No abstract

Background: Alemtuzumab is a humanized monoclonal antibody that targets the CD52 glycoprotein expressed on most lymphocytes, subsequently inducing complement-mediated and antibody-mediated cytotoxicity. Owing to its ability to induce profound immune depletion, alemtuzumab is frequently used in patients before allogeneic hematopoietic stem cell transplantation to prevent graft rejection and acute graft-versus-host disease. In this clinical context, a stable immunoassay with high sensitivity and specificity to determine alemtuzumab levels is essential for performing pharmacokinetic and pharmacodynamic analyses; however, the available methods have several limitations. Here, we report the successful development and validation of an efficient and highly sensitive enzyme-linked immunosorbent assay technique based on commercially available reagents to quantify alemtuzumab in human serum or plasma.Methods: This enzyme-linked immunosorbent assay technique was developed and validated in accordance with the European Medicines Agency guidelines on bioanalytical method validation.Results: The assay sensitivity (lower limit of quantification) is 0.5 ng$mL 21 , and the dynamic range is 0.78-25 ng$mL 21 . To accom-modate quantification of peak concentration and concentrations below the lympholytic level (,0.1 mcg$mL 21 ), patients' serum samples were prediluted 20-400 times according to the expected alemtuzumab concentration. The overall within-run accuracy was between 96% and 105%, whereas overall within-run precision (coefficient of variation) was between 3% and 9%. The between-run assessment provided an overall accuracy between 86% and 95% and an overall coefficient of variation between 5% and 14%. Conclusions:The developed assay provides accurate insight into alemtuzumab exposure and its effects on the clinical response to treatment, which is key to optimizing treatment strategies.

Alemtuzumab (anti-CD52 antibody) is frequently prescribed to children with non-malignant diseases undergoing allogeneic hematopoietic stem cell transplantation (HSCT) to prevent graft failure (GF) and acute graft-versus-host disease (aGvHD). This multicenter study aimed at the characterization of alemtuzumab population pharmacokinetics to perform a novel model-based exposure-response analysis in 53 children with non-malignant immunological or hematological disease and a median age of 4.4 years (IQR 0.8, 8.7). Median cumulative alemtuzumab dose was 0.6 mg/kg (IQR 0.6-1) administered over 2-7 days. A 2-compartment population pharmacokinetic model with parallel linear and non-linear elimination including allometrically scaled bodyweight [median 17.50 kg (IQR 8.76, 33.00)] and lymphocyte count at baseline [mean 2.24 10*9/L (SD 1.87)] as significant pharmacokinetic predictors was developed using non-linear mixed effects modelling (NONMEM). According to the model estimated median concentration at day of HSCT (0.77 µg/mL, IQR 0.33-1.82), patients were grouped into a low (</equal 0.77 µg/mL) or high (>0.77 µg/mL) exposure group. High alemtuzumab exposure at day of HSCT correlated with delayed CD4+ and CD8+ T-cell reconstitution (p-value <0.0001) and increased risk of graft failure (p-value=0.043). In contrast, alemtuzumab exposure did not significantly influence the incidence of aGvHD ≥ grade II, mortality, chimerism at 1-year, viral reactivations and autoimmunity at a median follow-up of 3.3 years (IQR 2.5-8.0). In conclusion, this novel population PK model is suitable for individualized intravenous precision dosing to predict alemtuzumab exposure in pediatric allogeneic HSCT for non-malignant diseases, aiming at the achievement of early T-cell reconstitution and prevention of graft failure in future prospective studies.