Anti-GITR with Radiation Therapy Enhances Anti-Tumor Abscopal Effects in Anti-PD1 Resistant Murine Model

Abstract

Although immunotherapy has demonstrated potential to cure metastatic NSCLC, many patients do not respond to such intervention. As a monotherapy, anti-PD1 response rates remain low, around 20%, in advanced 2L NSCLC. Previously, we demonstrated the ability of radiation to partially abrogate anti-PD1 resistance via upregulation of MHC I expression in an anti-PD1 resistant murine lung cancer model, suggesting that radiation therapy (RT) has potential to increase response rates. Herein, we sought to further improve these anti-tumor responses (local and abscopal) by targeting Tregs, which are upregulated by RT, through the addition of anti-GITR therapy. 129 Sv/Ev mice were inoculated with 344SQ resistant (344SQ_R) NSCLC cells into the right leg and, four days later, to the left leg. Tumor-bearing mice were treated with intravenous anti-PD1 and anti-GITR and RT to the primary tumor. Mice were followed for survival and tumor growth was recorded. Spleen, blood, draining lymph node and tumor infiltrating lymphocytes were isolated and analyzed by flow cytometry at various post-treatment time points. CD4 and CD8 depleting antibodies were administered to explore the cellular mechanism of treatment response. To evaluate the ability of triple therapy to generate a memory response, triple therapy mice were rechallenged with S344SQ_R cells 90 days after tumor clearance. Mice treated with triple therapy (anti-GITR, anti-PD1 and RT) had significantly improved survival compared to any dual therapy combination. Of note, anti-GITR therapy depleted Tregs at the tumor site but not in the spleen. Interestingly, half of the triple therapy mice became tumor free. Upon rechallenge with 344SQ_R cells, 100% (4/4) of these mice remained tumor free, showing a durable response. Mechanistically, we confirmed that this was related to generation of CD4 and CD8 T cell memory (CD44hi/CD62Llow). To confirm the observations, we did depletion studies and further demonstrated that the mechanism is largely dependent on CD4, and partially CD8 T cells. The addition of anti-GITR to anti-PD1 and local RT may circumvent a NSCLC anti-PD1 resistant phenotype through enhanced anti-tumor immunity via depletion of Tregs and generation of anti-tumor memory, thereby improving survival.