First-in-Human Clinical Trial to Determine the Safety and Potency of Inducible T Regulatory Cells after Allogeneic Hematopoietic Cell Transplantation
Authors: MacMillan Margaret L. , Hippen Keli Lee , McKenna David H. , DeFor Todd E. , Warlick Erica D. , Brunstein Claudio G , Miller Jeffrey S. , Weisdorf Daniel J. , Blazar Bruce R. , Wagner John E. 
Published: October 2019

Abstract
Introduction: Adoptive transfer of thymic derived regulatory T-cells (tTregs) or Treg type I cells that arise in the periphery can ameliorate GVHD and autoimmunity in mice. However, high Treg doses (~1:1 with donor T-cells) are required to reproducibly suppress disease. In the periphery, a population of non-Treg CD4+ T-cells can be induced to acquire a Treg phenotype and function. We defined a GMP-compliant method for selecting, inducing and expanding Tregs from peripheral blood CD4+25- T cells with IL-2, sirolimus and TGFß. Sirolimus/TGFß iTregs are as suppressive as cord blood derived tTregs and the potential yield is 50-fold higher in a 2 week culture period. We hypothesized that inducible regulatory T cells (iTregs) will be effective for acute GVHD (aGVHD) prevention without concomitantly increasing the risk of opportunistic infection and relapse.
Methods: We performed a phase I single center dose escalation study with an extension at the best available dose to determine the tolerability of inducible regulatory T cells (iTregs) given to adult patients undergoing reduced intensity HLA-identical sibling donor peripheral blood stem cell (PBSC) transplantation for high risk malignancy. The study used a fast track design with 1 patient per iTreg dose cohort (3.0 x 106/kg, 3.0 x 107/kg, 3.0 x 108/kg and 10.0 x 108/kg) along with CSA/MMF GVHD prophylaxis. An extension phase then occurred to total 10 patients at the best available dose, but using sirolimus/MMF as sirolimus is permissive of Treg expansion. HLA-identical sibling donors underwent 2 aphereses. On day -14, an unmobilized 15L apheresis was performed to collect cells for iTreg production. On day 0 (and +1 if needed), G-CSF mobilized 15L apheresis collected PBSCs. All patients received cyclophosphamide 50 mg/kg on day -6, fludarabine 30 mg/m2/day on days -6 to -2, and 200 cGy TBI as a single fraction on day -1. The iTregs were administered on day 0 at least 4 hours before the PBSC infusion.
Results: Sixteen patients were enrolled. Two did not receive iTregs as inadequate numbers of cells were produced. 14 patients (7 male,7 female; median age 63 years) received iTregs. One patient each received 3.0 x 106/kg, 3.0 x 107/kg, 3.0 x 108/kg iTregs with a corresponding T effector:iTreg ratio of 86:1, 8:1 and 1:2. As a ratio of 1:1 was considered sufficient to suppress GVHD in mice, dose escalation was halted at 3.0 x 108/kg considering manufacturing costs despite no observed dose limiting toxicities (DLT). After 3 patients received 3.0 x 108/kg with CSA/MMF immunoprophylaxis with no DLTs, 2 patients received 3.0 x 108/kg iTregs using sirolimus/MMF. Both patients developed grade III aGVHD, resulting in suspension of the sirolimus/MMF arm. An additional 7 patients (for a total of 10) received 3.0 x 108/kg iTregs along with CSA/MMF. No severe infusional toxicities occurred in the 14 patients who received iTregs. All achieved neutrophil engraftment at a median of 7 days. Of 10 patients who received 3.0 x 108/kg iTregs using CSA/MMF, 2 developed grade II and 1 grade III aGVHD by day 100. Circulating Foxp3+iTregs were detected early after adoptive transfer, but were not detectable beyond day 28. Higher numbers of iTregs were detected at day 3 in patients who received 3.0 x 108/kg iTregs along with CSA/MMF versus 3.0 x 106/kg or 3.0 x 107/kg (21% +/-3% vs 9% +/- 3% of CD4 cells were Foxp3+CD25+). On day 7, lower numbers were seen in all patients and by day 28 only 3% of CD4+ cells were Foxp3+CD25 in all patients. In a contemporary institutional cohort of 203 adult patients with malignancies receiving the same RIC and GVHD prophylaxis, but without iTregs, the probabilities of grade II-IV aGVHD was 41% (95% CI, 34-48%) and grade III-IV GVHD was 25% (95% CI, 19-31%) suggesting iTreg administration may suppress GVHD to some extent in this small trial (Figure).
Conclusions: HLA-identical sibling donor iTregs were safely administered in the setting of HLA-identical sibling PBSCT. 3 x 108/kg iTreg/kg was the best tested dose to suppress GVHD with CSA/MMF immunoprophylaxis. Sirolimus/MMF was ineffective as GVHD prophylaxis in this iTreg clinical trial. Future studies should test the combination of iTregs and tTregs which synergistically induce tolerance in mice.
Figure. Figure.

Disclosures
MacMillan: Angiocrine: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Consultancy; Equillium: Consultancy. Brunstein:Gamidacell: Research Funding. Weisdorf:FATE: Consultancy; SL Behring: Consultancy; Seattle Genetics: Consultancy; Pharmacyclics: Consultancy; Equillium: Consultancy. Blazar:Kadmon Corporation, LLC: Consultancy, Research Funding. Wagner:Magenta Therapeutics: Consultancy, Research Funding; Novartis: Research Funding.