细胞外囊泡（EV）正在成为治疗性生物制剂递送的有前景的载体。基因工程代表了将感兴趣的蛋白质加载到 EV 中的强大策略。富含 EV 的蛋白质的鉴定可提高蛋白质货物的装载效率。许多富含 EV 的蛋白质通过运输 (ESCRT) 依赖途径所需的内体分选复合物被分选到 EV 中。与此同时，病毒通过保守的晚期结构域基序劫持这种 EV 生物合成机制，以促进宿主细胞的流出。受 EV 和病毒生物发生相似性的启发，我们开发了一种基于 Late 域的合成 EV 支架蛋白，该蛋白能够在 EV 表面展示一组单链可变片段 (scFv)。我们将该支架命名为基于晚期域的外泌体抗体表面展示平台（LEAP）。我们应用LEAP支架对HEK293T细胞衍生的EV进行重编程，通过在EV表面同时展示αPD-L1和αCD3 scFv（表示为αPD-L1×αCD3双特异性T细胞接合外泌体，BiTExos）来引发T细胞抗肿瘤免疫。 ）。我们证明，αPD-L1×αCD3 BiTExos 主动重定向 T 细胞与 PD-L1 肿瘤细胞结合，促进 T 细胞激活、增殖和杀肿瘤细胞因子的产生。此外，αPD-L1×αCD3 BiTExos 促进 T 细胞浸润到肿瘤微环境中，从而减轻体内肿瘤负荷。我们的研究表明，LEAP 支架可以作为 EV 表面显示的平台，并可广泛应用于基于 EV 的生物医学应用。© 2024 作者。 《Journal of Extracellular Vesicles》由 Wiley periodicals LLC 代表国际细胞外囊泡学会出版。

Extracellular vesicles (EVs) are emerging as promising carriers for the delivery of therapeutic biologics. Genetic engineering represents a robust strategy for loading proteins of interest into EVs. Identification of EV-enriched proteins facilitates protein cargo loading efficiency. Many EV-enriched proteins are sorted into EVs via an endosomal sorting complex required for transport (ESCRT)-dependent pathway. In parallel, viruses hijack this EV biosynthesis machinery via conserved late domain motifs to promote egress from host cells. Inspired by the similarity of biogenesis between EVs and viruses, we developed a synthetic, Late domain-based EV scaffold protein that enables the display of a set of single chain variable fragments (scFvs) on the EV surface. We named this scaffold the Late domain-based exosomal antibody surface display platform (LEAP). We applied the LEAP scaffold to reprogramme HEK293T cell-derived EVs to elicit T-cell anti-tumor immunity by simultaneously displaying αPD-L1 and αCD3 scFvs on the EV surface (denoted as αPD-L1×αCD3 bispecific T-cell engaging exosomes, BiTExos). We demonstrated that αPD-L1×αCD3 BiTExos actively redirected T cells to bind to PD-L1+ tumor cells, promoting T-cell activation, proliferation and tumoricidal cytokine production. Furthermore, the αPD-L1×αCD3 BiTExos promoted T-cell infiltration into the tumor microenvironment to mitigate the tumor burden in vivo. Our study suggested that the LEAP scaffold may serve as a platform for EV surface display and could be applied for a broad range of EV-based biomedical applications.© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.