Biomarkers assays for T-lymphocyte activation and proliferation applied in a multicenter clinical trial of AEB071 in renal transplantation
J. M. Kovarik, K. Budde, J. Steiger, J. M. Grinyo, F. Straube, A. Groenewegen, A. Vitalit, J. Geissler, J. Klupp, M. J. Barten
Ann Transplant 2008; 13(1): 42-42
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 ï¬‚ow 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