Abstract A52: Radiation and dual PD-L1 and CTLA4 checkpoint blockade non-redundantly improves tumor resistance, response, and immunity
Authors: Rech Andrew J. , Twyman Christina , Maity Amit , Rengan Ramesh , Pauken Kristen E. , Stelekati Erietta , Xu Bihui , Dada Hannah , Odorizzi Pamela M. , Herati Ramin D. , Mansfield Kathleen D. , Patsch Dana , Amaravadi Ravi , Schuchter Lynn , Ishwaran Hemant , Mick Rosemarie , Pryma Daniel , Xu George , Feldman Michael , Gangadhar Tara C. , Hahn Stephen , Wherry E. John , Minn Andy J. , Vonderheide Robert H. 
Published: October 2015

Abstract
Background: Immune checkpoint inhibitors such as CTLA4 and PD-1 result in impressive clinical responses, but optimal results will require combination therapy. This raises fundamental questions about non-redundancy and mechanisms of resistance. Emerging data indicate that combining immune checkpoint inhibitors with radiation (RT) may hold promise. We therefore evaluated this combination for metastatic melanoma using parallel studies in mice and humans.
Methods: In a phase I clinical trial with 19 patients with multiple melanoma metastases, a single index lesion was irradiated with hypofractionated RT, delivered over two or three fractions, followed by four cycles of the anti-CTLA4 antibody ipilimumab. We reproduced this therapy in mice using the melanoma cell line B16-F10. For this, each flank of C57BL/6 mice was implanted with tumors to model multiple metastases. Mice received anti-CTLA4 (on days 5, 8, and 11), irradiation of one tumor using an image-guided micro-irradiator (20 Gy x 1 on day 8), or both treatments. Mechanistic studies were performed on material obtained from patients and mice at baseline and thereafter.
Results: Overall, treatment in the phase I study was well tolerated and toxicity was similar to that reported for anti-CTLA4. Major tumor regressions were observed in a subset of patients with metastatic melanoma treated with anti-CTLA4 + RT. In mice, although combined treatment enhanced the CD8 T cell to Treg ratios and improved responses in irradiated and unirradiated tumors, resistance was common. Genome-wide and unbiased analyses revealed that resistant tumors have increased PD-L1, interferon-stimulated genes, and exhausted T cells that depress the CD8/Treg ratio. Patients and mice with high PD-L1 tumors that were treated with RT + anti-CTLA4 poorly reinvigorated exhausted T cells, did not respond, and rapidly progressed. In mice, adding anti-PD-L1/PD-1 to RT + anti-CTLA4 reinvigorated exhausted T cells, leading to complete responses and immunity across multiple cancer types. The extent of T cell exhaustion/reinvigoration predicts response and can be assessed through peripheral blood.
Conclusions: Resistance to RT + anti-CTLA4 results from depression in the CD8/Treg ratio due to elevated tumor PD-L1 and persistent T cell exhaustion. Both clinical and pre-clinical data suggest that the combination of RT, anti-CTLA4, and anti-PDL1 is a rational and non-redundant approach to improving resistance, response, and immunity to melanoma and other cancer types.
Citation Format: Andrew J. Rech, Christina Twyman, Amit Maity, Ramesh Rengan, Kristen E. Pauken, Erietta Stelekati, Bihui Xu, Hannah Dada, Pamela M. Odorizzi, Ramin D. Herati, Kathleen D. Mansfield, Dana Patsch, Ravi Amaravadi, Lynn Schuchter, Hemant Ishwaran, Rosemarie Mick, Daniel Pryma, George Xu, Michael Feldman, Tara C. Gangadhar, Stephen Hahn, E. John Wherry, Andy J. Minn, Robert H. Vonderheide. Radiation and dual PD-L1 and CTLA4 checkpoint blockade non-redundantly improves tumor resistance, response, and immunity. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A52.