An In Vitro Assay to Evaluate the Anti-Tumor Activity of Tumor-Specific CAR T Cells

Take a multiwell plate containing B cell lymphoma tumor cells expressing a B cell-specific surface antigen and a cytoplasmic luciferase enzyme.

Introduce CAR T cells into selected wells and incubate. The cells express chimeric antigen receptors, or CARs, comprising an extracellular domain targeting the tumor antigen and intracellular signaling domains.

CAR binds to the tumor antigen, inducing activation of the CAR T cells.

The activated cells release cytokines that promote the cells' anti-tumor activity, triggering the release of toxins.

The toxins enter the tumor cells and induce cell death.

Spin down the cells and isolate the supernatant. Quantify the cytokines to assess CAR T cell activity.

Resuspend the cells in a buffer containing luciferin, a bioluminescent substrate.

Upon entry, luciferin is oxidized by luciferase in living tumor cells, emitting light.

Measure the bioluminescence to identify a decreased signal in the treated tumor cells, confirming CAR T cell-mediated cytotoxicity.