A GMP-Compliant Procedure for the Generation of Gene-Modified T cells

This protocol describes a simplified and GNP compliant process of transducing human primary T-cells with gene of interest. This particular technique has been designed to be cost-effective and GNP compliant without the use of expensive closed system manufacturing platforms currently available in many academic centers. This method could potentially be applied to the generation of gene-modified cell therapies, including but not limited to CAR T-cells, which have been a paradigm shift to tackle hematologic malignancies.

To isolate PBMC from the cells collected after leukapheresis of donor blood, perform a polysaccharide-based density gradient centrifugation of the sample, maintaining a reagent to sample ratio of 1:2, by centrifuging the mixture at 800 G for 30 minutes at room temperature with the brakes off. Then using a micropipette, collect the PBMCs into a clean 50 milliliter tube. Wash the cells twice with PBS by centrifugation.

Before resuspending the washed cells in complete hematopoietic media. Activate about 20 million of the obtained cells by adding 10 microliters of T-cell stimulation reagent for every 2 million cells. Next, after adding recombinant human interleukin-2 at 20 units per milliliter, mix the solution gently and transfer it into a six-well plate.

Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 72 hours before performing viral transduction. On day three, transfer the cell culture to a 50 milliliter conical tube and mix it thoroughly. Centrifuge the tube at 300G for 10 minutes at room temperature to remove the activation reagent.

Discard the supernatant completely and resuspend the pellet in one milliliter of complete medium before counting the cells. Adjust the cell suspension volume using complete medium to achieve a concentration that allows for plating 0.5 million cells per well in a 24 well plate. Add recombinant human interleukin-7 and recombinant human interleukin-15 to the cells at appropriate concentrations.

In a separate tube, add the desired amount of vectofusin-1 to Opti-MEM medium, considering the final viral volume to be used. Combine this mixture with the concentrated virus at a one-to-one ratio, ensuring thorough mixing. Next, add the resultant mixture containing the virus to the cells.

Adjust the total volume of each well to 400 microliters using complete medium if necessary. After covering the plate with a lid, seal it using a paraffin film before centrifuging it at 1000G for two hours at 32 degrees Celsius. Incubate the plate overnight at 37 degrees Celsius and 5%carbon dioxide.

On the next day, collect and pull the cells from each well into a 50 milliliter tube. Next, centrifuge the cells at 400G and room temperature for five minutes. After carefully removing the supernatant, fully resuspend the pellet in one milliliter of complete medium before counting the cells.

Then adjust the cell concentration with the media to achieve 1 million cells per milliliter and add human recombinant interleukin-7 and human recombinant interleukin-15 at 155 and 290 units per milliliter respectively before culturing the cells. Once the desired cell number is reached, harvest and transfer the cells into 50 milliliter tubes and centrifuge them. After removing the supernatant, resuspend the pellet in 10 milliliters of media to count the cells.

After centrifuging again, discard the supernatant completely and resuspend the pellet in a cryopreservation solution consisting of 50%HSA, and 40%HBSS, at a concentration of 10 million cells per milliliter per cryo vial. Transfer 0.9 milliliters of cell suspension each to the required numbers of cryo vials, and add 10%dimethyl sulfoxide to each cryo vial. Place the cryo vials on ice and quickly shift them into a controlled-rate freezer for cryo-preservation.

Then transfer the cryopreserved samples to a monitored liquid nitrogen tank for long-term storage. To evaluate transduction efficiency, add 500 microliters of FACS buffer to the sample in a fluorescence activated cell sorting or FACS tube. After centrifuging the sample at 400G and room temperature for five minutes, discard the supernatant completely using a pipette.

Resuspend the pellet in a one to five diluted solution of CD3 in FACS buffer. Vortex the sample and incubate for 30 minutes on ice in the dark before centrifuging as demonstrated previously. Next, after discarding the supernatant entirely, resuspend the pellet in a one to 20 diluted solution of 7-AAD in FACS buffer.

Vortex this mixture before incubating it for 10 minutes on ice in a dark setting. Finally, add 200 microliters of FACS buffer to it And proceed to analyze the sample using a flow cytometer. Viability analysis of the transduced cells revealed that on day 14, more than 95%of the cells were CD3 positive and alive after excluding 7-AAD positive cells, indicating successful T-cell activation and expansion.

The transduction efficiency within the CD3 positive population was measured to be 58.7%compared to the non-transduced cells, which exhibited a transduction efficiency of 0.51%When the transduced cells were expanded from day four until day 14, with sub-culturing every two days, the cells underwent a 25 fold expansion. The product underwent various quality control tests and met all the set criteria, indicating its suitability for further use. The whole procedure should be performed with strictly aseptic techniques.

And the most tricky step is the transduction process where one needs to take into account all the reagents to be added in order to maintain a specific total volume.