Intracellular expression of carboxypeptidase G2 in a nitrogen mu

Details Intracellular expression of carboxypeptidase G2 in a nitrogen mu Intracellular expression of carboxypeptidase G2 in a nitrogen mu

1997-01-30

1999-12-21

Crouch, Deborah

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

Genetically modified micro-organism, cell, or virus

424 932, 435 29, 4353201, 435325, 435357, A61K 4800, C12N 1500, C12Q 100

Patent

active

060045507

DESCRIPTION:

BRIEF SUMMARY
The present invention relates to gene directed enzyme prodrug therapy (GDEPT) and its use in the treatment of disease, including tumours.
A therapeutic approach termed "virus-directed enzyme prodrug therapy" (VDEPT) has been proposed as a method for treating tumour cells in patients using prodrugs. Tumour cells are targeted with a viral vector carrying a gene encoding an enzyme capable of activating a prodrug. The gene may be transcriptionally regulated by tissue specific promoter or enhancer sequences. The viral vector enters tumour cells and expresses the enzyme, in order that a prodrug is converted to an active drug within the tumour cells (Huber et al, Proc. Natl. Acad. Sci. USA (1991) 88, 8039). Alternatively, non-viral methods for the delivery of genes have been used. Such methods include calcium phosphate co-precipitation, microinjection, liposomes, direct DNA uptake, and receptor-mediated DNA transfer. These are reviewed in Morgan & French Anderson, Annu. Rev. Biochem., 1993,62;191. The term "GDEPT" (gene-directed enzyme prodrug therapy) is used to include both viral and non-viral delivery systems.
A number of different enzymes have been proposed as being suitable for use in GDEPT therapy. In general, the enzymes have been selected to be compatible with prodrugs which are known to be effective against the tumour type being treated. It is also important for the success of GDEPT that the drug is capable of entering the cell.
The enzyme carboxypeptidase G2 (CPG2) has been proposed for use in Antibody-Directed Enzyme Prodrug Therapy (ADEPT) systems, in combination with a number of types of prodrugs, including nitrogen mustard prodrugs. In ADEPT, the prodrug is activated outside the cell or on the cell surface, and thus the prodrug was believed to have no significant ability to enter the cell. Also the prodrug contains the hydrophilic glutamic acid. It has now surprisingly been found that such prodrugs not only enter the cell but can be activated intracellularly by CPG2. Furthermore, following activation in the cell, the drugs exhibit an effective bystander effect where surrounding cells are also killed by activated drug.


DISCLOSURE OF THE INVENTION

In order to overcome these problems, the present invention thus provides a two component system for gene-directed enzyme prodrug therapy which comprises: (a) a vector which encodes a carboxypeptidase which is expressed within a cell; and (b) a prodrug which can be converted into an active drug by said carboxypeptidase. The enzyme is preferably a bacterial carboxypeptidase, especially carboxypeptidase CPG2 (SEQ ID No. 1) or Pseudomonas .gamma.-glutamylhydrolase EC3.4.22.12 (Levy CC & Goldman P J. Biol. Chem. 242; p2933 (1967).
The vector may be a RNA or DNA vector. It may be derived from a viral vector, including any suitable vector available in the art for targeting tumour cells.
The invention also provides the system of the invention for use in a method of treatment of a patient, and a method of treating a tumour in a patient in need of treatment which comprises administering to said pat,ient an effective amount of a vector encoding a carboxypeptidase and a prodrug capable of being converted by said enzyme to an active drug.


BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (A-B) Expression of CPG2* in NIH3T3 cells (A) Immunoblot analysis. NIH3T3 cells were transfected with MLV.beta.plink (lane 1) or with MLVCPG2* (lane 2). The cells were extracted into buffer A and samples of each were electrophoresed in 10% w/v polyacrylamide gels, transferred to nitrocellulose and probed with a CPG2 specific polyclonal antibody. The position of migration of CPG2* is shown and the positions of migration of standard molecular mass markers (in kDa) are shown to the left of the figure. (B) CPG2 enzyme activity assay. The cell extracts from part (A) were analysed by CPG2 enzyme activity assay. Sample numbers correspond to lane numbers from part (A). The enzyme activity is expressed in arbitrary units, relative to buffer controls.
FIG. 2. Cytotoxicity assay NIH3T3 cells were tran

REFERENCES:
patent: 5665543 (1997-09-01), Foulkes et al.
(Mullen (1994) Pharm. Ther. 63, 199-207, 1994.
Cornell et al (1976) Biotech.Bioeng. vol. XVIII, 1171-1174, 1976.
Yasuda et al (1994) Bull. Mukogawa Univ. Nat. Sci. 42, 63-66, 1994.
Mullen (1994) Pharmac. Therap. 63, 199-207.
Ann. N.Y., Acad. Sci., vol. 716, May 31, 1994 pp. 115-125, K. Sikora et al, "Therapeutic strategies using c-34bB-2 promoter-controlled drug activation".
Trends in Biotechnology, vo. 11, No. 5, 3, Cambridge GB, pp. 197-201, K. Sikora, "Gene therapy for cancer".

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