Optimising Oncolytic Virotherapy and Immunotherapy for the treatment of Disseminated Colorectal Cancer

Colorectal cancer (CRC) is one of the most prevalent malignancies in the Western world with a 5 year survival rate of patients with metastatic disease of less than 10%. As such, there remains a pressing need for novel treatment strategies and modalities. Established treatments including anti-EGFR antibodies, for example cetuximab, have improved survival although disappointingly only 10-20% of patients obtain an objective clinical response. Advancing treatment modalities include oncolytic viruses (Reovirus, Vaccinia Virus (JX-594)), which preferentially replicate in cancer cells causing cell death and stimulate anti-tumour immunity, and BH3-mimetic inhibitors (ABT- 263), which antagonise the BCL-2 family of pro-survival proteins, may enhance CRC patient survival. Harnessing the potential immune and anti- cancer effects of these treatment modalities, alone and in combination, at primary tumour and sites of metastatic and micrometastatic (for example lymph nodes) disease could form the basis of successful clinical adjuvant strategies. This MD thesis aims to investigate the efficacy of two OV, Reovirus and JX- 594 vaccinia virus against CRC. In particular, the work outlined in this thesis has examined whether i) Reovirus directly kills CRC cell lines with differential mutational status, alone or in combination with BH3-mimetics and ii) can activate immune effector cells to enhance killing of EGFR targeted cells in order to optimise the use of anti-EGFR therapy; iii) delineate the mechanism of cell death induced by JX-594 treated CRC cells, and iv) test the ability of JX-594 to activate and induce an innate immune response in the blood and lymph nodes. Studies investigating Reovirus in combination with ABT-263 and Cetuximab were performed. ABT-263 in combination with Reovirus demonstrated that this strategy did not yield enhanced killing over either agent alone, with only additive effects observed in a single cell line, SW620. However, initial studies combining Cetuximab with OV did reveal the potential of Reovirus to increase EGFR-mediated ADCC against a KRAS mutant cell line, SW480, demonstrating the importance of OV immune activation in combination therapeutic approaches. To date, the immune potential of reovirus is well recognised however, less is known about the immune potential of JX-594, an OV currently in clinical testing at Leeds Teaching Hospitals NHS Trust. To test the ability of JX594 to activate immune cell populations, healthy donor blood, along with blood and lymph nodes from CRC patients, were collected and treated with JX-594. JX-594 treated NK cells from patient blood and lymph nodes demonstrated CD69 activation, enhanced degranulation and increased cytotoxicity against CRC cell line targets. In blood, NK cell activation was dependent on IFN production and the presence of CD14+ve monocytes however in lymph node mononuclear cells this was IFN independent and the mechanism remains to be elucidated. Importantly, OV activation of immune effector cells known to reside in LN is encouraging for targeting distant micrometastatic disease. OV hold promise as a novel treatment modality. Direct tumour-specific lysis, transgene expression and the induction of tumour specific innate immunity in isolation, or in combination with adjunct antitumour treatment modalities, means that they may provide a two-pronged attack against the tumour at different disease sites.