A Perspective on the CD47-SIRPA Axis in High-Risk Neuroblastoma
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Neuroblastoma is a pediatric cancer with significant clinical heterogeneity. Despite extensive efforts, it is still difficult to cure children with high-risk neuroblastoma. Immunotherapy is a promising approach to treat children with this devastating disease. We have previously reported that macrophages are important effector cells in high-risk neuroblastoma. In this perspective article, we discuss the potential function of the macrophage inhibitory receptor SIRPA in the homeostasis of tumor-associated macrophages in high-risk neuroblastoma. The ligand of SIRPA is CD47, known as a “don’t eat me” signal, which is highly expressed on cancer cells compared to normal cells. CD47 is expressed on both tumor and stroma cells, whereas SIRPA expression is restricted to macrophages in high-risk neuroblastoma tissues. Notably, high SIRPA expression is associated with better disease outcome. According to the current paradigm, the interaction between CD47 on tumor cells and SIRPA on macrophages leads to the inhibition of tumor phagocytosis. However, data from recent clinical trials have called into question the use of anti-CD47 antibodies for the treatment of adult and pediatric cancers. The restricted expression of SIRPA on macrophages in many tissues argues for targeting SIRPA on macrophages rather than CD47 in CD47/SIRPA blockade therapy. Based on the data available to date, we propose that disruption of the CD47-SIRPA interaction by anti-CD47 antibody would shift the macrophage polarization status from M1 to M2, which is inferred from the 1998 study by Timms et al. In contrast, the anti-SIRPA F(ab’)2 lacking Fc binds to SIRPA on the macrophage, mimics the CD47-SIRPA interaction, and thus maintains M1 polarization. Anti-SIRPA F(ab’)2 also prevents the binding of CD47 to SIRPA, thereby blocking the “don’t eat me” signal. The addition of tumor-opsonizing and macrophage-activating antibodies is expected to enhance active tumor phagocytosis.Keywords:
CD47
Abstract Despite the improvement in clinical outcome with 13- cis -retinoic acid (13- cis RA) + anti-GD2 antibody + cytokine immunotherapy given in first response ~40% of high-risk neuroblastoma patients die of recurrent disease. MYCN genomic amplification is a biomarker of aggressive tumors in the childhood cancer neuroblastoma. MYCN expression is downregulated by 13- cis RA, a differentiating agent that is a component of neuroblastoma therapy. Although MYC amplification is rare in neuroblastoma at diagnosis, we report transcriptional activation of MYC medicated by the transcription factor OCT4, functionally replacing MYCN in 13- cis RA-resistant progressive disease neuroblastoma in large panels of patient-derived cell lines and xenograft models. We identified novel OCT4-binding sites in the MYC promoter/enhancer region that regulated MYC expression via phosphorylation by MAPKAPK2 (MK2). OCT4 phosphorylation at the S111 residue by MK2 was upstream of MYC transcriptional activation. Expression of OCT4, MK2, and c-MYC was higher in progressive disease relative to pre-therapy neuroblastomas and was associated with inferior patient survival. OCT4 or MK2 knockdown decreased c-MYC expression and restored the sensitivity to 13- cis RA. In conclusion, we demonstrated that high c-MYC expression independent of genomic amplification is associated with disease progression in neuroblastoma. MK2-mediated OCT4 transcriptional activation is a novel mechanism for activating the MYC oncogene in progressive disease neuroblastoma that provides a therapeutic target.
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CD47
Cancer Immunotherapy
Internalization
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Abstract CD47 binds to SIRPα on the surface of macrophages and delivers a “do not eat” signal that suppresses phagocytosis. There is strong evidence that many liquid and solid tumors exploit the CD47-SIRPα pathway to escape macrophage-mediated destruction. Blockade of CD47 using a soluble SIRPα-Fc fusion protein (SIRPαFc) has emerged as a promising strategy to neutralize the suppressive effects of CD47 and promote the eradication of tumor cells. We have previously reported data demonstrating that human SIRPαFc binds strongly to tumor cells but very poorly to human red blood cells (RBCs), despite abundant surface expression of CD47 on RBCs and strong reactivity with CD47-specific antibodies. Here we expand upon these early findings and assess inter-species differences in RBC binding. Our results, based on a panel of 43 human donors, clearly show that SIRPαFc binds very poorly to human RBCs regardless of gender, ABO blood group or Rh antigen status. Consistent with this finding, SIRPαFc was unable to induce agglutination of RBCs in vitro, although hemagglutination was triggered by CD47-blocking antibodies. Curiously, although the binding affinity of human SIRPαFc to cynomolgus macaque CD47 is approximately 10-fold lower than the binding to the human target, it binds strongly to cyno RBCs. This indicates that affinity alone does not predict the ability to bind erythrocytes. Instead, we hypothesized that the mobility of CD47 in the RBC membrane is a key determinant of SIRPαFc binding and thus compared the detergent solubilization profile of CD47 in human and cyno RBCs. CD47 was observed to segregate largely into the detergent-soluble fraction in monkey erythrocytes but was localized primarily to the insoluble pellet fraction in human RBCs, suggesting greater membrane mobility in cyno compared to human RBCs. This finding is consistent with a model in which CD47 mobility is required to form high affinity clusters with SIRPαFc, and indeed we have previously observed that pre-clustering CD47 with a non-blocking antibody converts human RBCs into strong SIRPαFc binders. Finally, the consequences of cyno RBC binding were assessed in vivo. Significant depletion of RBCs was evident following intravenous infusions of SIRPαFc in cyno monkeys. We speculate that similar anemia is likely to occur in humans treated with CD47-blocking antibodies that bind to human erythrocytes, but not with a low RBC-binding SIRPαFc therapeutic. In conclusion, human SIRPαFc binds very poorly to human RBCs but is highly reactive with cyno erythrocytes. This unusual pattern of species cross-reactivity may result from species-specific differences in CD47 membrane mobility and is consistent with a model in which SIRPαFc binding requires mobile CD47 to form high affinity clusters. We predict that anemia, which occurs in monkeys following SIRPαFc administration, is not likely to occur in human patients where significant RBC binding is absent. Citation Format: Penka S. Petrova, Karen Dodge, Tanya Prasolava, Vien Chai, Xinli Pang, Robert A. Uger. Lack of CD47 membrane mobility contributes to the poor erythrocyte binding of SIRPαFc, a novel CD47-blocking cancer immunotherapeutic. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4271. doi:10.1158/1538-7445.AM2015-4271
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Freshly isolated human red blood cells (RBC) bind to the signal regulatory protein alpha (SIRPα) on macrophages in a CD47-dependent manner (CD47 = integrin-associated protein). This interaction provides a ‘do not eat me signal’ such that these RBC are not phagocytosed and remain in circulation as exemplified for mouse RBC by Oldenborg and collaborators [1, 2]. Recently, Burger et al. have reported [3] and reviewed in this journal [4] that CD47 can function like a switch and then induces rather than inhibits phagocytosis of ‘experimentally aged human RBC’. In reality the studied RBC were oxidatively damaged by treating with CuSO4 and ascorbic acid. The ‘eat me signal’ could be induced by blocking CD47 on oxidized RBC with F(ab')2 anti-CD47. Data from others [5] suggested that thrombospondin-1, known to interact with CD47, may act similarly. Hence, the authors studied whether a particular peptide of thrombospondin-1 (4N1K) promoted the interaction of CD47 with SIRPα. Experiments with human red pulp macrophages and oxidized RBC showed that phagocytosis increased upon addition of the 4N1K decapeptide, but not when supplemented with an irrelevant peptide from thrombospondin-1. The 4N1K peptide was half as effective as blocking CD47 with F(ab’)2 anti-CD47. The F(ab’)2 fragment of the CD47-specific antibody prevented CD47 from interacting with SIRPα and this was sufficient to induce an ‘eat me signal’, because the inhibitory signal could not be induced by SIRPα. The authors think that binding of the 4N1K peptide to oxidized CD47 induces an ‘eat me signal’ not by blocking the interaction with SIRPα, but by conveying to oxidized CD47 the ability to interact in a new, so far unknown way with SIRPα which then induces an ‘eat me signal’. The authors suggest that thrombospondin-1 binding to CD47 can switch the role of CD47 to a promoter of erythrophagocytosis, which may even be responsible for in vivo clearance of aged RBC. This, however, is highly questionable, because the 4N1K peptide of thrombospondin-1 was applied at 3 × 10−5 mol/l, a concentration that exceeds the thrombospondin-1 concentration in plasma by a factor of 103 to 104 [6]. Correspondingly, Head et al. [7] found that the 4N1K peptide at the very same high concentration (50 µg/ml) binds to CD47 without the need to impose a conformational change by e.g. oxidation. Moreover, incubation of RBC with 50 µg/ml 4N1K peptide for 24 h induced phosphatidylserine exposure on these RBC, amounting to an annexin binding that was 5 times higher than in controls and resulted in 40% loss of viable RBC.
The two sets of findings may explain the extra RBC destruction during vaso-occlusive crisis in sickle cell anemia, where local concentrations of thrombospondin may be considerably higher and the plasma concentration is 2–3 times higher than normal [6]. This type of induced RBC destruction is random and does not affect a particular RBC subpopulation. Otherwise the findings of Burger et al. [3, 4] can in no way provide mechanistic details on how aged RBC are selectively cleared in vivo at the end of their life span of 120 days. The CD47/thrombospondin/SIRPα interactions lack the subtleties required to signal a preferential clearance of senescent RBC at a controlled pace. One reason is that about 40% of CD47 are mobile within the plane of the membrane of RBC of any cell age [8]. Hence, oxidative damage, aggregation, and the altered conformation of CD47 are induced in an unrestricted manner. Furthermore, binding of thrombospondin-1 or its active peptide stabilizes the new CD47 conformation at any concentration above a minimal dose having sufficient affinity. Thus, the suggested recognition principle lacks means to selectively tag a particular RBC subpopulation. This becomes evident by comparing the properties of the CD47/thrombospondin/SIRPoα-induced RBC removal with those operating through a naturally occurring antibody (NAb) to band 3 protein [for review see 9]. In this system recognition of senescent or oxidatively stressed RBC depends on bivalent binding of anti-band 3 NAbs to band 3 oligomers, but not to preexisting band 3 dimers. Anti-band 3 NAbs [10] have a low affinity and require that their target is presented in form of oligomers. Cross-linkable band 3 oligomers represent a minute fraction of band 3 protein of 1.5 ± 0.3% on young and 1.9 ± 0.3% on senescent RBC (different at a confidence level of 0.06) despite a million copies of band 3 per cell [11]. Band 3 oligomers are formed upon detachment of band 3 protein from the cytoskeleton via selective phosphorylation [12] and binding of oxidatively generated hemichromes to the cytoplasmic portion of band 3 protein, promoting clusterization [13]. Finally, the few anti-band 3 NAbs associating with oligomerized band 3 protein represent an insufficient number to induce phagocytosis [14]. The low number of firmly bound anti-band 3 NAbs is, however, compensated by a massive deposition of C3b induced by bound anti-band 3 NAbs. The reason is that bound anti-band 3 NAbs have a unique affinity for C3 within their Fab arm [15] and therefore preferentially generate C3b2-IgG complexes in the presence of active complement [16]. C3b2-IgG complexes subsequently stimulate alternative complement pathway C3b deposition because these complexes first bind properdin that greatly enhances factor B binding [17]. This sequence of well controlled processes favors a selective opsonization of in vivo aged and oxidatively stressed RBC and at the same time prevents an excessive opsonization.
CD47
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Transgenic CD47 overexpression is an encouraging approach to ameliorating xenograft rejection and alloresponses to pluripotent stem cells, and the efficacy correlates with the level of CD47 expression. However, CD47, upon ligation, also transmits signals leading to cell dysfunction or death, raising a concern that overexpressing CD47 could be harmful. Here, we unveiled an alternative source of cell surface CD47. We showed that extracellular vesicles, including exosomes, released from normal or tumor cells overexpressing CD47 (transgenic or native) can induce efficient CD47 cross-dressing on pig or human cells. Like the autogenous CD47, CD47 cross-dressed on cell surfaces is capable of interacting with SIRPα to inhibit phagocytosis. However, ligation of the autogenous, but not cross-dressed, CD47 induced cell death. Thus, CD47 cross-dressing provides an alternative source of cell surface CD47 that may elicit its anti-phagocytic function without transmitting harmful signals to the cells. CD47 cross-dressing also suggests a previously unidentified mechanism for tumor-induced immunosuppression. Our findings should help to further optimize the CD47 transgenic approach that may improve outcomes by minimizing the harmful effects of CD47 overexpression.
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CD47
Cancer Immunotherapy
Chemoimmunotherapy
Immune checkpoint
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Abstract CD47 is a ubiquitously expressed transmembrane glycoprotein that plays a complex role in regulation of cell survival and function. We have previously shown that the interspecies incompatibility of CD47 plays an important role in triggering rejection of cellular xenografts by macrophages. However, the role of CD47 in solid organ transplantation remains undetermined. Here, we explored this question in mouse models of heart allotransplantation. We observed that the lack of CD47 in donor hearts had no deleterious effect on graft survival in syngeneic or single MHC class I‐mismatched recipients, in which both wild‐type (WT) and CD47 knockout (CD47 KO) mouse hearts survived long term with no sign of rejection. Paradoxically, elimination of donor CD47 was beneficial for graft survival in signal MHC class II‐ and class I‐ plus class II‐mismatched combinations, in which CD47 KO donor hearts showed significantly improved survival compared to WT donor hearts. Similarly, CD47 KO donor hearts were more resistant than WT hearts to humoral rejection in α1,3‐galactosyltransferase‐deficient mice. Moreover, a significant prolongation of WT allografts was observed in recipient mice treated with antibodies against a CD47 ligand thrombospondin‐1 (TSP1) or with TSP1 deficiency, indicating that TSP1‐CD47 signaling may stimulate vascularized allograft rejection. Thus, unlike cellular transplantation, donor CD47 expression may accelerate the rejection of vascularized allografts.
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