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

Manuel Wiesinger, Diane Stoica, Susanne Roessner, Carmen Lorenz, Anika Fischer, Raja Atreya, Clemens F Neufert, Imke Atreya, Alexander Scheffold, Beatrice Schuler-Thurner, Markus F Neurath, Gerold Schuler, Caroline J Voskens, Manuel Wiesinger, Diane Stoica, Susanne Roessner, Carmen Lorenz, Anika Fischer, Raja Atreya, Clemens F Neufert, Imke Atreya, Alexander Scheffold, Beatrice Schuler-Thurner, Markus F Neurath, Gerold Schuler, Caroline J Voskens

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.

Keywords: autoimmunity; expansion; good manufacturing practice; lot-release; regulatory T cell.

Figures

Figure 1
Figure 1
Flowchart of the production of the regulatory T cell (Treg) drug substance and Treg drug product.
Figure 2
Figure 2
Assessment of identity and cellular composition. (A) Representative FACS plots showing the gating strategy. (B) Representative FACS plots gated on PI− cells showing CD4, CD25, CD127, CD8, CD19, and CD56/CD16 expression after CD25+ cell enrichment at day 0 and after thawing the day 21 regulatory T cell (Treg) drug product. (C) Proportion of PI− cells expressing CD4, CD25, CD127, CD8, CD19, and CD56/CD16 after CD25+ cell enrichment at day 0 (n = 4, white bars) and after thawing the day 21 Treg drug product (n = 4, filled bars). ND, not determined.
Figure 3
Figure 3
Assessment of regulatory T cell (Treg) drug product purity. (A) Representative FACS plots gated on PE+/APC+ anti-CD3/anti-CD28 expander beads in a control sample containing no anti-CD3/anti-CD28 expander beads (=background sample), a control sample containing 750 anti-CD3/anti-CD28 expander beads (=negative control sample), a control sample containing 1,000 anti-CD3/anti-CD28 expander beads (=standard sample), and a control sampler containing 3,000 anti-CD3/anti-CD28 expander beads. (B) FACS plots gated on PE+/APC+ anti-CD3/anti-CD28 expander beads in a thawed Treg drug product from consistency run 1 (=Con1), consistency run 2 (=Con2), consistency run 3 (=Con3), and consistency run 4 (=Con4).
Figure 4
Figure 4
Assessment of regulatory T cell (Treg) drug product function. (A) Representative FACS plots showing the gating strategy defining CD25− responder cell proliferation. (B) Representative histograms gated on CD8+/CFSE+ responder cells showing percentage of responder cell proliferation within the most divided cell generation in the presence of the thawed day 21 Treg drug product cells at a Treg to responder cell ratio of 1 + 1, 1 + 5, and 1 + 10, respectively. (C) Proportion of the first generation of responder cells (n = 4) showing proliferation in the presence of no anti-CD3/anti-CD28 beads (=negative control), in the presence of anti-CD3/anti-CD28 beads (=positive control) and at a Treg to responder cell ratio of 1 + 1 (mean 1.09%), 1 + 5 (mean 2.37%), and 1 + 10 (mean 6.62%), respectively. (D) Amount of suppression in the first generation of divided cells at a Treg to responder cell ratio of 1 + 1 (mean 94.7%), 1 + 5 (mean 88.3%), and 1 + 10 (mean 70.7%).
Figure 5
Figure 5
The regulatory T cell (Treg) drug product is polycloncal and hypomethylated at intron 1 of the FoxP3 locus. (A) Proportion of Treg expressing indicated T cell receptor Vβ subtype after CD25+ cell enrichment at day 0 (n = 4, white bars) and after 21 days of ex vivo expansion (n = 4, filled bars). (B) Percentage hypomethylation at intron 1 of the FoxP3 locus after CD25+ cell enrichment at day 0 (n = 4, white bar) and after 21 days of ex vivo expansion (n = 4, filled bar). NS, not significant.
Figure 6
Figure 6
Markers associated with homing are significantly expressed on the regulatory T cell (Treg) drug product. Proportion of CD25+ cells at day 0 (white bars) and day 21 Treg drug product cells (filled bars) expressing CCR4 (n = 4), PSG-1 (n = 4), CCR9 (n = 4), CCR5 (n = 4), α4β7 (n = 4), CD103 (n = 4), CD62L (n = 4), CXCR3 (n = 4), CCR8 (n = 4), and GPR15 (n = 4). NS, not significant, *P < 0.05.

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