Enzyme prodrug therapy designed to target l-methioninase to the tumor vasculature

Annexin V-Directed Enzyme Prodrug Therapy Plus Docetaxel for the Targeted Treatment of Pancreatic Cancer

“…To address the vascular targeting capabilities of these fusion protein systems, we have previously shown that all three systems bind tightly to phosphatidylserine expressing human abdominal aorta endothelial cells in vitro, with dissociation constants ranging from 0.5 to 1.5 nM. [18][19][20] We aimed to take advantage of the phosphatidylserine exposure effect of docetaxel without unleashing its broad-spectrum antitumor properties, as to establish the benefit of docetaxel only with respect to fusion protein binding. This was in fact demonstrated because all of the no treatment controls with docetaxel did not show any significant deviations in viability compared to the no treatment controls without docetaxel.…”

“…To mimic in vivo EPT, cells were treated with a saturating concentration of fusion protein (100 nM) every 3 days for 2 hours at 37°C in accordance with previous binding stability studies. [18][19][20] Each day, the medium was replaced with a medium containing varying concentrations of prodrug (SM and 5-FC from Fisher Scientific, Waltham, MA and FD from VWR, Radnor, PA) or drug analog (2-FA from Fisher Scientific and 5-FU from Sigma-Aldrich, St. Louis, MO) both containing appropriate concentrations of docetaxel. An Alamar Blue (Invitrogen, Grand Island, NY) assay was preformed every 2 days to measure cell viability.…”

“…Dissociation constants were determined as described previously. [18][19][20] Briefly, cells were fixed with glutaraldehyde, treated with bovine serum albumin, and incubated with varying concentrations (0-20 nM) of biotin-labeled fusion proteins, which after washing were then allowed to react with streptavidin-horseradish peroxidase. Subsequently, binding was quantified with the chromogenic substrate o-phenylenediamine by measuring absorbance at 450 nm.…”

“…[18][19][20] Briefly, the polymerase chain reaction was used to amplify the genes encoding each enzyme and AV, as well as a fusion site containing a 6-residue flexible linker and an N-or C-terminal His 6 tag with a human rhinovirus protease cleavage site immediately downstream or upstream, respectively. Plasmids containing each fusion protein were created via transformation of NovaBlue competent cells and then expressed in E. coli BL21 (DE3) cells.…”

“…We have previously reported the vascular targeting and killing capabilities of each of these systems on nonconfluent human abdominal aorta endothelial cells as mimics of tumor vasculature in vitro. [18][19][20] We have also validated the safety and efficacy of the MT-AV system in vivo for the treatment of MDA-MB-231 breast cancer xenografts in mice. 4 To date, the efficacy of our AV-directed EPT systems on PDAC remains unexplored.…”