Macrophage checkpoint blockade: results from initial clinical trials, binding analyses, and CD47-SIRPα structure-function

Abstract

The macrophage checkpoint is an anti-phagocytic interaction between signal regulatory protein alpha (SIRPα) on a macrophage and CD47 on all types of cells - ranging from blood cells to cancer cells. This interaction has emerged over the last decade as a potential co-target in cancer when combined with other anti-cancer agents, with antibodies against CD47 and SIRPα currently in preclinical and clinical development for a variety of hematological and solid malignancies. Monotherapy with CD47 blockade is ineffective in human clinical trials against many tumor types tested to date, except for rare cutaneous and peripheral lymphomas. In contrast, pre-clinical results show efficacy in multiple syngeneic mouse models of cancer, suggesting that many of these tumor models are more immunogenic and likely artificial compared to human tumors. However, combination therapies in humans of anti-CD47 with agents such as the anti-tumor antibody rituximab do show efficacy against liquid tumors (lymphoma) and are promising. Here, we review such trials as well as key interaction and structural features of CD47-SIRPα.

Keywords: CD47; SIRPα; immune checkpoint; phagocytosis.

Figure 1

Phagocytosis is maximized by inhibiting CD47 on ‘self’ cells (the target) or SIRPα on macrophages in combination with antibodies that opsonize the target. CD47 binding to SIRPα signals “don’t eat me” to the macrophage (leftmost). Neither antibody blockade of CD47-SIRPα nor antibody opsonization of a target is sufficient to make target engulfment efficient (middle two), whereas the combination maximizes phagocytosis (rightmost).

Figure 2

Novel re-analysis of TTI-621 binding and phagocytosis data from Ref. [72]. (A) Molecular partition function (ξ) fitting to the hemagglutination data. K₁ and K₂ are the association constants, inversely related to dissociation constants or EC50. Schematic of possible binding states of various CD47 affinity agents is shown for two apposed RBC membranes. Magrolimab (5F9) and BRIC126 both exhibit high hemagglutination and show cross-bridging, which can be fit (5F9: K₁ = 1.2 10⁻² nM⁻¹, K₂ = 0.24 nM⁻¹; BRIC126: K₁ = 6.6 10⁻² nM⁻¹; K₂ = 1.9 nM⁻¹), whereas TTI-621, B6H12, and 2D3 do not. (B) TTI-621 shows non-zero binding to RBCs, which is weaker than anti-CD47 antibodies but consistent with past reports of sub-μM affinity between CD47 and SIRPα [39]. Inset: same data plotted with y-axis on log scale. Note that the plot follows the same color scheme as in (A). (C) TTI-621 binding data show sub-μM affinity for white blood cells, primary tumor samples, and human tumor cell lines. Phagocytosis of the human tumor lines requires less binding for effective phagocytosis. (D) TTI-621 binding affinities do not predict phagocytic efficiency across various cancer cell types. BR.C: breast cancer, AML: acute myeloid leukemia, BCL: B cell lymphoma, MM: multiple myeloma, and TCL: T-cell lymphoma.

Figure 3

Sequence alignment and crystal structures reveal constant contact residues critical for cross-species reactivity and ligand binding for both CD47 and SIRPα. (A) Sequence overlays of CD47 and SIRPα, respectively, reveal conserved residues across different species. Green highlighted residues are conserved relative to human wildtype sequence. Blue highlighted residues are non-conserved mutations relative to human wildtype; however, maintain H-bonding. Red highlights are non-conserved mutations. Porcine CD47 binds human SIRPα and this can be seen from the conservation of most of the contact residues. Based on this, monkey CD47, which shares the same contact residues as human CD47, and dog CD47, which shares the same contact residues as pig CD47, should bind to human SIRPα. Likewise, when comparing SIRPα variants across different species, the conservation of contact residues among the sequences of NOD mice and pig SIRPα with human SIRPα provide some rationale as to why human CD47 interacts with these variants. Based on this, human CD47 should interact with monkey and dog SIRPα. Crystal structures of various (B) CD47 and (C) SIRPα bound inhibitors. For all antibody bound structures, only the first 100 residues in each of the heavy and light chains are shown. CD47 and SIRPα contact residues in each complex are highlighted in red. Inset tables list all contact residues in the respective receptors and how many times each contact residue is involved in binding across the various complexes. (B) PDB codes 2JJS (CD47/SIRPαV2), 5IWL (CD47/magrolimab), 5TZ4 (CD47/B6H12), 4KJY (CD47/FD6), 5TZ2 (CD47/C47B222), and 5TZT (CD47/C47B161). (C) PDB codes 2JJS (SIRPαV2/CD47), 6NMR (SIRPαV1/FAB 119), 4CMM (SIRPαV1/CD47), and 6BIT (SIRPαV1/KWAR23).