Foreign particles and cells are cleared from the body by phagocytes that must also recognize and avoid clearance of “self” cells. The membrane protein CD47 is reportedly a “marker of self” in mice that impedes phagocytosis of self by signaling through the phagocyte receptor CD172a. Minimal “Self” peptides were computationally designed from human CD47 and then synthesized and attached to virus-size particles for intravenous injection into mice that express a CD172a variant compatible with hCD47. Self peptides delay macrophage-mediated clearance of nanoparticles, which promotes persistent circulation that enhances dye and drug delivery to tumors. Self-peptide affinity for CD172a is near the optimum measured for human CD172a variants, and Self peptide also potently inhibits nanoparticle uptake mediated by the contractile cytoskeleton. The reductionist approach reveals the importance of human Self peptides and their utility in enhancing drug delivery and imaging.

Phagocytes engulf foreign cells but not ‘self’ in part because self cells express CD47 as a ligand for signal regulatory protein SIRPα, which inhibits phagocytosis. Motivated by reports of upregulation of CD47 on both normal and cancerous stem cells [1] and also by polymorphisms in SIRPα [2], we show here that inhibition of engulfment correlates with affinity of CD47 for SIRPα – but only at low levels of CD47. One common human polymorph of SIRPα is studied and binds more strongly to human-CD47 than to mouse-CD47 (Kd ≈ 0.12 μM and 6.9 μM, respectively) and does not bind sheep red blood cells (RBC) – which are well-established targets of human macrophages; in comparison, a common mouse polymorph of SIRPα binds with similar affinity to human and mouse CD47 (Kd ≈ 0.22 μM). Using immunoglobulin (IgG)-opsonized particles with varying levels of either human- or mouse-CD47, the effective inhibition constants Ki for blocking phagocytosis are then determined with both human- and mouse-derived macrophages. Only human phagocytes show significant differences in man versus mouse Ki's and only at CD47 levels below normal densities for RBCs. While phospo-signaling through human-SIRPα shows similar trends, consistent again with the affinity differences, saturating levels of CD47 (> Ki) can signal and inhibit phagocytosis regardless of man versus mouse. Quantitative analyses here prompt more complete characterizations of both CD47 levels and SIRPα polymorphisms when attempting to study in vivo effects of these key proteins in innate immunity.

The macrophage checkpoint receptor SIRPα signals against phagocytosis by binding CD47 expressed on all cellsincluding macrophages. Here, we found that inhibiting cis interactions between SIRPα and CD47 on the same macrophage increased engulfment ('eating') by approximately the same level as inhibiting trans interactions. Antibody blockade of CD47, as pursued in clinical trials against cancer, was applied separately to human-derived macrophages and to red blood cell (RBC) targets for phagocytosis, and both scenarios produced surprisingly similar increases in RBC engulfment. Blockade of both macrophages and targets resulted in hyper-phagocytosis, and knockdown of macrophage-CD47 likewise increased engulfment of 'foreign' cells and particles, decreased the baseline inhibitory signaling of SIRPα, and linearly increased binding of soluble CD47 in trans, consistent with cis-trans competition. Many cell types express both SIRPα and CD47, including mouse melanoma B16 cells, and CRISPR-mediated deletions modulate B16 phagocytosis, consistent with cis-trans competition. Additionally, soluble SIRPα binding to human CD47 displayed on Chinese hamster ovary (CHO) cells was suppressed by SIRPα co-display, and atomistic computations confirm SIRPα bends and binds CD47 in cis. Safety and efficacy profiles for CD47-SIRPα blockade might therefore reflect a disruption of both cis and trans interactions.

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