Dataset Information

Polymorphisms in the human inhibitory signal-regulatory protein ? do not affect binding to its ligand CD47.

ABSTRACT: CD47 is a widely distributed membrane protein that interacts with signal-regulatory protein ? (SIRP?), an inhibitory receptor on myeloid cells that gives a "don't-eat-me" signal. Manipulation of the interaction is of considerable interest in the immunotherapy of cancer and in xenotransplantation. The amino-terminal ligand binding domain of SIRP? is highly polymorphic in contrast to the single Ig-like domain of CD47. There is confusion as to whether the polymorphisms will affect ligand binding, but this is an important point for this interaction and other paired receptors being considered as targets for therapy. We show by x-ray crystallography that one human SIRP? allele differing in 13 amino acid residues has a very similar binding site and that several different alleles all bind CD47 with similar affinity as expected because the residues are mostly surface-exposed and distant from the binding site. A peptide from the binding site of CD47 has been reported to mimic the CD47 interaction with SIRP?, but we could find no binding. We discuss the possible pitfalls in determining the affinity of weak interactions and also speculate on how SIRP? polymorphisms may have been selected by pathogens and how this may also be true in other paired receptors such as the KIRs.

SUBMITTER: Hatherley D

PROVIDER: S-EPMC3974974 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Polymorphisms in the human inhibitory signal-regulatory protein α do not affect binding to its ligand CD47.

Hatherley Deborah D Lea Susan M SM Johnson Steven S Barclay A Neil AN

The Journal of biological chemistry 20140218 14

CD47 is a widely distributed membrane protein that interacts with signal-regulatory protein α (SIRPα), an inhibitory receptor on myeloid cells that gives a "don't-eat-me" signal. Manipulation of the interaction is of considerable interest in the immunotherapy of cancer and in xenotransplantation. The amino-terminal ligand binding domain of SIRPα is highly polymorphic in contrast to the single Ig-like domain of CD47. There is confusion as to whether the polymorphisms will affect ligand binding, b ...[more]

PMID: 24550402

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Project description: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.

Dataset Information

Polymorphisms in the human inhibitory signal-regulatory protein ? do not affect binding to its ligand CD47.

Publications

Polymorphisms in the human inhibitory signal-regulatory protein α do not affect binding to its ligand CD47.

Similar Datasets

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