Abstract 163: Evaluation of ETS2101 and its combination with anti-mCTLA4 monoclonal antibody on CD4 and CD8 TILs in a subcutaneous colorectal cancer model of CT-26
Authors: Sherry Lorcan , Anderson Mark , Waller John , Sardar Adam , Flores Victoria , Hynes Daniel 
Published: July 2017

Abstract
ETS2101, (HU-211, Dexanabinol) is a synthetic cannabinoid with limited affinity to cannabinoid receptors 1 and 2 (CB1 and CB2), reducing its psychotropic potential in comparison to other molecules in the cannabinoid class. ETS2101 has been found to have effects on immune cell function in vitro. The presence of Tumor Infiltrating Lymphocytes (TILs) has been previously shown to correlate with better patient outcomes during various antitumor therapies in a multitude of cancers. This study utilized immunohistochemistry (IHC) combined with digital image analysis to quantify CD4+ and CD8+ TILs within CT-26 tumors treated with ETS2101 in combination with a checkpoint inhibitor, anti-mCTLA4.
A CT-26 colon carcinoma tumor line was implanted s.c. into immune-competent mice. Ten days post implantation groups were treated with ETS2101 administered at 200 mg/kg QD in combination with anti-mCTLA-4 administered as 10 mg/kg i.p. BiW. Thirty seven days post treatment, tumors were collected and IHC was performed for CD4+ and CD8+ TILs on FFPE tumor sections (n=12-15 per group). Whole slide images were generated per IHC section and customized algorithms developed within the Indica Labs HALO platform to classify viable tumor present across each section and to quantify CD4+ or CD8+ stain area within the viable tumor region of interest (ROI).
Anti-mCTLA4 monotherapy resulted in regression of 44% tumors (7% CR) and anti-mCTLA4 + ETS2101 combination therapy resulted in regression of 74% tumors (36% CR). There was a significant increase in CD8+ % staining of viable tumor observed following anti-mCTLA4 monotherapy treatment versus control (2.16% v. 1.16%, P&lt;0.0027) and anti-mCTLA4 + ETS2101 combination therapy (2.18% v. 1.10%, P&lt;0.0019) compared to control. A similar trend was observed for CD4+ % staining of viable tumor with a significant increase observed following anti-mCTLA4 monotherapy treatment versus control (1.82% v. 0.49%, P&lt;0.0083) and anti-mCTLA4 + ETS2101 combination therapy (1.93% v. 0.41%, P&lt;0.0028) compared to control. Although, there was no significant difference between anti-mCTLA4 monotherapy treatment and anti-mCTLA4 + ETS2101 combination therapy for either marker alone, following treatment with the combination a statistically significant relationship between best overall response and the ratio of CD8+:CD8+ TILs was observed (P=0.0186).
In summary, there was an observed difference in the number of mice exhibiting a partial/complete regression in tumor volume for the combination arm 74% (36% CR) versus the antibody monotherapy arm 44% (7% CR). A statistically significant relationship (p=0.0186) was observed between the best overall tumor response and the ratio of CD8+:CD4+ TILs. IHC combined with image analysis can be utilized to evaluate therapeutic response on TILs within the context of their tumor microenvironment.
Citation Format: Lorcan Sherry, Mark Anderson, John Waller, Adam Sardar, Victoria Flores, Daniel Hynes. Evaluation of ETS2101 and its combination with anti-mCTLA4 monoclonal antibody on CD4 and CD8 TILs in a subcutaneous colorectal cancer model of CT-26 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 163. doi:10.1158/1538-7445.AM2017-163