Expansion and Enrichment of Gamma-Delta (γδ) T Cells from Apheresed Human Product

The Gamma-Delta T cells have powerful cytotoxic activity against many cancer type, including acute myeloid leukemia. However, circulating Gamma-Delta T cells in a blood are relatively rare, particularly in patients with malignancies. The infusion of donor derived, X-vivo expanded Gamma-Delta T cells, have promise in reducing the recurrence of leukemia, and improving survival of patients with acute myeloid leukemia.

The proposed method achieves greater than 200, 000 fold Gamma-Delta T cell expansion, reaching therapeutically relevant doses of a highly pure Gamma-Delta T cell product, in three easy steps. So ligating an IEL to expansion, alpha beta T cell depletion by magnetic sorting and bounty secondary expansion with AAPCs. The proposed method expands the utility of Gamma-Delta T cells for diseases such as cancer and autoimmune diseases by rapidly creating a large number of cells that can be used off the shelf.

This process could be adapted to expand Gamma-Delta cells using different enrichment methods and adding additional manipulations such as transduction. Demonstrating the procedure will be lithed in LA, a cell therapy technologist, three from my laboratory. Begin by elutriation of the effervescence product sample, on the counterflow centrifugation device, using a primary medium of Hanks balanced salt solution, with 1%human serum albumin and the secondary medium of saline solution or DPBS.

With the elutriation centrifugation speed at 900 times G, collect fractions based on flow rate and time. Collect the samples from fraction two to perform the sterility testing, cell count and cell phenotyping by flow cytometry. From fraction two, expand a pure lymphocyte fraction of cells in culture at 10 times 10 to the sixth cells per centimeter squared, with five micromoles per liter of zoledronic acid and 300 units per milliliter of Interleukin-2 in a one liter closed system bioreactor.

Incubate the system for seven days at 37 degrees celsius, with 5%carbon dioxide. After incubation, harvest cells from the one liter closed system bioreactor flask. To do so, sterile weld a one liter transfer pack to the red line of the bioreactor, and use the appropriate pharmaceutical pump to transfer the cells into the transfer pack.

Take the samples for testing of spent medium sterility, cell count and cell phenotyping by flow cytometry. From the remaining sample, re-suspend the cells at five times 10 to the eighth cells per milliliter, in PBS-EDTA buffer containing 0.5%HSA and biotinylated T cell receptor, alpha beta specific antibody. And incubate the cells at two to eight degrees Celsius with shaking.

After 15 minutes, wash the cells with 600 milliliters of PBS-EDTA buffer containing 0.5 HSA and centrifuge at 200 to 500 times G for 15 minutes, at two to eight degrees Celsius, to remove the unbound antibody. Then, re-suspend the cells at approximately five times 10 to the eighth cells per milliliter, in PBS-EDTA buffer containing 0.5%HSA and 7.5 milliliters per vial of anti-biotin specific microbeads. As described earlier, incubate the cells with shaking, before centrifuging the cell suspension to remove the unbound microbeads.

Re-suspend six times 10 to the seventh cells per milliliter in PBS-EDTA buffer with 0.5%HSA. Collect them into a transfer pack bag. Spike it when instructed by the instrument.

Next, install a tubing set in the clinical grade magnetic cell separation device. Place the packs with the PBS-EDTA buffer, and the transfer pack with the cell product into the instrument. For the depletion of the labelled alpha beta T cells, select the depletion 1.2 protocol in the software.

Then, centrifuge the cell product to collect the target fraction enriched with Gamma-Delta T cells. Re-suspend the collected cells in the medium, supplemented with 10%human AB serum. Perform a cell count viability and flow cytometry phenotyping post depletion.

Bring cells to a final concentration of approximately one times 10 to the sixth cells per milliliter. Irradiate five times 10 to the seventh Artificial Antigen-presenting Cells or AAPCs per flask, at 100 gray on the x-ray generating instrument. Prepare a co-culture, by placing the irradiated AAPCs and Gamma-Delta T cells at the ratio of 10 is to one, in one liter closed system bioreactor flasks.

With one liter of culture medium, supplemented with 10%human AB serum, seed up to 10 flasks. Incubate the culture at 37 degrees Celsius, and 5%carbon dioxide for 10 days, with a screening of glucose and lactate levels every three to four days using strips, glucose and a lactate meter. If glucose level drops to 215 milligrams per deciliter, reduce the volume in the flask to 200 milliliters.

Mix the cells in the remaining 200 milliliters of media, and remove a 0.5 milliliter sample for cell counting and viability measurement by acridine orange or propidium iodide staining. If the cell count is equal to, or more than 10 to the ninth, split the contents of one flask into two and fill each flask upto one liter with AIM-V medium, supplemented with 10%human AB serum. If the cell count is less than 10 to the ninth, feed the cells with a fresh one liter of culture medium, supplemented with 10%human AB serum, and return the flasks to the incubator.

At the end of 10 days in co-culture, harvest the bioreactor flasks one at a time, and pull all the cells into a transfer pack of appropriate size. After removing the quality control samples, centrifuge the cells at 200 to 500 times G for 15 minutes at room temperature, and discard the supernatant. Wash the cells in a solution of balanced crystalloid solution, supplemented with 0.5%HSA at 200 to 500 times G for 15 minutes at room temperature.

Re-suspend them in a target volume of 100 to 300 milliliters of balanced crystalloid solution with 0.5%HSA. The cells separated from the post counterflow centrifugation fraction. Two yielded a pure lymphocyte population, with an excellent average viability of 95.80%The Gamma-Delta T cells specific expansion, with zoledronic acid depended on the initial percentage of natural killer cells present in the lymphocyte fraction after elutriation.

The enrichment of Gamma-Delta T-cell drug substance, with T-cell receptor alpha beta depletion was consistent. The Gamma-Delta T-cell drug product manufactured from three healthy donors, met release criterion of less than 1%of T-cell receptor alpha beta positive T cells, with an average of 0.11%CD20 positive B cells, and 0.00%T-cell receptor alpha beta positive T cells. The average percentage of natural killer cells in the final product was around 17.06%which met the release criterion of less than 35%Cell surface staining and flow cytometric analysis, were utilized to characterize the identity, purity and process impurities of the drug substance and drug product.

The second re-expansion achieved from the co-culture of the AAPC and Gamma-Delta T-cell drug substance, generated a Gamma-Delta T-cell drug product that met all release criteria. Pathons which enrich the T-cell population impact the expansion and characteristics of the product. Sufficient consideration and investigation of enrichment methods is required, prior to initiation of their enrichment process.