Engineering of regulatory T cells by means of mRNA electroporation in a GMP-compliant manner

Abstract

Regulatory T cells (Tregs) are crucial in inducing and maintaining tolerance. This unique capacity of Tregs, in combination with proof-of-principle in preclinical studies, highlights the potential clinical use of Tregs for the treatment of autoimmunity and transplant rejection. Although proven to be safe and well tolerated in the first clinical trials, only modest clinical results were observed. In this regard, it has been hypothesized that current challenges lie in the development of antigen-specific Tregs. Here, we present an innovative, good manufacturing practices (GMP)-compliant manufacturing protocol for Tregs applicable in a clinical-grade setting, allowing efficient and safe redirection of Treg specificity. First, a soluble polymer conjugated with antibodies to CD3 and CD28 and high amounts of exogenous IL-2 for in vitro Treg expansion resulted in a >70-fold and 185-fold increase of a pure population of CD4⁺CD127^-CD25^hi Tregs and CD4⁺CD127^-CD25⁺CD45RA⁺ Tregs, respectively. Next, as a proof-of-principle, expanded Tregs were engineered by means of TCR-encoding mRNA electroporation to generate antigen-specific Tregs. This resulted in an expression of the newly introduced TCR in up to 85% of Tregs. Moreover, we did not observe a negative effect on the phenotype of Tregs, as demonstrated by the expression of FOXP3, Helios, CTLA-4 and CCR4, nor on the TSDR methylation status. Importantly, mRNA-engineered Tregs were still able to induce in vitro suppression of effector T cells and produced anti-inflammatory, but not pro-inflammatory, cytokines when activated. In conclusion, our findings demonstrate that high numbers of stable and functional Tregs can be obtained with high purity and successfully engineered for gain of function, in a GMP-compliant manner. We envisage that this clinical-grade protocol will provide solid basis for future clinical application of mRNA-engineered Tregs.

Keywords: T cell receptor; adoptive cell therapy; antigen specificity; good manufacturing practice; mRNA-electroporation; regulatory T cells.