MA04.01 Development of an in Vivo Platform to Identify Novel Mechanisms Governing Lung Cancer Response to Immunotherapy

Abstract

Immune checkpoint inhibitors (ICIs) have emerged as a promising therapy for the treatment of advanced stage lung cancer. While these agents elicit durable responses in some patients, most do not respond. An improved understanding of the mechanisms governing ICI response in a complex in vivo setting has potential to reveal novel therapeutic combinations to enhance ICI efficacy and associated biomarkers indicative of response. We have developed a syngeneic lung tumour model for in vivo CRISPR/Cas9 screens to deduce novel tumour-intrinsic mediators of response to anti-PD1 therapy. To delineate physiologically relevant ICI response mechanisms, an in vivo, syngeneic and orthotopic lung tumour model with an intact immune system is required. For this purpose, we have developed the KRAS-mutant Lewis Lung Carcinoma (LLC) model. Flow cytometry and immunohistochemistry were used to characterize immune cell composition in resected tumours and in vivo experiments were conducted to determine the effects of anti-PD1 treatment on LLC tumour growth. Luciferase-tagged LLC cells were implanted into the lungs of syngeneic C57BL/6 mice using orthotopic injection and mice reached humane endpoints 10-14 days post injection. Immunophenotyping of dissociated tumours revealed changes in the proportions of myeloid and lymphocyte populations relative to tumour-naïve lungs. CD8+ T-cells were present and tumour cells expressed PDL1 suggesting LLC has the capacity to respond to ICI. Consistent with these observations, orthotopic LLC growth was delayed in mice treated with anti-PD1 therapeutic antibody compared to anti-IgG2a isotype control, demonstrating that LLC is an appropriate model for identifying mechanisms that confer sensitivity and/or resistance to ICI therapy. Based on these findings, we generated LLC-Luciferase cells stably expressing Cas9. Since genome-wide screens are not feasible with this in vivo tumour model, we are synthesizing a custom, focussed guide RNA (gRNA) library. Genomic analyses have identified ∼500 candidate immunomodulatory genes expressed in LLC and clinical lung tumours that will be targeted to determine the effects of inactivating these candidates on anti-PD1 response. This platform will enable high-throughput genetic screens to elucidate novel tumour-intrinsic determinants of ICI response in vivo. Our discoveries will have potential to inform novel biomarkers predictive of response, and putative targets for new combination therapies to enhance the anti-tumour effects of ICIs. Collectively, this work will improve our understanding of the biological mechanisms governing ICI sensitivity, thereby stimulating the development of new strategies to maximize therapeutic benefit from ICIs in lung cancer patients.