Biomarkers assays for T-lymphocyte activation and proliferation applied in a multicenter clinical trial of AEB071 in renal transplantation

Background: AEB071 is a novel protein kinase C inhibitor that blocks the activation of T-lymphocytes.
Material/Methods: In a multicenter phase 2 trial, de novo kidney transplant recipients received AEB071 200 mg twice-daily with basiliximab, tacrolimus, and corticosteroids. We collected blood samples in a subset of 28 patients before transplantation and on day 8 posttransplantation to quantify biomarkers of T-lymphocyte activation and proliferation. The study sponsor provided analytical reagents and trained the site personnel. Within 16 hours of collection, heparinized blood was stimulated with PMA and anti-CD28 for T-cell activation and frozen. For T-cell proliferation, heparinized blood was stimulated with lectin followed by addition of 3H-thymidine and then frozen. Frozen samples were sent to the analyzing laboratory via a central collection agency. T-cell activation was quantified by flow cytometry to yield the percent T-cells double positive for IL2 and TNF (IL2+TNF+). T-cell proliferation was quantified by scintillation counting of thymidine uptake. Data are medians [interquartile range].
Results: Before transplantation, the percent IL2+TNF+ T-cells was 2.0 [0.5-5.9]%. This was reduced by 100% posttransplant to 0 [0-0.1]% (p=0.002). Thymidine uptake was 5.0 [2.9-14.7] cpm - 103 before transplantation. This was reduced by 84% posttransplant to 0.7 [0.4-1.4] cpm - 103 (p=0.003).
Conclusions: Despite the complexity of these assays, they were successfully implemented in the context of an international clinical trial. These biomarkers yielded individualized, quantitative evidence that T-cell activation and proliferation were inhibited by the immunosuppressive regimen and will contribute to the overall interpretation of the study results.

Keywords: resistant bacteria, T-Lymphocytes, Tacrolimus