In recent years, the exploration of regulatory T cell (Treg)-based cellular therapy has become an attractive strategy to ameliorate inflammation and autoimmunity in various clinical settings. The main obstacle to the clinical application of Treg in human is their low number circulating in peripheral blood. Therefore, ex vivo expansion is inevitable. Moreover, isolation of Treg bears the risk of concurrent isolation of unwanted effector cells, which may trigger or deteriorate inflammation upon adoptive Treg transfer. Here, we present a protocol for the GMP-compliant production, lot-release and validation of ex vivo expanded Tregs for treatment of patients with autoimmune and inflammatory disorders. In the presented production protocol, large numbers of Treg, previously enriched from a leukapheresis product by using the CliniMACS® system, are ex vivo expanded in the presence of anti-CD3/anti-CD28 expander beads, exogenous IL-2 and rapamycin during 21 days. The expanded Treg drug product passed predefined lot-release criteria. These criteria include (i) sterility testing, (ii) assessment of Treg phenotype, (iii) assessment of non-Treg cellular impurities, (iv) confirmation of successful anti-CD3/anti-CD28 expander bead removal after expansion, and (v) confirmation of the biological function of the Treg product. Furthermore, the Treg drug product was shown to retain its stability and suppressive function for at least 1 year after freezing and thawing. Also, dilution of the Treg drug product in 0.9% physiological saline did not affect Treg phenotype and Treg function for up to 90 min. These data indicate that these cells are ready to use in a clinical setting in which a cell infusion time of up to 90 min can be expected. The presented production process has recently undergone on site GMP-conform evaluation and received GMP certification from the Bavarian authorities in Germany. This protocol can now be used for Treg-based therapy of various inflammatory and autoimmune disorders.

ObjectiveUlcerative colitis (UC) is a chronic, debilitating immune-mediated disease driven by disturbed mucosal homeostasis, with an excess of intestinal effector T cells and an insufficient expansion of mucosal regulatory T cells (Tregs). We here report on the successful adoptive transfer of autologous, ex vivo expanded Tregs in a patient with refractory UC and associated primary sclerosing cholangitis (PSC), for which effective therapy is currently not available.DesignThe patient received a single infusion of 1×106 autologous, ex vivo expanded, polyclonal Tregs per kilogram of body weight, and the clinical, biochemical, endoscopic and histological responses were assessed 4 and 12 weeks after adoptive Treg transfer.ResultsThe patient showed clinical, biochemical, endoscopic and histological signs of response until week 12 after adoptive Treg transfer, which was associated with an enrichment of intestinal CD3+/FoxP3+ and CD3+/IL-10+ T cells and increased mucosal transforming growth factor beta and amphiregulin levels. Moreover, there was marked improvement of PSC with reduction of liver enzymes. This pronounced effect lasted for 4 weeks before values started to increase again.ConclusionThese findings suggest that adoptive Treg therapy might be effective in refractory UC and might open new avenues for clinical trials in PSC.Trial registration numberNCT04691232.

IntroductionAccumulating evidence suggests that the adoptive transfer of ex vivo expanded regulatory T cells (Treg) may overcome colitogenic immune responses in patients with inflammatory bowel diseases. The objective of the ER-TREG 01 trial is to assess safety and tolerability of a single infusion of autologous ex vivo expanded Treg in adults with ulcerative colitis.Methods and analysisThe study is designed as a single-arm, fast-track dose-escalation trial. The study will include 10 patients with ulcerative colitis. The study intervention consists of (1) a baseline visit; (2) a second visit that includes a leukapheresis to generate the investigational medicinal product, (3) a third visit to infuse the investigational medicinal product and (4) five subsequent follow-up visits within the next 26 weeks to assess safety and tolerability. Patients will intravenously receive a single dose of 0.5×106, 1×106, 2×106, 5×106 or 10×106 autologous Treg/kg body weight. The primary objective is to define the maximum tolerable dose of a single infusion of autologous ex vivo expanded Treg. Secondary objectives include the evaluation of safety of one single infusion of autologous ex vivo expanded Treg, efficacy assessment and accompanying immunomonitoring to measure Treg function in the peripheral blood and intestinal mucosa.Ethics and disseminationThe study protocol was approved by the Ethics Committee of the Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany (number 417_19 Az). In addition, the study was approved by the Paul-Ehrlich Institute, Federal Institute for Vaccines and Biomedicines, Langen, Germany (number 3652/01). The study is funded by the German Research Foundation (DFG, KFO 257 project 08 and SFB/TransRegio 241 project C04). The trial will be conducted in compliance with this study protocol, the Declaration of Helsinki, Good Clinical Practice and Good Manufacturing Practice. The results will be published in peer-reviewed scientific journals and disseminated in scientific conferences and media.Trial registration numberNCT04691232.

Background Ulcerative colitis (UC) is marked by impaired mucosal homeostasis with predominance of intestinal effector T cells and insufficient expansion of mucosal regulatory T cells (Tregs), thereby providing a scientific rationale for Treg-based immunotherapy in UC. Methods We developed a protocol to generate large numbers of autologous CD25+ cells under aegis of IL-2, rapamycin and anti-CD3/anti-CD28 expander beads ex vivo from CD25+ precursors derived from peripheral blood cells, intended for clinical use under good manufacturing practice (GMP) conditions. We initiated a single-centre, open-label, fast-track dose-escalation, phase-1 clinical trial with a single adoptive transfer of autologous ex vivo expanded CD4+CD25+CD127−/lo Tregs in patients with refractory UC. The primary objective of the trial was to define safety and the maximum tolerable dose of a single intravenous administration of autologous expanded Tregs according to the fast-track dosing principle. Adoptive transfer was escalated from the starting dose of 0.5×106 Tregs/kg body weight to the next dose level (1×106, 2×106, 5×106 and 10×106 Tregs/kg body weight) in each consecutive patient, respectively, if no dose-limiting toxicity occurred. We here report interim study data of the first 8 patients that received Treg transfer and were followed up over 12 weeks. There were no changes in concomitant medication during the study period. Results The 8 patients received 0.5 x106 (n=1), 1×106 (n=1), 2×106 (n=1), 2.8x106 (n=1), 5×106 (n=1) and 10×106 (n=3) Treg/kg body weight, respectively. The patients did not show any dose-limiting toxicity and there were no adverse events related to Treg transfer. 87.5% (7/8) of patients had previously received treatment with at least 2 different substance classes of advanced therapies, such as biologics and JAK inhibitors and the mean Mayo Score (MS) at baseline was 9.1±1.6. The mean modified MS (mMS) dropped significantly from 6.6±1.2 at baseline to 4.5±2.2 at week 4 (p=0.006) and 4.6±2.0 at week 12 (p=0.01). There was reduction of mean faecal Calprotectin levels from 1708±397 µg/g at baseline, to 1411±471 µg/g at week 4 and 919±332 µg/g at week 12. Clinical response was reached by 62.5% (5/8) of patients at week 4 and also week 12. Clinical response predominantly occurred in patients receiving the highest number of 10×106 Tregs/kg body weight. Clinical remission was achieved by 12.5% (1/8) of patients at week 4 and also week 12. Conclusion Autologous regulatory T cell transfer was well tolerated up to the maximal tested dose of 10×106 Tregs/kg body weight. Study results suggest that adoptively transferred polyclonal Tregs might be effective in refractory ulcerative colitis, warranting further clinical studies.