Abstract 4258: Both NGR and TNF portions contribute to the biological activity of NGR-TNF vascular targeting agent in vivo

NGR-TNF, presently in advanced clinical development, is a direct-acting vascular targeting agent coupling the CNGRCG peptide (NGR), homing to angiogenic blood vessels, and tumour necrosis factor alpha (TNF). To elucidate its mechanism of action, we investigated the NGR-TNF homing and downstream effects using doses comparable to those used in clinical trials. Targeting studies with NGR-quantum dots administered in vivo demonstrate that the binding to CD13 occurs only in neo-angiogenic tissues, sparing CD13-expressing normal tissues. NGR-TNF binding is highly specific targeting the tumour vessel of human colon cancer specimens, whereas the binding does not occur in the normal colon tissue of the very same patients. To investigate the interaction between NGR and CD13, we incubated human primary mesangioblasts with biotinylated peptides, cross-linked with BS3 to form covalent bounds between interacting molecules, lysed the cells and pulled down biotinylated proteins by streptavidin-coated magnetic beads. Only biotinylated NGR pulls down CD13 in monomeric and dimeric forms. After CD13 immunoprecipitation, the CD13 immunoprecipitated from NGR-treated cells is biotinylated, unlike CD13 immunoprecipitated from control peptide. Depriving CD13 completely in the total lysate of cells incubated with NGR, the biotinylated bands at 150 kDa disappears. Overall these data demonstrate that biotinylated NGR directly interacts with CD13 on the cell membrane. To address whether NGR binding affects the TNF-TNFR interaction, we used TNF conjugated to an Ig Fc portion so that it can be visualised onto target cells, and we tested the ability of both TNF and NGR-hTNF to compete with this binding. Interestingly, while in NGR-non-binder cells the affinity between TNF and NGR-TNF is the same, NGR-hTNF clearly displays an higher affinity than TNF in NGR-binder cells. It is worth noting that the 1-log increase in NGR-hTNF affinity is actually due to the binding of NGR to CD13 because this is overcome with increasing amounts of free NGR. We then investigated how NGR-hTNF affects signaling pathways in human endothelial cells. Interestingly, we found that, over hTNF, NGR-hTNF impairs cell survival a) by inhibiting the activation of the pro-survival Ras pathway inducing a down-modulation of Raf, MEK, Erk, and Akt, along with preventing VEGFR2 mRNA up-regulation, and b) by activating the pro-apoptotic caspases (3, 8 and 9). Interestingly, if we stimulate endothelial cells with TNF and a neu