Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-09-10
2001-01-16
Fredman, Jeffrey (Department: 1655)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C435S252300, C435S455000, C435S471000, C435S069100, C435S325000, C435S320100
Reexamination Certificate
active
06175003
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to Ribonucleases (RNases), and more particularly relates to ranpirnase. In its most immediate sense, the invention relates to nucleic acids that encode proteins that can be used to produce ranpirnase and an RNase that is highly homologous to it.
Ranpirnase is the generic name of an RNase that is produced by Alfacell Corporation (assignee herein) under the registered trademark ONCONASE. Ranpirnase is a protein
104
residues long, with a blocked N-terminal of pyroglutamic acid (<Glu) that is produced by autocyclization of glutamine (Gln). It is disclosed in U.S. Pat. No. 5,559,212. As is stated therein, ranpirnase is presently produced from eggs of the rana pipiens frog. It would be advantageous to produce ranpirnase using recombinant DNA technology instead of processing biological material.
Additionally, work done at the direction of Dr. Richard Youle of the National Institute of Health has suggested that there would be an advantage to modifying ranpirnase in a particular manner. Dr. Youle is a pioneer in the field of “cysteinizing” therapeutically active RNases (specifically, human pancreatic RNase) with the object of increasing their effectiveness. Dr. Youle conceived the idea of re-engineering an RNase so it could be more easily attached to a targeting molecule, thereby making it possible for the RNase to be delivered to a particular cell receptor where it might be most effective. To achieve this objective, he utilized a property of the amino acid cysteine.
Cysteine has a single reactive sulfhydryl (“SH”) group. The availability of this group facilitates the chemical linking of a targeting molecule to the cysteine residue. Dr. Youle realized that by conservatively substituting a cysteine residue at an appropriate location in an RNase, the RNase could easily be linked to a targeting moiety (such as a monoclonal antibody) that targets a predetermined cell receptor. This would permit the RNase to be delivered to the precise location where it might be most therapeutically effective.
Accordingly, it would be advantageous to produce a cysteinized ranpirnase, i.e. a modified ranpirnase in which an amino acid residue at an appropriate location was conservatively replaced by cysteine.
One object of the invention is to provide a nucleic acid that encodes ranpirnase, and to provide a method of synthesizing that nucleic acid.
Another object of the invention is to provide a nucleic acid that encodes cysteinized ranpirnase, and to provide a method of synthesizing that nucleic acid.
In accordance with the invention, two nucleic acids are produced. Each of these nucleic acids encodes a corresponding protein. One protein is converted to ranpirnase by cleavage of an N-terminal methionine residue at position −1 and autocyclization of a glutamine residue at position 1. The other protein (after a like cleavage and autocyclization) is converted to a cysteinized ranpirnase in which the methionine residue at position 23 is replaced by a residue of leucine, and in which the serine residue at position 72 is replaced by a residue of cysteine. (The substitution of methionine at position 23 does not appear to adversely affect the bioactivity of the resulting RNase.) The cysteine residue provides a location at which a targeting moiety (such as a monoclonal antibody) can be attached, to deliver the cysteinized ranpirnase to that receptor site where it can most efficiently be used.
In accordance with preferred embodiments of the invention, synthesis of both nucleic acids begins with a recombinant plasmid originally synthesized in Dr. Youle's laboratory. This recombinant plasmid, named pET11d-rOnc(Q1, M23L) and made up of a rOnc(Q1, M23L) gene cloned in a pET-11d vector, encodes a protein. The protein is highly homologous to ranpirnase, but a) has an N-terminal residue of methionine at position −1 followed by a residue of glutamine at position 1, and b) has a leucine residue at position 23 instead of a methionine residue (as ranpirnase has). An overlapping PCR protocol is used to mutate the rOnc(Q1, M23L) gene. In accordance with a first preferred embodiment of the invention, the rOnc(Q1, M23L) gene is modified to encode a protein that, after cleavage of its N-terminal methionine residue and autocyclization of an adjacent glutamine residue, is ranpirnase. In accordance with a second preferred embodiment of the invention, the rOnc(Q1, M23L) gene is modified to encode a protein that, after a like cleavage and autocyclization, is a cysteinized ranpirnase wherein the leucine residue at position 23 is left in place and the serine residue at position 72 is changed to a residue of cysteine.
The expressed protein from each of these preferred embodiments has an N-terminal residue of methionine at position −1 followed by a residue of glutamine at position 1. When in each instance the methionine residue is cut off (or “cleaved”), the glutamine autocyclizes to form pyroglutamic acid (which is also located at position 1 in ranpirnase).
REFERENCES:
patent: WO 97/31116 (1997-08-01), None
Boix et al.,J. Mol. Biol.(1996) 257, 992-1007.
Alfacell Corporation
Einsmann Juliet C.
Fredman Jeffrey
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