免疫检查点抑制剂 (ICIs) 引发的抗肿瘤作用已经改变了癌症治疗方法。然而，这些治疗引起的严重免疫相关不良事件（irAE）限制了 ICI 的应用。为了克服不良事件，我们开发了一种肿瘤病变选择性 pro-PD-1Ig，它可以被肿瘤中过度表达的蛋白酶激活。我们通过 MMP 底物序列将白蛋白与修饰的 PD-1Ig（以下称为 mutPD-1Ig）的 N 末端进行基因连接，形成 Alb-hinge-mutPD-1Ig。我们证明，未消化的 Alb-hinge-mutPD-1Ig 的结合活性比 mutPD-1Ig 低约 11 倍。然而，IV 型胶原酶的消化将 Alb-hinge-mutPD-1Ig 的结合活性恢复到与天然 mutPD-1Ig 相当的水平。为了提高Alb-mutPD-1Ig的掩蔽效率，我们通过生物信息学工具模拟了多种MMP底物连接体在不同起始位置连接白蛋白和PD-1的效果。我们的验证实验表明 Alb-hinge-mutPD-1Ig 在所有模拟构建体中显示出最佳的掩蔽效率。我们的研究表明，白蛋白可能最适合掩盖其结合基序集中且位于蛋白质 N 末端附近的目标蛋白。© 2023 作者。由美国化学会出版。

The antitumor effects elicited by immune checkpoint inhibitors (ICIs) have transformed cancer treatments. However, severe immune-related adverse events (irAEs) resulting from these treatments have restricted the application of ICIs. To overcome the adverse events, we developed a tumor lesion-selective pro-PD-1Ig that is activated by proteases overexpressed in tumors. We genetically linked albumin to the N-terminus of a modified PD-1Ig (termed mutPD-1Ig hereafter) via an MMP substrate sequence to form Alb-hinge-mutPD-1Ig. We demonstrate that the binding activity of nondigested Alb-hinge-mutPD-1Ig is approximately 11-folds lower than mutPD-1Ig. However, digestion by type IV collagenase restored the binding activity of Alb-hinge-mutPD-1Ig to a level comparable to that of native mutPD-1Ig. In order to enhance the masking efficiency of Alb-mutPD-1Ig, we simulated the effects of diverse MMP substrate linkers for connecting albumin and PD-1 at various starting positions by bioinformatics tools. Our validation experiments indicate Alb-hinge-mutPD-1Ig displayed the best masking efficiency among all simulated constructs. Our study suggests that albumin may be best applicable to mask a target protein whose binding motif is centralized and in the proximity of the N-terminus of the protein.© 2023 The Authors. Published by American Chemical Society.