Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1998-06-10
2003-11-18
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S252330, C435S320100, C536S023200, C536S023100, C536S023500
Reexamination Certificate
active
06649393
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to methods and compositions for the recombinant production of Onc, a cytotoxic ribonucleolytic protein having anti-tumor and anti-viral properties. In particular, the invention relates to a recombinant Onc protein having an amino terminal methionine and comprising an Onc polypeptide.
BACKGROUND OF THE INVENTION
ONCONASE, or Onc, is a ribonuclease purified from
Rana pipiens
oocytes. While Onc is homologous to pancreatic RNases in amino acid sequence (Ardelt et al.,
J. Biol. Chem
. 266:245-251 (1991)) and three dimensional structure (Mosimann et al.,
J. Mol. Biol
. 236:1141-1153 (1994)), its pharmacological properties are quite unique. Onc displays cytostatic and cytotoxic activity against numerous cancer cell lines in vitro (Darzynkiewicz et al.,
Cell Tissue Kinet
. 21:169-182 (1988)), is up to five-thousand times more toxic to animals than is the homologous protein, RNase A (Newton et al.,
J. Neurosci
. 14(2):538-44 (1994)), and displays anti-tumor activity in vivo (Mikulski et al.,
J. Natl. Canc. Inst
. 82:151-152 (1990);
Int. J. Oncol
. 3:57-64 (1993). Moreover, Onc has been found to specifically inhibit HIV-1 replication in infected H9 leukemia cells at non-cytotoxic concentrations (Youle et al.,
Proc. Natl. Acad. Sci. USA
91:6012-6016 (1994)). Such promising pharmacologic properties explain why this protein is currently the subject of phase III clinical trials.
Unfortunately, since Onc is isolated from oocytes, procurement of an adequate supply is uncertain. Recent concerns regarding the availability of the anti-cancer compound taxol illustrate some of the problems of obtaining natural products for use as pharmaceuticals. Similarly, availability of Onc is increasingly problematic in light of the declining population of
R. pipiens
and the seasonal variation in the supply of its oocytes.
Accordingly, what is needed in the art is a means to produce Onc by recombinant methods so as to meet demand for this therapeutic and alleviate the impact on its native source. Further, what is needed is a means to derivatize and alter the sequence of Onc to provide more efficacious compounds. Quite surprisingly, the present invention provides these and other advantages.
SUMMARY OF THE INVENTION
The present invention is directed to an rOnc protein, comprising a polypeptide of SEQ ID NO:1 or conservatively modified variant thereof. Preferably, the polypeptides of the invention have a glutamine residue at position +1 of the polypeptide. Even more preferably, the glutamine residue is at the amino terminus of the rOnc protein.
In one embodiment, the polypeptide comprises a hydrophobic residue at position 23. In a further embodiment, the polypeptide comprises the amino acid leucine at position 23. Preferably, the polypeptides of the present invention also have a lysine at position 9, a histidine at position 10, a histidine at position 97, a lysine at position 31, a phenylalanine at position 98, and a threonine at position 35.
In another embodiment, the amino acid sequence of rOnc protein is generally modified so that it is not susceptible to cleavage by cyanogen bromide. Preferably, upon cyclization of the amino terminal glutamine to pyroglutamyl, the polypeptide of SEQ ID NO:1 or conservative variants thereof have a relative IC
50
in U251 cells at least 50% that of the polypeptide of SEQ ID NO:2. Polypeptides of the present invention may be joined to a ligand binding moiety such as an immunoglobulin.
In another aspect of the present invention, a rOnc protein is provided. The rOnc protein comprises a polypeptide of SEQ ID NO:1 or conservatively modified variant thereof, preferably with a glutamine residue at position 1, and an amino terminal methionine. Nucleic acids encoding for the rOnc protein of the present invention are also provided. In a preferred embodiment, the amino terminal methionine is directly linked to the polypeptides of the present invention. The amino terminal methionine may also be linked to the polypeptides of the present invention via less than 50 amino acid residues.
In another aspect of the present invention, a method of making a rOnc protein is provided. The method comprises expressing in a host cell a nucleic acid encoding a rOnc protein comprising a polypeptide of SEQ ID NO:1 or conservatively modified variant thereof and an amino terminal methionine; cleaving the amino terminal methionine with a cleaving agent; and causing the glutamine residue at position 1 of SEQ ID NO:1 or conservative variant thereof to cyclize to a pyroglutamyl residue. In one embodiment, the nucleic acid encodes a hydrophobic residue at position 23 of the polypeptide. Preferably, the nucleic acid encodes a leucine at position 23 when the cleaving agent is cyanogen bromide. The cleaving agent is typically a peptidase or cyanogen bromide.
In another aspect of the present invention, a host cell is provided that expresses a nucleic acid coding for an rOnc protein. The rOnc protein comprises a polypeptide of SEQ ID NO:1 or conservatively modified variant thereof, and wherein the polypeptide has a glutamine at position 1; and an amino terminal methionine. An expression vector encoding a polypeptide of SEQ ID NO:1 or conservatively modified variant thereof, wherein the polypeptide has a glutamine at position 1, and an amino terminal methionine is also provided. In one embodiment, the expression vector encodes a leucine at position 23 of the polypeptide. In another, the expression vector encodes methionine or another hydrophobic residue at position 23 of the polypeptide.
The present invention has utility in providing a means to recombinantly produce rOnc for use as an anti-cancer, anti-tumor, and anti-viral composition. Additionally, the rOnc proteins of the present invention also have use as a cell culture selection agent against cancerous or tumorigenic cells thereby providing, for example, a means to select and identify gene therapy compositions which inhibit tumorigenic growth.
DESCRIPTION OF THE PREFERRED EMBODIMENT
Introduction
The present invention is directed to recombinant Onc (rOnc), a potent anti-tumor and anti-viral compound derived from P-30 Protein, an oocyte protein of
Rana pipiens
(Ardelt et al.,
J. Biol. Chem
. 266:245-251 (1991)) and exemplified by the product ONCONASE, a registered tradename of the Alfacell Corporation, Bloomfield, N.J. The invention provides, inter alia, rOnc compositions, and compositions and methods for making rOnc.
The rOnc polypeptide of the present invention is altered in amino acid sequence relative to the native P-30 Protein (nOnc) such that recombinant production and subsequent conversion of this protein to its pharmacologically active form is readily achieved. Recombinant Onc has in vivo and in vitro utility. Recombinant Onc may be employed as an anti-cancer, anti-tumor, and anti-viral composition, or, for example, as a selection agent in cell culture work against tumorigenic cells. Thus, in some embodiments rOnc may be employed to inhibit HIV-1 replication, or to treat pancreatic cancer.
In contrast to homologous RNases, rOnc lacks appreciable ribonuclease activity or cytotoxicity when expressed with a methionine residue at the amino terminus. When chimeric proteins composed of rOnc and human pancreatic RNase (hRNase) sequences are constructed, they yield enzymes with similar substrate specificity and activity to that of Onc; however, they too lack appreciable cytoxicity. Thus, the features lending cytotoxicity to rOnc were not readily discernible. We have discovered that the amino terminal pyroglutamyl residue of rOnc plays a part in the cytotoxicity of this enzyme. Our identification of the role of this residue, and means to modify Onc for recombinant production while maintaining the desired cytotoxic activity provides, in part, the invention as described herein.
Definitions
Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al. (1994)
D
Ardelt Wojciech
Boix Ester
Vasandani Veena M.
Wu Yon-Neng
Youle Richard J.
Klarquist & Sparkman, LLP
Nashed Nashaat T.
The United States of America as represented by the Department of
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