Multiple Administration Routes, Including Intramuscular Injection, of Oncolytic Tanapoxvirus Variants Significantly Regress Human Melanoma Xenografts in BALB/c Nude Mice Reconstituted with Splenocytes from Normal BALB/c Donors

Abstract

Human melanoma is the most aggressive form of skin cancer and is responsible for the most deaths of all skin cancers. Localized tumors, and those which have limited spread, have 5-year survival rates of over 90%, with those numbers steadily rising over the past decade. However, metastatic melanomas have a sharp decrease in 5-year survival rates and are still an area of need for new, successful therapies. Immuno-oncolytic viruses (OVs) have emerged as a promising class of immunovirotherapy that can potentially address this disease. The Food and Drug Administration in the United States has approved one oncolytic herpes simplex virus expressing granulocyte-macrophage colony-stimulating factor (Talimogene Laherparepvec) for the treatment of metastatic melanoma, and others could soon follow for this and other cancers. In previous studies, Tanapoxvirus (TPV) recombinants expressing mouse interleukin-2 (mIL-2) and another expressing bacterial flagellin from Salmonella typhimurium (FliC) have demonstrated anti-tumor efficacy in nude mouse models. TPV replicates only in humans and monkeys, including tumor cells, which makes the use of syngeneic tumor models impossible for the study of this OV in a standard immunocompetent system. In this study, TPV/Δ66R/mIL-2 and TPV/Δ2L/Δ66R/FliC were tested for their ability to treat human melanoma xenografts (SK-MEL3) in a BALB/c nude mouse model reconstituted with splenocytes from genetically compatible, normal BALB/c donor mice. Two SK-MEL3 tumors were transplanted into each flank of BALB/c nude mice, and the larger tumor was treated intratumorally (IT) with virus or mock injection. In one set of animals, mice received adoptive transfers of splenocytes from BALB/c mice on day 4 to reconstitute their immune systems and allow for adaptive immune responses to occur in a xenograft model. Direct IT injection of TPV/Δ66R/mIL-2 led to significantly greater rates of tumor regression compared to reconstituted control (RC) mice in the primary and distant tumor sites, whereas TPV/Δ2L/Δ66R/FliC treatment led to significantly greater rates of tumor regression in distant tumor sites only. A second experiment used TPV/Δ66R/mIL-2 to test whether TPV could be administered intravenously (IV), intramuscularly (IM), or both routes simultaneously to exert similar anti-tumor effects in an indirectly treated tumor. A single SK-MEL3 tumor was transplanted into one flank of BALB/c nude mice and was treated either into the tail vein, the nearest rear leg to the tumor, or both. All mice then received adoptive transfers of splenocytes in the same way as previously described on day 4 and received an additional TPV treatment on day 14. The results demonstrated that TPV/Δ66R/mIL-2 treatment IV or IM had significantly greater rates of tumor regression than RC-treated mice but failed to exert this effect when both routes were used simultaneously. Data obtained through these experiments support the continued development of Tanapoxvirus for the treatment of human melanoma and using immune reconstitution to create intact adaptive immunity in a xenograft context, which can allow other tropism-limited OVs to be studied against human cancers.

Keywords

• melanoma
• immuno-oncolytic viruses
• xenograft
• adoptive transfer
• immune reconstitution
• tanapoxvirus