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Interactions of CD47 and RhAG and the Rh proteins are visualized between one another and with the cytoskeleton of intact erythrocytes. In a first study, CD47 is labeled with a phycoerythrin (PhE)- tagged antibody, which generates discrete spots that reflect induced clusters of CD47. Rh and RhAG colocalize with each other and to these induced cluste...
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... labeling of erythrocytes at subsaturating concentrations of BRIC126 conjugated with R-phycoerythrin (PhE-BRIC126) leads to discrete surface spots (Figure 1). At the concentrations of PhE-BRIC126 used for these experiments, the average cluster size on normal red cells appears to be 520 130 nm in diameter spaced by 1017 248 nm. ...
Similar publications
CD47-specific antibodies and fusion proteins that block CD47–SIRPα signaling are employed as antitumor agents for several cancers. Here, we investigated the synergistic antitumor effect of simultaneously targeting CD47 and autophagy in non–small cell lung cancer (NSCLC). SIRPαD1-Fc, a novel CD47-targeting fusion protein, was generated and was found...
CD47-targeting immune checkpoint inhibitors have been investigated for immunotherapy of several cancers, glioblastoma, one of the most common tumors in brain, was still a challenge for CD47-targeting therapy. Herein, we reported novel strategies for glioblastoma therapy via blocking CD47-SIRPα by SIRPα-Fc alone or in combination with autophagy inhi...
Thrombospondin is a potent inhibitor of angiogenesis and might therefore be important in controlling tumour growth. TSP interacts with a number of proteases and receptors and in this way inhibits stimulation of angiogenesis. An earlier study showed that thrombospondin is expressed in benign prostatic hyperplasia (BPH) and high-grade prostatic intra...
Membrane rafts may act as platforms for membrane protein signalling. Rafts have also been implicated in the sorting of membrane components during membrane budding. We have studied by fluorescence microscopy cross-linking of ganglioside GM1 in the human erythrocyte membrane, and how membrane proteins CD47 and CD59 distribute in GM1 patched discoid c...
CD47, a "don't eat me" signal for phagocytic cells, is expressed on the surface of all human solid tumor cells. Analysis of patient tumor and matched adjacent normal (nontumor) tissue revealed that CD47 is overexpressed on cancer cells. CD47 mRNA expression levels correlated with a decreased probability of survival for multiple types of cancer. CD4...
Citations
... CD38) with the highest expression on D-negative RBCs. 34 Free drug in patient plasma causes robust agglutination with all RBCs tested and can interfere in ABO typing. 21 Patients receiving anti-CD47 often experience anaemia and thrombocytopenia which increases the possibility that they may require transfusion. ...
The last decade has seen significant growth in the application of DNA‐based methods for extended antigen typing, and the use of gene sequencing to consider variation in blood group genes to guide clinical care. The challenge for the field now lies in educating professionals, expanding accessibility and standardizing the use of genotyping for routine patient care. Here we discuss applications of genotyping when transfusion is not straightforward including when compatibility cannot be demonstrated by routine methods, when Rh type is unclear, when allo‐ and auto‐antibodies are encountered in stem cell and organ transplantation, for prenatal testing to determine maternal and foetal risk for complications, and Group A subtyping for kidney and platelet donors. We summarize current commercial testing resources and new approaches to testing including high‐density arrays and targeted next‐generation sequencing (NGS).
... 21,22 CD47 is highly expressed on RBC membranes and is a member of the Rh complex. [23][24][25] Binding of anti-CD47 to RBCs and free circulating anti-CD47 in the plasma during and after treatment have been reported to interfere with pretransfusion serological tests, including blood typing (ABO and Rh), direct and indirect antiglobulin tests (DAT and IAT, respectively), antigen typing, and platelet antibody tests, depending on the circulating drug level and test method ( Figure 2). 12,26 Here, we review the interference of magrolimab in RBC typing and serological testing and outline potential mitigation strategies. ...
... CD47 is a component of the Rh blood group complex and is linked to the RBC cytoskeleton. 11,25,34 The anti-CD47 therapy magrolimab binds CD47 on RBCs, 12 resulting in transient anemia 21,35 and interference with pretransfusion F I G U R E 1 Mechanism of action of magrolimab. CD47 is an antiphagocytic signal that is overexpressed in multiple cancers, including acute myeloid leukemia, leading to immune evasion. ...
... Key to understanding the relationship between the structure of the MAS and cellular properties is the ability to manipulate the cell mechanically and image the resulting deformation and structural reorganizations that accompany cell deformation. The most advanced studies have combined fluorescence labeling of membrane components and controlled mechanical deformation of the cell (fluorescence imaged microdeformation, FIMD) to gain insights into how specific membrane components are redistributed during deformation (Lee et al., 1999;Picart et al., 2000;Dahl et al., 2003;Huang et al., 2017). Here, we applied this approach to document and quantify the changes in the mechanical stability of the MAS in primary mammalian erythroid cells at different stages of maturation. ...
... Firstly, on the membrane of young RBCs, CD47 is localised in a distinct complex with RhAG, protein 4.2, Band 3 and the cytoskeleton, which prevents CD47 from clustering. 21 When RBCs age, CD47 can bind to thrombospondin (TSP) or TSP-like peptide (4N1K) and thereby colocalise with gangliosides (a component of lipid rafts) and clusters. On the surface of tumour cells, CD47 binds TSP during the whole lifespan, 2,22 creating a distinct binding profile that may enable 'untargeting' of young RBCs. ...
Background:
The CD47-signal regulatory protein alpha (SIRPα) 'don't eat me' signalling axis is perhaps the most prominent innate immune checkpoint to date. However, from initial clinical trials, it is evident that monotherapy with CD47-SIRPα blocking has a limited therapeutic effect at the maximum tolerated dose. Furthermore, treatment is associated with severe side effects, most notably anaemia, that are attributable to the ubiquitous expression of CD47. Nevertheless, promising clinical responses have been reported upon combination with the tumour-targeting antibody rituximab or azacytidine, although toxicity issues still hamper clinical application.
Main body:
Here, we discuss the current state of CD47-SIRPα blocking therapy with a focus on limitations of current strategies, such as depletion of red blood cells. Subsequently, we focus on innovations designed to overcome these limitations. These include novel antibody formats designed to selectively target CD47 on tumour cells as well as tumour-targeted bispecific antibodies with improved selectivity. In addition, the rationale and outcome of combinatorial approaches to improve the therapeutic effect of CD47 blockade are discussed. Such combinations include those with tumour-targeted opsonizing antibodies, systemic therapy, epigenetic drugs, other immunomodulatory T-cell-targeted therapeutics or dual immunomodulatory CD47 bispecific antibodies.
Conclusion:
With these advances in the design of CD47-SIRPα-targeting therapeutic strategies and increasing insight into the mechanism of action of this innate checkpoint, including the role of adaptive immunity, further advances in the clinical application of this checkpoint can be anticipated.
... Band 3 (the anion exchanger) enhances the expression of the Rh antigens in transfected cells, suggesting that band 3 may also be associated with the Rh core complex. 47 The Rh core complex is linked to the membrane skeleton through interactions between CD47 and protein 4.2 48 and through a Rh/RhAG-ankyrin cytoskeleton connection. 49 Rhnull RBCs also have reduced expression of CD47, an integrinassociated protein (IAP) that has wide tissue distribution, binds β3 integrins, and is required for integrin-regulated Ca 2+ entry into endothelial cells. ...
In this chapter, Rhesus (Rh) and Landsteiner‐Wiener (LW) antigens are incorporated together based on a historic serologic connection and evidence that they are physically associated within the red cell membrane. The Rh system is second only to the ABO system in importance in transfusion medicine because Rh antigens, especially D, are highly immunogenic, and antibodies to these antigens cause hemolytic disease of the fetus and newborn and hemolytic transfusion reactions. The Rh proteins exist in the red cell membrane as complexes with several other proteins, with Rh and Rh‐associated glycoprotein serving as the core of the complex. Genotyping is important in the prenatal setting to determine whether the fetus has inherited the paternal antigen to which the mother has a clinically significant antibody. The LW antigens are the original so‐called “Rhesus” antigens shared by humans and the rhesus monkey.
... 26,27 CD47 forms part of the Rh complex in the RBC membrane, and expression level varies in different Rh phenotypes with highest expression on Rh(D)-negative RBCs. 28 Owing to the high expression of CD47 on RBCs, anti-CD47 can cause interference with blood bank testing, potentially leading to excessive workups and delays in obtaining compatible blood products if information regarding anti-CD47 therapy is unknown to the blood bank. A common pretransfusion challenge in samples from non-group O patients receiving anti-CD47 therapy involves ABO reverse typing discrepancy when antibodies in the patient's plasma appear to bind reagent type A and B RBCs. ...
... Key to understanding the relationship between the structure of the MAS and cellular properties is the ability to manipulate the cell mechanically and image the resulting deformation and structural reorganizations that accompany cell deformation. The most advanced studies have combined fluorescence labeling of membrane components and controlled mechanical deformation of the cell (fluorescence imaged microdeformation, FIMD) to gain insights into how specific membrane components are redistributed during deformation (Lee et al., 1999;Picart et al., 2000;Dahl et al., 2003;Huang et al., 2017). Here, we applied this approach to document and quantify the changes in the mechanical stability of the MAS in primary mammalian erythroid cells at different stages of maturation. ...
The combined use of fluorescence labeling and micro-manipulation of red blood cells has proven to be a powerful tool for understanding and characterizing fundamental mechanisms underlying the mechanical behavior of cells. Here we used this approach to study the development of the membrane-associated cytoskeleton (MAS) in primary embryonic erythroid cells. Erythropoiesis comes in two forms in the mammalian embryo, primitive and definitive, characterized by intra- and extra-vascular maturation, respectively. Primitive erythroid precursors in the murine embryo first begin to circulate at embryonic day (E) 8.25 and mature as a semi-synchronous cohort before enucleating between E12.5 and E16.5. Previously, we determined that the major components of the MAS become localized to the membrane between E10.5 and E12.5, and that this localization is associated with an increase in membrane mechanical stability over this same period. The change in mechanical stability was reflected in the creation of MAS-free regions of the membrane at the tips of the projections formed when cells were aspirated into micropipettes. The tendency to form MAS-free regions decreases as primitive erythroid cells continue to mature through E14.5, at least 2 days after all detectable cytoskeletal components are localized to the membrane, indicating continued strengthening of membrane cohesion after membrane localization of cytoskeletal components. Here we demonstrate that the formation of MAS-free regions is the result of a mechanical failure within the MAS, and not the detachment of membrane bilayer from the MAS. Once a “hole” is formed in the MAS, the skeletal network contracts laterally along the aspirated projection to form the MAS-free region. In protein 4.1-null primitive erythroid cells, the tendency to form MAS-free regions is markedly enhanced. Of note, similar MAS-free regions were observed in maturing erythroid cells from human marrow, indicating that similar processes occur in definitive erythroid cells. We conclude that localization of cytoskeletal components to the cell membrane of mammalian erythroid cells during maturation is insufficient by itself to produce a mature MAS, but that subsequent processes are additionally required to strengthen intraskeletal interactions.
... Because CD47 can be heavily glycosylated with five potential NXT/S sequences in its extracellular IgV domain [38] and/or modified by addition of glycosaminoglycans, differing patterns or extents of carbohydrate additions in different cell types could also explain the differential binding of CD47 antibodies [39]. A further explanation for the differential binding of CD47 mAbs to cancer versus normal cells lies in potential differences in surface mobility of CD47 on different cell types [40] or different densities or distribution of CD47 in the lipid rafts [41]. Furthermore, the binding of CD47 mAbs to CD32a on macrophage can play dual roles: inducing FcγR-mediated phagocytosis of cancer cells and as a scaffold introducing CD47-mediated death signals into tumor cells [42]. ...
Much progress has been made in targeting CD47 for cancer immunotherapy in solid tumors (ST) and hematological malignancies. We summarized the CD47-related clinical research and analyzed the research trend both in the USA and in China. As of August 28, 2021, there are a total 23 related therapeutic agents with 46 clinical trials in the NCT registry platform. Among these trials, 29 are in ST, 14 in hematological malignancies and 3 in both solid tumor and hematological malignancy. The ST include gastric cancer, head and neck squamous cell carcinoma and leiomyosarcoma, while the hematological malignancies include non-Hodgkin's lymphoma, acute myeloid leukemia, myelodysplastic syndrome, multiple myeloma and chronic myeloid leukemia. Majority of the CD47-related clinical trials are at the early phases, such as 31 at phase I, 14 at phase II and 1 at phase III in the USA and 9, 6, 1, in China, respectively. The targets and spectrums of mechanism of action include 26 with mono-specific and 20 with bi-specific targets in the USA and 13 with mono-specific and 3 with bi-specific targets in China. The new generation CD47 antibodies have demonstrated promising results, and it is highly hopeful that some candidate agents will emerge and make into clinical application to meet the urgent needs of patients.
... CD47, formerly known as IAP, is expressed ubiquitously on the cell membrane [48]. It is an Ig V-like domain that interacts with various proteins and participates in various biologic processes such as leukocytes motility, platelet activation, and regulation of apoptotic cell clearance [48]. ...
... CD47, formerly known as IAP, is expressed ubiquitously on the cell membrane [48]. It is an Ig V-like domain that interacts with various proteins and participates in various biologic processes such as leukocytes motility, platelet activation, and regulation of apoptotic cell clearance [48]. CD47 is highly expressed in malignancies and is considered to be an adverse clinical prognostic factor [49]. ...
Background: Oral squamous cell carcinoma (OSCC) has a high prevalence and predicted global mortality rate of 67.1%, necessitating better therapeutic strategies. Moreover, the recurrence and resistance of OSCC after chemo/radioresistance remains a major bottleneck for its effective treatment. Molecular targeting is one of the new therapeutic approaches to target cancer. Among a plethora of targetable signaling molecules, PDK1 is currently rising as a potential target for cancer therapy. Its aberrant expression in many malignancies is observed associated with glycolytic re-programming and chemo/radioresistance. Methods: Furthermore, to better understand the role of PDK1 in OSCC, we analyzed tissue samples from 62 patients with OSCC for PDK1 expression. Combining in silico and in vitro analysis approaches, we determined the important association between PDK1/CD47/LDHA expression in OSCC. Next, we analyzed the effect of PDK1 expression and its connection with OSCC orosphere generation and maintenance, as well as the effect of the combination of the PDK1 inhibitor BX795, cisplatin and radiotherapy in targeting it. Results: Immunohistochemical analysis revealed that higher PDK1 expression is associated with a poor prognosis in OSCC. The immunoprecipitation assay indicated PDK1/CD47 binding. PDK1 ligation significantly impaired OSCC orosphere formation and downregulated Sox2, Oct4, and CD133 expression. The combination of BX795 and cisplatin markedly reduced in OSCC cell’s epithelial-mesenchymal transition, implying its synergistic effect. p-PDK1, CD47, Akt, PFKP, PDK3 and LDHA protein expression were significantly reduced, with the strongest inhibition in the combination group. Chemo/radiotherapy together with abrogation of PDK1 inhibits the oncogenic (Akt/CD47) and glycolytic (LDHA/PFKP/PDK3) signaling and, enhanced or sensitizes OSCC to the anticancer drug effect through inducing apoptosis and DNA damage together with metabolic reprogramming. Conclusions: Therefore, the results from our current study may serve as a basis for developing new therapeutic strategies against chemo/radioresistant OSCC.
... components of the Rh complex in the erythrocyte membrane) [1]. Conversely, AO-176 binding may depend on a specific lateral CD47-interacting protein in cancer cells, differences in the surface mobility of CD47 [102], differential densities, or differential partitioning of CD47 into lipid rafts between normal and tumor cells [103]. ...
CD47 is a ubiquitously expressed cell surface glycoprotein that functions as a signaling receptor for thrombospondin-1 and as the counter-receptor for signal regulatory protein-α (SIRPα). Engaging SIRPα on macrophages inhibits phagocytosis, and CD47 thereby serves as a physiological marker of self. However, elevated CD47 expression on some cancer cells also protects tumors from innate immune surveillance and limits adaptive antitumor immunity via inhibitory SIRPα signaling in antigen presenting cells. CD47 also mediates inhibitory thrombospondin-1 signaling in vascular cells, T cells, and NK cells, and blocking inhibitory CD47 signaling on cytotoxic T cells directly increases tumor cell killing. Therefore, CD47 functions as an innate and adaptive immune checkpoint. These findings have led to the development of antibodies and other therapeutic approaches to block CD47 functions in the tumor microenvironment. Preclinical studies in mice demonstrated that blocking CD47 can limit the growth of hematologic malignancies and solid tumors and enhance the efficacy of conventional chemotherapy, radiation therapy, and some targeted cancer therapies. Humanized CD47 antibodies are showing promise in early clinical trials, but side effects related to enhanced phagocytic clearance of circulating blood cells remain a concern. Approaches to circumvent these include antibody preloading strategies, development of antibodies that recognize tumor-specific epitopes of CD47, SIRPα antibodies, and bivalent antibodies that restrict CD47 blockade to specific tumor cells. Preclinical and clinical development of antibodies and related biologics that inhibit CD47/SIRPα signaling are reviewed, including strategies to combine these agents with various conventional and targeted therapeutics to improve patient outcome for various cancers.
