Human monoclonal antibodies to the hepatitis B surface antigen

Details Human monoclonal antibodies to the hepatitis B surface antigen Human monoclonal antibodies to the hepatitis B surface antigen

1998-12-10

2001-07-03

Wortman, Donna C. (Department: 1645)

Drug, bio-affecting and body treating compositions

Immunoglobulin, antiserum, antibody, or antibody fragment,...

Monoclonal antibody or fragment thereof

C424S142100, C424S133100, C435S339000, C435S005000, C530S388150, C530S388300, C530S388100

Reexamination Certificate

active

06254867

ABSTRACT:

FIELD OF THE INVENTION
The present invention concerns a process for obtaining hybridoma cell lines which produce human antibodies capable of binding to the hepatitis B virus surface antigen, the hybridoma cell lines, antibodies produced by the cell lines, and various uses thereof.
BACKGROUND OF THE INVENTION
Hepatitis B virus (HBV) infection is a major worldwide health problem. Approximately 5% of the world population is infected by HBV and chronically infected patients carry a high risk of developing cirrhosis and hepatocellular carcinoma. (Progress in Hepatitis Research: Hepatitis B virus (HBV), Hepatitis C virus (HCV) and Hepatitis Delta virus (HDV) Ed. O. Crivelli, Sorin Biomedica, 1991).
The immune response to HBV-encoded antigens includes both a cellular immune response which is active in the elimination of HBV infected cells, as well as a humoral antibody response to viral envelope antigens which contributes to the clearance of circulating virus particles. The dominant cause of viral persistence during HBV infection is the development of a weak antiviral immune response.
Recombinant HBV vaccines provide a safe and effective means for active immunization against HBV, however, they do not always induce a sufficient and rapid antibody response.
Interferon-&agr; has been used in the therapy of Hepatitis B infection showing an efficacy of only 30-40% in highly selected patients.
In addition, passive immunization with human polyclonal anti Hepatitis B antisera has been shown to be effective in delaying and even preventing recurrent HBV infection (Wright, T. L. and Lau, J. Y. N. The Lancet 342:1340-1344, (1993)). Such human polyclonal antisera are prepared from pooled plasma of immunized donors. These preparations are very expensive and available in relatively small amounts. Furthermore, pooled plasma may contain contaminated blood samples and thus treatment with such antisera increases the patient's risk to contract other viral infections such as hepatitis C or HIV.
An alternative approach for the treatment of HBV infection is the use of monoclonal antibodies (MoAb).
PCT patent application PCT/NL94/00102 discloses human monoclonal antibodies directed against Hepatitis B surface antigen HBVsAg which are secreted by the hybridoma cell lines Mab 4-7B and Mab 9H9. The monoclonal antibody secreted by the cell line Mab 4-7B recognizes a linear epitope of HBVsAg and is different from the Mab 9H9 monoclonal antibody which recognizes a conformational epitope. The antibodies are claimed for simultaneous use in the treatment of chronic Hepatitis B infections.
PCT patent application PCT/US92/09749 discloses human monoclonal antibodies against HBVsAg which are secreted by the hybridoma cell lines PE1-1, ZM1-1, ZM1-2, MD3-4 and LO3-3. The antibodies bind to different HBV epitopes and are used for reducing the level of circulating HBVsAg.
Japanese Patent Application JP 93066104 discloses a hybridoma of a human lymphocyte cell strain TAW-925 and a human lymphocyte transformed by Epstein-Barr virus. The hybridoma produces a human monoclonal antibody against HBVsAg.
U.S. patent application Ser. No. 4,883,752 discloses preparation of human-derived monoclonal antibody to HBVsAg, by administration of HBVsAg vaccine to humans, recovering their lymphocytes, stimulating the lymphocytes in vitro by a non specific stimulator, fusing said cells with a myeloma cell, and selecting for hybridomas with secrete anti HBVsAg antibodies.
Ichimori et al,
Biochem. and Biophysic. Research Communications
129(1):26-33, 1985 discloses a hybridoma secreting human anti HBVsAg monoclonal antibodies which recognize the a-determinant of HBVsAg. Later, Ichimori, et al., supra 142(3):805-812, 1987 disclosed another hybridoma which stably secretes human monoclonal antibody against HbsAg.
The abovementioned antibodies were all developed by in vitro immortalization of antibody-producing cells from individuals positive for anti-HBV antibodies.
A new approach enabling adaptive transfer of human peripheral blood mononuclear cells (PBMC) into lethally irradiated normal strains of mice radioprotected with severe combined immune deficieny (SCID) bone marrow was recently described (Lubin I., et al.,
Blood,
83:2368, 1994). Secondary humoral responses to various recall antigens as well as a primary humoral response to other antigens were shown to be generated effectively in such human/mouse chimeras (Marcus H., et al,
Blood
86:398-406, 1995).
SUMMARY OF THE INVENTION
In accordance with the present invention, it was found that hybridoma cell lines secreting human antibodies capable of binding to the Hepatitis B surface antigen (HBVsAg) may be obtained using the above mentioned human/mouse chimeras. In accordance with the present invention, human peripheral blood lymphocytes (PBL) from human donors positive for anti HBVsAg antibodies are engrafted into normal strains of mice which were lethally irradiated and radioprotected wits SCID bone marrow. After immunization of such chimeric mice with HBVsAg, human cells are obtained from the mice spleens and fused in vitro with heteromyeloma cells to generate hybridomas secreting human antibodies having a high affinity and specificity to HBVsAg.
The present invention thus provides a process for obtaining human monoclonal antibodies (hMoAb) capable of binding to Hepatitis B virus surface antigen (HBVsAg) comprising:
(a) immunizing a chimeric rodent M4 having xenogeneic hematopoietic cells with Hepatitis B surface antigen (HBVsAg) such that xenogeneic antibody-producing cells are produced in said rodent, wherein said rodent M4 is a rodent M1, the hematopoietic cells of which have been substantially destroyed, said rodent M1 having transplanted therein hematopoietic cells derived from a mouse M2 having a hematopoietic deficiency, and xenogeneic hematopoietic cells derived from human M3;
(b) removing and immortalizing said antibody-producing cells;
(c) selecting and cloning the immortalized antibody producing cells producing the antibodies capable of binding to HBVsAg and;
(d) isolating the antibodies produced by the selected, cloned immortalized antibody producing cells.
In accordance with the invention, spleens of the immunized chimeric rodent M4 are removed between 12 and 20 days after human PBL transplantation, preferably at day 14 after transplantation thereof. Cell suspensions are prepared from the spleens and the antibody producing cells obtained from the immunized a heteromyeloma by techniques well known in the art (e.g. Kohler & Milstein, chimeric rodent M4 are fused preferably with a human-mouse fusion partner such as
Nature,
256:495-497, 1975). In order to isolate the antibodies produced by the selected hybridoma cell lines in accordance with the invention, the hybridoma cell lines are either cultured in vitro in a suitable medium wherein the desired monoclonal antibody is recovered from the supernatant or, alternatively, the hybridoma cell lines may be injected intraperitoneally into mice and the antibodies harvested from the malignant ascitis or serum of these mice. The supernatant of the hybridoma cell lines are first screened for production of human IgG antibodies by any of the methods known in the art such as enzyme linked immunosorbent assay (ELISA) or radioimmuno assay (RIA). Hybridomas testing positive for human IgG are then further screened for production of anti HBVsAg antibodies by their capability to bind to HBVsAg.
The M1 rodent in accordance with the invention is preferably a rodent conventionally used as a laboratory animal, most preferably a rat or a mouse.
The mouse M2 may have any hematopoietic deficiency including genetic hematopoietic deficiencies as well as induced hematopoietic deficiencies. Non limiting examples of hematopoietic deficiencies include SCID, Bg, Nu, Xid or mice having any combination of the abovementioned hematopoietic deficiencies. In addition, the hematopoietic deficiency may also be a result of gene deletion or transgenic mice may be used.
The hematopoietic cells derived from the donor mouse M2 are preferably bone marrow cells either u

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