In vitro CAR-T cell killing: validation of the potency assay.

Area of Science:

• Cellular immunology
• Biotechnology
• Pharmaceutical development

Background:

• Potency assays are crucial for advanced therapy medicinal products.
• International guidelines mandate assay development and validation.
• Chimeric antigen receptor (CAR) T-cell therapies require robust characterization.

Purpose of the Study:

• To validate a killing assay for autologous anti-CD19 CAR T-cell potency evaluation.
• To ensure reliable and consistent data for product characterization.
• To meet regulatory requirements for advanced therapy medicinal products.

Main Methods:

• Utilized anti-CD19 CAR T-cells as effector cells and CD19+ (REH) or CD19- (MOLM-13) cell lines as target cells.
• Co-cultured cells for 24 hours, followed by flow cytometry analysis using 7-AAD, anti-CD3, and anti-CD19 antibodies.
• Assessed assay specificity, linearity, accuracy, robustness, and precision (intra-assay, inter-assay, inter-day, inter-analyst).

Main Results:

• The killing assay demonstrated specificity for CD19+ targets.
• Established acceptance criteria for linearity (r² ≥ 0.97) and accuracy (average relative error ≤ 10%).
• Confirmed assay robustness, precision, and inter-analyst reliability (intra-class correlation coefficient > 0.4).

Conclusions:

• The developed killing assay is validated for evaluating anti-CD19 CAR T-cell potency.
• The assay is suitable for use throughout product development and quality control.
• This validated assay supports the reliable characterization of CAR T-cell products.