Abstract 2116: Phosphorylated extracellular signal-regulated kinase (pERK) activation in T effector cells as a target engagement biomarker for the DGKζ inhibitor BAY2965501 in clinical trials

Abstract

Abstract Introduction: BAY2965501 is a small molecule that is a potent selective inhibitor of diacylglycerol kinases zeta (DGKζ). DGK are a family of enzymes that catalyzes the phosphorylation of the membrane lipid sn-1,2 diacylglycerol (DAG) to form phosphatidic acid. In T-cells, DGKζ is considered to play a key role in T-cell receptor (TCR) signaling. One of the signaling pathways controls T-cell proliferation and activation in response to antigen receptor engagement is controlled by extracellular-regulated kinase ERK1 and ERK2 (ERK1/2). The RASGRP/ERK pathway plays a crucial part the polarization of microtubule-organizing center in the cytotoxic T-cells, which is required to deliver granzyme to the targeted cells. Restoring T-cell cellular DAG level and activation of ERK1/2 in anergic T-cells via DGK inhibition has been considered a viable mechanism to assist T-cell mediate tumor cell kill. BAY2965501 showed that pERK could be activated in preclinical models. Phosphorylation of ERK (pERK) inhibition in tumor cells and its difficulty in clinical implementation has been largely acknowledged in oncology community. Circulating T-cell pERK changes as a target engagement biomarker may provide a plausible solution in clinical trial. Here, we report a development of a fit-for-purpose pERK+CD8+ assay. Method: Feasibilities to detect BAY2965501 induced pERK activation was tested to minimize the expected clinical hurdles by using various conditions such as various blood collection tubes (BCT), CD3/CD28 concentrations, and incubation times were evaluated. Whole blood was stimulated with BAY2965501 followed by activation with anti-CD3/anti-CD28 cocktail with subsequent lysis and fixation procedure including freezing. Samples were then stained with antibodies against T-cell cell-surface markers and anti-pERK antibody followed by analyzing on a flow cytometer. Results: A pERK+CD8+ cell frequency increase was successfully observed after stimulation with BAY2965501 at different concentrations (e.g., 12.89% spiked vs. 30.42% non-spiked). A peak of pERK+CD8+ cell frequency was observed with incubation of anti-CD3/CD28 at 15 min compared to 5 min or 30 min. Titration of anti-CD3/anti-CD28 cocktail resulted in saturation observed at a concentration of 2 µg/mL (range 1-4 µg/ml). Three collection tubes were tested; 1) Sodium heparin (SoHep); 2) Citrate and 3) TruCulture. SoHep showed the highest pERK+ CD8+ cell frequency (8.29%) compared to Citrate BCT or TruCulture. Activated and stimulated samples are stable at -80°C for at least 6 days (D0 30.42% vs D6-80°C 29.52%) although samples are not stable at ambient or cooled conditions (D0 12.38% vs D1RT 3.02% vs D14°C 7.66%). Conclusion: These findings support implementing the pERK+CD8+ assay in T cells as a target engagement biomarker during early phase clinical trials. Citation Format: Nicole Schubert, Dennis Kirchhoff, Joumana Zeidan, Joanie Duchesne, Philip Boulais, Yuko Ishii. Phosphorylated extracellular signal-regulated kinase (pERK) activation in T effector cells as a target engagement biomarker for the DGKζ inhibitor BAY2965501 in clinical trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2116.