Therapeutic cell transfer using regulatory T cells (Tregs) in transplant recipients may reduce the need for drug based immunosuppression (IS) and improve long term graft survival. In late 2014 we initiated a Phase 1 trial of autologous, polyclonally expanded Tregs in living donor kidney transplants (KTx) (NCT 02145325, IND 15898). This is a nonrandomized dose-ranging study with 4 tiers of cell dosing (0.5, 1, 5, and 8 x 10E9 cells infused, n=3 subjects/tier). Enrolled subjects underwent a nonmobilized leukopheresis at least 2 weeks pre-KTx. This leukopheresis product was cryopreserved. We have demonstrated the ability to isolate and manufacture phenotypically pure, functional, GMP grade Tregs from cryopreserved pheresis product in support of our IND. Briefly, we enrich for natural Tregs through sequential negative (CD8, CD19) and positive (CD25) immunomagnetic selection (CliniMACS, Miltenyi Biotec). The enriched Tregs undergo a 3 week expansion culture using CD3/CD28 Exp-Act beads (Miltenyi Biotec), IL2, and sirolimus. Resultant cells need to meet the following release criteria: > 70% viable; > 70% CD4+CD25+; < 10% CD8+ and CD19+; <3000 Exp-Act® beads/10E8 cells; endotoxin < 5.0EU/kg; negative aerobic/anaerobic/fungal sterilities, negative mycoplasma /negative gram stain. KTx recipients receive alemtuzumab induction to achieve lymphodepletion (deemed important for the later effectiveness of TRACT therapy) and tacrolimus and mycophenolate based IS. Pts are converted from tacrolimus to sirolimus 30 days post KTx to provide an IS milieu conducive to infused Tregs. Tregs are infused day +60.

9 pts have been enrolled and 5 have received TRACT. We have successfully isolated and expanded Tregs from all pheresis products. All manufactured product has met release criteria. There have been no serious adverse events attributable to TRACT. Protocol biopsies performed one month after TRACT have not shown rejection. No pt has developed DSA. There have been no infectious complications. Preliminary immunophenotypic analysis shows a significant (9-20 fold) increase in % of circulating CD4+CD127-CD25HighFOXP3+ cells in peripheral blood one month post TRACT. Subject recruitment/treatment is ongoing.

Conclusion: TRACT using autologous polyclonally expanded Tregs appears safe. Plans for a Phase 2 trial are underway.

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