the collected B lymphocytes to a PCR tube. 15 μL cell lysate in cells (final concentration when diluted to 25 μL: 1x 1 ^st strand buffer (attached to GIBCO-BRL, SuperScript III), 0.2 mM dNTP, 0.25% NP-40, 0.1 mg / Add mL BSA, 10 mM DTT, 0.05 μM Random Primer, 1U / μL RNasin (Promega, Madison, WI), and add reverse transcriptase SuperScriptIII (GIBCO-BRL, Rockville, MD) (5 μL, 50U) CDNA was synthesized from mRNA by reaction at 37 ^° C. for 1 hour.

[First amplification of antibody gene]
Using the synthesized human antibody gene cDNA as a template, PCR reaction was performed using the following primers designed to amplify the V regions of the H chain and L chain of the antibody.

Primers for heavy chains of human antibodies (IgM and IgG)

For PCR-1
Primer1 5 'mix
hVH17a.1 atggactgsayytggagvdtc (SEQ ID NO: 17)
hVH2a.1 tccacrctcctgctrctgac (SEQ ID NO: 18)
hVH3a.1 gggcygagstggvttttyct (SEQ ID NO: 19)
hVH4a.1 tcctcctsctggtggcagct (SEQ ID NO: 20)
hVH5.1 tcaaccgccatcctcgccct (SEQ ID NO: 21)
hVH6.1 ctccttcctcatcttcctgcc (SEQ ID NO: 22)
Primer1 3 'mix
hIGHG1-4out agtccttgaccaggcagccca (SEQ ID NO: 23)
hIGHMout attctcacaggagacgagggg (SEQ ID NO: 24)

Primer for human antibody L chain

For PCR-1
Primer1 5 'mix
hKV12.1 atgaggstcccygctcagctc (SEQ ID NO: 25)
hKV3.1 ctcttcctcctgctactctggc (SEQ ID NO: 26)
hKV45.1 ctsttsctytggatctctg (SEQ ID NO: 27)
hKV6.1 tgggtttctgctgctctggg (SEQ ID NO: 28)
hKV7.1 atagggtccggggctcctttg (SEQ ID NO: 29)
hLV12.1 cykctsctcctcactctcctc (SEQ ID NO: 30)
hLV3.1 ttctcctcctcggcctcctct (SEQ ID NO: 31)
hLV4.2-2 ccagcytgtgctgactcaatc (SEQ ID NO: 32)
hLV789.2 tcycagmctgtgstgacycag (SEQ ID NO: 33)
hLV6.1 ttttatgctgactcagcccc (SEQ ID NO: 34)
hLV7.1 ggcctggactcctctctttctg (SEQ ID NO: 35)
hLV8.1 ggcctggatgatgcttctcctc (SEQ ID NO: 36)
hLV9.1 tcctctgctcctcaccctcct (SEQ ID NO: 37)
hLV10.1 cctgggtcatgctcctcctga (SEQ ID NO: 38)
hLV11.1 gcctgggctccactacttctc (SEQ ID NO: 39)
Primer1 3 'mix
hIGK1 ctgctcatcagatggcggga (SEQ ID NO: 40)
hIGL1 gacacacyagtgtggccttgt (SEQ ID NO: 41)

  A PCR reaction was performed, and the H chain and L chain obtained by amplifying the cDNA of the V region of the antibody H chain and L chain of the obtained cells were amplified using separate tubes. PCR reaction was performed using 2 μL of the above cDNA in 18 μL of PCR mix 1 (final concentration: 1x TAKARA ExTaq buffer, 0.25 mM dNTP, Primer 1 5 ′ mix 0.5 μM each, Primer 1 3 ′ mix 0.5 μM each, 0.05 U / μL ExTaq (Takara, Kyoto) ), 94 ° C, 5 min; (94 ° C, 30 sec; 60 ° C, 1 min; 72 ° C, 1 min 30 sec) x 40 cycles; 72 ° C, 5 min. By this PCR reaction, DNA from the leader sequence of the antibody gene to the constant region is amplified.

[Second amplification of antibody gene]
Since cDNA was not sufficiently amplified by one PCR, the second nested PCR was performed. In the second PCR reaction, 2 μL of the PCR reaction solution obtained in the first round was added to 18 μL of PCR mix2 (final concentration: 1x TAKARA ExTaq buffer, 0.25 mM dNTP, Primer2 5 ′ mix 0.5 μM each, Primer2 3 ′ mix 0.5 μM each, 0.05 U / μL ExTaq), 94 ^° C., 5 min; (94 ° C., 30 sec; 60 ° C., 1 min; 72 ° C., 1 min 30 sec) × 40 cycles; 72 ° C., 5 min. By the second round of PCR, the cDNA sequence from the variable region to the constant region of the antibody gene amplified in the first round is amplified. The primer sequences used are shown below.

Primers for heavy chains of human antibodies (IgM and IgG)

For PCR-2
Primer2 5 'mix
hVH17a.2 ggtgcagctkgtrcartctgg (SEQ ID NO: 42)
hVH2a.2 caccttgarggagtctggtcc (SEQ ID NO: 43)
hVH3a.2 aggtdcarctgktggagtcyg (SEQ ID NO: 44)
hVH4a.2 ggtcctgtctcaggtgcagct (SEQ ID NO: 45)
hVH5a.2 gtgcagctggtgcagtctgg (SEQ ID NO: 46)
hVH6.2 gcagcagtcaggtccaggact (SEQ ID NO: 47)
Primer2 3 'mix
hIGHG1-4S aagacsgatgggcccttggtg (SEQ ID NO: 48)
hIGHMin aagggttgggcggatgcact (SEQ ID NO: 49)

Primer for human antibody L chain

For PCR-2
Primer1 5 'mix
hKV1.2 ccagatgacccagtctccatc (SEQ ID NO: 50)
hKV2.2 ccagtggggatattgtgatgac (SEQ ID NO: 51)
hKV3.2 cagtctccagccaccctgtct (SEQ ID NO: 52)
hKV4.2 gtgatgacccagtctccagac (SEQ ID NO: 53)
hKV 5.2 acactcacgcagtctccagca (SEQ ID NO: 54)
hKV67.2 ttgtgctgacycagtctccag (SEQ ID NO: 55)
hLV1.2 agtctgtgctgacgcagccgc (SEQ ID NO: 56)
hLV23.2 tgactcagccwcyctcmgtgtc (SEQ ID NO: 57)
hLV4.2-3 caatcatcctctgcmtctgc (SEQ ID NO: 58)
hLV5.2-2 gactcagccaacctccctctc (SEQ ID NO: 59)
hLV6.2 gactcagccccactctgtgtc (SEQ ID NO: 60)
hLV789.2 tcycagmctgtgstgacycag (SEQ ID NO: 61)
hLV1011.2 tgactcagccmcmctckgtgtc (SEQ ID NO: 62)
Primer1 3 'mix
hIGK2 gacagatggtgcagccacagt (SEQ ID NO: 63)
hIGL2 cttgragctcctcagaggaggg (SEQ ID NO: 64)

[Determination of the base sequence of the amplified antibody gene]
The PCR product was analyzed and purified using an agarose gel, cloned into pT7Blue-T vector (Novagen, Madison, Wis.), The nucleotide sequence of the antibody gene was determined, and it was confirmed that it was translated into protein. The nucleotide sequence of the H chain V region cDNA is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. Further, the base sequence of the cDNA of the V region of the L chain is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4.

Example 2
[Production of antibody protein, analysis of antigen binding ability]
The antibody H chain and L chain variable region genes amplified by RT-PCR were incorporated into an antibody protein expression vector (FIG. 3). That is, the gene fragment of the amplified antibody H chain variable region is the VH portion of the HEF-HBs-gγ1 vector, and the L chain variable region gene fragment is 5 of the constant region (Human Cκ) of the HEFs-gκ vector. 'Introduced using a restriction enzyme site on the side. In order to express antibody proteins from the prepared H chain and L chain expression vectors, both expression vectors were simultaneously introduced into human fetal kidney-derived cell 293T cells. Gene transfer was performed using the calcium phosphate method in accordance with a conventional method (cell engineering separate volume 8 new cell engineering experiment protocol, Institute of Medical Science, University of Tokyo, edited by Cancer Research Department, Shujunsha Co., Ltd.). The cell supernatant was collected after 3 days. The binding ability of antibodies in the culture supernatant to HBs antigen was measured by ELISA using a 96 well plate coated with HBs antigen. Measurement by ELISA was performed as follows. That is, 50 μL of HBs antigen (10 μg / mL) diluted with phosphate buffered saline (PBS) was dispensed into each well in a 96-well plate, and allowed to react overnight at 4 ° C. for adsorption. The wells were washed with PBS, and 400 μL of PBS containing 3% BSA and 0.05% Tween-20 was dispensed into each well to remove non-specific binding, followed by blocking at room temperature for 2 hours. As a negative control well, a well blocked without antigen adsorption was also prepared. Thereafter, the wells were washed with PBS containing 0.1% Tween-20 (PBS-T), and 50 μL of the culture supernatant of the 293T cells was dispensed into each well and allowed to react at room temperature for 2 hours. The wells were washed with PBS-T, and alkaline phosphatase (ALP) -labeled anti-human immunoglobulin diluted 1000-fold was dispensed into each well at 50 μL and reacted at room temperature for 2 hours. The wells were washed with PBS-T, and 1 mg / mL p-nitrophenyl phosphate (ALP substrate) dissolved in ALP substrate buffer (100 mM NaCl, 5 mM MgCl ₂ 100 mM Tris pH 9.5) was dispensed into each well at 50 μL. After 20 minutes of reaction at room temperature, the absorbance at 405 nm was measured with a microplate reader. Furthermore, in order to check the specificity of the binding of the monoclonal antibody of the present invention to the antigen, the soluble HBs antigen and the culture supernatant were preincubated when the cell culture supernatant containing the antibody was added to the well. . The mixed solution was added to the well, and it was examined whether the binding of the antibody to the HBs antigen adsorbed to the well was competitively inhibited by the soluble HBs antigen. The results are shown in FIG.

  As shown in FIG. 4, the prepared 116 antibody bound to HBs antigen, and the antibody did not bind to wells not bound to HBs antigen (data not shown). Further, in order to examine the specificity of binding, a mixture of 116 antibody and soluble HBs antigen was added to wells bound with HBs antigen, and ELISA was performed in the same manner. It was observed that binding of 116 antibody to well bound HBs antigen was dose-dependently and competitively inhibited by soluble HBs antigen. From this result, it was shown that the 116 antibody specifically binds to the HBs antigen.

Determination of epitope to which 116 antibody binds
Using an ELISA system, the 116 antibody binds to the epitope a, d, or r of the adr type HBs antigen, and the monoclonal antibody that binds to each epitope competes for the binding of the 116 antibody to the adr type HBs antigen. It examined by investigating whether it inhibits. First, 50 μL of adr-type HBs antigen (10 μg / mL) diluted with phosphate buffered saline (PBS) was dispensed into each well in a 96-well plate and allowed to react overnight at 4 ^° C. Adsorbed. Wash wells with PBS, dispense 400 μL of PBS containing 3% BSA and 0.05% Tween-20 (PBS-T / BSA) to remove non-specific binding, and react at room temperature for 2 hours. Blocked. As a negative control well, a well blocked without antigen adsorption was also prepared. The wells were then washed with PBS containing 0.1% Tween-20 (PBS-T), and a mouse monoclonal antibody that recognizes epitope-a of PBS-T / BSA or 2 μg / mL (anti-a antibody) and a mouse monoclonal that recognizes epitope d 50 μL of antibody (anti-d antibody) or mouse monoclonal antibody (anti-r antibody) recognizing epitope r was dispensed into each well and allowed to react at room temperature for 1 hour. 5 μL of 0.5 μg / mL biotinylated 116 antibody was dispensed into each well and further reacted at room temperature for 2 hours. The wells were washed with PBS-T, and alkaline phosphatase (ALP) -labeled streptavidin diluted 1000-fold was dispensed into each well in an amount of 50 μL and reacted at room temperature for 2 hours. Wash wells with PBS-T and add 50 μL of 1 mg / mL p-nitrophenyl phosphate (ALP substrate) dissolved in ALP substrate buffer (100 mM NaCl, 5 mM MgCl ₂ , 100 mM Tris pH 9.5) to each well. After dispensing for 20 minutes at room temperature, the absorbance at 405 nm was measured with a microplate reader. The results are shown in FIG.

  The well that did not adsorb antigen did not develop color (column 1), and the well that adsorbed antigen developed color (column 2), indicating that the 116 antibody was specifically bound to the adr type HBs antigen. Recognize. Anti-a antibody inhibited binding of 116 antibody to antigen (column 3), whereas anti-d and anti-r antibodies did not inhibit binding of 116 antibody to antigen (columns 4 and 5). These results indicate that the 116 antibody does not recognize epitope d or epitope r but recognizes epitope a common to HB viruses.

  In the present invention, in producing the 116 antibody, the HBs vaccine is of the adr type, and screening is also performed using the adr HBs antigen. Therefore, the 116 antibody binds to an adr type HBs antigen. As to where epitope 116 recognizes epitope a, epitope d, and epitope r, when measuring binding of 116 antibody to adr-type HBs antigen by ELISA as described above, epitope a, epitope d, epitope r When anti-a antibody, anti-d antibody, and anti-r antibody that are known to bind to are added and competed simultaneously, they compete with anti-a antibody and inhibit binding, but with anti-d antibody and anti-r antibody Is not competitive and binding is not inhibited. Thus, it can be seen that, instead of recognizing different epitopes d and r depending on the virus, epitope a common to all viruses is recognized. Thus, the antibody of the present invention is an HBs antigen-specific antibody that can have neutralizing activity against all types of HBV.

Example 3
[Human hepatocyte chimeric mice]
The method for producing a human hepatocyte chimeric mouse is described in detail in the following paper. [C Tateno, Y Yoshizane, N Saito, M Kataoka, R Utoh, C Yamasaki, A Tachibana, Y Soeno, K Asahina, H Hino, T Asahara, T Yokoki, T Furukawa, K Yoshizato. Near completely humanized liver in mice shows Am J Pathol, 165: 901-912, 2004] Briefly, uPA mice (B6SJL-TgN (Alb1Plau) 144Bri, The Jackson Laboratory, Bar Harbor, ME) and scid mice (Fox Chase SCID CB-17 / lcr-scid Jcl, Clea Japan Inc., Tokyo, Japan) was used to produce uPA / SCID mice. For human hepatocytes, hepatic non-parenchymal cells, including small hepatocytes, were isolated from liver tissue collected from donors with consent and used for transplantation. One day before transplantation of small hepatocytes, anti-asialo GM1 antibody was intraperitoneally administered to 20-30 days old uPA / SCID mice. One day later, 5-7 × 10 ⁵ live hepatocytes were transplanted through the portal vein. The success or failure of transplantation was evaluated by measuring the human albumin level in mouse serum.

[Analysis of virus neutralizing ability of anti-HBs-antibody]
Since HBV only infects human or chimpanzee hepatocytes, whether anti-HBs-antibodies can block HBV infection has been performed using chimpanzees. Recently, an experimental system in which fresh liver tissue is collected from volunteers and infected with HBV in vitro using the hepatocytes has also been reported. However, since the collected hepatocytes easily lose their susceptibility to infection by continuing the culture in vitro, it is necessary to always use fresh hepatocytes, and it has been difficult to actually perform it because of the burden on humans. However, the above human hepatocyte chimeric mouse can retain hepatocytes for a long time without losing cell properties such as HBV infection susceptibility of the transplanted hepatocytes, and further, a chimeric mouse is prepared using human hepatocytes derived from the chimeric mouse. This makes it possible to supply an HBV infection experiment system easily, stably, and at a low price. In this experiment, HBV infection experiments were performed using human hepatocyte chimeric mice, and whether the 116 antibody could block HBV infection was examined using the experimental system. The outline of the experiment is as follows.

Sera from patients infected with hepatitis B virus (containing 1 x 10 ⁸ copies / mL of hepatitis B virus (HBV) genome) 50 µL and 116 antibodies (20 µg / animal) mixed in a plastic tube and reacted at room temperature for 30 minutes Then, the mixture was injected from the subclavian vein of human hepatocyte chimeric mice (2 mice). As a positive control for infection, only patient serum was administered to two human hepatocyte chimeric mice. As a negative control of 116 antibody, human IgG (20 μg / mouse) incubated with patient serum was administered to two human hepatocyte chimeric mice. Sera from mice were collected at 2 weeks, 4 weeks, and 6 weeks after administration, and the presence of hepatitis B virus genomic gene was quantified using quantitative PCR.

  The results are shown in FIG. FIG. 6 shows that human chimeric mice (1-7) were injected with only HBV (1-3), HBV and control IgG (4, 5), and HBV and 116 antibody (6, 7). It is the result of having measured the HBV genomic DNA in the mouse | mouth serum of the 1st and 6th week by quantitative PCR method. The lower column shows the amount of human albumin in the serum of the human hepatocyte chimeric mouse, indicating the presence of human hepatocytes in the chimeric mouse.

  As shown in FIG. 6, when only patient serum (HBV) was administered, viral genomic DNA was detected from 2 weeks after administration, and the amount of DNA increased as the 4th and 6th weeks passed. This result indicates that HBV is infected and proliferated in transplanted human hepatocytes since HBV does not infect mouse cells. Similar results were obtained when human IgG was used as a negative control antibody. On the other hand, when a mixture of antibody 116 and HBV was administered to human hepatocyte chimeric mice, a small amount of the viral genome gene was detected in 2 weeks in one mouse, but in 4th and 6th weeks. In both mice, no viral genome genes were detected. The above results indicate that the 116 antibody retains an activity (neutralizing activity) that inhibits virus infection in cells.

Example 4
Single chain Fv (ScFv)
Gene construction
116 antibody gene sequences and control antibody gene sequences, respectively as a template, the bound gene was constructed by PCR by a linker (GGGGS) ₃ to the 5 'side of the side and V _L genes 3' V _H gene. In addition, a T7 promoter sequence on the 5 'side of this V _H- (GGGGS) ₃ -V _L sequence, an ε sequence to increase translation efficiency, an SD sequence that is a ribosome binding site and an initiation codon, and an epitope tag on the 3' side An HA tag (amino acid sequence: YPYDVPDYA) and a stop codon were added by PCR (FIGS. 7 and 8).

scFv protein synthesis Cell-free protein synthesis reaction solution PURESYSTEM SS (Post Genome Laboratory, Tokyo) 50 μL was added with 1 pmol of the PCR product as a template, and protein synthesis was performed at 37 ° C. for 60 minutes. After the reaction, the tube was transferred to ice and used in the following experiments as scFv116 and scFvcont.

Examination of antigen specificity of scFv116
The binding of scFv to the antigen was examined by ELISA. 50 μL of adr type HBs antigen (10 μg / mL) diluted with PBS was dispensed into each well in a 96-well plate and reacted overnight at 4 ^° C. for adsorption. Wash wells with PBS, dispense 400 μL of PBS containing 3% BSA and 0.05% Tween-20 (PBS-T / BSA) to remove non-specific binding, and react at room temperature for 2 hours. And blocked. After washing the wells with PBS containing 0.1% Tween-20 (PBS-T), 50 μL of scFv116 or scFvcont diluted with PBS-T / BSA was added to each well and allowed to react at room temperature for 1 hour. Thereafter, the wells were washed with PBS-T, and then a mouse anti-HA monoclonal antibody diluted 1000 times was dispensed into each well by 50 μL and reacted at room temperature for 1 hour. After washing with PBS-T, alkaline phosphatase (ALP) -labeled goat anti-mouse IgG antibody diluted 2000-fold was dispensed at 50 μL per well and allowed to react at room temperature for 2 hours. The wells were washed with PBS-T, and 1 mg / mL p-nitrophenyl phosphate (ALP substrate) dissolved in ALP substrate buffer (100 mM NaCl, 5 mM MgCl ₂ , 100 mM Tris pH 9.5) was dispensed in 50 μL each well. After injecting and reacting at room temperature for 20 minutes, the absorbance at 405 nm was measured with a microplate reader. As shown in FIG. 9, the scFv protein (scFvcont) constructed from the control gene sequence does not bind to the HBs antigen at all, and the scFv constructed from the 116 gene sequence (scFv116) binds to the HBs antigen in a concentration-dependent manner. It has been shown.

  To examine the specificity of scFv116 binding to HBs antigen, the above ELISA was performed after reacting scFv116 at a constant concentration with HBs antigen at various concentrations (0, 10, 100, 400, 1,000 ng / mL). went. As shown in FIG. 10, the binding of scFv116 to HBs antigen was inhibited depending on the concentration of HBs antigen. This result indicates that the binding of scFv116 and HBs antigen is specific.

Furthermore, in order to investigate whether the epitope recognized by scFv116 and the original IgG116 is the same, various concentrations (0, 40, 200, 1,000 ng / mL) of IgG116 were added to a certain concentration of scFv116. The ELISA was performed. As a result, the binding of scFv116 was inhibited depending on the concentration of added IgG116 (FIG. 11). This result shows that the IgG type and scFv type of HL116 antibody recognize the same epitope.
As described above, prepared based on the 116 antibody gene

Such an antibody against hepatitis B virus (the antibody of the present invention) is expected to be effective for the treatment of a needle stick accident as described above, but is also expected to be effective at the time of liver transplantation. In other words, in patients infected with hepatitis B virus, hepatitis develops due to reinfection of hepatitis B virus in the newly transplanted liver even after liver transplantation, and the liver function is likely to deteriorate. The meaning of liver transplantation may be lost. In this case, it is expected that hepatitis B virus infection in the transplanted liver can be protected by neutralizing and removing hepatitis B virus in the patient's body with an antibody against hepatitis B virus at the time of liver transplantation. Thus, antibodies having the ability to neutralize hepatitis B virus, particularly recombinant antibodies, are considered to be very useful for preventing infection with hepatitis B virus.

The schematic diagram of the structure of an antibody gene. The schematic diagram of structures, such as single chain Fv (scFv). Antibody γ chain expression vector and antibody κ chain expression vector. Results of specific binding test of 116 antibody to HBs antigen. The test result of the epitope which 116 antibody recognizes. The HBV neutralization ability test result of 116 antibody. scFv gene construction and scFv protein synthesis scheme. The base sequence of the 116 antibody scFv gene. The base sequence of the 116 antibody scFv gene. Results of binding test of scFv protein (scFvcont) to HBs antigen. Specificity test results for HBs antigen of scFv116. Specificity test results of scFv116 to IgG116. Explanatory drawing of the method of preparing an antibody using combining a CDR gene.

Claims (21)

A gene consisting of any of the following DNA.
(1) DNA having the base sequence of SEQ ID NO: 1,
(2) It has a base sequence having a deletion, substitution and / or addition of one to several bases in the base sequence of SEQ ID NO: 1, and encodes the heavy chain variable region of a monoclonal antibody against HBs antigen of hepatitis B virus DNA to perform, and
(3) The heavy chain variable region of a monoclonal antibody against the HBs antigen of hepatitis B virus, which has the base sequence of SEQ ID NO: 1 or a base sequence that hybridizes with a base sequence complementary to this base sequence under stringent conditions DNA encoding A gene encoding any of the following proteins.
(1) a protein having the amino acid sequence of SEQ ID NO: 2,
(2) A heavy chain variable region of a monoclonal antibody against hepatitis B virus HBs antigen having an amino acid sequence having a deletion, substitution and / or addition of 1 to several amino acids in the amino acid sequence of SEQ ID NO: 2. Protein, and
(3) A protein which has an amino acid sequence having 90% or more homology with the amino acid sequence of SEQ ID NO: 2 and is a heavy chain variable region of a monoclonal antibody against hepatitis B virus HBs antigen. A gene consisting of any of the following DNA.
(1) DNA having the sequence of SEQ ID NO: 3,
(2) It has a base sequence having a deletion, substitution and / or addition of 1 to several bases in the base sequence of SEQ ID NO: 3 and encodes the light chain variable region of a monoclonal antibody against HBs antigen of hepatitis B virus DNA to perform, and
(3) The light chain variable region of a monoclonal antibody against the HBs antigen of hepatitis B virus, which has a base sequence of SEQ ID NO: 3 or a base sequence that hybridizes with a base sequence complementary to this base sequence under stringent conditions DNA encoding A gene encoding any of the following proteins.
(1) a protein having the amino acid sequence of SEQ ID NO: 4,
(2) A light chain variable region of a monoclonal antibody against hepatitis B virus HBs antigen having an amino acid sequence having a deletion, substitution and / or addition of 1 to several amino acids in the amino acid sequence of SEQ ID NO: 4 Protein, and
(3) A protein which has an amino acid sequence having 90% or more homology with the amino acid sequence of SEQ ID NO: 4 and is a light chain variable region of a monoclonal antibody against hepatitis B virus HBs antigen. Any of the following proteins.
(1) a protein having the amino acid sequence of SEQ ID NO: 2,
(2) A heavy chain variable region of a monoclonal antibody against hepatitis B virus HBs antigen having an amino acid sequence having a deletion, substitution and / or addition of 1 to several amino acids in the amino acid sequence of SEQ ID NO: 2. Protein, and
(3) A protein which has an amino acid sequence having 90% or more homology with the amino acid sequence of SEQ ID NO: 2 and is a heavy chain variable region of a monoclonal antibody against hepatitis B virus HBs antigen. Any of the following proteins.
(1) a protein having the amino acid sequence of SEQ ID NO: 4,
(2) A light chain variable region of a monoclonal antibody against hepatitis B virus HBs antigen having an amino acid sequence having a deletion, substitution and / or addition of 1 to several amino acids in the amino acid sequence of SEQ ID NO: 4 Protein, and
(3) A protein which has an amino acid sequence having 90% or more homology with the amino acid sequence of SEQ ID NO: 4 and is a light chain variable region of a monoclonal antibody against hepatitis B virus HBs antigen. Any of the following genes.
(1) A monoclonal antibody against the hepatitis B virus HBs antigen having the base sequence encoding the 76th to 105th base sequence in the base sequence of SEQ ID NO: 1 or the 26th to 35th amino acid sequence of the amino acid sequence of SEQ ID NO: 3 A gene encoding heavy chain complementarity determining region 1 (CDR-H1) of an antibody.
(2) Monoclonal antibody against hepatitis B virus HBs antigen having a nucleotide sequence of 148 to 198 in the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence encoding the 50th to 66th amino acid sequence of the amino acid sequence of SEQ ID NO: 3 A gene encoding the heavy chain complementarity determining region 2 (CDR-H2).
(3) Monoclonal antibody against hepatitis B virus HBs antigen having a nucleotide sequence of 295 to 333 in the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence encoding amino acids 99 to 111 in the amino acid sequence of SEQ ID NO: 3 A gene encoding the heavy chain complementarity determining region 3 (CDR-H3). Any of the following genes.
(1) A monoclonal antibody against a hepatitis B virus HBs antigen having a base sequence encoding 69-102 in the base sequence of SEQ ID NO: 3, or a base sequence encoding 24-34 in the amino acid sequence of SEQ ID NO: 4 A gene encoding light chain complementarity determining region 1 (CDR-L1).
(2) A monoclonal antibody against a hepatitis B virus HBs antigen having a base sequence encoding 148 to 168 in the base sequence of SEQ ID NO: 3 or a base sequence encoding 50 to 56 of the amino acid sequence of SEQ ID NO: 4 A gene encoding light chain complementarity determining region 2 (CDR-L2).
(3) A monoclonal antibody against a hepatitis B virus HBs antigen having a nucleotide sequence encoding 265 to 291 in the nucleotide sequence of SEQ ID NO: 3 or an amino acid sequence of 89 to 97 in the amino acid sequence of SEQ ID NO: 4. A gene encoding light chain complementarity determining region 3 (CDR-L3). Any of the following peptides.
(1) a peptide which is a heavy chain complementarity determining region 1 (CDR-H1) of a monoclonal antibody against HBs antigen of hepatitis B virus having an amino acid sequence of 26 to 35 in the amino acid sequence of SEQ ID NO: 2;
(2) a peptide that is a heavy chain complementarity determining region 2 (CDR-H2) of a monoclonal antibody against HBs antigen of hepatitis B virus, having an amino acid sequence of 50 to 66 in the amino acid sequence of SEQ ID NO: 2;
(3) A peptide that is a heavy chain complementarity determining region 3 (CDR-H3) of a monoclonal antibody against hepatitis B virus HBs antigen, having an amino acid sequence of 99 to 111 in the amino acid sequence of SEQ ID NO: 2. Any of the following peptides.
(1) A peptide that is a light chain complementarity determining region 1 (CDR-L1) of a monoclonal antibody against hepatitis B virus HBs antigen, having an amino acid sequence of 24-34 in the amino acid sequence of SEQ ID NO: 4,
(2) a peptide that is a light chain complementarity determining region 2 (CDR-L2) of a monoclonal antibody against hepatitis B virus HBs antigen, having an amino acid sequence of 50 to 56 in the amino acid sequence of SEQ ID NO: 4,
(3) A peptide which is a light chain complementarity determining region 3 (CDR-L3) of a monoclonal antibody against hepatitis B virus HBs antigen, having an amino acid sequence of 89 to 97 in the amino acid sequence of SEQ ID NO: 4. A monoclonal antibody against the hepatitis B virus HBs antigen, comprising a heavy chain comprising a protein having the amino acid sequence of SEQ ID NO: 2 as a variable region and a light chain comprising a protein having the amino acid sequence of SEQ ID NO: 4 as a variable region. A heavy chain comprising peptides having amino acid sequences of 26 to 35, 50 to 66 and 99 to 111 in the amino acid sequence of SEQ ID NO: 2 as complementarity determining regions 1 to 24 and 24 to 34, 50 to 50 in the amino acid sequence of SEQ ID NO: 4 A monoclonal antibody against the HBs antigen of hepatitis B virus, comprising a light chain comprising peptides having amino acid sequences of 56 and 89 to 97 as complementarity determining regions 1 to 3. A single chain antibody against hepatitis B virus HBs antigen, wherein a protein having the amino acid sequence of SEQ ID NO: 2 and a protein having the amino acid sequence of SEQ ID NO: 4 are linked by a linker peptide. A protein comprising peptides having amino acid sequences of 26 to 35, 50 to 66 and 99 to 111 in the amino acid sequence of SEQ ID NO: 2 as complementarity determining regions 1 to 3, and 24 to 34, 50 to 56 in the amino acid sequence of SEQ ID NO: 4 And a protein comprising a peptide having an amino acid sequence of 89 to 97 as a complementarity determining region 1 to 3 and linked with a linker peptide, a single chain antibody against hepatitis B virus HBs antigen. A dimer of the single-chain antibody according to claim 13 or 14. The trimer of the single chain antibody according to claim 13 or 14. A hepatitis B virus wherein a protein having the amino acid sequence of SEQ ID NO: 2 and a protein having the amino acid sequence of SEQ ID NO: 4 are linked by a linker peptide, and the CH3 region is bound to the protein having the amino acid sequence of SEQ ID NO: 2. Minibody against HBs antigen. A protein comprising peptides having amino acid sequences of 26 to 35, 50 to 66 and 99 to 111 in the amino acid sequence of SEQ ID NO: 2 as complementarity determining regions 1 to 3, and 24 to 34, 50 to 56 in the amino acid sequence of SEQ ID NO: 4 And a protein comprising the peptide having the amino acid sequence of 89 to 97 as complementarity determining regions 1 to 3 and linked to each other by a linker peptide, and the amino acids of 26 to 35, 50 to 66 and 99 to 111 in the amino acid sequence of SEQ ID NO: 2 A minibody against hepatitis B virus HBs antigen, in which a CH3 region is bound to the protein comprising a peptide having a sequence as complementarity determining regions 1 to 3. The protein according to claim 5 or 6, the peptide according to claim 9 or 10, the monoclonal antibody according to claim 11 or 12, the single-chain antibody according to claim 13 or 14, and the two according to claim 15. A method for assaying hepatitis B virus using a trimer, a trimer according to claim 16, and a minibody according to claim 17 or 18. The protein according to claim 5 or 6, the peptide according to claim 9 or 10, the monoclonal antibody according to claim 11 or 12, or the single-chain antibody according to claim 13 or 14, A method for diagnosing hepatitis B using a dimer, a trimer according to claim 16, and a minibody according to claim 17 or 18. The protein according to claim 5 or 6, the peptide according to claim 9 or 10, the monoclonal antibody according to claim 11 or 12, or the single-chain antibody according to claim 13 or 14, A method for treating hepatitis B using a dimer, a trimer according to claim 16, and a minibody according to claim 17 or 18.