Targeted BRAF inhibitors: Immunological effects and combination with immunotherapy

Metastatic melanoma is the most aggressive form of skin cancer, associated with a poor prognosis, and the incidence worldwide is increasing. Recently, selective mutant BRAF inhibitors and checkpoint blockade immunotherapy have advanced clinical treatment of metastatic melanoma. However, efficacy of these therapies individually is limited. Combining treatments may allow BRAF inhibition to augment immunotherapy by increasing tumour antigen availability and improving immune system targeting of tumours. The success of this approach depends upon fully elucidating immunological interactions of BRAF inhibitors, and optimizing combination strategies.  To study the immunological effects of BRAF inhibitors and their combination with immunotherapy, novel murine BrafV600E Pten-/- Cdkn2a cell lines were characterized. These were found to be moderately sensitive to BRAF inhibition compared with the widely used human BRAFV600E cell lines A375 and SK-mel-5. In vitro targeted BRAF inhibition was shown to induce cell death through apoptosis, and partially reverse melanoma-mediated immunosuppression by human melanoma cell lines.   Utilising subcutaneously injected syngeneic, murine BRAFV600E cell lines, the BRAF inhibitor PLX4720 was shown to decrease tumour growth in vivo. Host immune involvement in BRAF inhibitor efficacy was determined by comparing PLX4720 treatment in NOD/Scid and C57BL/6 mice. PLX4720 control of tumour growth was significantly less effective in immunocompromised mice, resulting in reduced survival advantage. These findings demonstrate that the anti-tumour effects of mutant BRAF inhibitors are partially immune dependent, although the nature of this immune involvement remains to be defined. It was further shown that BRAFV600E inhibition directly affected immune responses. In vitro, both human and murine T cell activation were boosted by low concentrations of mutant BRAF inhibitors. This was confirmed in vivo, with antigen-specific T cell proliferation significantly increased by PLX4720 treatment.  The final chapters of this thesis explored the combination of active immunotherapy with targeted BRAF inhibition. A vaccine was devised that consisted of irradiated, autologous tumour cells loaded with the adjuvant α-galactosylceramide. This vaccine was shown to be effective in both prophylactic and therapeutic settings in a BRAFV600E melanoma model. Mechanistically, vaccine increased effector T cell responses and decreased frequencies of Tregs. Vaccine efficacy was CD4⁺ T cell-dependent, and did not require CD8⁺ T cells. Combination of vaccine with targeted BRAF inhibition was investigated in different settings. A combination therapy strategy was developed that achieved additive, but not synergistic benefit. Additionally, targeting specific aspects of the tumour microenvironment that may confer tumour resistance to BRAF inhibitor-mediated cell death was investigated. Both depletion of Tregs and inhibition of TNFα were explored, but did not result in a significant improvement in therapy.   In summary, the studies undertaken in this thesis demonstrate that BRAF inhibitors can augment vaccine-induced T cell responses. Moreover, this research revealed the anti-tumour efficacy of BRAFV600E inhibition is partially immune dependent and can be improved by combination with active immunotherapy. These discoveries generated a combination therapy strategy with improved efficacy over single agent treatment. Further studies are needed to realise the full potential of this combination therapy approach, and achieve a synergistic benefit.