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Efficient Production of Chimeric Antigen Receptor (CAR) T Cells with Transgenes Exceeding 10 kb Using Lentiviral
Philipp C Rommel1, Nils W Engel2, Bruce L Levine3
1Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania; Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania; prommel@pennmedicine.upenn.edu.
Abstract:
Engineering multiple receptors into chimeric antigen receptor (CAR) T cells has emerged as a powerful strategy to prevent antigen-negative relapse and reduce on-target/off-tumor toxicities. However, manufacturing multi-receptor CAR T cells remains challenging, as increasing lentiviral transgene sizes significantly reduces viral titers and T cell transduction rates. Current production workflows often rely on cell sorting to enrich transduced T cells from low-yield productions. Yet, cell sorting techniques do not increase the absolute number of CAR T cells and add further complexity to the already elaborate manufacturing process. Consequently, these limitations impede the clinical translation of multi-receptor CAR T cells and restrict the development of next-generation immunotherapies. Here, we present a detailed, step-by-step production workflow optimized for generating CAR T cells with large lentiviral transgenes. Using this workflow, we demonstrate size-dependent increases in transduction efficiency across a range of transgene sizes, with the most pronounced enhancement of up to 14.8-fold observed for a 10.1 kb lentiviral vector. Importantly, the workflow supports robust T cell expansion and eliminates the need for cell sorting. By overcoming current size limitations in lentiviral gene transfer, this workflow enables the efficient generation of multi-receptor CAR T cells, thereby facilitating the development of advanced immunotherapies.
Insights
Developing multi-receptor chimeric antigen receptor (CAR) T cells is promising but faces manufacturing challenges. This study presents an optimized workflow to efficiently produce large-transgene CAR T cells, overcoming lentiviral size limitations.
Area of Science:
- Immunotherapy
- Gene Therapy
- Cellular Engineering
Background:
- Chimeric antigen receptor (CAR) T cells engineered with multiple receptors offer enhanced efficacy and safety.
- Manufacturing challenges, including reduced viral titers and transduction rates with larger lentiviral transgenes, hinder clinical translation.
- Current methods rely on cell sorting, which does not increase CAR T cell numbers and complicates production.
Purpose of the Study:
- To develop and validate an optimized, step-by-step production workflow for generating CAR T cells with large lentiviral transgenes.
- To overcome the limitations of lentiviral vector size in CAR T cell manufacturing.
- To facilitate the clinical translation of multi-receptor CAR T cell immunotherapies.
Main Methods:
- Detailed optimization of a lentiviral gene transfer production workflow.
- Evaluation of transduction efficiency across various lentiviral transgene sizes.
- Assessment of T cell expansion and the necessity of cell sorting.
Main Results:
- Demonstrated size-dependent increases in transduction efficiency for lentiviral vectors.
- Achieved up to a 14.8-fold enhancement in transduction for a 10.1 kb lentiviral vector.
- The workflow supported robust T cell expansion and eliminated the need for cell sorting.
Conclusions:
- The optimized workflow overcomes lentiviral size limitations for CAR T cell production.
- Enables efficient generation of multi-receptor CAR T cells.
- Facilitates the development of next-generation immunotherapies by improving manufacturing processes.
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