Genecradle Therapeutics Inc., Beijing 100176, China.
Clinical Pharmacology Research Center, State Key Laboratory of Complex Severe and Rare Diseases, NMPA Key Laboratory for Clinical Research and Evaluation of Drug, Beijing Key Laboratory of Clinical PK and PD Investigation for Innovative Drugs, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China.
Viruses. 2024 Sep 24;16(10):1512. doi: 10.3390/v16101512.
Anti-AAV neutralizing Abs (NAbs) titer is usually measured by cell-based microneutralization (MN) assay and is crucial for patient screening in AAV-based gene therapy clinical trials. However, achieving uniform operation and comparable results among different laboratories remains challenging. Here, we established a standardized MN assay for anti-AAV9 NAbs in human sera or plasma and transferred the method to the other two research teams. Then, we validated its parameters and tested a set of eight human samples in blind across all laboratories. The end-point titer, defined by a transduction inhibition of 50% (IC), was calculated using curve-fit modelling. A mouse neutralizing monoclonal antibody in human negative serum was used for system quality control (QC), requiring inter-assay titer variation of <4-fold difference or geometric coefficient of variation (%GCV) of <50%. The assay demonstrated a sensitivity of 54 ng/mL and no cross-reactivity to 20 μg/mL anti-AAV8 MoAb. The intra-assay and inter-assay variation for the low positive QC were 7-35% and 22-41%, respectively. The titers of the blind samples showed excellent reproducibility within and among laboratories, with a %GCV of 18-59% and 23-46%, respectively. This study provides a commonly transferrable MN assay for evaluating anti-AAV9 NAbs in humans, supporting its application in clinical trials.
抗 AAV 中和抗体 (NAb) 滴度通常通过基于细胞的微量中和 (MN) 测定来测量，这对于 AAV 为基础的基因治疗临床试验中的患者筛选至关重要。然而，在不同实验室中实现统一操作和可比结果仍然具有挑战性。在这里，我们建立了一种标准化的人血清或血浆中抗 AAV9 NAb 的 MN 测定法，并将该方法转移到另外两个研究小组。然后，我们验证了其参数，并在所有实验室中以盲法测试了一组 8 个人类样本。终点滴度，由转导抑制 50%（IC）定义，使用曲线拟合模型计算。用人阴性血清中的抗 AAV8 中和单克隆抗体进行系统质量控制 (QC)，要求实验内变异<4 倍差异或几何变异系数 (%GCV) <50%。该测定法的灵敏度为 54ng/ml，与 20μg/ml 的抗 AAV8 MoAb 无交叉反应。低阳性 QC 的实验内和实验间变异分别为 7-35%和 22-41%。盲样的滴度在实验室内部和实验室之间具有极好的重现性，%GCV 分别为 18-59%和 23-46%。本研究提供了一种可普遍转移的 MN 测定法，用于评估人类中的抗 AAV9 NAb，支持其在临床试验中的应用。
