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344 Comparative assessment of product quality attributes associated with production of chimeric antigen receptor T cells expressing scFv CD-19 and scFv IL-13Ra2 by two manufacturing platforms
  1. Sujin Hwang,
  2. Heba Degheidy,
  3. Mondona McCann,
  4. Steven Bauer,
  5. Raj Puri and
  6. Bharat Joshi
  1. Food and Drug Administration, Silver Spring, MD, USA


Background The recent advances in adoptive immunotherapy of cancer have led to the FDA approval of chimeric antigen receptor modified T (CAR-T) cells targeting CD19 antigen on advanced hematological malignancies, which has resulted in dramatic responses in large numbers of patients. Because of several challenges and complexities in manufacturing for characterizing CAR-T cell products, it is important to optimize their manufacturing and critical quality attributes (CQAs). Herein, we performed a comparative assessment of scFv-CD-19-CAR-T and IL-13Ra2-CAR-T cells and manufactured using conventional and G-REX platforms.

Methods We manufactured lentiviral vectors (LVV) by employing common molecular biology techniques using HEK 293T as a producer cell line, transfected with scFVIL-13Ra2 or CD19 transgene plasmids along with three helper plasmids. The LVVs were used for transducing PBMCs from normal human blood donors after their activation and were expanded in T cell growth medium supplemented with cytokines including IL-2 or IL-2 + IL-15, IL-7 + IL-15 for specified durations. A battery of activation and exhaustion phenotype markers was studied at the end of expansion along with their potency to lyse the target cells and secrete Interferon-gamma in a co-culture assay.

Results Our results showed that the T cell activation was efficient by commercially available reagents such as anti-CD3/CD28 coated magnetic beads or nanoparticles and transduction either by retronection or Vectofusin. The CAR-T cell yields were at least two logs higher in the G-REX platform. Analysis of phenotype markers for T cells (CD3, CD4, CD8), T cell activation (CD25, CD45, CD69)/exhaustion markers (PD-1, LAG-3, TIM-3) and Central memory T cells, {CD45RA low, CD62L (CCR7 high} revealed better health and yield of CAR-T cell products manufactured in G-REX platform.

Conclusions We conclude that G-REX platform was superior to conventional methods for manufacturing both types of CAR-T cells. The data also suggest that better activation and transduction by specific reagents and expansion in combination with selected cytokines are key issues associated in manufacturing healthy and potent CAR-T cell products.

Acknowledgements We thank Pamela Leland, former lab member in the Center for Biologics Evaluation and Research, FDA, Silver Spring, for her performance of the experiment.

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