PF-07257876是一种旨在治疗某些晚期或转移性实体肿瘤的双特异性抗体。为了支持PF-07257876的临床开发，中和抗体(NAb)测定方案被开发为分层免疫原性测试的一部分。由于PF-07257876同时靶向CD47和PD-L1，确定NAb反应的结构特异性可能提供有关PK、疗效和安全性的额外见解。由于功能性细胞系统的局限性，我们开发了两种基于细胞结合的电化学发光法来检测结构特异性的NAb。虽然这两种NAb测定方法都采用了细胞结合的方法，并且有一些共同的要求，比如对潜在干扰物的敏感性和耐受性，但是每种方法的开发都面临着独特的挑战。在所遇到的障碍中，尽管保持针对CD47的结构特异性而实现药物耐受性反应是一个特别具有挑战性的问题。因此，需要一个样品预处理程序来从潜在干扰物中分离NAb。所开发的样品预处理程序是基于一种磁珠萃取和酸解离(BEAD)方法的。然而，使用标准的BEAD方法结合整个药物来捕获NAb导致了在某些情况下NAb检测的丧失。具体而言，在细胞结合测定中，包含对双特异性抗体两个结合结构域的NAb阳性对照的模拟样品产生了假阴性结果。对标准BEAD方法进行的改进恢复了结构特异性的NAb检测，并在存在高达400 µg/mL的临床相关药物耐受水平的条件下，也有助于测定灵敏度为1 µg/mL。© 2023。作者。

PF-07257876 is a bispecific antibody being developed for the treatment of certain advanced or metastatic solid tumors. To support clinical development of PF-07257876, neutralizing antibody (NAb) assays were developed as part of a tiered immunogenicity testing approach. Because PF-07257876 targets both CD47 and PD-L1, determination of domain specificity of a NAb response may provide additional insight relating to PK, efficacy, and safety. Due to limitations of functional cell systems, two cell-based binding assays were developed using electrochemiluminescence to detect domain-specific NAb. While both NAb assays utilized a cell-based binding approach and shared certain requirements, such as sensitivity and tolerance to potentially interfering substances, the development of each assay faced unique challenges. Among the hurdles encountered, achieving drug tolerance while preserving domain specificity for CD47 proved particularly challenging. Consequently, a sample pretreatment procedure to isolate NAb from potentially interfering substances was necessary. The sample pretreatment procedure developed was based on a bead-extraction and acid dissociation (BEAD) approach. However, the use of the standard BEAD approach with whole drug to capture NAb resulted in loss of NAb detection under certain circumstances. Specifically, mock samples containing a mixture of NAb positive controls against both binding domains of the bispecific antibody produced false-negative results in the cell-based binding assay. An adaptation made to the standard BEAD approach restored domain-specific NAb detection, while also contributing to an assay sensitivity of 1 µg/mL in the presence of a clinically relevant drug tolerance level of up to 400 µg/mL.© 2023. The Author(s).