Abstract 635: Antigen-specific immune responses in melanoma using stereotactic radiotherapy combined with anti-PD1 checkpoint blockade
Authors: Sharabi Andrew , Nirschl Christopher , Ceccato Tina , Francica Brian , Alme Angela , Nirschl Thomas , Velarde Esteban , DeWeese Theodore , Drake Charles 
Published: May 2015

Abstract
Purpose: Multiple pre-clinical studies and case reports have described potential synergy between radiotherapy and immunotherapy, including checkpoint blockade with anti-PD1 and anti-CTLA-4 antibodies. However, further understanding of how radiotherapy contributes to immune mediated cell death, especially with regards to timing and dose per fraction, is needed to guide protocols for clinical trials. Here we investigated the development of radiation induced antigen-specific immune responses (RASIR) using stereotactic image guided small animal radiotherapy combined with anti-PD1 checkpoint blockade in a B16 melanoma model.
Experimental Design: MC38 colorectal carcinoma, B16, and B16-OVA melanoma were cultured in complete RPMI media. A gamma-cell irradiator was used for in-vitro irradiation of cell suspensions. For in-vivo experiments 6-8 week old C57BL/6 mice were injected with 1x10^5 or 5x10^5 B16-OVA cells into the flank. Tumors were irradiated using a stereotactic CT guided small animal irradiator to treat a 1cmx1cm field prescribed to isocenter. For adoptive transfer experiments mice received 2x10^6 purified OT1 T-cells via retro-orbital injection. Anti-PD1 antibody was given intraperitoneally in three scheduled doses of 200ug. Cells were processed, stained, and analyzed by Flow cytometry on BD FACSCaliber or LSR II for indicated cell surface markers or intracellular cytokines.
Results: We found that irradiation with 10Gy or 20Gy increased cell surface expression of MHC Class I, CCR7, CXCR3, and FAS in MC38 colorectal carcinoma and B16 melanoma at 24 and 48 Hours. We observed in-vivo that stereotactic radiotherapy of B16-OVA melanoma tumors increased CFSE labeled proliferation and Interferon-gamma activation of adoptively transferred OT1 T-cells in the draining lymph node and spleen. 18 Gy of radiotherapy resulted in increased activation and proliferation of antigen-specific T-cells when compared to 12Gy suggesting a possible dose response. Furthermore, when radiotherapy was combined with scheduled anti-PD1 antibody there was near eradication of B16-OVA melanoma tumors accompanied by increased development of endogenous antigen-specific immune responses.
Conclusions: Radiotherapy increased expression of immunogenic cell surface markers in MC38 colorectal carcinoma and B16 melanoma. Stereotactic radiotherapy induced endogenous CD8 mediated antigen-specific immune responses when combined with scheduled anti-PD1 immunotherapy and resulted in near eradication of established B16-OVA melanoma. This study provides important pre-clinical evidence to support and guide clinical trials combining radiotherapy with anti-PD1 checkpoint blockade in melanoma. Future goals include analyzing development of RASIR using fractionated stereotactic radiotherapy with combined checkpoint blockade in multiple different tumor types.
Citation Format: Andrew Sharabi, Christopher Nirschl, Tina Ceccato, Brian Francica, Angela Alme, Thomas Nirschl, Esteban Velarde, Theodore DeWeese, Charles Drake. Antigen-specific immune responses in melanoma using stereotactic radiotherapy combined with anti-PD1 checkpoint blockade. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 635. doi:10.1158/1538-7445.AM2014-635