Efficient and Rapid Generation of CAR-T and Cytokine-Induced Killer Cells in GMP-scalable Devices

We describe a G-Rex protocol for the efficient and scalable expansion of adoptive cell therapy products, including CAR-T and CIK cells. The core challenge lies in setting up a protocol to support high-density cultures all while ensuring minimal user intervention and robust clinical scalability. To begin, resuspend 2 times 10 to the power of 6 freshly isolated peripheral blood mononuclear cells in 2 milliliters of complete culture medium, supplemented with 5%FBS referred to as CCMF.

Transfer the suspension into the wells of a 24-well G-Rex culture vessel. Add 4 microliters of anti-CD3 antibody and 20 microliters of anti-CD28 antibody directly into each well to activate the T cells and incubate at 37 degrees Celsius with 5%carbon dioxide. After overnight stimulation, harvest and count the activated T cells using the Trypan Blue exclusion assay to assess viability, calculate 1 times 10 to the power of 6 viable activated cells to seed for each well of the G-Rex culture vessel.

Transfer the cells into a tube and centrifuge at 300 g for 5 minutes. Now, prepare the transduction medium for each well by combining the required lentiviral vector volume with 5 microliters of an 8 milligram per milliliter stock solution of protamine sulfate. Use CM medium supplemented with 5%FBS and without penicillin or streptomycin to bring the total volume to 0.8 milliliters per well.

Then, resuspend the pelleted cells in 0.8 milliliters of the prepared transduction medium and transfer the suspension into a new well of the G-Rex culture vessel. Perform the same procedure for the untransduced control condition, avoiding the addition of the lentiviral vector. Incubate the cells overnight at 37 degrees Celsius with 5%carbon dioxide.

On day 2, increase the culture volume to a total of 8 milliliters per well by adding 7.2 milliliters of fresh CCMF supplemented with human interleukin-7 and interleukin-15. Next, on day 5, refresh the culture medium by removing 6 milliliters of conditioned medium from each well without disturbing the cells at the bottom. Replace it with 6 milliliters of fresh CCMF containing human interleukin-7 and interleukin-15.

On day 9, gently remove and discard 5 milliliters of conditioned medium from each well. Using a 1000-microliter pipette, resuspend the CAR-T cells in the remaining 3 milliliters of medium and transfer them to a 15-milliliter tube. Count the CAR-T cells and check their viability using the Trypan Blue exclusion assay.

Seed 1 times 10 to the power of 6 freshly isolated peripheral blood mononuclear cells for each well of the G-24 culture vessel in 8 milliliters of CCM supplemented with recombinant human interferon-gamma. Incubate the culture vessel at 37 degrees Celsius with 5%carbon dioxide. On day 1, after 22 to 24 hours, add 4 microliters of anti-CD3 antibody directly into each well and 4 microliters of recombinant human interleukin-2.

Incubate the cells again at 37 degrees Celsius with 5%carbon dioxide. Then, on days 5, 9, and 13, remove 6 milliliters of conditioned medium from each well without disturbing the settled cells. Replace it with 6 milliliters of fresh CCM supplemented with recombinant human interleukin-2.

On day 14, gently remove and discard 5 milliliters of conditioned medium from each well. Resuspend the cytokine-induced killer cells in the remaining 3 milliliters of medium and transfer them into a 15-milliliter tube. Assess the total cell number and viability using the Trypan Blue exclusion assay.

For CAR-T cell expansion, perform activation of peripheral blood mononuclear cells on day 0 and lentiviral transduction on day 1 as demonstrated earlier. Maintain all concentrations and seeding densities but scale up all volumes to match the G-6M device format. On day 2, bring the total culture volume in each well to 100 milliliters using CCMF medium, calculate and add human interleukin-7 and human interleukin-15 to reach final concentrations of 10 nanograms per milliliter each.

On day 9, remove approximately 90 milliliters of medium from each well using a serological pipette. Resuspend the CAR-T cells, the remaining 10 milliliters of medium, and transfer the cell suspension to a 15-milliliter tube for subsequent assays. For CIK cell expansion, perform seeding of peripheral blood mononuclear cells on day 0 following the CIK cell expansion protocol described previously.

On day 1, bring the total culture volume to 100 milliliters using CCM and add recombinant human interleukin-2 and anti-CD3 antibody. Every 3 to 4 days, supplement each well with recombinant human interleukin-2 at a final concentration of 500 international units per milliliter without changing the medium. On day 14, remove approximately 90 milliliters of medium from each well using a serological pipette.

Finally, resuspend the cytokine-induced killer cells in the remaining 10 milliliters of medium and transfer the cells into a 15-milliliter tube for further assays. Both CD19 CAR-T and CIK cells expanded in parallel from healthy donor peripheral blood mononuclear cells in G-24 and G-6M devices exhibited high fold expansion and viability with comparable proliferation between the two device formats. For CD19 CAR-T cells, CAR expression and the CD4 and CD8 positive subsets were comparable in G-24 and G-6M devices.

For the bulk CIK cell population, the frequency of T, NK, and CIK populations as well as the expression of the activating receptor NKG2D were equal across scales. The differentiation profile of CD19 CAR-T and CIK cells, including naive, central memory, effector memory, and effector memory CD45RA-positive subpopulations, was similar between G-24 and G-6M expansion formats. CD19 CAR-T cells expanded in both G-24 and G-6M devices displayed potent cytolytic activity against Burkitt's lymphoma target cells, confirming functional preservation across scale.

CIK cells cultured in both G-24 and G-6M devices demonstrated strong cytotoxicity against MCF-7 breast cancer target cells with overlapping lysis curves across 24 hours. Our G-Rex protocol overcomes complex scaling hurdles, enabling a seamless and proportional transition of cell product manufacturing from preclinical to clinical. Our protocol is highly reproducible and less labor-intensive than standard preclinical cell culture systems, leading to consistent high yields across donors.

We advance adoptive cell therapy to clinical application by standardizing the protocol and enabling an easier translational use for patients.