A novel vascular endothelial growth factor-directed therapy that selectively activates cytotoxic prodrugs.

We have generated fusion proteins between vascular endothelial growth factor (VEGF) and the bacterial enzyme carboxypeptidase G2 (CPG2) that can activate the prodrug 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamic acid (CMDA). Three asparagine residues of CPG2 were mutated to glutamine (CPG2(Q)3) to prevent glycosylation during secretion, and truncations of VEGF(165) were fused to either the C- or N-terminal of CPG2. The K(m) of the fusion proteins (37.5 microM) was similar to that of secreted CPG2(Q)3 (29.5 microM) but greater than that of wild-type CPG2 (8 microM). The affinity of the fusion proteins for VEGF receptor-2 (VEGFR2) (K(d)=0.5-1.1 nM) was similar to that of [(125)I]VEGF (K(d)=0.5 nM) (ELISA) or slightly higher (K(d)=1.3-9.6 nM) (competitive RIA). One protein, VEGF(115)-CPG2(Q)3-H(6), possessed 140% of the enzymic activity of secreted CPG2(Q)3, and had a faster half-maximal binding time for VEGFR2 (77 s), than the other candidates (330 s). In vitro, VEGF(115)-CPG2(Q)3-H(6) targeted CMDA cytotoxicity only towards VEGFR-expressing cells. The plasma half-life of VEGF(115)-CPG2(Q)3-H(6) in vivo was 3 h, comparable to equivalent values observed in ADEPT. We conclude that enzyme prodrug therapy using VEGF as a targeting moiety represents a promising novel antitumour therapy, with VEGF(115)-CPG2(Q)3-H(6) being a lead candidate.

Appropriate subcellular localisation of prodrug-activating enzymes has important consequences for suicide gene therapy.

Escherichia coli B nitroreductase (NR) has been expressed stably in MDA-MB-361 human breast adenocarcinoma cells either as the wild-type protein (wtNR), which is distributed evenly between the cytoplasmic and nuclear compartments, or targeted to the mitochondrion (mtNR). Whereas bacterial NR is active as a dimer, a proportion of wtNR is monomeric. In contrast, mtNR is mostly dimeric, suggesting that it adopts a more stable, native conformation. Despite this, when tested in gene-directed enzyme prodrug therapy cell cytotoxicity studies, cells expressing wtNR or mtNR had similar sensitivity to the prodrug CB1954 and mounted similar bystander killing effects. Furthermore, when short prodrug exposures were given, wtNR was more efficient at killing cells than mtNR. These data demonstrate that the site of enzyme expression and prodrug activation is an important variable that requires consideration in suicide gene therapy approaches.

Detection and identification of novel actinomycetes.

The actinomycetes are well known as a group of filamentous, Gram-positive bacteria that produce many useful secondary metabolites, including antibiotics and enzymes. Although they have been intensively studied for both theoretical and practical objectives, there is much scope for developing our basic knowledge of the means of detection and isolation of these microbes. This session concentrated on new methods for the detection and identification of novel actinomycetes from a range of environments. Approaches to the detection of actinomycetes ranged from investigations of neglected habitats and extreme environments (e.g. alkaline soils and oil drills) to the analysis of DNA extracted from the environment and use of specific phages. The continuing problems of the identification of actinomycete isolates were also considered. Topics discussed included use of phage typing, DNA probes, and correlation between phenetic and genotypic species of Streptomyces.

Viable but non-culturable salmonellas in soil.

An enzyme-linked immunosorbent assay (ELISA) and a microwell fluorescent antibody (FA) direct count method have been developed for the monitoring of salmonellas in soil. Both methods have a minimum detection level of ca 10(6) cells per gram of soil. The FA direct count method gave a linear recovery for the inoculum range 10(6)-10(9) cells per gram of soil. When monitored by plate counts the survival of salmonellas was greater in a sterile than in a non-sterile soil. Evidence was found for the production of viable but non-culturable salmonellas in non-sterile soil; plate counts dropped rapidly with time, but FA direct counts and ELISA remained level. The salmonella cells became progressively smaller and rounder with time. Dead salmonella cells introduced into soil rapidly disappeared.

An ion-exchange based extraction method for the detection of salmonellas in soil.

A method that uses a cation-exchange resin (Chelex 100) and differential centrifugation for the extraction and detection of salmonellas in soil was developed. The extraction efficiencies of a range of materials were examined and Chelex plus polyethylene glycol was identified as the best combination. Shake speeds, shake times and differential centrifugation speeds were selected to give an optimum salmonella recovery. The Chelex method accurately enumerated 1 cell per 10 g of nonsterile soil within 24 h. Addition of glycerol to soil samples enabled storage at -70 degrees C for 85 d without significant decreases in salmonella numbers. The Oxoid Salmonella Rapid Test (SRT) could be used to pre-screen large numbers of soil samples for the presence of salmonellas, prior to analysis by the Chelex method. The SRT method detected Salmonella typhimurium at levels as low as 2.5 cells per 10 g of nonsterile soil.