表面暴露的钙网蛋白（CRT）通过向巨噬细胞产生“吃我”信号，成为免疫原性细胞凋亡的关键细胞损伤相关分子模式。针对精准免疫疗法，我们打算用重组CRT以靶向方式在体内人工标记肿瘤细胞。为此，我们构建了一种 CRT 融合蛋白，旨在表面附着 CXCR4 癌细胞，以刺激其免疫破坏。作为 CRT 构建体的靶向配体并驱动其特异性细胞粘附，我们使用了肽 V1，它是 vMIP-II 细胞因子的衍生物和 CXCR4 的拮抗剂。在离子锌的辅助下，模块化蛋白质倾向于自组装为常规的 16 nm 纳米粒子。通过体内和体外实验，我们确定 CRT 本身赋予构建体克服 V1 的细胞靶向能力，而 V1 的细胞靶向能力只是中等。特别是，CRT 在没有进一步内化的情况下通过完全独立于 CXCR4 的途径结合 HeLa 细胞。此外，通过 THP-1 细胞的细胞计数，我们观察到该蛋白质的结合优先于死细胞而不是活细胞，这一事实不能与单纯的人为吸附相关。这些数据是在 CRT 的寡聚特性以及用这种新型细胞表面配体功能化的蛋白质和蛋白质材料的潜在临床适用性的背景下讨论的。版权所有 © 2024。由 Elsevier B.V 出版。

Surface-exposed calreticulin (CRT) serves as a crucial cell damage-associated molecular pattern for immunogenic apoptosis, by generating an "eat me" signal to macrophages. Aiming at precision immunotherapies we intended to artificially label tumoral cells in vivo with a recombinant CRT, in a targeted way. For that, we have constructed a CRT fusion protein intended to surface attach CXCR4+ cancer cells, to stimulate their immunological destruction. As a targeting ligand of the CRT construct and to drive its specific cell adhesion, we used the peptide V1, a derivative of the vMIP-II cytokine and an antagonist of CXCR4. The modular protein tends to self-assemble as regular 16 nm nanoparticles, assisted by ionic Zn. Through both in vivo and in vitro experiments, we have determined that CRT itself confers cell targeting capabilities to the construct overcoming those of V1, that are only moderate. In particular, CRT binds HeLa cells in absence of further internalization, by a route fully independent of CXCR4. Furthermore, by cytometry in THP-1 cells, we observed that the binding of the protein is preferential for dead cells over live cells, a fact that cannot be associated to a mere artefactual adsorption. These data are discussed in the context of the oligomerizing properties of CRT and the potential clinical applicability of proteins and protein materials functionalized with this novel cell surface ligand.Copyright © 2024. Published by Elsevier B.V.