Abstract 896: Enhanced sensitivity detection of replication competent lentivirus by qPCR

Abstract

Abstract Introduction: Chimeric antigen receptor T (CAR T) cells are an important emerging therapy for hematologic malignancies and are currently being investigated for other cancer types as well. CAR-T cells require a retrovirus or lentivirus to introduce the chimeric antigen receptor gene into the activated T cells. Safety measures are put in place by design to eliminate any chance to create replication competent virus. Multiple plasmids carrying specific components of the virus are used to reduce the chances of creating a replication competent lentivirus. Despite these measures, safety concerns have been posed by the FDA regarding use of a lentivirus for this treatment due to the potential for replication competent lentiviruses (RCL) to arise. FDA requires monitoring of both CAR T products as well as patients undergoing therapy. It has proven to be difficult to achieve acceptable detection sensitivity of the VSV-G sequence in a background of gDNA due to the high level of homology to many areas in gDNA. Methods: We have developed a new rapid and sensitive qPCR assay targeting the vesicular stomatitis virus G glycoprotein (VSV-G), an envelope protein on lentiviral vectors. Incorporation of the envelope gene would be required to generate a replication competent virus. The intended use of the validated assay is to detect and monitor for RCL presence in human blood samples collected during clinical monitoring. A validated reference assay targeting RPPH1 will be used to quantify the amount of amplifiable genomic DNA in each extracted DNA sample. The size of the amplicons are 66 and 87 base pairs for VSV-G and RPPH1 respectively. The VSV-G assay test includes one positive and one negative control per assay run. The VSV-G positive control is composed of plasmid containing the VSV-G target sequence spiked into a background of VSV-G negative human gDNA. The negative control is composed of negative gDNA without VSV-G plasmid. Results: Improved sensitivity without non-specific amplification was achieved by optimized primer and probe selection. Additionally sensitivity and specificity were enhanced by employing a step-down PCR protocol whereby the annealing and extension temperature is lowered in stepwise fashion over the first five cycles of PCR. Genomic DNA inputs up to 200ng per reaction showed high efficiency of amplification from 10 to 1e6 copies target sequence per reaction. Conclusion: This assay will provide a rapid and sensitive method for safety monitoring of patients undergoing CAR-T therapy with therapeutic cells generated using lentiviral vectors. Citation Format: David Burke, Kristine Drafahl, Clark Fjeld, Chad Galderisi, Cindy Spittle. Enhanced sensitivity detection of replication competent lentivirus by qPCR [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 896.