Application of pharmacodynamic assays in a phase Ia trial of Apo2L/TRAIL in patients with advanced tumors

Abstract

3535 Background: Recombinant human (rh) Apo2L/TRAIL is a dual pro-poptotic receptor agonist (PARA) that induces apoptosis by binding to pro-apoptotic receptors DR4 and DR5, which recruit a death inducing signaling complex upon ligand binding. This results in activation of the effector caspase 3/7, that subsequently cleaves intracellular substrates to execute cellular apoptosis. A Phase1a trial is underway to evaluate the safety and tolerability of rhApo2L/TRAIL in patients with advanced tumors. The aim of this study was to develop and validate high-throughput pharmacodynamic assays to monitor rhApo2L/TRAIL activity in easily accessible patient samples such as serum. Methods: To monitor rhApo2L/TRAIL activity in patients, we optimized assays to measure the release of the apoptotic markers caspase 3/7, cytokeratin 18 (CK18), and genomic DNA (gDNA) in serum. Serum caspase 3/7 levels were monitored using the Caspase Glo kit, which generates a luminescent signal upon cleavage of a caspase 3/7 substrate; cleavage of the caspase substrate CK18 was measured using an optimized form of the M30 ELISA assay; gDNA was measured using a β-actin-specific TaqMan real-time PCR assay. Mice bearing Colo205 xenografts were treated with rhApo2L/TRAIL and sera were collected and assayed for apoptotic markers. Upon validating these assays, we monitored the levels of apoptotic markers in cancer patients who received rhApo2L/TRAIL. Results: We detected transient increases in apoptotic markers in mouse sera 8–24 hr after treatment with rhApo2L/TRAIL. This increase was dose-dependent and correlated with active caspase 3 detected by IHC in Colo205 tumors. In the phase Ia study, increases in serum caspase 3/7 and gDNA levels were observed in >50% of colorectal, lung and sarcoma patients evaluated. Preliminary analyses show the percentage of increase correlates using both analytes and is dose-dependent. Conclusions: These findings support the use of serum-based pharmacodynamic assays as a means to monitor rhApo2L/TRAIL activity in patients with advanced tumors. A complete analysis of all patient serum samples from the ongoing phase Ia trial will be reported. No significant financial relationships to disclose.