Development of a novel ddPCR assay for nonclinical and clinical pharmacokinetic characterization of ICVB-1042.

Abstract

e14693 Background: ICVB-1042 is a novel chimeric oncolytic Adenovirus (Ad) with potential for intravenous administration. A key component of the clinical assessment of ICVB-1042 will be pharmacokinetic (PK) viral genome analysis by Polymerase Chain Reaction (PCR) to understand plasma viral kinetics and tumor replication. Droplet Digital PCR (ddPCR) is more sensitive and more accurately quantifies a lower template concentration compared to traditional quantitative PCR. Our ddPCR assay targets ICVB-1042 and will not amplify other Ad5 or Ad34 viruses. The aim is to show that this assay specifically measures ICVB-1042 and may be used as a clinical biomarker. Methods: PCR primers and probe were designed to target the Ad5/Ad34 chimeric fiber of ICVB-1042. Template ICVB-1042 was spiked into representative human and murine sample matrices. Preclinical studies tested assay performance in vitro using patient derived disassociated tumor cells (DTCs) and in vivo using tumor bearing and non-tumor bearing mouse models. Viral replication and PK were assessed in vitro by analyzing supernatant of infected cells at multiple timepoints and in vivo with serial bleeds from different animals. Viral distribution was assessed by isolating viral DNA from homogenized tissue samples taken at multiple timepoints. Results: Analysis of ICVB-1042 by ddPCR showed linear quantification over a wide range of viral concentrations (5E5-5E12 viral genomes/mL) with sensitivity to low concentrations of virus (lower limit of quantification = 1.11E2 total viral genomes). Viral genomes were detected and quantified in human plasma samples spiked with ICVB-1042. Non-spiked samples were negative for signal. ICVB-1042 viral genomes were also detectible in tumor sections from ICVB-1042-treated NSG mice. Increasing viral genomes could be detected in vitro at days 6 and 9 post ICVB-1042 infection in the supernatants of several DTCs (2 donors each). The measured increase in viral genomes was accompanied by viral mediated cell death. ICVB-1042 could be detected in vivo in the plasma of non-tumor bearing CD-1 mice 1 and 24 hours post ICVB-1042 dosing but was cleared and not detected out to 90 days post dose. ICVB-1042 was detected post dose in the plasma of tumor bearing NSG mice and was again cleared. ICVB-1042 was detectable in plasma samples and accompanied by anti-tumor efficacy at later timepoints. Studies directly comparing tumor bearing to non-tumor bearing NSG mice have shown that the virus present in the plasma is due to tumor specific replication. Tissue viral genome distribution differences could also be detected in C57BL/6 mice expressing huCD46, the viral tropism of ICVB-1042. Conclusions: ICVB-1042 viral genomes can be detected using ddPCR. This assay can be used to assess viral replication in vitro and PK and distribution in vivo. Use of ddPCR to detect ICVB-1042 in patient samples will be key to the characterization of clinical efficacy.