title: "Velcro" engineering of high affinity CD47 ectodomain as signal regulatory protein α (SIRPα) antagonists that enhance antibody-dependent cellular phagocytosis.
abstract: 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 α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα 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-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. 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 SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα 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 SIRPα 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.
authors.affiliation: From the Department of Bioengineering, Stanford University School of Engineering, Departments of Molecular and Cellular Physiology, Pathology, and Structural Biology, Institute for Stem Cell Biology and Regenerative Medicine, and.
authors.forename: Chia Chi M
authors.initials: CC
authors.lastname: Ho
authors.affiliation: Pathology, and Institute for Stem Cell Biology and Regenerative Medicine, and.
authors.forename: Nan
authors.initials: N
authors.lastname: Guo
authors.affiliation: Departments of Molecular and Cellular Physiology, Structural Biology.
authors.forename: Jonathan T
authors.initials: JT
authors.lastname: Sockolosky
authors.affiliation: Departments of Molecular and Cellular Physiology, Structural Biology.
authors.forename: Aaron M
authors.initials: AM
authors.lastname: Ring
authors.affiliation: Pathology, and Institute for Stem Cell Biology and Regenerative Medicine, and.
authors.forename: Kipp
authors.initials: K
authors.lastname: Weiskopf
authors.affiliation: Departments of Molecular and Cellular Physiology, Structural Biology, Howard Hughes Medical Institute.
authors.forename: Engin
authors.initials: E
authors.lastname: Özkan
authors.affiliation: Pathology, and Institute for Stem Cell Biology and Regenerative Medicine, and Stanford Cancer Institute, Stanford University School of Medicine, Stanford, California 94305.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Mori
authors.affiliation: Pathology, and Institute for Stem Cell Biology and Regenerative Medicine, and Stanford Cancer Institute, Stanford University School of Medicine, Stanford, California 94305 Ludwig Center for Cancer Stem Cell Research and Medicine.
authors.forename: Irving L
authors.initials: IL
authors.lastname: Weissman
authors.affiliation: Departments of Molecular and Cellular Physiology, Structural Biology, Howard Hughes Medical Institute, kcgarcia@stanford.edu.
authors.forename: K Christopher
authors.initials: KC
authors.lastname: Garcia
mesh_terms: D000818:Animals; D000906:Antibodies; D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology; D051928:CD47 Antigen / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D045744:Cell Line, Tumor; D006801:Humans; D007167:Immunotherapy; D008264:Macrophages / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D009369:Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D010587:Phagocytosis / Q000187:drug effects*; D011485:Protein Binding; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011971:Receptors, Immunologic / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D000906:Antibodies; D000943:Antigens, Differentiation; D000970:Antineoplastic Agents; D051928:CD47 Antigen; C497894:CD47 protein, human; C497895:Cd47 protein, mouse; C501922:Sirpa protein, mouse; D011971:Receptors, Immunologic; C501920:SIRPA protein, human
keywords: CD47; SIRPα; cancer therapy; directed evolution; don't-eat-me signal; immunotherapy; phagocytosis; protein engineering
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