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Published on: June 19, 2013
Development of a replication-competent lentivirus assay for dendritic cell-targeting lentiviral vectors
Daniel C Farley1, Laura McCloskey1, Barbara A Thorne2
1Oxford BioMedica (UK) Limited, Windrush Court, Transport Way , Oxford, UK.
Abstract:
It is a current regulatory requirement to demonstrate absence of detectable replication-competent lentivirus (RCL) in lentiviral vector products prior to use in clinical trials. Immune Design previously described an HIV-1-based integration-deficient lentiviral vector for use in cancer immunotherapy (VP02). VP02 is enveloped with E1001, a modified Sindbis virus glycoprotein which targets dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) expressed on dendritic cells in vivo. Vector enveloped with E1001 does not transduce T-cell lines used in standard HIV-1-based RCL assays, making current RCL testing formats unsuitable for testing VP02. We therefore developed a novel assay to test for RCL in clinical lots of VP02. This assay, which utilizes a murine leukemia positive control virus and a 293F cell line expressing the E1001 receptor DC-SIGN, meets a series of evaluation criteria defined in collaboration with US regulatory authorities and demonstrates the ability of the assay format to amplify and detect a hypothetical RCL derived from VP02 vector components. This assay was qualified and used to test six independent GMP production lots of VP02, in which no RCL was detected. We propose that the evaluation criteria used to rationally design this novel method should be considered when developing an RCL assay for any lentiviral vector.
Insights
A new assay was developed to detect replication-competent lentivirus (RCL) in VP02 lentiviral vectors, ensuring product safety for cancer immunotherapy clinical trials. This novel method successfully confirmed the absence of RCL in multiple production lots.
Area of Science:
- Biotechnology
- Virology
- Immunotherapy
Background:
- Lentiviral vectors are crucial for cancer immunotherapy but require stringent safety testing.
- Standard replication-competent lentivirus (RCL) assays are unsuitable for novel vectors like VP02 due to specific envelope proteins (E1001) and target cell tropism (DC-SIGN).
Purpose of the Study:
- To develop and qualify a novel assay for detecting RCL in clinical lots of the lentiviral vector VP02.
- To ensure the safety of VP02 for use in cancer immunotherapy clinical trials by demonstrating the absence of detectable RCL.
Main Methods:
- Developed a custom RCL assay using a murine leukemia virus positive control and a 293F cell line engineered to express the E1001 receptor, DC-SIGN.
- Evaluated the assay against criteria established in collaboration with US regulatory authorities.
- Utilized the qualified assay to test six Good Manufacturing Practice (GMP) production lots of VP02.
Main Results:
- The novel assay demonstrated the capacity to amplify and detect a hypothetical RCL derived from VP02 vector components.
- The assay met predefined evaluation criteria set in collaboration with regulatory agencies.
- Testing of six GMP lots of VP02 confirmed the absence of detectable RCL in all batches.
Conclusions:
- A robust and qualified assay has been established for RCL detection in VP02 lentiviral vector products.
- The developed assay ensures the safety of VP02 for clinical applications in cancer immunotherapy.
- The evaluation criteria used for this assay development can serve as a model for creating RCL assays for other novel lentiviral vectors.
