A Quantitative Approach to Potency Testing for Chimeric Antigen Receptor-Encoding Lentiviral Vectors and Autologous...

Area of Science:

• Biotechnology
• Immunotherapy
• Cellular Therapy

Background:

• Potency testing is crucial for the commercial approval of clinical-grade lentiviral vectors (LVVs).
• Developing a suitable potency test during clinical development is essential for CAR-T therapies.
• Current methods may have limitations in precision and direct mechanism of action evaluation.

Purpose of the Study:

• To develop an affordable, quantitative potency test for CAR19-LVV based on transgene functional activity.
• To validate the assay for precision, specificity, robustness, linearity, and accuracy.
• To assess the assay's adaptability to other CAR-encoding LVVs and autologous CAR-T cell products.

Main Methods:

• Explored functional activity indicators in a co-culture system of CAR-transduced Jurkat cells and CD19-expressing target cells.
• Selected and developed a quantitative assay measuring CD69 expression (T-cell activation marker) via flow cytometry.
• Validated the assay and tested its adaptability to CARBCMA-LVV and autologous CAR-T cell products, including cytotoxicity assessment.

Main Results:

• CD69 expression on CAR-transduced cells is a specific indicator of CAR functionality.
• Quantification of CD69 via mean fluorescence intensity (MFI) with standard curve calibration yielded a precise and accurate assay.
• The CD69 assay demonstrated adaptability to CARBCMA-LVV and autologous CAR-T cells, reflecting cytotoxicity and showing reduced variability compared to IFNγ assays.

Conclusions:

• A validated, quantitative, in vitro cell-based potency test using flow cytometry for CAR19-LVV was successfully developed.
• The assay measures transgene functionality by detecting T-cell activation upon CAR-antigen binding and is adaptable to other LVVs and CAR-T products.
• This CD69-based assay offers reduced variability and simultaneous evaluation of mechanism of action, including cytotoxicity, for autologous CAR-T products.