An Ex Vivo Technique to Generate Tumor-Specific Chimeric Antigen Receptor T Cells

Take retroviral vectors mixed with primary spleen cells.

The vectors contain transgenic RNA encoding a chimeric antigen receptor or CAR, which targets a tumor-specific antigen.

The cytokine interleukin-2 in the media selectively promotes T cell proliferation while the other cells eventually die.

Transfer the mixture to a microplate coated with recombinant fibronectin fragments. 

Centrifuge to facilitate the fibronectin fragments binding the vectors and T cells, mediating virus-host cell interaction, and incubate.

Upon fusion of the vectors' envelope with the cell membrane, the viral enzymes reverse-transcribe the released RNA into DNA and incorporate it into the host genome.

The transduced T cells express surface-bound CARs, consisting of an extracellular antibody variable fragment targeting the tumor-specific antigen and intracellular signaling regions to respond to the antigen.

Resuspend the cells before transferring them to a tube.

Centrifuge to discard the supernatant containing non-internalized viruses, and resuspend the cells in a buffer for downstream assays.

On the day after the splenectomy, prepare PBS containing 25 micrograms per milliliter recombinant human Fibronectin f