Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 25 or more amino acid residues in defined sequence
Reexamination Certificate
1999-10-08
2001-09-25
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
25 or more amino acid residues in defined sequence
C530S326000, C514S012200, C514S013800
Reexamination Certificate
active
06294649
ABSTRACT:
TECHNICAL FIELD
The invention relates to polypeptides having cytotoxic activity, methods of inhibiting cells, and in particular to methods of inhibiting cells using polypeptides derived from ant venom.
BACKGROUND ART
The present inventors reported the molecular cloning and characterisation of a major allergen from the venom of the Australian jumper ant Myrmecia pilosula. The results of this work was reported in Donovan et al. 1993 Biochemica et Biophysica Acta, 1171: 272-280. This major allergen was originally called Myr p I.
Since the report on this major allergen from Myrmecia pilosula, the present inventors have made the surprising discovery that several peptides derived from the Myr p I amino acid sequence, including a polypeptide called Pilosulin 1, have potent cytotoxic activity to a wide range of cells.
DISCLOSURE OF THE INVENTION
In a first aspect, the present invention consists in an isolated polypeptide having cytotoxic activity comprising the amino acid sequence of Pilosulin 1 shown in
FIG. 1
from residues 1 to 56, an active portion thereof. or a functionally equivalent amino acid sequence thereof.
Preferably, the polypeptide is Pilosulin 1, an active portion thereof, or a functionally equivalent amino acid sequence thereof. More preferably the polypeptide is Pilosulin 1.
In a second aspect, the present invention consists in an isolated polypeptide having cytotoxic activity comprising the amino acid sequence from residues 1 to 22 (SEQ ID NO:2) shown in
FIG. 1
, an active portion thereof, or a functionally equivalent amino acid sequence thereof.
In one preferred form, the polypeptide includes the amino acid sequence from residues 1 to 22 (SEQ ID NO:2) shown in FIG.
1
.
As used herein the term “functionally equivalent amino acid sequence” is intended to cover minor variations in the amino acid sequence described which results in a polypeptide having relative cytotoxic activity which is not substantially less than that of the corresponding native polypeptide. Preferably, a polypeptide having ail altered amino acid sequence from its sequence shown in
FIG. 1
has substantially the same or greater cytotoxic activity than that of the native polypeptide. This may be achieved by various changes in the sequence, such as insertions, deletions and substitutions.
Polypeptides including the amino acids from residues 1 to 22 (SEQ ID NO:2) shown in
FIG. 1
, or Pilosulin 1, or active portions thereof, or functionally equivalent amino acid sequences thereof, can be produced recombinantly or produced synthetically by standard methods known to the art. Furthermore, modifications of the polypeptides can be carried out by known techniques after production of the polypeptides.
It will also be appreciated that the polypeptides of the present invention may have at least some of the peptide bonds between the amino acids replaced by peptide bond mimics (mimetics) to form pseudopeptide analogues. Such mimics are well known to the art and include N-methyl isosteres, hydroxyl isosteres, reduced peptide bonds and retro-inverso-peptomimetics. Alterations of the peptides in this manner may reduce or eliminate their antigenicity so as to reduce the chance of stimulating the immune response in subjects when administered with the peptides over a prolonged period.
In a third aspect, the present invention consists in a method of inhibiting the growth of a cell comprising exposing the cell to an effective amount of a polypeptide according to the first or second aspects of the present invention.
It will be appreciated that the method according to the present invention is suitable to inhibit any cell type sensitive to the inhibitory or cytotoxic action of polypeptides according to the present invention including the amino acid sequence from residues 1 to 22 (SEQ ID NO:2) shown in
FIG. 1
, or Pilosulin 1, an active portion thereof, or functionally equivalent amino acid sequence thereof.
Preferably the cell is a tumour cell, a cancer cell, a B-lymphocyte or a T-lymphocyte.
The effective amount of the cytotoxic polypeptides according to the present invention will often depend on the cell type being inhibited. Concentrations of Pilosulin 1, for example, of approximately 0.02 &mgr;M have been found to inhibit cells but lower concentrations of other polypeptides may also be suitable to inhibit certain cell types. In one preferred form, concentrations of at least about 0.2 &mgr;M, preferably at least about 2 &mgr;M, of the peptides according to the present invention are used to inhibit the growth of cells.
Preferably, the inhibition causes cell death.
Polypeptides including the amino acid sequence from residues 1 to 22 (SEQ ID NO:2) shown in
FIG. 1
, or Pilosulin 1, or active portions thereof, or functionally equivalent amino acid sequences thereof, may be used to inhibit cells in vitro, for example in laboratory applications, or in vivo, for example, by administration to a subject.
The method according to the third aspect of the present invention may be carried out by administering the active polypeptide to a subject by any known delivery method or system. For example, the delivery may be topically, parentally or via the alimentary canal.
In a fourth aspect, the present invention consists in a composition for use in inhibiting the growth of a cell, the composition comprising a polypeptide according to the first or second aspects of the present invention, together with a pharmaceutically acceptable diluent.
In order that the present invention may be more clearly understood, preferred forms will be described with reference to the following examples and figures.
REFERENCES:
patent: 4883661 (1989-11-01), Daly
patent: 5576351 (1996-11-01), Yoshimura
patent: 5604244 (1997-02-01), DeSantis
patent: 5668112 (1997-09-01), Lipsky
patent: 5766873 (1998-06-01), Noble
patent: 5858365 (1999-02-01), Faller
Donovan, Biochimica et Biophysica Acta 1171 272-280, 1993.*
Biochemistry and Molecular Biology International, vol. 39, No. 5, Aug. 1996. Donovan, Gregory et al., “Expression of Jumper Ant (Myrmecia Pilosula) venom allergens: post-transational processing of allergen gene products.”, pp. 877-885.
Electrophoresis, vol. 16, No. 5, May 1995. Donovan, Gregory et al., “Separation of jumper ant (Murmecia pilosula) venom allergens: A novel group of highly basic proteins.”, pp. 804-810.
Baldo Brian
Donovan Gregory
Browdy and Neimark
Low Christopher S. F.
Lukton David
Northern Sydney Area Health Service
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