Safety and tolerability of a single infusion of autologous ex vivo expanded regulatory T cells in adults with ulcerative colitis (ER-TREG 01): protocol of a phase 1, open-label, fast-track dose-escalation clinical trial.

INTRODUCTION: Accumulating 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 ANALYSIS: The 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 DISSEMINATION: The 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 NUMBER: NCT04691232.

Cryopreservation impairs 3-D migration and cytotoxicity of natural killer cells.

Natural killer (NK) cells are important effector cells in the immune response to cancer. Clinical trials on adoptively transferred NK cells in patients with solid tumors, however, have thus far been unsuccessful. As NK cells need to pass stringent safety evaluation tests before clinical use, the cells are cryopreserved to bridge the necessary evaluation time. Standard degranulation and chromium release cytotoxicity assays confirm the ability of cryopreserved NK cells to kill target cells. Here, we report that tumor cells embedded in a 3-dimensional collagen gel, however, are killed by cryopreserved NK cells at a 5.6-fold lower rate compared to fresh NK cells. This difference is mainly caused by a 6-fold decrease in the fraction of motile NK cells after cryopreservation. These findings may explain the persistent failure of NK cell therapy in patients with solid tumors and highlight the crucial role of a 3-D environment for testing NK cell function.

Good Manufacturing Practice-Compliant Production and Lot-Release of Ex Vivo Expanded Regulatory T Cells As Basis for Treatment of Patients with Autoimmune and Inflammatory Disorders.

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.

Characterization and Expansion of Autologous GMP-ready Regulatory T Cells for TREG-based Cell Therapy in Patients with Ulcerative Colitis.

BACKGROUND: A local imbalance between regulatory (Treg) and effector T cells is believed to play a major role in gut-specific inflammation, including ulcerative colitis (UC). Restoration of this balance through an adoptive Treg transfer is an attractive new treatment approach in patients who are refractory to current standard therapies. It was our goal to develop a Good Manufacturing Practices (GMP)-conform protocol for expansion of UC Treg cells as a rational backbone for future studies on Treg therapy in UC. METHODS: CD25 blood T cells derived from patients with UC were ex vivo expanded in the presence of IL-2, rapamycin, and anti-CD3/anti-CD28 expander beads using a GMP-conform protocol. Cells were subsequently assessed for stability and function. RESULTS: Patient-derived ex vivo rapamycin-expanded GMP-ready CD25 cells were polyclonal, hypomethylated at intron 1 of the FoxP3 locus, and suppressive in carboxyfluorescein succinimidyl ester-dilution assays against autologous peripheral blood-derived and allogeneic colon-derived responder cells. Function was mediated by soluble factors, including toxic granules. In addition to CD4 T cells, suppressive hypermethylated CD8 T-cell subsets were also induced during the expansion process. CONCLUSIONS: Patient-derived rapamycin-expanded CD25 cells are stable and functional, and as such, ready to serve in a phase I dose-escalation safety study in UC.