A Rapamycin-Based GMP-Compatible Process for the Isolation and Expansion of Regulatory T Cells for Clinical Trials

Abstract

The concept of regulatory T cell (Treg)-based immunotherapy has enormous potential for facilitating tolerance in autoimmunity and transplantation. Clinical translation of Treg cell therapy requires production processes that satisfy the rigors of Good Manufacturing Practice (GMP) standards. In this regard, we report our findings on the implementation of a robust GMP compliant process for the ex vivo expansion of clinical grade Tregs, demonstrating the feasibility of this developed process for the manufacture of a final product for clinical application. This Treg isolation procedure ensured the selection of a pure Treg population that underwent a 300-fold expansion after 36 days of culture, while maintaining a purity of more than 75% CD4+CD25+FOXP3+ cells and a suppressive function of above 80%. Furthermore, we report the successful cryopreservation of the final product, demonstrating the maintenance of phenotype and function. The process outlined in this manuscript has been implemented in the ONE study, a multicenter phase I/IIa clinical trial in which cellular therapy is investigated in renal transplantation.

Methods to manufacture regulatory T cells for cell therapy

“…Beyond CD25 there is considerable variation in the methods used to isolate clinical grade T regs . For example, groups using magnetic selection to isolate T regs from peripheral blood deplete CD8 + cells , whereas groups isolating T regs from UCB do not . Groups with access to flow cytometric sorting capability typically include additional markers such as positive selection for CD45RA or negative selection for CD127 .…”

“…~100 international units (IU)/ml , but many groups now add this cytokine at concentrations up to 1000 IU/ml) . Notably, protocols differ not only in the concentration of IL‐2 added, but also in the addition timing, with some groups adding IL‐2 from the start and others only 3 or 4 days post‐stimulation . Some groups also use the immunosuppressive drug rapamycin in T reg expansion protocols because it can limit the growth of effector T cells .…”

“…Notably, protocols differ not only in the concentration of IL‐2 added, but also in the addition timing, with some groups adding IL‐2 from the start and others only 3 or 4 days post‐stimulation . Some groups also use the immunosuppressive drug rapamycin in T reg expansion protocols because it can limit the growth of effector T cells . There is evidence that rapamycin may increase the suppressive function of T regs , so culturing T regs with rapamycin may result in a purer, more potent product.…”

“…Some groups also use the immunosuppressive drug rapamycin in T reg expansion protocols because it can limit the growth of effector T cells . There is evidence that rapamycin may increase the suppressive function of T regs , so culturing T regs with rapamycin may result in a purer, more potent product. Although rapamycin improves purity it also limits T reg proliferation, so it is most commonly used by groups who do not employ flow cytometric sorting as part of their protocol, such that maintaining sufficient T reg purity is a greater challenge and more important than maximal expansion.…”

Future prospects for CD8+ regulatory T cells in immune tolerance

“…The purity of cell product, the expression of key markers, and the cytotoxic and suppressive functions will be controlled. Fortunately, the manufacturing processes of CD4 + Tregs and CD8 + TILs used in ongoing clinical trials are helpful to set up the good manufacturing practices manufacturing of CD8 + Tregs product 140,[146][147][148][149][150]. In addition, these processes canbe upgraded by technical advances, such as clinical grade flow cytometry cell sorting.…”