PO-400 Dual targeting of TIM3 and adenosine produces synergistic improvement in anti-tumour immunity

Introduction CD8 +Cytotoxic T Lymphocytes (CTL) are able to kill tumour cells. However, their killing is frequently suppressed within the tumour microenvironment, due to the interaction of immune checkpoint receptors on tumour infiltrating CTL with tumour-derived ligands. Drugs which block checkpoints have shown great promise in controlling tumour growth. Nevertheless, there is a high rate of immune-related adverse effects associated with these drugs, thus limiting their use. Moreover, in response to blockade of one receptor, tumours may escape treatment by upregulating other checkpoint pathways. As such, there is a need to identify novel tumour-specific checkpoints, to mitigate autoimmune side-effects; and to target these in combination, to prevent tumour immune evasion. Material and methods BALB/c mice bearing subcutaneous murine renal carcinomas, expressing Haemagglutinin (HA) from Influenza A/PR/8 as a model tumour antigen were treated as follows: a) Adoptive Transfer of activated TcR transgenic, HA-specific CD8 +T cells (CL4 cells) b) A2a Adenosine-receptor antagonist 200 ug q2d (ZM241385, Santa Cruz) c) Anti-TIM3 mAb (Clone RMT-3, BioXcell), 100 ug q2d. Vehicle and isotype controls were used. Tumour growth was measured and the volume calculated, Volume=0.5 (Length x (Width 2 )). Flow cytometric analysis of checkpoint expression was carried out using a BD Fortessa and FACS DIVA software. Data was analysed using FlowJo (Treestar). Principal component analysis and ANCOVA were performed using SPSS and R-Studio. Results and discussions In our model, although blockade of the A2aR Adenosine receptor produces partial control of tumour growth, tumours do not completely regress. Principal component analysis was used to evaluate expression of other immune checkpoints across 43 treated and untreated tumours. Analyses showed that expression of T-cell immunoglobulin and mucin-domain containing-3 (TIM3) contributed strongly to the variation between treated and control groups. These data suggest that upregulation of TIM3 represents a mechanism of immune evasion in response to A2aR blockade. Critically, co-blockade of A2aR and TIM3 produces complete and durable tumour regression. Conclusion Expression of TIM3 represents a key mechanism of tumour-mediated immune evasion in response to A2aR blockade. The fact that co-blockade of A2aR and TIM3 mediates complete and durable tumour regression, with no obvious adverse effects, strongly supports the use of such dual blockade in the treatment of cancer.