A Phase I Clinical Trial with Ex Vivo Expanded Recipient Regulatory T cells in Living Donor Kidney Transplants

Abstract

There is considerable interest in therapeutic transfer of regulatory T cells (Tregs) for controlling aberrant immune responses. Initial clinical trials have shown the safety of Tregs in hematopoietic stem cell transplant recipients and subjects with juvenile diabetes. Our hypothesis is that infusion(s) of Tregs may induce transplant tolerance thus avoiding long-term use of toxic immunosuppressive agents that cause increased morbidity/mortality. Towards testing our hypothesis, we conducted a phase I dose escalation safety trial infusing billions of ex vivo expanded recipient polyclonal Tregs into living donor kidney transplant recipients. Despite variability in recipient's renal disease, our expansion protocol produced Tregs which met all release criteria, expressing >98% CD4⁺CD25⁺ with <1% CD8⁺ and CD19⁺ contamination. Our product displayed >80% FOXP3 expression with stable demethylation in the FOXP3 promoter. Functionally, expanded Tregs potently suppressed allogeneic responses and induced the generation of new Tregs in the recipient's allo-responders in vitro. Within recipients, expanded Tregs amplified circulating Treg levels in a sustained manner. Clinically, all doses of Treg therapy tested were safe with no adverse infusion related side effects, infections or rejection events up to two years post-transplant. This study provides the necessary safety data to advance Treg cell therapy to phase II efficacy trials.

Figure 1

Clinical Protocol and subjects in the Phase I safety trial: (A) Outline of the clinical trial design from initiation to one-year follow-up. Although not listed patients were given intravenous corticosteroids of 500 mg pre-operatively, and weaned to 250 mg, 125 mg and 0 mg on post-operative days 1, 2 and 3 respectively. (B) The inclusion and exclusion criteria. The target population was adult recipients of living donor renal allografts that would not require hemodialysis during the first week following renal transplantation. (C) Demographics of living donor kidney transplant recipients receiving expanded autologous Tregs. Number of Tregs received by the Subjects: 1–3: 0.5 × 10⁹, 4–6: 1 × 10⁹, and 7–9: 5 × 10⁹.

Figure 2

Expansion and Profile of nine expanded Treg products: (A) Outline of clinical good manufacturing expansion protocol established at the Mathews Center for Cellular Therapy, Northwestern Memorial Hospital. (B) Absolute cell number of all nine Treg products throughout expansion protocol; black bar represents grand median of all products (n=9). (C) Representative clinical phenotyping of Treg products, specifically CD4⁺CD25⁺FOXP3⁺ expression on post CD25 enrichment (day 0) as well as days 14 and 21 of culture. (D) Represents the average (±SD) of expression of Treg markers CD4; CD25; and FOXP3; also non-Treg markers CD8, CD20, and CD127 throughout expansion protocol (n=9). (E) Heat map of clinical Treg products and controls (day 0) depicting the percentage of methylation within 9 CpG sites of the conserved non-coding sequence 2 of FOXP3 gene in the expanded Tregs (TRK- 01–09; n=4). (F) Average (+SD) of the percent methylation in day 0 control products and day 21 expanded Treg products (n=4). ***p < 0.005, *p < 0.05; GM  =  growth medium (described under Materials).

Figure 3

In process testing of Tregs suppressive function: Suppression assays were performed. (R) denotes recipient PBMC’s; (Sx) denotes irradiated healthy allogeneic PBMC’s (stimulators); (Rx) denotes irradiated responder cells used as a control to keep cell number consistent across groups. (A) Graph shows raw count per minutes (±SD in triplicate cultures) of thymidine incorporation assay after six days of culture (representative). (B) Average (±SD) of percent suppression (n = 9) at varying Treg: Tresp ratios throughout clinical expansion protocol. Percent suppression calculated as described in materials and methods.

Figure 4

Immune monitoring of recipients post Alemtuzumab induction and Treg infusion. Absolute cell numbers (symbols) and grand median (- line) of T, B, and NK, Tregs per uL of blood from pre-transplant to 12 months post-transplant in all nine recipients (n = 9). Note: Alemtuzumab was given on day 0 and 2 of transplant, and Tregs were infused at two months post-transplant.

Figure 5

Immune monitoring for Tregs in recipients post Alemtuzumab induction and Treg infusion. (A) Absolute cell numbers per uL of blood, and (B) percentage of Tregs from pre-transplant to 12 months post-transplant in all nine recipients (n = 9). (C) Fold Change in the percentage of Tregs within all nine recipients post Treg infusion when normalized against the pre-transplant percentages. (D) The controls were 10 renal transplant recipients treated with the same conditioning and immunosuppression but did not receive Tregs.

Figure 6

Recipient’s recall response to various mitogens and antigens: Stimulation indices calculated from the thymidine incorporation assays of recipient PBMCs plus antigen versus recipient PBMCs plus media only. Individual patient responses are displayed as symbols (n=9) and the grand median as –line at indicated time points post-transplant. Pre-Tx = pre-transplant.