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Dual targeting to immune checkpoints has achieved a better therapeutic efficacy than single targeting due to synergistic extrication of tumour immunity. However, most dual targeting strategies are usually antibody dependent which facing drawbacks of antibodies, such as poor solid tumour penetration and unsatisfied affinity. To meet the challenges, we engineered a cell membrane displaying a fusion protein composed of SIRPa and PD-1 variants, the high-affinity consensus (HAC) of wild-type molecules, and with which prepared nanovesicles (NVs). Through disabling both SIRPa/CD47 and PD-1/PD-L1 signalling, HAC NVs significantly preserved the phagocytosis and antitumour effect ofmacrophages and T cells, respectively. In vivo study revealed that HAC NVs had better tumour penetration than monoclonal antibodies and higher binding affinity to CD47 and PD-L1 on tumour cells compared with the NVs expressing wild-type fusion protein. Exhilaratingly, dual-blockade of CD47 and PD-L1 with HAC NVs exhibited excellent therapeutic efficacy and biosafety. This study provided a novel biomaterial against tumoural immune escape and more importantly an attractive biomimetic technology of protein delivery formulti-targeting therapies. [ABSTRACT FROM AUTHOR]

Blockade of immune checkpoints as a strategy of cancer cells to overcome the immune response has received ample attention in cancer research recently. In particular, expression of PD‐L1 by various cancer cells has become a paradigm in this respect. Delivery of PD‐L1 to its site of action occurs either by local diffusion, or else by transport via small extracellular vesicles (sEVs, commonly referred to as exosomes). Many steps of sEVs formation, their packaging with PD‐L1 and their release into the extracellular space have been studied in detail. The likely dependence of release on Ca2+‐signaling, however, has received little attention. This is surprising, since the intracellular Ca2+‐concentration is known as a prominent regulator of many secretory processes. Here, we report on the roles of three Ca2+‐dependent proteins in regulating release of PD‐L1‐containing sEVs, as well as on the growth of tumors in mouse models. We show that sEVs release in cancer cell lines is Ca2+‐dependent and the knockdown of the gene coding the Ca2+‐channel protein ORAI1 reduces Ca2+‐signals and release of sEVs. Consequently, the T cell response is reinvigorated and tumor progression in mouse models is retarded. Furthermore, analysis of protein expression patterns in samples from human cancer tissue shows that the ORAI1 gene is significantly upregulated. Such upregulation is identified as an unfavorable prognostic factor for survival of patients with non‐small‐cell lung cancer. We show that reduced Ca2+‐signaling after knockdown of ORAI1 gene also compromises the activity of melanophilin and Synaptotagmin‐like protein 2, two proteins, which are important for correct localization of secretory organelles within cancer cells and their transport to sites of exocytosis. Thus, the Ca2+‐channel ORAI1 and Ca2+‐dependent proteins of the secretion pathway emerge as important targets for understanding and manipulating immune checkpoint blockade by PD‐L1. [ABSTRACT FROM AUTHOR]