A comparison of automated perfusion- and manual diffusion-based human regulatory T cell expansion using a soluble activator complex

Abstract

Background & Aim Human regulatory T cells (hTregs) play a central role in the maintenance of self-tolerance and suppression of autoimmune responses. The absence or reduced frequency of hTregs can limit the control of immune inflammatory responses, autoimmunity and the success of transplant engraftment. Clinical studies indicate that the use of hTregs as immunotherapeutics would require billions of cells per dose; e.g., Phase I studies by Mathew et al (2018) suggest that hTreg doses in the range of 0.5 to 5.0\u202f×\u202f109 cells are needed to facilitate kidney transplantation and clinical studies by Bluestone et al (2015) indicate that polyclonal hTreg doses in the range of 5\u202f×\u202f106 to 2.6\u202f×\u202f109 cells are well tolerated in type 1 diabetes patients. The Quantum Cell Expansion System is a hollow-fiber (HFM) bioreactor system that has been used to grow billions of functional T-cells in a short timeframe (8 to 9 days). We have now focused our protocol development on the ex vivo expansion of selected hTregs in the Quantum system using a soluble activator, composed of an anti-CD3/CD28/CD2 tetrameric mAb complex. Methods, Results & Conclusion hTreg CD4+CD25+CD127− cells from 3 unrelated healthy donors, previously isolated by the vendor via column-free Imag positive selection, were thawed and grown under static conditions and subsequently seeded into Quantum system HFM bioreactors and into T225 control flasks in an identical culture volume of 124 mL PRIME-XV® XSFM medium supplemented with 100 IU/mL of rhIL-2 and antibiotics for scale-up expansion over 9 days to compare the effects of automated perfusion with manual diffusion-based culture. Cell growth, viability, and phenotype of the hTregs were compared. The hTreg cell harvest from 3 parallel expansion runs produced an average of 4.0\u202f×\u202f108 (range 1.9 to 5.6\u202f×\u202f108) hTregs in the flask arm with an average viability of 71.3% versus 7.0\u202f×\u202f109 (range 3.6 to 13.0\u202f×\u202f109) hTregs in the Quantum arm with an average viability of 91.8%. This translates into an average 17.7-fold increase in hTreg cell yield for the Quantum system over that obtained in flasks, coupled with a higher cell viability in the Quantum system. In addition, these 2 culture processes gave rise to cells with an hTreg CD4+CD25+FoxP3+ phenotype of 76.5% for the flask arm versus 80.3% for the Quantum system arm. The results suggest that an automated perfusion HFM bioreactor can support the scale-up expansion of hTreg cells more efficiently than diffusion-based flask culture.