Abstract 2256: Low dose brain radiotherapy enhances the efficacy of an extracranial in situ vaccine regimen against melanoma brain metastases in a pre-clinical murine model
Authors: Sriramaneni Raghava N. , Clark Paul A. , Bates Amber M. , Anderson Bryce R. , Jin Wonjong , Jagodinsky Justin C. , Rakhmilevich Alexander L. , Morris Zachary S. 
Published: September 2020

Abstract
Brain metastases develop in &gt;60% of advanced melanoma patients, resulting in considerable morbidity and mortality. We previously showed that a regimen combining radiation (RT) and intra-tumoral (IT) injection of immunocytokine (IC: αGD2 antibody fused to IL2) resulted in enhanced response to immune checkpoint inhibition (ICI: αCTLA4 Ab) in mice bearing immunologically cold murine melanoma tumors. Here, we used the immunologically cold B78 murine melanoma model to investigate the efficacy of this in situ vaccine (ISV) regimen (RT + IT-IC + ICI) in mice bearing melanoma tumors in the brain and at extracranial sites. B78 (GD2+) cells were implanted into the right and left flanks and the right striatum of the brain in syngeneic C57BL/6 mice. Right flank tumors (150-200 mm3) were treated following a previously optimized ISV regimen [RT (12Gy, day 1), IT-IC (days 6-10) and αCTLA4 (days 3, 6, 9)]. ISV eliminated both flank tumors [22/22 complete response (CR)] compared to αCTLA-4 alone (0/8 CR). However, ISV had only modest effect in prolonging survival in mice with a B78 brain melanoma tumor and all mice succumbed to progressive brain tumor burden. Comparing tumors from the left flank and brain of mice treated with ISV to a right flank tumor, qPCR demonstrated no difference in the expression of Mhc-1 or of various checkpoint receptors/ligands (including Ctla4, Tigit, Pd-L1, Lag3). Immunohistochemistry and flow cytometry at day 15 after ISV demonstrated decreased regulatory T cells (Tregs; CD4+, FOXP3+, CD25+) and a higher ratio of CD8+ T cells:Tregs in the left flank tumor compared to the brain tumor. In contrast, these levels did not differ between left flank and brain tumors in untreated mice. Following ISV, CD8+ T cell:Treg ratio at both the left flank and brain tumor sites significantly increased compared to these respective tumor sites in untreated mice. Multiplex profiling of 32 cytokines demonstrated indistinguishable cytokine expression between untreated tumors in the left flank and brain but showed a distinct pattern of response at these locations following right flank ISV with reduced activation of pro-inflammatory cytokines in the brain melanoma tumor. Given the capacity of low dose RT to temporarily deplete Tregs and increase production of pro-inflammatory cytokines, we hypothesized that low dose whole brain (WB) RT (4 Gy x 1) would enhance ISV response of melanoma brain tumors. Addition of WBRT at day 15 after ISV significantly increased survival of mice with a melanoma tumor in the brain compared to ISV or WBRT alone with 2/11 exhibiting durable CR compared to 0/10 with either ISV or WBRT alone. Interestingly, WBRT administered concurrently on day 1 with ISV had no survival benefit compared to ISV alone - suggesting a critical immunomodulatory role for WBRT at day 15. With WBRT + ISV, we observed an increased ratio of CD8+:FOXP3+ T cells in melanoma brain tumors compared to ISV alone. These results suggest that low dose brain RT may enhance the response to an extracranial ISV at immunologically cold brain metastases.
Citation Format: Raghava N. Sriramaneni, Paul A. Clark, Amber M. Bates, Bryce R. Anderson, Wonjong Jin, Justin C. Jagodinsky, Alexander L. Rakhmilevich, Zachary S. Morris. Low dose brain radiotherapy enhances the efficacy of an extracranial in situ vaccine regimen against melanoma brain metastases in a pre-clinical murine model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2256.