Abstract
We have engineered an attenuated measles virus derived from the vaccine Edmonton strain to display the venom peptide echistatin with M28L mutation for targeting multiple myeloma tumors and associated angiogenesis. Echistatin binds irreversibly to integrin αVβ3 that has been shown by others to be expressed on activated endothelial cells and also on multiple myeloma tumor cells. We have chemically synthesized the echistatin gene and cloned it into p(+)MVGFP, a plasmid used to rescue measles virus, at the C-terminus of the gene coding for the viral attachment protein, H. Incorporation of the echistatin gene was confirmed by RT-PCR and the successful display of the protein as a C-terminal extension of H was demonstrated in replicating viruses by immunoblotting. Specific targeting of the virus was confirmed by infection of B10 cells (transformed Chinese Hamster Ovary cells) expressing the cognate receptor αVβ3. The recombinant measles virus displaying echistatin produced characteristic green syncytial rings, thereby, the vector was named emerald ring virus (ERV). Soluble echistatin peptide competitively inhibited ERV infection of B10 cells in a dose dependent manner. The growth kinetics of ERV showed similar characteristics to the wild type virus MV-Edm, but the titer was consistently about one log lower. In vitro, ERV infected primary cow pulmonary endothelial (CPAE) cells plated on matrigel more efficiently than the control virus, MV-Edm. Both MV-Edm and ERV infected human umbilical vein endothelial cells (HUVEC) in monolayer cultures, but when HUVEC formed capillary-like structures on matrigel, ERV infected the tubules more efficiently than MV-Edm. Although echistatin constitutes a toxin in snake venom, no toxicity was observed in ERV treated mice. ERV was injected intravenously or subcutaneously into SCID mice without any apparent toxicity. Chick chorioallantoic membrane (CAM) assay showed that ERV can infect bFGF-stimulated blood vessels. In an in vivo pilot study, ERV showed significant oncolytic activity against MM1 myeloma xenografts which are completely resistant to therapy with unmodified MV-Edm. In conclusion, we have explored the possibility of using a ligand derived from snake venom to display on measles virus. The ligand, echistatin, targets both activated endothelial cells and multiple myeloma cells.