Abstract 3826: GITRL-Fc biomarker and mechanism study: GITRL-Fc reduces Treg frequency in tumors and requires effector function for inhibition of tumor growth
Authors: Argast Gretchen M. , Cancilla Belinda , Cattaruzza Fiore , Yeung Pete , Lahmy Reyhaneh , Scolan Erwan Le , Harris Rose , Brunner Alayne , Wang Min , Axelrod Fumiko , Monteon Jorge , Elechko Jennifer , Lam Andrew , Xie MingHong , Lowe Earth Light , O'Young Gilbert , Gurney Austin , Kapoun Ann M. 
Published: August 2018

Activation of the co-stimulatory receptor GITR (Glucocorticoid-Induced Tumor Necrosis Factor Receptor) by GITR-Ligand (GITRL) promotes proliferation and activation of effector T cells (T eff) and inhibits suppressive activity of regulatory T cells (Treg). Here, we have further characterized the mechanism of action of a single-gene GITRL trimer fused to an immunoglobulin Fc domain (GITRL-Fc, 336B3) by examining pharmacodynamic (PD) biomarkers in time course studies. Mice bearing CT26.WT colon tumors were treated with weekly GITRL-Fc and sacrificed 24 hours, 7 and 14 days after the first dose. Immuno-phenotyping of tumor-associated immune cells revealed a reduction in Treg frequency in tumor by 24 hours post-dose that was maintained at 7 and 14 days. Furthermore, GITRL-Fc treatment increased activation markers on tumor-associated CD4+ and CD8+ T cells, suggesting an increased cytotoxic environment within the tumor. This was supported by significant and sustained increase in CD8+ T cell:Treg ratio in the tumor after GITRL-Fc treatment. To determine whether intratumoral (IT) injection of GITRL-Fc is an effective route of administration, we compared efficacy, pharmacodynamic (PD) markers and pharmacokinetics in IT- and intraperitoneal (IP)-injected mice bearing bilateral CT26.WT tumors. Both routes of administration showed similar tumor growth inhibition (TGI) and PD markers in both the treated and the abscopal tumors, but IT injection resulted in a significantly lower serum GITRL-Fc concentration, suggesting that IT administration may be an alternative route of administration to IP with similar efficacy. The GITRL-Fc molecule 336B3 is effector function competent and able to induce cell-mediated cytotoxicity upon binding. To determine whether this effector function is required for GITRL-Fc-induced TGI, we treated CT26.WT tumor-bearing mice with 336B3 and 336B22, a GITRL-Fc molecule deficient in effector function. The effector function-competent 336B3 induced significant TGI and a more robust activation of Teff cells and reduction in Treg frequency, when compared to 336B22, suggesting that effector function is important for efficacy. To identify biomarkers for GITRL-Fc, we performed microarray analyses on multiple syngeneic mouse models treated with GITRL-Fc and developed GITRL gene signatures from blood and from tumors. We also developed multiplexed immunohistochemistry panels designed to quantify frequency of GITR and GITRL expression (GITR+CD8, GITR+FOXP3) in tumors. In conclusion, we have examined the effects of GITRL-Fc on preclinical mouse models. Biomarker analysis showed that loss of Tregs, activation of T cells and Fc-mediated effector function are key elements in the mechanism of action of the molecule. We have identified potential biomarkers to be used for PD and potential predictive analysis in clinical trial patient samples.

Citation Format: Gretchen M. Argast, Belinda Cancilla, Fiore Cattaruzza, Pete Yeung, Reyhaneh Lahmy, Erwan Le Scolan, Rose Harris, Alayne Brunner, Min Wang, Fumiko Axelrod, Jorge Monteon, Jennifer Elechko, Andrew Lam, MingHong Xie, Earth Light Lowe, Gilbert O'Young, Austin Gurney, Ann M. Kapoun. GITRL-Fc biomarker and mechanism study: GITRL-Fc reduces Treg frequency in tumors and requires effector function for inhibition of tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3826.