Role of Radiation Therapy in Inducing Antigen Specific Antitumor Immune Responses When Combined With Anti-PD1 Checkpoint Blockade: Mechanism and Clinical Implications

Abstract

Immunotherapy with anti-CTLA-4 and anti-PD1 antibodies (Ab) has gained significant interest due to activity in multiple tumor types and high rates of systemic control in metastatic melanoma. Preclinical studies and case reports have described potential synergy between radiation therapy (RT) and immunotherapy (IT); however, the direct role that RT may play in augmenting anti-tumor immune responses remains unclear. Here we report a mechanistic role for RT in inducing antigen-specific immune responses when combined with anti-PD1 Ab in melanoma and breast carcinoma. Stereotactic image-guided RT was delivered to B16-OVA and 4T1-HA flank tumors via the small animal radiation research platform (SARRP). Mice received three IP injections of 200 ug anti-PD1 and/or anti-CTLA-4 Ab before, during, and after RT. Cell surface markers and development of antigen-specific immune responses was analyzed by flow cytometry on LSR II. Adoptive transfer studies were performed to validate protective immunity and in vitro studies elucidated a molecular mechanism of action. Stereotactic RT modified the immunophenotype by increasing the surface expression of tumor associated antigens, MHC, and Fas. RT of B16-OVA enhanced tumor associated antigen presentation in-vitro and in the draining lymph node (DLN) in-vivo. Additionally, hypofractionated RT of 18 Gy x 1 or 7 Gy x 3 increased the proliferation and activation of antigen specific CD8 T-cells in the DLN (IFN-gamma positive: mock 20.8 +/- 3.6% vs 18 Gy 45.6 +/- 1.9%, p < 0.01). We observed significant increases in the development of endogenous SIINFEKL Pentamer positive anti-tumor immune responses when RT was combined with anti-PD1 Ab (RT 0.6% vs RT + anti-PD1 1.3%, p < 0.05). Furthermore, combination therapy resulted in primary tumor control, increased survival of treated mice, and elicited the abscopal effect (B16-OVA: IT 372 +/- 138 cc, RT 121 +/- 43 cc, RT + IT 57 +/- 31 cc∗, p < 0.05; 4T1-HA: IT 663 +/- 23 cc, RT 351 +/- 150 cc, RT + IT 99 +/- 51 cc∗, p < 0.05). Mechanistically, we identified that RT upregulates specific chemokine receptors which may drive development of antigen specific immune responses. RT altered the immunophenotype of tumor cells and enhanced tumor associated antigen presentation. Combination RT + IT resulted in significantly increased antigen-specific immune responses, primary tumor control, and abscopal effects outside of the radiation field. These data provide critical evidence supporting the ability of stereotactic RT to induce anti-tumor immune responses and augment the efficacy of anti-PD1 checkpoint blockade in melanoma and breast carcinoma. These findings have important implications for the novel treatment paradigm of RT combined with IT and provide mechanistic rational for clinical trials of combined therapy in the definitive and metastatic setting.