Custom CAR-T Cell Development

A Milestone-Measured Process from Concept to CAR-T

The development of custom CAR-T cells is a complex process that requires a stepwise workflow, implemented in stages:

• Design and construct lentivector for scFv-CAR of different varieties including mono-scFv, dual scFv, and tandem scFv CARs.
• Transfect lentivector and packaging plasmids in 293T cells to make high titer, concentrated virus.
• Validate CAR construct by transducing into the NFAT reporter Jurkat cell line to check for expression of CAR by flow cytometry and for functional activity by reporter assay.

• Isolate, activate and expand primary T cells (from healthy donors).
• Transduce T cells and check the expression of CAR by flow cytometry.
• Establish target cells stably expressing antigen of interest in a luciferase reporter CHO cell line.
• Measure IFNγ production by CAR-T cells in co-culture assays using BPS Bioscience’s IFNγ ELISA kit.
• Assess cytotoxic cell killing using co-culture of target cells and effector CAR-T cells.

Customization to Meet Specific CAR-T Aims

• Compound and antibody screening using CAR-T cells or CAR/NFAT reporter Jurkat cells
• Intracellular costimulatory and activation domain comparisons
• CAR-T signaling in the tumor microenvironment
• Screening CAR-T donor variations
• Evaluate Treg and T memory cells in the CAR-T setting

Functional Validation