Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
1995-04-25
2001-11-06
Budens, Robert D. (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S455000, C435S471000, C435S472000, C435S476000, C536S023200
Reexamination Certificate
active
06312939
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a DNA coding for a therapeutically suitable glutaminase, a polypeptide which has the activity of a therapeutically suitable glutaminase, as well as their use in antiviral and anticancer therapy.
BACKGROUND OF THE INVENTION
Use of glutaminase to deplete glutamine in tumor-bearing hosts offers an attractive method for attacking cancer cells. Glutamine occupies an important role in the biosynthesis of a large number of cellular metabolites. Compared with normal tissues, some neoplasms have been shown to operate at a marginal level of glutamine availability because of decreased synthesis and stepped-up utilization (Levintow, 1954,
J. Natl. Cancer Inst.
15:347-352; Roberts, et al., 1960,
Amino Acids, Proteins and Cancer Biochemistry
(J. T. Edsall, ed.), Academic Press. New York, N.Y. pp. 121-145; Weber, G., 1983.,
Cancer Res.
43:3466-3492; Sebolt, et al., 1984,
Life Sci.
34:301-306). Experiments have revealed a negative correlation between glutamine content and the growth rate of transplanted rat hepatoma tumors. The in vivo concentration of glutamine in hepatoma 3924A was found to be 9-fold lower (0.5 mM) than in liver (4.5 mM) and lower than in any other rat tissues (2 to 5 mM) (Weber, 1983,
Cancer Res.
43:3466-3492). In recent years accumulated data indicate that glutamine may be an important fuel source of cellular energy in a variety of neoplasms, including hematopoietic tumors, hepatomas, Ehrlich carcinoma, and HeLa cells (Abou-Khalil, et al., 1983,
Cancer Res.
43:1990-1993; Kovacevic, et al., 1972,
J. Biol. Chem.
33:326-333; Kovacevic, 1971,
Biochem. J.
125:757-763; Reitzer, et al., 1979,
J. Biol. Chem.
254:2669-2676).
L-asparaginase, the first enzyme to be intensively studied as an antitumor agent in man, is highly effective in the treatment of acute lymphoblastic leukemia. This enzyme, however, has little or no activity against any other neoplasms in humans. The enzyme glutaminase has activity against a much broader range of cancers than asparaginase.
Several mammalian and microbial glutaminase and glutaminase-asparaginase enzymes have been purified and characterized. Of these Pseudomonas 7A glutaminase-asparaginase appears to be best suited for therapeutic use because of its low K
M
for glutamine (micromolar range), good stability and activity in a physiological milieu, and long plasma half-life in tumor-bearing hosts (Roberts, 1976,
J. Biol. Chem.
251:2119-2123, and Roberts, et al., 1979,
Cancer Treat. Rep.
63:1045-1054).
The known mammalian glutaminase enzymes are not suitable for use as therapeutic agents because of their high K
M
values (millimolar range), and their requirement for phosphate esters or malate for activation. The
E. coli
glutaminases (A and B) are also unsuited for therapeutic use because of their high K
M
values (millimolar range), low activity at physiological pH (glutaminase A), or requirement for special activating substances (glutaminase B).
Pseudomonas 7A glutaminase-asparaginase is composed of four identical subunits with a molecular weight of approximately 35,000. Active enzyme sedimentation studies indicate that the catalytic activity is associated with the tetramer; no smaller active species are observed (Holcenberg, et al., 1976,
J. Biol. Chem.,
251:5375-5380). The purified enzyme has a ratio of glutaminase to asparaginase activity of approximately 2:1. Binding studies with C
14
-labelled analogs of glutamine (6-diazo-5-oxo-L-norleucine; DON) and asparagine (6-diazo-5-oxo-L-norvaline; DONV) suggest that the two analogs may react preferentially with hydroxyl groups at two different sites on the protein, and that the two binding sites act cooperatively as part of the active site (Holcenberg, et al., 1978.,
Biochemistry
17:411-417).
Pseudomonas 7A glutaminase-asparaginase was shown to have considerable antineoplastic activity against a variety of rodent leukemia (L1210, C1498, EARAD/1), ascites tumors (Taper liver, Ehrlich carcinoma, meth A sarcoma, S 180) and certain solid tumors (Walker 256 carcinosarcoma, B16 melanoma). Additionally, antagonism of glutamine by glutamine analogs and glutaminase was found to be strongly inhibitory to human colon, breast and lung carcinomas growing in athymic mice (McGregor, 1989,
Proc. Amer. Assoc. Cancer Res.
30:578; Roberts, 1979,
Cancer Treat. Rep.
63:1045-1054; Ovejera, 1979,
Cancer Res.
39:3220-3224; Houchens, 1979,
Cancer Treat. Rep.
63:473-476; Duvall, 1960,
Cancer Chemother. Rep.
7:86-98).
An important characteristic of glutaminase therapy is that resistant strains do not develop after repeated treatments with this enzyme (Roberts, 1979,
Cancer Treat. Rep.
63:1045-1054). Treatment with glutaminase was also shown to delay development of resistance against methotrexate (Roberts, 1979,
Cancer Treat. Rep.
63:1045-1054).
A bioactive glutaminase-asparaginase has been shown to inhibit mouse retroviral disease. Glutamine depletion strongly inhibits the replication of Rauscher murine leukaemia retrovirus (RLV) in vitro. Pseudomonas 7A glutaminase-asparaginase (PGA), capable of depleting glutamine and asparagine for prolonged periods, was used to determine the therapeutic effectiveness, of glutamine depletion in mice infected with RLV or Friend virus. During PGA treatment of viremic animals, serum reverse transcriptase activity fell to control levels and infected animals did not develop splenomegaly. The therapeutic results obtained with PGA compare favorably with those of azidothymidine given intraperitoneally at 30 mg/kg/day (Roberts, 1991,
Journal of General Virology,
72:299-305).
Despite the promise of glutaminase as a therapeutic agent, there are currently no therapeutically useful glutaminases available which can be produced cheaply and with little or no contamination by other substances, for example by endotoxins of a host microorganism. Moreover, a suitable enzyme is not available in quantities which are large enough to allow for wide-spread clinical trails.
SUMMARY OF THE INVENTION
It is an object of the invention to provide a method of inhibiting the replication of HIV in infected cells.
It is another object of the invention to provide a method of inhibiting the growth of cancer cells.
It is yet another object of the invention to provide an
E. coli
cell which comprises a therapeutically suitable glutaminase.
It is still another object of the invention to provide a DNA molecule encoding a therapeutically suitable glutaminase.
It is an object of the invention to provide a therapeutically suitable glutaminase free of Pseudomonas endotoxin.
It is another object of the invention to provide methods of treating transformed cells in a body.
It is still another object of the invention to provide a therapeutic composition for treating neoplastic cells.
These and other objects of the invention are provided by one or more of the embodiments which are described below. In one embodiment a method of inhibiting the replication of HIV in HIV-infected cells is provided. The method comprises administering a therapeutically suitable glutaminase to HIV-infected cells in an amount sufficient to inhibit replication of HIV in said cells.
In another embodiment of the invention a method of inhibiting the growth of cancer cells is provided. The method comprises administering a bound complex to tumor cells which express a tumor-associated antigen, the amount of said complex administered being sufficient to inhibit DNA synthesis in said tumor cells. The complex comprises: (a) a therapeutically suitable glutaminase and (b) an antibody immunoreactive with a tumor associated antigen.
In yet another embodiment of the invention an
E. coli
cell is provided which comprises a Pseudomonas 7A glutaminase-asparaginase gene.
In still another embodiment of the invention an isolated and purified DNA molecule is provided. The molecule comprises a nucleotide sequence coding for a therapeutically suitable glutaminase.
In one embodiment of the invention a cell-free preparation of a therapeutically suitable glutaminase is provided. Th
Freeman Abbie G.
MacAllister Thomas W
Roberts Joseph
Sethuraman Natarajan
Budens Robert D.
Foley & Lardner
ME Medical Enzymes AG
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