Abstract
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.
Highlights
Regulatory T cells (Treg) play a critical role in maintaining immune homeostasis and limiting autoimmune responses by modulation of both innate and adaptive immunity [1]
The following materials are used during the Treg production process: Autologous leucapherisate Autologous plasma MACS® good manufacturing practice (GMP) ExpAct Treg Kit Human serum albumin MACS® GMP Rapamycin CliniMACS® CD8 Reagent CliniMACS® CD19 Reagent CliniMACS® CD25 Reagent l-Glutamine CliniMACS® PBS/EDTA IL-2 (Proleukin®) X-VIVO15 Dimethyl sulfoxide (DMSO) Glucose solution 40% (Glucosteril 40%)
With use of the protocol, leukapheresis-derived CD25+ cells expanded greater than 2 orders of magnitude with an average cell number of 113 × 106 at day 7, 501 × 106 at day 11, 635 × 106 at day 14, 986 × 106 at day 18, and 1,076 × 106 at day 21 after anti-CD3/anti-CD28 expander bead removal (Figures 2A,B)
Summary
Regulatory T cells (Treg) play a critical role in maintaining immune homeostasis and limiting autoimmune responses by modulation of both innate and adaptive immunity [1]. Defined Treg are characterized by their constitutive expression of CD4, CD25, and FoxP3 [2] and nearly absent expression of CD127 [3, 4]. They can be divided in (i) natural Treg originating from the thymus and peripherally induced Treg, which differentiate from naïve T cells when self or non-self antigen is encountered under tolerogenic conditions [5, 6]. Isolation of Treg bears the risk of concurrent isolation of unwanted effector cells
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