Abstract A41: Combination immunotherapy leads to decreased tumor growth
Authors: Gartrell Robyn , Blake Zoe , Simoes Ines , Fu Yichun , Sato Takuro , Qian Yingzhi , Zhang James , Lu Yan , Saenger Yvonne 
Published: March 2017

Abstract
Introduction/Background: Talimogene Laherparepvec (T-Vec) is the first oncolytic virus to be FDA approved for the treatment of cancer. T-Vec, a modified Herpes Simplex Type I virus (HSV I), has two mechanisms of action: direct cell lysis and immune activation. Combination immunotherapy using T-Vec and check-point blockade has shown promise in clinical trials.1 In preliminary work, our lab has shown that T-Vec causes up-regulation of programmed cell death protein 1 (PD-1) on infiltrating T cells in mice, suggesting potential synergy of T-Vec and anti-PD1 (α-PD1). The purpose of our study is to evaluate the effectiveness of combination immunotherapy to treat melanoma tumors in a transgenic BRAF melanoma model and study the immune microenvironment of these tumors post treatment.
Materials and Methods: In a temporally and spatially regulated murine model of BRAFCA PTEN-/- spontaneous melanoma2, tumors are induced on the shaved right flank of mice. When tumors reach ≥5mm in diameter, mice are randomized into 6 treatment groups comparing combinations of BRAF inhibition (BRAFi) or control chow, intraperitoneal injections of α-PD1 or control (2A3), and intratumoral injections of T-Vec or control (PBS) by intratumoral injection.
Treatment Groups:
1: Control + 2A3 + PBS
2: BRAFi + 2A3 + PBS
3: BRAFi + α-PD1 + PBS
4: BRAFi + 2A3 + T-Vec
5: BRAFi + α-PD1 + T-Vec
6: Control + α-PD1 + T-Vec
Tumor growth is measured biweekly until end of study. Flow cytometry is performed on tumor, lymph node, and spleen to assess immune microenvironment. Multiplex immunohistochemistry will also be performed and analyzed with Mantra, using 6 immune biomarkers.
Results: Mean tumor volume and survival was plotted to compare groups. Mice treated with combination T-Vec + BRAFi with or without α-PD1 have decreased tumor growth and longer survival compared to mice treated with control or single drug arms. Flow cytometry shows an increase in percent CD3+ cells in tumors of mice treated with combination α-PD1 + T-Vec compared to the control and single drug arms. Further analysis revealed very few CD45+ cells in tumors of the control group whilst mice receiving combination immunotherapy without BRAFi have higher CD45+ cells in tumor. Percent CD8+/CD3+ cells in tumors treated with immunotherapy appears to be increased compared to the control and BRAFi only group. Additionally, percent of CD4+/FOXP3+ cells in tumors appears to be decreased in groups receiving T-Vec while no change in CD4+/FOXP3+ populations was observed in draining lymph node or spleen.
Conclusions: Initial findings show that combination therapy of BRAFi + α-PD1 + T-Vec is more effective than any single treatment. Combination immunotherapy increases infiltration of T cells into tumor. Furthermore, oncolytic virus appears to decrease CD4+/FOXP3+ (regulatory T cells) infiltrating tumor. This study is ongoing and further analysis will continue as we further evaluate the immune microenvironment using flow cytometry and immunohistochemistry.
Acknowledgements: The study was funded by the Melanoma Research Alliance and Amgen (Amgen-CUMC-MRA Established Investigator Academic-Industry Partnership Award).
References:
1. Andtbacka, R.H.I., et al. OPTiM: A randomized phase III trial of talimogene laherparepvec (T-VEC) versus subcutaneous (SC) granulocyte-macrophage colony-stimulating factor (GM-CSF) for the treatment (tx) of unresected stage IIIB/C and IV melanoma. ASCO Meeting Abstracts 31, LBA9008 (2013).
2. Dankort, D., et al. Braf(V600E) cooperates with Pten loss to induce metastatic melanoma. Nature genetics 41, 544-552 (2009).
Citation Format: Robyn Gartrell, Zoe Blake, Ines Simoes, Yichun Fu, Takuro Sato, Yingzhi Qian, James Zhang, Yan Lu, Yvonne Saenger. Combination immunotherapy leads to decreased tumor growth. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A41.