Forced revision can still inform - Lessons and Questions from the “original” TWO Study

Kidney transplantation is the preferred treatment for patients with end stage kidney disease but the life-long immunosuppression required remains a source of long-term complications. One approach to minimize or eliminate immunosuppression that has gained traction recently is the use of cellular therapies. The One Study, a multi-center phase 1 trial demonstrated the safety of CD4+Foxp3+ regulatory T cells (Tregs) in the setting of kidney transplantation [1]. But the specifics of which cells to use, how to isolate them and how to administer them are active areas at several centers internationally. In the accompanying manuscript [2], Brook and co-workers describe results from a limited study with lymphodepletion and delayed administration of ex vivo expanded polyclonal Tregs.

The Transplantation Without Over-immunosuppression (TWO) Study [3] was originally designed as a Phase2b randomized control trial of delayed administration of autologous Treg therapy in living donor kidney transplant recipients. Study subjects underwent induction with alemtuzumab (anti-CD52) lymphodepleting therapy followed by maintenance immunosuppression with mycophenolate mofetil (MMF) and tacrolimus. Subjects in the cell therapy arm had progressive MMF reduction starting at 12 weeks post-transplant with full discontinuation of MMF at six months at which time they also received 5-10 x 10⁶ Treg cells/kg; control subjects were maintained on both drugs and received no cell therapy. Provided protocol biopsies done at 38 weeks post-transplant showed no acute rejection, the dose of tacrolimus was reduced from a goal of 5-10ng/dL to 4-6ng/dL in the cell therapy arm. Subjects were followed out to 18 months post-transplant with clinical assessment and immune monitoring performed longitudinally. Unfortunately, the COVID-19 pandemic led to authorities in the United Kingdom to suspend use of alemtuzumab limiting enrollment to a total of only nine subjects, four in the cell therapy arm, three in the control arm and two were withdrawn due to COVID-19 related delays in transplantation. The TWO study was subsequently substantially modified [3], so these seven represent a unique cohort to which there will be no additional subjects.

Overall, lymphodepletion followed by delayed administration of CD4+Foxp3+ Tregs was well tolerated and there were no safety concerns. All three patients were weaned to single drug immunosuppression with “low dose” tacrolimus without evidence of acute rejection. However, despite the lower tacrolimus target post-infusion in the cell thearpy arm, drug levels were not statistically different from those in control subjects. Protocol biopsies carried out at 9 months post-transplant/12 weeks post-infusion demonstrated a lack of cellular infiltrate and minimal tubulointerstitial fibrosis. Notably, 1 patient in each group developed mild clinical CMV disease that was treated with 3 weeks of valganciclovir leading to full resolution; neither group had any subjects with evidence of BK virus nephropathy. Immune monitoring studies showed a trend toward an increase in Treg number and frequency, but only during the first 1-2 weeks post-infusion. While there was transient depletion of B cells following alemtuzumab, naïve and marginal zone B cell numbers were similar between the two groups at the time of cell infusion and at the 72-week endpoint of the study.

The overall findings of safety and a lack of acute rejection are reassuring. An earlier phase 1 study by the Northwestern group showed that Tregs could be safely given 2 months after Alemtuzumab [4] but those patients remained on MMF whereas the current study with Treg infusion delayed until 6 months post-transplant suggests that minimization to tacrolimus monotherapy can be achieved using this approach. Similarly, the STEADFAST study is a phase 1/2a trial of CAR-Treg with delayed administration at three months following rATG induction [5]. Based on data publicly presented but not yet published, this study has had no significant safety concerns at this time. In combination with the current study, there is ever increasing evidence that autologous Treg can be safely administered after lymphodepletion. Regarding infectious complications, unlike the ONE Study where the cell therapy arm had a noticeable absence of CMV viremia [1], one of four patients in the cell therapy arm had CMV reactivation; similar to the frequency of one of the three control subjects. While not a safety signal, and only small numbers of subjects, these data may decrease the optimism surrounding the correlation between cellular therapy and protection from viral reactivation. From a mechanistic stance, the authors provide data for major T and B cell populations including longitudinal Treg frequencies, with no statistical differences between the cell therapy and control groups. Unfortunately, the authors were not able to quantitate changes in donor reactive T cells after the infusion due to technical challenges. With the biopsy tissue obtained as part of the protocol and the group’s growing expertise in spatial profiling, it will be interesting to see high dimensional data from the tissues. Will there be differences in innate or adaptive cell infiltration or signals that could point toward markers consistent with successful immunosuppression tapering? These types of studies combined with planned gene expression studies as well as cytokine and metabolic profiling (per the original protocol provided in the Supplement) are sure to provide the community with invaluable mechanistic insights.

Overall, the conclusions that can be drawn from this study are limited due to the numbers enrolled, the authors should be acknowledged and applauded for disseminating the data on this unique set of patients as it has the potential to guide the planning of new trials involving lymphodepletion and delayed administration of Treg post-kidney transplantation.