Velcro Engineering of High Affinity CD47 Ectodomain as Signal Regulatory Protein α(SIRPα) Antagonists That Enhance Antibody-dependent Cellular Phagocytosis

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.