Structure of the human marker of self 5-transmembrane receptor CD47.

CD47 is the only 5-transmembrane (5-TM) spanning receptor of the immune system. Its extracellular domain (ECD) is a cell surface marker of self that binds SIRPα and inhibits macrophage phagocytosis, and cancer immuno-therapy approaches in clinical trials are focused on blocking CD47/SIRPα interaction. We present the crystal structure of full length CD47 bound to the function-blocking antibody B6H12. CD47 ECD is tethered to the TM domain via a six-residue peptide linker (114RVVSWF119) that forms an extended loop (SWF loop), with the fundamental role of inserting the side chains of W118 and F119 into the core of CD47 extracellular loop region (ECLR). Using hydrogen-deuterium exchange and molecular dynamics simulations we show that CD47's ECLR architecture, comprised of two extracellular loops and the SWF loop, creates a molecular environment stabilizing the ECD for presentation on the cell surface. These findings provide insights into CD47 immune recognition, signaling and therapeutic intervention.

Therapeutic modulation of phagocytosis in glioblastoma can activate both innate and adaptive antitumour immunity.

Tumour cell phagocytosis by antigen presenting cells (APCs) is critical to the generation of antitumour immunity. However, cancer cells can evade phagocytosis by upregulating anti-phagocytosis molecule CD47. Here, we show that CD47 blockade alone is inefficient in stimulating glioma cell phagocytosis. However, combining CD47 blockade with temozolomide results in a significant pro-phagocytosis effect due to the latter's ability to induce endoplasmic reticulum stress response. Increased tumour cell phagocytosis subsequently enhances antigen cross-presentation and activation of cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) in APCs, resulting in more efficient T cell priming. This bridging of innate and adaptive responses inhibits glioma growth, but also activates immune checkpoint. Sequential administration of an anti-PD1 antibody overcomes this potential adaptive resistance. Together, these findings reveal a dynamic relationship between innate and adaptive immune regulation in tumours and support further investigation of phagocytosis modulation as a strategy to enhance cancer immunotherapy responses.

Targeting CD47 in Sézary syndrome with SIRPαFc.

Sézary syndrome (SS), the leukemic variant of cutaneous T-cell lymphoma, has limited treatment options and rare occurrences of long-term remission, thus warranting research into new treatment approaches. CD47 has emerged as a promising target for multiple tumor types, but its role in SS remains unknown. Here, we show that CD47 is highly expressed on Sézary cells in the peripheral blood and skin, and the high level of CD47 expression correlates with worse overall survival (OS) in patients with SS. We also demonstrate that CD47 expression on Sézary cells is under the influence of interleukin 4 (IL-4), IL-7, and IL-13. Signal regulatory protein αFc (SIRPαFc; TTI-621), a novel CD47 decoy receptor, triggers macrophage-mediated phagocytosis of Sézary cells and, when administered in clinical trial settings, results in significant tumor load reduction. We conclude that inhibition of the CD47-SIRPα signaling pathway has therapeutic benefit for patients with SS. This trial was registered at www.clinicaltrials.gov as #NCT02663518.

Anti-SIRPα antibodies as a potential new tool for cancer immunotherapy.

Tumor cells are thought to evade immune surveillance through interaction with immune cells. Much recent attention has focused on the modification of immune responses as a basis for new cancer treatments. SIRPα is an Ig superfamily protein that inhibits phagocytosis in macrophages upon interaction with its ligand CD47 expressed on the surface of target cells. Here, we show that SIRPα is highly expressed in human renal cell carcinoma and melanoma. Furthermore, an anti-SIRPα Ab that blocks the interaction with CD47 markedly suppressed tumor formation by renal cell carcinoma or melanoma cells in immunocompetent syngeneic mice. This inhibitory effect of the Ab appeared to be mediated by dual mechanisms: direct induction of Ab-dependent cellular phagocytosis of tumor cells by macrophages and blockade of CD47-SIRPα signaling that negatively regulates such phagocytosis. The antitumor effect of the Ab was greatly attenuated by selective depletion not only of macrophages but also of NK cells or CD8+ T cells. In addition, the anti-SIRPα Ab also enhances the inhibitory effects of Abs against CD20 and programmed cell death 1 (PD-1) on tumor formation in mice injected with SIRPα-nonexpressing tumor cells. Anti-SIRPα Abs thus warrant further study as a potential new therapy for a broad range of cancers.

Durable antitumor responses to CD47 blockade require adaptive immune stimulation.

Therapeutic antitumor antibodies treat cancer by mobilizing both innate and adaptive immunity. CD47 is an antiphagocytic ligand exploited by tumor cells to blunt antibody effector functions by transmitting an inhibitory signal through its receptor signal regulatory protein alpha (SIRPα). Interference with the CD47-SIRPα interaction synergizes with tumor-specific monoclonal antibodies to eliminate human tumor xenografts by enhancing macrophage-mediated antibody-dependent cellular phagocytosis (ADCP), but synergy between CD47 blockade and ADCP has yet to be demonstrated in immunocompetent hosts. Here, we show that CD47 blockade alone or in combination with a tumor-specific antibody fails to generate antitumor immunity against syngeneic B16F10 tumors in mice. Durable tumor immunity required programmed death-ligand 1 (PD-L1) blockade in combination with an antitumor antibody, with incorporation of CD47 antagonism substantially improving response rates. Our results highlight an underappreciated contribution of the adaptive immune system to anti-CD47 adjuvant therapy and suggest that targeting both innate and adaptive immune checkpoints can potentiate the vaccinal effect of antitumor antibody therapy.

Molecular Pathways: Activating T Cells after Cancer Cell Phagocytosis from Blockade of CD47 "Don't Eat Me" Signals.

Recent advances with immunotherapy agents for the treatment of cancer have provided remarkable, and in some cases, curative results. Our laboratory has identified CD47 as an important "don't eat me" signal expressed on malignant cells. Blockade of the CD47:SIRP-α axis between tumor cells and innate immune cells (monocytes, macrophages, and dendritic cells) increases tumor cell phagocytosis in both solid tumors (including, but not limited to, bladder, breast, colon, lung, and pancreatic) and hematologic malignancies. These phagocytic innate cells are also professional antigen-presenting cells (APC), providing a link from innate to adaptive antitumor immunity. Preliminary studies have demonstrated that APCs present antigens from phagocytosed tumor cells, causing T-cell activation. Therefore, agents that block the CD47:SIRP-α engagement are attractive therapeutic targets as a monotherapy or in combination with additional immune-modulating agents for activating antitumor T cells in vivo.

Activation of parenchymal CD47 promotes renal ischemia-reperfusion injury.

Ischemia-reperfusion injury (IRI) contributes to decreased allograft function and allograft rejection in transplanted kidneys. Thrombospondin-1 is a stress protein typically secreted in response to hypoxia and the ligand activator for the ubiquitously expressed receptor CD47. The function of activated CD47 in IRI remains completely unknown. Here, we found that both CD47 and its ligand thrombospondin-1 were upregulated after renal IRI in mice. CD47-knockout mice were protected against renal dysfunction and tubular damage, suggesting that the development of IRI requires intact CD47 signaling. Chimeric CD47-knockout mice engrafted with wild-type hematopoietic cells had significantly lower serum creatinine and less tubular damage than wild-type controls after IRI, suggesting that CD47 signaling in parenchymal cells predominantly mediates renal damage. Treatment with a CD47-blocking antibody protected mice from renal dysfunction and tubular damage compared with an isotype control. Taken together, these data imply that CD47 on parenchymal cells promotes injury after renal ischemia and reperfusion. Therefore, CD47 blockade may have therapeutic potential to prevent or suppress ischemia-reperfusion-mediated damage.

Thrombospondin-1/CD47 blockade following ischemia-reperfusion injury is tissue protective.

BACKGROUND: Nitric oxide has prosurvival effects that can limit ischemia-reperfusion injuries. However, the matrix glycoprotein thrombospondin-1 is induced following ischemia-reperfusion injury and limits nitric oxide signaling by engaging its cell surface receptor CD47. In this article, the authors examine whether postinjury blocking of this inhibitory signal can protect from ischemia-reperfusion injury in a rat flap model. METHODS: A total of 40 tissue flaps were created in rats based solely on the deep inferior epigastric vessels. Microvascular clamps were used to create 45 minutes of ischemia time to the flaps. The flaps were then treated using a monoclonal antibody to CD47 or an isotype-matched control immunoglobulin G1 5 or 30 minutes after clamp removal. Twenty-four or 72 hours postoperatively, the necrotic area of the flap was determined, and serum, deep inferior epigastric vessels, and flaps were harvested for analysis from five rats in each respective group. RESULTS: Treatment with a CD47 antibody 5 minutes after reperfusion significantly reduces flap necrosis compared with immunoglobulin G1 control (9 percent versus 43 percent; p < 0.01). The protective effect is even more dramatic when treatment is delayed until 30 minutes after reperfusion (10 percent versus 88 percent for control; p < 0.01). Markers of neutrophil and endothelial cell activation along with total leukocytes are reduced in CD47 antibody-treated flaps, as are tissue malondialdehyde levels. Levels of cyclic guanosine monophosphate are elevated 72 hours postoperatively in the CD47 antibody-treated deep inferior epigastric vessels versus the control flaps. CONCLUSIONS: Therapies targeting the thrombospondin-1 receptor CD47 offer potential for increasing tissue survival in ischemia-reperfusion injuries. The ability to protect when given after ischemia-reperfusion injury enables a broader clinical applicability.

Gene silencing of CD47 and antibody ligation of thrombospondin-1 enhance ischemic tissue survival in a porcine model: implications for human disease.

BACKGROUND: Insufficient tissue perfusion underlies many acute and chronic diseases. Tissue perfusion in turn requires adequate blood flow, determined in large part by the relative state of relaxation or constriction of arterial vessels. Nitric oxide (NO) produced by vascular cells modulates blood flow and tissue perfusion by relaxing and dilating arteries. Recently, we reported that the secreted protein thrombospondin-1 (TSP1), through its cell surface receptor CD47, limits the ability of NO to relax and dilate blood vessels and thus decreases tissue perfusion. In the present study, we tested the hypothesis that blocking TSP1-CD47 signaling increases ischemic tissue survival in random cutaneous porcine flaps. METHODS: Random cutaneous flaps 2 x 10 cm2 were raised in white hairless Yucatan miniature pigs and were treated with a monoclonal antibody to TSP1, an antisense morpholino oligonucleotide to CD47 or control agents and tissue survival assessed. Primary vascular smooth muscle cell cultured from Yucatan pigs were also treated with the same agents +/- and an NO donor (DEA/NO) and cGMP quantified. RESULTS: Antibody blockade of TSP1 or morpholino suppression of CD47 dramatically enhanced survival of random tissue flaps. These responses correlated with increased blood vessel patency and tissue blood flow on vessel injection studies. NO-stimulated cGMP flux in Yucatan vascular smooth muscle cell was abrogated after antibody or morpholino treatment. CONCLUSION: Antibody ligation of TSP1 or antisense morpholino knock down of CD47 greatly increased tissue survival to ischemia. Given the similarity between porcine and human soft tissues these results suggest significant therapeutic potential for people.

Mechanisms of neutrophil transmigration across renal proximal tubular HK-2 cells.

BACKGROUND: Adhesion of intratubular leukocytes to proximal tubules in biopsies of patients with rapidly progressive glomerulonephritis and the appearance of leukocytes in the urine in interstitial nephritis suggest interactions between leukocytes and tubular epithelia in renal diseases. The aim of this study was to investigate the effect of cytokines and endotoxin on leukocyte migration through proximal tubular epithelial cells and also to determine the role of the transmembrane adhesion molecules ICAM-1 and CD47 in this process. METHODS: Experiments determined transepithelial migration (TEM) of PMN (polymorphonuclear) leukocytes through monolayers of HK-2. Expression of ICAM-1 and CD47 was assessed via confocal immunofluorescence, FACS analysis and western blotting. The effect of antibodies against ICAM-1 and CD47 on TEM was examined. Furthermore measurements of cytokine release (IL- 6 and IL-8) were performed. RESULTS: Preincubation of HK-2 cells with either TNFalpha or LPS resulted in stimulation of PMN migration through monolayers of HK-2 cells. There was no preferred direction of transmigration. ICAM-1 was expressed by HK-2 cells and expression was increased after 4 h stimulation with TNFalpha or LPS. Application of ICAM-1 antibodies inhibited TEM. CD47 was expressed in both HK-2 cells and PMN. CD47 antibodies inhibited predominantly basolateral-to-apical TEM. HK-2 cells released IL-8 and IL-6 preferably into the apical compartment. Additionally, we showed that fMLP induced transmigration through monolayers of HK-2 cells was associated with significant increased CD47 expression on PMN cell surfaces. CONCLUSIONS: Inflammatory mediators stimulate TEM of PMN through monolayers of HK-2 cells without a clearly discernible preference of direction. Mechanisms involved in TEM stimulated by cytokines or endotoxin appear to be mainly changes in surface receptor densities of HK-2 cells with ICAM-1 and CD47 playing an essential role.