Sculpting the tumour microenvironment by combining radiotherapy and ATR inhibition for curative-intent adjuvant immunotherapy

Results of combining radiotherapy/chemoradiotherapy and immune checkpoint blockade have been disappointing in patients with locally advanced head and neck squamous cell carcinoma (HNSCC). For such a potentially radiocurable disease, there remains an imperative to explore novel combination approaches. Here, we show that combining ATR inhibition with radiotherapy (ATRi/RT) increases the frequency of activated NKG2A/PD-1 double-positive T cells in animal models of HNSCC. Addition of dual anti-NKG2A/-PD-L1 blockade to ATRi/RT in the adjuvant, post-radiotherapy setting induces a robust antitumour response. Efficacy of the combination relies on CD40/CD40L costimulatory-mediated infiltration of activated/proliferative/memory CD8 and CD4 T cells with persistent or new T cell receptor (TCR) signalling, respectively. In this favourable therapeutic context, we reveal increased richness of the TCR repertoire and the emergence of numerous and large TCR clusters that share antigen specificity in response to combination therapy. Collectively, our data point towards promising combination approaches for future clinical testing in HNSCC. To investigate therapeutic effects of combination of ATR inhibition with radiotherapy (ATRi/RT) and dual anti-NKG2A and anti-PD-1/-PD-L1 antibodies, we used 8- to 14-week-old females C57BL/6 WT mice, inoculated with head and neck cancer cells from several cell lines. We used models of murine immunocompetent HPV-negative and HPV-positive head and neck cancer, MOC1/MOC2 and mEER, respectively. Mice were inoculated subcutaneously (ectopic) on the right flank with 4 × 10^6 cells for the MOC1 or 10^6 cells for the MOC2 and mEER models. Mice were further treated with combination of the ATR inhibitor AZD6738 and radiotherapy OR with anti-NKG2A/anti-PD-L1 antibodies OR with both. Additionally, vehicle-treated mice were added as a control group. Each treatment group consisted of four biological replicates. We then performed gene expression profiling analysis using data obtained from RNA-seq of tumour cells from the treated mice.