Clinical Manufacturing of Regulatory T Cell Products For Adoptive Cell Therapy and Strategies to Improve Therapeutic Efficacy

Figures & data

Table 1. Published manufacturing protocols for regulatory T cells assessed in kidney transplantation phase I/II clinical trials.

Kidney transplantation
Trial ID/Name	Phase/Study status	Ref.	Treg product (name)	Target dose	Isolation method	Expansion/Culture conditions	Activation reagent	Expansion length	Purity or release criteria	Outcome
NCT02129881 (ONE)*	I/II (Completed)	59, 98, 114, 115	polyTreg	1–6 x 10^6/kg	CliniMACS CD8^+ depletion & CD25^+ Enrichment	TexMACS media + 5% HS + 500 IU/mL IL-2 + 100 nM Rapa	MACS GMP ExpAct Treg kit (Miltenyi)	36 days w/ restim d12 and 24	74.7–88.2% CD4^+CD25^+FoxP3^+	No adverse effects in 12 patients.
NCT02088931 (TASK)*	I (Completed)	28	polyTreg	3.2x10^8 total cells	FACS for CD4^+CD25^highCD127^low	X-Vivo 15 + 10% HS + 300 IU/mL IL-2 (day 2)	anti-CD3/28 beads (Invitrogen)	14 days w/ restim d9	93–96.7% FoxP3^+	Infused Tregs were stable and persistent. No adverse events or infusion reactions. Tregs peaked in circulation in the first week and were detectable during first month dropping off at 3 months post infusion.
NCT02145325 (TRACT)*	I (Completed)	29	polyTreg	0.5–5.0 × 10^9 total cells	CliniMACS CD8^+ depletion & CD25^+ Enrichment	TexMACS + 5% HS + 1 ug/mL TGF-B + 1000 IU/mL (on day 0) + 100 ng/mL Rapa (days 0–9)	MACS GMP ExpAct Treg kit (Miltenyi)	21 days w/ restim d7	>80% FoxP3^+	All doses of Tregs were safely infused with no infusion related side effects. No infections or rejection events up to two years post Tx.
NCT02371434 (ONEnTreg13) *	I/II (Completed)	98, 114	polyTreg	0.5–3 x 10^6/kg	CliniMACS CD8^+ depletion & CD25^+ Enrichment	X-Vivo 15 + 10% HS + 500 U/mL IL-2 + 100 nM Rapa & several re-stims	MACS GMP ExpAct Treg kit (Miltenyi)	23 days	>90% CD4+ CD25+ FoxP3+ of total cells	No dose limiting toxicity observed. Treg cell therapy is achievable and safe in living-donor kidney transplant recipients.
NCT02244801 (ONE) (DART)*	I/II (Completed)	89, 98	darTreg	3.0 or 9.0 × 10^8 total cells	FACS for CD4^+CD25^highCD127^low	OpTimizer Medium + GlutaMAX + Penicillin/Streptomycin + 2% HS or X-Vivo15 medium + 10% HS with 300 IU/mL IL-2	sBcs (4:1 sBC:T cell) or GMP anti-CD3/28 beads (1:1 bead:cell)	Restim d9 or d11	Not specified	Part of the ONE study. Treg therapy is safe in living-donor kidney transplant recipients.
NCT02091232 (ONE)*	I/II (Completed)	98	Belatacept-conditioned darTreg	3.0 or 9.0 x 10^8	Magnetic bead–selected Tregs generated by ex vivo belatacept-mediated CSB	PBMCs were co-cultured for 72hrs with an equal number of irradiated kidney donor PBMNCs + Belatacept and restim without CSB	Not specified	Not specified	Not specified	Part of the ONE study. Treg therapy is safe in living-donor kidney transplant recipients.
NCT02711826 (TASK)*	I (Recruiting)	28, 117	polyTreg or darTreg	3.0–5.0 × 10^6 total cells	Not specified	Not specified	Not specified	Not specified	Not specified	n.a.
ISRCTN11038572 (TWO)*	IIB (Recruiting)	24, 59	polyTreg	5–10 x 10^6/kg	CliniMACS CD8^+ depletion & CD25^+ Enrichment	Not specified	anti-CD3/28 GMP-grade expansion beads	Not specified	Not specified	n.a.
NCT03284242*	I/II (Recruiting)	118	polyTreg	Unknown	CliniMACS CD4^+CD25^high Enrichment	TexMACS + 2% HS + 500 IU/mL IL-2 + 100 nM Rapa or Evermolis	MACS GMP ExpAct Treg kit (2:1 bead:cell)	Not specified	Not specified	n.a.

*Details of expansion protocol and data obtained from referenced publication describing preclinical or clinical protocol development. Ref. = References; Treg = regulatory T cell; Foxp3 = forkhead box protein 3; CSB = co-stimulatory blockade; sBc = stimulated B cell; Tx = transplant; dar-Treg = donor alloantigen reactive; polyTreg = polyclonal regulatory T cells; w/ = with; restim = restimulation; n.a. = not applicable; HS = suman serum; KTx = Kidney Transplantation; LTx = Liver Transplantation; HTx = Heat Transplantation.

Table 2. Published manufacturing protocols for regulatory T cells assessed in liver and heart transplantation phase I/II clinical trials.

Liver Transplantation
Trial ID/Name	Phase/Study status	Ref.	Treg product (name)	Target dose	Isolation method	Expansion/Culture conditions	Activation reagent	Expansion length	Purity or release criteria	Outcome
NCT02166177 (ThRIL)*	I (Completed)	33, 45	polyTreg	0.5–6.5 × 10^6 cells/kg	CliniMACS CD4^+CD25^high Enrichment	TexMACS + 5% HS + 500 IU/mL IL-2 (day 4) + 100 nM Rapa	MACS GMP ExpAct Treg kit (Miltenyi)	36 days w/ restim every 10–12 days	Mean 89.2% ±2.61% CD4+ CD25+ FoxP3+	Treg administration was safe and well tolerated. Therapy increased the pool of circulating Tregs and reduced anti-donor T cell responses.
UMIN-000015789*	I (Completed)	85	donor-specific iTreg-enriched	0.34–6.37 × 10^6 cells/kg	Recipient lymphocytes separated by Ficoll-Paque	Lymphocytes co-cultured with irradiated donor PBMCs + 10ug/mL anti-CD80/86 antibodies for 2 weeks in AlyS505N-0 B10 medium and recipient HS + CSB	Not specified	14 days	2.6–16.9% of cell population	Treg infusion was safe and immunosuppression withdrawal achieved in 7/10 patients with no significant adverse events.
NCT02474199 (ARTEMIS)	I/II (Completed with results)	25, 94, 116, 117	darTreg	3.0–5.0 × 10^8 cells	FACS for CD4^+CD25^highCD127^low	Medium + deuterated glucose in co-cultures with donor sBcs	anti-CD3/28 GMP beads for secondary stimulation	16 days	>95% CD4, >60% FOXP3, <5% CD8, <1% CD19 by flow cytometry; >85% viable	Treg infusion was not associated with adverse events. Insufficient number of recipients were treated for assessing efficacy. Four of nine Treg products failed to meet minimal infusible dose.
NCT03577431 (LITTMUS-MGH)	I/II (Recruiting)	25, 33	Alloantigen reactive Treg (arTreg-CSB)	2.5–500 x 10^6	CD4^+CD25^high Treg	Not specified	Not specified	Not specified	Not specified	n.a.
NCT03654040 (LITTMUS-UCSF)	I/II (Recruiting)	25, 116, 117	darTreg	1.0–5.0 × 10^7 total cells	FACS for CD4^+CD25^highCD127^low	Not specified	Not specified	Not specified	Not specified	n.a.
NCT02188719 (deLTa)*	I (Terminated)	25, 89, 116, 117	darTreg	25–960 x 10^6 total cells	FACS for CD4^+CD25^highCD127^low	300 IU/mL IL-2	sBcs (4:1 sBcs:Treg) and anti-CD3/28 beads (1:1 bead:cell)	14 days w/ restim on d9 or d11	≥95% CD4+, ≥60% FOXP3+, ≤5% CD8+, ≤1% CD19+	Enrollment was terminated due to high number of ineligible subjects, slow enrollment, and manufacturing difficulties within the constraints of the funding period.
Heart transplantation
NCT04924491 (THYTECH)*	I/II (Recruiting)	46	polyTreg	1.0–2.0 × 10^7 thyTreg/kg	CliniMACS CD25 Enrichment	TexMACS GMP medium + 600 IU/mL IL-2	T cell TransAct (Miltenyi)	7–10 days	70.10% – 98.41% of xfCD25+ FOXP3+ cells	n.a.

*Details of expansion protocol and data obtained from referenced publication describing preclinical or clinical protocol development. Ref. = References; Treg = regulatory T cell; Foxp3 = forkhead box protein 3; CSB = co-stimulatory blockade; sBc = stimulated B cell; Tx = transplant; dar-Treg = donor alloantigen reactive; polyTreg = polyclonal regulatory T cells; w/ = with; restim = restimulation; n.a. = not applicable; HS = suman serum; KTx = Kidney Transplantation; LTx = Liver Transplantation; HTx = Heat Transplantation.

Figure 1. Overview of clinical manufacturing of regulatory T cell (Treg) products. Tregs can be isolated from peripheral blood, umbilical cord blood, leukapheresis, or G-CSF mobilized peripheral blood by magnetic cell separation and/or flow cytometric sorting using GMP grade closed systems. Isolated Tregs are then ex vivo expanded using anti-CD3/CD28 magnetic expander beads or artificial antigen-presenting cells (K562 64/86 aAPCs) in the presence of interleukin (IL)-2 with or without rapamycin. AlloAg-specific Tregs can be generated by culturing recipient Tregs with donor AlloAg-expressing APCs. Current in vivo and ex vivo strategies are being used to improve Treg ACT persistence and migration. Figure created with BioRender.com.

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