Analysis of Anti-Tumor Immune Responses with Radiation Combined with Anti-PD-L1 Immunotherapy in an HPV Specific Head & Neck Cancer Model.

Abstract

Anti-PD-1 checkpoint blockade immunotherapy (CBI) was recently approved for recurrent or metastatic cisplatin refractory Head and Neck Squamous Cell Carcinoma (HNSCC); however, response rates remain low. Multiple pre-clinical studies and emerging clinical studies have described synergistic interactions between radiation (RT) and CBI. We previously defined the role of RT in enhancing T-cell activation and proliferation via antigen cross-presentation in the draining lymph node (DLN) when combined with anti-PD-1 CBI. However, the ability for RT to enhance local control and anti-tumor immune responses when combined with anti-PD-L1 CBI in HPV specific HNSCC models is understudied. Here we used novel flank and orthotopic HNSCC tumor models including AT-84 expressing HPV E7 to study the effects of RT+CBI. Focused RT (8-12Gyx1) was delivered to AT84-E7 flank tumors and orthotopic tongue tumors via the JL Shepard CS-137. Cohorts of C3H or B6 mice received either no treatment or three IP injections of 200ug anti-PD-L1 Ab immediately before, and at 3 and 6 days post RT. Tumor diameter was measured every 2 to 3 days using electronic caliper and reported as volume using the formula (m1 x m22)/2. Cell surface markers and development of antigen-specific immune responses were analyzed in tumor draining lymph node (DLN) and matched tumor infiltrating lymphocytes (TIL) by flow cytometry on BD FACSAria II. RT + anti-PD-L1 CBI resulted in a statistically significant improvement in local tumor control compared to RT alone in our HNSCC orthotopic tumor models (RT 16.5 +/- 8.9 mm3 vs RT + CBI 1.2 +/- 0.6 mm3, p<0.001). RT + CBI also significantly increased CD8+ and CD4+ activated T-cells in the TIL population (CD8: RT 2.3 +/- 0.7% vs RT + IT 4.1 +/- 1.2%, p < 0.05; CD4: RT 0.7 +/- 0.3% vs RT + IT 4.1 +/- 2.2%, p< 0.05). Additionally, RT + CBI enhanced IFNg production and development of antigen specific anti-tumor immunity in the DLN compared to RT or CBI alone. Mechanistically we observed significant changes in cell adhesion molecules including ICAM-1 and VCAM-1 which may contribute to enhanced T-cell infiltration. Response rates to single agent CBI remain low in HNSCC and combinatorial strategies are required to improve outcomes. Here we used novel HPV specific flank and orthotopic HNSCC models and observed that RT + anti-PD-L1 CBI results in a significant improvement in local control and development of anti-tumor immune responses compared to RT or CBI alone. These findings indicate that combination RT + CBI could be an effective therapy for HPV positive head and neck cancer, and provide additional rationale for clinical trials in this space.