An Ex Vivo Technique to Manufacture Chimeric Antigen Receptor T Cells

Take primary T cells, mixed with antibody-conjugated magnetic beads in an interleukin-2-supplemented medium, and incubate.

The antibodies bind to CD3 in the T cell receptor complex and the co-stimulatory receptor CD28, while interleukin-2 binds to its cognate receptor, causing T cell activation and proliferation.

Introduce lentiviral vectors with a transgenic RNA encoding a chimeric antigen receptor or CAR, and incubate.

Upon vector-cell membrane fusion, the viral RNA undergoes reverse transcription into DNA, integrating into the host genome.

The transduced T cells express surface-bound CARs.

Harvest the cells at defined intervals.

For cryopreservation, take an aliquot of cells, detach the bound beads by pipetting, and magnetically separate them.

Spin down the cells and resuspend them in a cryopreservation medium.

Freeze the cells and store them at a cryogenic temperature.

To assess CAR expression, take another aliquot of cells, spin down the cells, and resuspend them in a buffer.

Introduce a CAR-specific fluorescently-tagged antibody and detect CAR expression using flow cytometry.

Activate fresh or cryopreserved primary human T cells by mixing them with anti-CD3/CD28 magnetic beads at a ratio of 3 beads per T cell in the wells of the culture dishes. Culture the T cells in X-VIVO 15 medium supplemented with normal human AB serum, L-glutamine, HEPES, and IL-2.

Activate the T cells at a concentration of 1 million T cells per milliliter during expansion, and culture them at 37 degrees Celsius with 20% oxygen, 5% carbon dioxide, and 95% humidity. After overnight stimulation, add lentiviral supernatant to the activated T cells. Calculate the volume of supernatant necessary to achieve a multiplicity of infection between 3 and 5. Continue culturing the cells using the same conditions.

On day 3, collect a representative aliquot of the cells of cryopreservation. Prior to cryopreservation, remove the magnetic beads by gently pipetting and magnetic separation. Prepare the freezing medium which contains PBS with 0.5% DMSO and store it at 4 degrees Celsius until ready to use.

Next, centrifuge the T cells at 300 times g for 5 minutes. Discard the supernatant and add 5 milliliters of PBS. Centrifuge the cells again at 300 times g for 5 minutes and discard the PBS. Resuspend the pellet in 1 milliliter of cold cryopreservation medium. Freeze the T cells in a chilled freezing container and store at minus 80 degrees Celsius for 48 hours.

After this, transfer the frozen cells to liquid nitrogen. Wash the rest of the T cells once in 5 milliliters of PBS to eliminate any residual vector. Centrifuge at 300 times g for 5 minutes, then decant the PBS and resuspend the cell pellet in T cell culture medium at a concentration of 500,000 cells per milliliter.

Split the T cells into two cultures designed for day 5 and 9. Count the T cells by flow cytometry using counting beads and monoclonal antibodies to human CD4 and CD8 as well as a viability dye. Refeed the cultures every other day to maintain them at a concentration of 500,000 cells per milliliter.

On day 5, counter and cryopreserve the day 5 cultures as previously described. On day 7, wash 500,000 cells in PBS and resuspend them in 100 microliters of fluorescence-activated cell sorting buffer. Then detect CAR surface protein expression by immunostaining with a fluorescently-conjugated anti-CAR19 idiotype by flow cytometry. On day 9, count and cryopreserved the day 9 cultures as previously described.