Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Culture medium – per se
Reexamination Certificate
1998-11-06
2003-09-30
Kunz, Gary (Department: 1647)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Culture medium, per se
C435S325000, C435S405000
Reexamination Certificate
active
06627440
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to cell growth and cell culture.
Mammalian alpha-fetoprotein (AFP) is a 70,000-Da glycoprotein of primarily yolk sac and hepatic origin which is present in fetal blood in milligram amounts during perinatal life. At birth serum AFP levels begin a gradual decline to the low nanogram amounts normally found in the adult. Chemical analysis has shown that AFP molecules are composed of single polypeptide chains containing about 4% carbohydrate.
SUMMARY OF THE INVENTION
I have discovered that unglycosylated recombinant human alpha-fetoprotein made in a prokaryote (e.g.,
E. coli
) is a cell proliferative agent, e.g., promotes the growth of bone marrow in vitro.
In general, the invention features a cell culture medium including recombinant human alpha-fetoprotein or a cell-stimulating fragment or analog thereof. Preferably, such recombinant human alpha-fetoprotein is produced in a prokaryotic cell (
E. coli
) and is unglycosylated.
Accordingly, the invention features a method of cell culture, said method including (a) providing a cell culture medium including recombinant human alpha-fetoprotein; (b) providing a cell; (c) and growing the cell in the medium, where the cell proliferates, and is maintained. Preferably, the cell is a mammalian cell. Examples of such mammalian cells include bone marrow cells (e.g., a T cells, a natural killer cell, a lymphocyte, etc.), hybridomas or a genetically-engineered cell line. Examples of other cells include hematopoietic cells such as stem cells, blast cells, progentior cells (e.g., erythroid progenitor cells such as burst-forming units and colony-forming units), myeloblasts, macrophages, monocytes, macrophages, lymphocytes, T-lymphocytes, B-lymphocytes, eosinophils, basophils, tissue mast cells, megarkaryocytes (see e.g.,
Best and Taylor's Physiological Basis of Medical Practice
, John B. West, ed., Williams & Wilkins, Baltimore). In other preferred embodiments the method involves ex vivo cell culture.
In another aspect, the invention features a method for inhibiting myelotoxcity in a mammal (e.g., a human patient) involving administering to the mammal a therapeutically effective amount of recombinant human alpha-fetoprotein or a myelotoxic-inhibiting analog or fragment thereof. Preferably, the recombinant human alpha-fetoprotein is produced in a prokaryotic cell (
E. coli
) and is unglycosylated.
In another aspect, the invention features a method of inhibiting suppression of bone marrow cell proliferation in a mammal, the method involving administering to the mammal an effective amount of recombinant alpha-fetoprotein or an anti-suppressive fragment or analog thereof. Preferably, the recombinant human alpha-fetoprotein is produced in a prokaryotic cell (e.g.,
E. coli
) and is unglycosylated.
In another aspect, the invention features a method of promoting bone marrow cell proliferation in a mammal, involving administering to the mammal an effective amount of recombinant human alpha-fetoprotein or a cell-stimulating fragment or analog thereof. Preferably, the recombinant human alpha-fetoprotein is produced in a prokaryotic cell (e.g.,
E. coli
) and is unglycosylated.
In another aspect, the invention features a method of preventing bone marrow cell transplantation rejection in a mammal, involving administering to the mammal an effective amount of recombinant human alpha-fetoprotein or an anti-rejection fragment or analog thereof. Preferably, the recombinant human alpha-fetoprotein is produced in a prokaryotic cell (e.g.,
E. coli
) and is unglycosylated.
By “cell-stimulating” is meant increasing cell proliferation, increasing cell division, promoting cell differentiation and/or development, or promoting cell longevity.
By “therapeutically effective amount” is meant a dose of unglycosylated recombinant human alpha-fetoprotein or an cell-stimulating fragment or analog thereof capable of stimulating the proliferation of a cell.
By “recombinant human alpha-fetoprotein” is meant a polypeptide having substantially the same amino acid sequence as the protein encoded by the human alpha-fetoprotein gene (
FIG. 1
, SEQ ID NO: 1) as described by Morinaga et al.,
Proc. Natl. Acad. Sci., USA
80: 4604 (1983). The method of producing recombinant human alpha-fetoprotein in a prokaryotic cell is described in U.S. Pat. No. 5,384,250.
By “myelotoxic-inhibiting” is meant inhibiting myeloablation.
According to the invention, administration of recombinant human alpha-fetoprotein (“rHuAFP”) (or a fragment or analog thereof) can be an effective means for promoting and boosting cell growth in vitro, ex vivo, or in vivo. Administration of such rHuAFP can also be an effective means of preventing or treating or ameliorating myleotoxcemia in a mammal.
The use of rHuAFP is especially advantageous since there are no known adverse side effects related to human alpha-fetoprotein and it is believed that relatively high doses can be safely administered. Furthermore, the use of rHuAFP as a principal component of tissue culture media is advantageous since there is little potential for contamination with pathogens.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
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Rudinger, inPeptide Hormones,J.A. Parsons, ed., University Park Press, Baltimore, 1976, pp. 1-6.*
Abramsky et al., Alpha-fetroprotein Suppresses Experimental Allergic Encephalomyelitis, Journal of Neuroimmunology 2:1-7 (1982).
Abramsky et al., Annals New York Academy of Sciences, pp. 108-115 (1983).
Aoyagi et al., “Differential Reactivity of &agr;-Fetoprotein with Lectins and Evaluation of Its Usefuleness in the Diagnosis of Hepatocellular Carcinoma”, Gann 75:809-815 (1984).
Biddle et al., “Specific cytoplasmic alpha-fetoprotein binding protein in MCF-7 human breast cancer cells and primary breast cancer tissue,” Breast Cancer Research and Treatment 10:279-286 (1987).
Boismenu et al., “Expression of Domains of Mouse Alpha-Fetoprotein inEscherichia Coli,” Life Sciences 43:673-681 (1988).
Brenner et al., “Influence of alpha-fetoprotein on the in vitro and in vivo immune response to acetylcholine receptor,” Annals New York Academy of Sciences 377:208-221 (1981).
Brenner et al., “Inhibitory effect of &agr;-fetoprotein on the binding of myasthenia gravis antibody to acetylcholine receptor,” Proc. Natl. Acad. Sci. USA 77:3635-3639 (1980).
Brenner et al., “Immunosuppression of Experimental Autoimmune Myasthenia Gravis by Alpha-Fetoprotein Rich Formation,” Immunology Letters 3:163-167 (1981).
Buamah et al., “Serum alpha fetoprotein heterogeneity as a means of differentiating between primary hepatocellular carcinoma and hepatic secondaries,” Clinica Chimica Acta. 139:313-316 (1984).
Buschman et al., “Experimental Myasthenia Gravis Induced in Mice by Passive Transfer of Human Myasthenic Immunoglobulin,” Journal of Neuroimmunology, 13:315-330 (1987).
Cohen et al., “Suppression by Alpha-Fetoprotein of Murine Natural Killer Cell Activity Stimulated in Vitro and in Vivo by Interferon and Interleukin 2,” Scand. J. Immunol. 23:211-223 (1986).
Dattwyler et al., “Binding of &agr;-foetoprotein to murine T cells,” Nature 256:656-657 (1975).
Gershwin et al., The Influence of &agr;-Fetoprotein on Moloney Sarcoma Virus Oncogenesis: Evidence For Generation of Antigen Nonspecific Suppressor T Cells, The Journal of Immunology 121:2292-2297 (1978).
Glazier et al., “Graft-Versus-Host Disease in Cyclosporin A-Treated Rats After Syngeneic and Autologous Bone Marrow Reconstitution,” J. Exp. Med. 158:
Clark & Elbing LLP
Gucker Stephen
Kunz Gary
Martinex R & D Inc.
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