title: A novel vascular endothelial growth factor-directed therapy that selectively activates cytotoxic prodrugs.
abstract: 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.
authors.affiliation: Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, UK.
authors.forename: R A
authors.initials: RA
authors.lastname: Spooner
authors.forename: F
authors.initials: F
authors.lastname: Friedlos
authors.forename: K
authors.initials: K
authors.lastname: Maycroft
authors.forename: S M
authors.initials: SM
authors.lastname: Stribbling
authors.forename: J
authors.initials: J
authors.lastname: Roussel
authors.forename: J
authors.initials: J
authors.lastname: Brueggen
authors.forename: B
authors.initials: B
authors.lastname: Stolz
authors.forename: T
authors.initials: T
authors.lastname: O'Reilly
authors.forename: J
authors.initials: J
authors.lastname: Wood
authors.forename: A
authors.initials: A
authors.lastname: Matter
authors.forename: R
authors.initials: R
authors.lastname: Marais
authors.forename: C J
authors.initials: CJ
authors.lastname: Springer
mesh_terms: D000230:Adenocarcinoma / Q000473:pathology; D016228:Endothelial Growth Factors / Q000235:genetics / Q000494:pharmacology*; D004727:Endothelium / Q000166:cytology; D005260:Female; D005971:Glutamates / Q000494:pharmacology*; D005973:Glutamine; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000494:pharmacology*; D008222:Lymphokines / Q000235:genetics / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D009389:Neovascularization, Pathologic; D009588:Nitrogen Mustard Compounds / Q000494:pharmacology*; D010051:Ovarian Neoplasms / Q000473:pathology; D010957:Plasmids; D017354:Point Mutation; D011355:Prodrugs / Q000494:pharmacology*; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000187:drug effects / Q000235:genetics*; D042442:Vascular Endothelial Growth Factors; D011623:gamma-Glutamyl Hydrolase / Q000235:genetics / Q000494:pharmacology*
chemical_list: D016228:Endothelial Growth Factors; D005971:Glutamates; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D009588:Nitrogen Mustard Compounds; D011355:Prodrugs; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; C467480:vascular endothelial growth factor A, mouse; D005973:Glutamine; C092574:4-((2-chloroethyl)(2-mesyloxyethyl)amino)benzoylglutamic acid; D040301:Vascular Endothelial Growth Factor Receptor-2; D011623:gamma-Glutamyl Hydrolase