"Velcro" Engineering of High Affinity CD47 Ectodomain as Signal Regulatory Protein α(SIRPα) Antagonists That Enhance Antibody-dependent Cellular Phagocytosis
"Velcro" Engineering of High Affinity CD47 Ectodomain as Signal Regulatory Protein α(SIRPα) Antagonists That Enhance Antibody-dependent Cellular Phagocytosis
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DOI：
10.1074/jbc.m115.648220
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发表时间：
2015-05-15
影响因子：
4.8
通讯作者：
Garcia, K. Christopher
中科院分区：
文献类型：
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作者：
Ho, Chia Chi M.;Guo, Nan;Garcia, K. Christopher
CD47是一种细胞表面蛋白，当与其受体信号调节蛋白α(SIRPα)接触时，它会向巨噬细胞传递反吞噬信号，即“不要吃我”信号。通过与CD47结合而拮抗CD47-SIRPα相互作用的分子，如抗CD47抗体和工程SIRPα变异体CV1，已被证明促进巨噬细胞介导的抗肿瘤反应。然而，这些针对CD47的策略由于CD47的普遍表达而受到体内巨大的抗原沉着和不分青红皂白的细胞结合的阻碍。这些因素降低了生物利用度，增加了毒性风险。在这里，我们提出了一种替代策略，通过设计针对限制组织表达的SIRPα的高亲和力CD47变体来对抗CD47-SIRPα途径。CD47被证明是针对其与SIRPα结合界面的传统亲和成熟技术的折射率。因此，我们开发了一种新的工程方法，通过N-端肽延伸来扩大现有的接触界面，被称为“尼龙搭扣”工程。高亲和力变异体(Velcro-CD47)与两个最重要的人类SIRPα等位基因结合，与野生型CD47相比亲和力大大提高，并有效地拮抗CD47与人巨噬细胞上SIRPα的结合。魔术贴-CD47与肿瘤特异性单抗协同作用，在体外增强巨噬细胞对肿瘤细胞的吞噬作用，其效力与CV1相似。最后，魔术贴-CD47与人类血液中的一部分髓系细胞特异地相互作用，而CV1与所有询问的髓系、淋巴系和红系群体结合。这与SIRPα相对CD47的限制性表达是一致的。在这里，我们证明了“尼龙搭扣”工程是一种强大的蛋白质工程工具，具有潜在的应用于其他系统，并且尼龙搭扣-CD47可以作为CD47靶向制剂的替代佐剂用于癌症免疫治疗。
CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the "don't-eat-me" signal, to macrophages upon engaging its receptor signal regulatory protein alpha (SIRP alpha). Molecules that antagonize the CD47-SIRP alpha interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRP alpha variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRP alpha pathway by engineering high affinity CD47 variants that target SIRP alpha, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRP alpha. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined "Velcro" engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRP alpha alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRP alpha on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRP alpha compared with CD47. Herein, we have demonstrated that "Velcro" engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy.