Development Of A B16f10 Cell Line Expressing Mnectin1 To Study The Activity Of Oncovexmgm-Csf In Murine Syngeneic Melanoma Models

Background: Talimogene laherparepvec is an oncolytic immunotherapy based on a modified herpes simplex virus type 1 (HSV-1), designed to kill neoplastic cells through two mechanisms: a) direct lysis and b) stimulation of a tumor antigen-specific adaptive immune response by way of GM-CSF expression. In order to support mechanism of action studies in a mouse melanoma model relevant to talimogene laherparepvec9s approved U.S. indication, we expressed the HSV-1 entry receptor Nectin1 on B16F10 melanoma cells. B16F10 cells have previously been shown to be resistant to HSV-1 due to lack of entry receptor expression (Miller et al., Mol Ther, 2001). The goal of our work was to determine if mNectin1 expression in B16F10 melanoma cells renders them sensitive to talimogene laherparepvec treatment. For preclinical studies we used OncoVEX mGM-CSF , an HSV-1 modified similarly to talimogene laherparepvec except that murine GM-CSF is expressed instead of human GM-CSF. Herein we describe studies to assess the efficacy of OncoVEX mGM-CSF in a novel B16F10-mNectin1 syngeneic melanoma model. Methods: Parental B16F10 cells were transduced with lentivirus expressing either mNectin1, or eGFP as a control. In vitro sensitivity was determined by plating both cell types in 96-well plates, infecting with serially diluted OncoVEX mGM-CSF and then measuring ATP 72 hours later. For efficacy studies, B16F10 or B16F10-mNectin1 cells were injected subcutaneously on the right flank of female C57BL/6 mice. On day 10, mice were randomized into treatment groups based on tumor volume (n = 10/group). Talimogene laherparepvec was delivered intratumorally (5×10 6 PFU/dose,) every 3 days during the first week. Tumor growth and body weight were measured twice per week throughout the experiment. Survival analysis was performed using a Kaplan-Meier estimator. Results: B16F10 cells were insensitive to OncoVEX mGM-CSF up to 100 MOI (multiplicity of infection). In contrast, two other syngeneic cell lines, A20 and CT-26, showed MOI IC 50 of 1 and 20. B16F10 cells expressing mNectin1 showed high sensitivity to OncoVEX mGM-CSF with an MOI IC 50 of 0.01. In contrast, B16F10 cells expressing eGFP were insensitive up to an MOI of 100. In vivo, B16F10 mNectin1 cells showed a similar growth pattern as the parental B16F10 when injected subcutaneously. However, OncoVEX mGM-CSF treatment of B16F10-mNectin1 tumors caused a highly significant (p mGM-CSF treatment were shown to maintain mNectin1 expression suggesting other mechanisms of OncoVEX mGM-CSF resistance were involved. Conclusion: Expression of mNectin 1 in the B16F10 melanoma cell line confers sensitivity to OncoVEX mGM-CSF in vitro and extends median overall survival of mice bearing B16F10-muNectin1 tumors. Citation Format: Keegan Cooke, Juan Estrada, Jinghui Zhan, Petia Mitchell, Yannick Bulliard, Pedro J. Beltran. Development of a B16F10 cell line expressing mNectin1 to study the activity of OncoVEXmGM-CSF in murine syngeneic melanoma models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2351.