846. In Vivo and In Vitro Cell Targeting with Library-Selected Protease-Activatable Replication-Competent Retroviruses

Top of pageAbstract Viruses conditionally replicating in cancer cells form an attractive novel class of anti-tumoural agents. To engineer such viruses we couple infectivity with proteolytic activity of the target cell by modifying the envelope (Env) protein of murine leukemia virus (MLV) with blocking domains that prevent cell entry unless they are cleaved off by tumor-associated proteases like the matrix metalloproteases (MMP). Here we show that MLV variants selectively spreading through MMP-positive cells can be evolved from virus libraries, in which a standard MMP-2 substrate peptide connecting the blocking domain CD40L with the Env protein was diversified. Passaging the virus library on a human fibrosarcoma cell line resulted in the selection of about 10 virus clones of which the two most frequent ones were reconstituted as molecular virus clones and characterized. While the origin of the activating protease for the AKGLYK linker peptide encompassing virus remained elusive, the PSGLYQ linker peptide encompassing virus was cleaved by MMP-2 in vitro and could be inhibited by a broad-range MMP inhibitor. Yet, both linker peptides conferred a remarkable advantage in the spreading through MMP-positive tumor cell lines. The in vivo spreading behavior of these viruses was monitored in SCID mice transplanted with HT1080 tumor cells. In a first experimental setting we applied mixtures of HT1080 cells infected with the selected viruses or the parental virus, respectively, and uninfected HT1080 cells. The AKGLYK linker peptide encompassing virus was able to spread through the complete tumor mass within two weeks when 0.2% infected cells were injected. The parental virus, in contrast, reverted to wildtype virus within the same period by deletion of the blocking domain, which is most likely enhanced by its low spreading efficiency. Similar results were obtained when the selected viruses were injected intratumorally into established HT1080 tumor tissue. Moreover, initial biodistribution data indicate, that the selected viruses, in contrast to the wildtype virus, show a clear preference for tumour tissue. Thus, the growth advantage of MMP-activatable viruses engineered by directed evolution observed in vitro was confirmed in vivo in mouse tumor models. The data suggest that retroviral protease substrate libraries form a potent tool for the engineering of viruses conditionally replicating in a given cancer cell type.