Journal: Nature

Article Title: CD47 blocking antibodies restore phagocytosis and prevent atherosclerosis

doi: 10.1038/nature18935

Figure Lengend Snippet: ( a ). Cytoscape network visualization of the genes which are significantly correlated with CD47 expression in both human and murine atherosclerotic plaque reveals a high number of TNF-α-related factors (indicated in blue), including ligands, receptors, and downstream signaling factors. ( b ). PANTHER pathway analysis of those genes which were (a) significantly associated with CD47 expression in mouse and human vascular tissue and (b) have been previously associated with atherosclerosis through the STAGE study , identifies “ inflammation mediated by chemokine and cytokine signaling pathway ” as the most over-abundant pathway associated with CD47 expression in vascular tissue. ( c ). Using the Hybrid Mouse Diversity Panel (HMDP), which correlates aortic gene expression with Luminex cytokine array data of plasma samples from over 100 inbred strains of mice, we found that vascular CD47 expression is positively correlated with three inflammatory cytokines in vivo, including TNF-α, IL-2 and CXCL1. Correlation data shown for CD47 and TNF-α. ( d ). Co-expression studies confirm that TNF-α and CD47 expression are positively correlated in human carotid endarterectomy samples from the BiKE validation study. The Pearson correlation coefficient was determined assuming a Gaussian distribution and P values were determined using a two-tailed test. ( e ). Experiments with primarily cultured mouse aortic SMCs indicate that TNF-α reproducibly induces CD47 mRNA upregulation, while a number of other classical pro-atherosclerotic stimuli have no significant effect. Notably, CXCL1, IL4, TGFβ and IL-2 fail to induce CD47 expression in vitro, as assessed by ANOVA. ( f ). Additional studies suggest that the effect of TNF-α on CD47 expression persists in the presence of oxidized LDL, as occurs in the atherosclerotic plaque. ( g ). Western blotting confirms that TNF-α induces CD47 expression in vascular cells at the protein level. For gel source data, see . ( h ). Immunocytochemistry studies of HCASMCs confirm that CD47 expression is induced on the cell surface of TNF-α treated cells. TNF-α effect is assessed by co-staining for HMGB1, and antibody specificity is confirmed with isotype control and recombinant CD47 peptide quenching assays. ( i ). Multiple assays (including FACS, Taqman and immunocytochemistry studies) reveal that CD47 expression is downregulated on vascular SMCs during programmed cell death, as has previously been observed with inflammatory cells. ( j ). Confirmatory assays in cultured human coronary artery SMC reveal that TNF-α induces changes similar to those observed in murine cells , including an induction of CD47 under physiological conditions and a blunting of its expected downregulation during apoptosis. ( k ). TNF-α’s capacity to impair CD47 downregulation during programmed cell death is also observed in mouse SMCs simultaneously exposed to pro-apoptotic stimuli and oxidized LDL. ( l ). No correlation between CD47 and other candidate cytokines was observed in the BiKE biobank, further supporting a specific relationship between CD47 and TNF-α. ( m ). Representative FACS-based apoptosis panels from cells exposed to the conditions used in confirm that TNF-α suppresses efferocytosis despite increasing programmed cell death. Comparisons made by two-tailed t tests, unless otherwise specified. *** = P < 0.001, * = P < 0.05. Error bars represent the SEM.

Article Snippet: In some experiments, membranes loaded with protein were incubated with anti-CD47 Ab that had been preabsorbed with CD47 peptide (R&D systems, 1866-CD, 1:5 dilution) for 16 hours, to determine the specificity of the primary Ab.

Techniques: Expressing, Gene Expression, Luminex, Clinical Proteomics, In Vivo, Biomarker Discovery, Two Tailed Test, Cell Culture, In Vitro, Western Blot, Immunocytochemistry, Staining, Control, Recombinant

Journal: Nature

Article Title: CD47 blocking antibodies restore phagocytosis and prevent atherosclerosis

doi: 10.1038/nature18935

Figure Lengend Snippet: In vivo serological data and additional in silico and bioinformatic data ( a ). Complete serological studies (including blood count, liver function studies, basic metabolic panel, and fasting glucose) from the 4 week apoE −/− -AngII atherosclerosis model indicate that anti-CD47 Ab induces a significant reduction in hemoglobin and compensatory reticulocytosis, consistent with prior reports 4 , 7 . The erythrophagocytosis of senescent RBCs appears to be self-limited, and no anemia was observed in the chronic atherosclerosis model or the reduced dose model (P = 0.54 and 0.57, respectively). No significant difference in any other serum marker is observed except for an increase in serum creatinine, which does not deviate outside of the reference range. Metabolic parameters and leukocyte differential data from the 12 week chronic atherosclerosis model are displayed at the bottom of the table. ( b ). Additional Upstream Regulator Analysis (URA) bioinformatic analyses of the Cytoscape data displayed in Extended Data Figure 7a performed within the Ingenuity Pathway Analysis (IPA) software identifies a number of TNF-α related factors (indicated in red) which are predicted to mediate transcriptional regulatory roles in the gene network shown in that panel. P-values were determined from Fisher’s Exact Test by comparing overlap of co-expressed genes with known upstream regulators from the Ingenuity Knowledge Base. ( c ). Several additional DAVID -based bioinformatics analyses including ( KEGG , SMART , PANTHER and GO analyses) confirm the association between CD47 and inflammatory signaling related to the TNF-α pathway (indicated in red). Blue panels indicate the –logp10 value for each identified factor. ( d ). Transcription factor binding site prediction algorithms identify several putative NFKB family binding sites within the CD47 promoter, as displayed in Extended Data Figure 8a . ( d ). List of primers used in this study.

Article Snippet: In some experiments, membranes loaded with protein were incubated with anti-CD47 Ab that had been preabsorbed with CD47 peptide (R&D systems, 1866-CD, 1:5 dilution) for 16 hours, to determine the specificity of the primary Ab.

Techniques: In Vivo, In Silico, Marker, Software, Binding Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Combinatorial Analysis of AT-Rich Interaction Domain 1A and CD47 in Gastric Cancer Patients Reveals Markers of Prognosis

doi: 10.3389/fcell.2021.745120

Figure Lengend Snippet: Expression of ARID1A and CD47 and their relationship in GC tissues. (A,B) Representative IHC images of ARID1A staining, ARID1A-preserved expression (A) , ARID1A-loss expression (B) . (C,D) Representative IHC images of CD47 staining, CD47 high expression (C) , CD47 low expression (D) . (E) Relationship between loss/preserved expression of ARID1A and CD47 expression ( n = 154). (F) Relationship between mutated/wild-type expression of ARD1A and CD47 expression (data from TCGA, n = 375). * p < 0.05, *** p < 0.001.

Article Snippet: In brief, equal amounts of cells were harvested, were Fc-blocked, and were incubated with anti-CD47 antibody (PE) (cat. 12283-MM07-P; SinoBiological) or isotype control for 30 min at room temperature.

Techniques: Expressing, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Combinatorial Analysis of AT-Rich Interaction Domain 1A and CD47 in Gastric Cancer Patients Reveals Markers of Prognosis

doi: 10.3389/fcell.2021.745120

Figure Lengend Snippet: The correlation between ARID1A status and CD47 expression and clinicopathological characteristics.

Article Snippet: In brief, equal amounts of cells were harvested, were Fc-blocked, and were incubated with anti-CD47 antibody (PE) (cat. 12283-MM07-P; SinoBiological) or isotype control for 30 min at room temperature.

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Combinatorial Analysis of AT-Rich Interaction Domain 1A and CD47 in Gastric Cancer Patients Reveals Markers of Prognosis

doi: 10.3389/fcell.2021.745120

Figure Lengend Snippet: Loss of ARID1A increases CD47 expression. (A,B) Protein and mRNA expression levels of ARID1A and CD47 among various GC cell lines. (C–E) Knockdown of ARID1A significantly increased total and membranous CD47 protein levels, and CD47 mRNA level measured by western blot, flow cytometry and qRT-PCR in GC cell lines. (F) Schematic illustration of the relative positions of qPCR probes to putative CD47 promoter region for ChIP-qPCR experiments. (G) ARID1A binding to the promoter region of CD47 was determined by ChIP-qPCR in BGC-823 and MKN28 cells, respectively. *** p < 0.001, **** p < 0.0001.

Article Snippet: In brief, equal amounts of cells were harvested, were Fc-blocked, and were incubated with anti-CD47 antibody (PE) (cat. 12283-MM07-P; SinoBiological) or isotype control for 30 min at room temperature.

Techniques: Expressing, Western Blot, Flow Cytometry, Quantitative RT-PCR, Binding Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Combinatorial Analysis of AT-Rich Interaction Domain 1A and CD47 in Gastric Cancer Patients Reveals Markers of Prognosis

doi: 10.3389/fcell.2021.745120

Figure Lengend Snippet: Kaplan–Meier curves under various conditions. (A,B) Survival curves according to ARID1A status or CD47 expression ( n = 154). (C) Survival curves according to ARID1A status combined with CD47 expression level ( n = 154). (D–G) Survival curves according to ARID1A status or CD47 expression with HER2 positive or negative.

Article Snippet: In brief, equal amounts of cells were harvested, were Fc-blocked, and were incubated with anti-CD47 antibody (PE) (cat. 12283-MM07-P; SinoBiological) or isotype control for 30 min at room temperature.

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Combinatorial Analysis of AT-Rich Interaction Domain 1A and CD47 in Gastric Cancer Patients Reveals Markers of Prognosis

doi: 10.3389/fcell.2021.745120

Figure Lengend Snippet: Characterization of tumor-infiltrating immune cells based on ARID1A and CD47 expression. (A) Estimation of tumor-infiltrating immune cells in different subgroups stratified by ARID1A status and CD47 expression. (B) Multiplex immunofluorescent images illustrate GC tissues with ARID1A loss CD47 high or ARID1A preserved CD47 low expression that express CD11c, CD163, and Foxp3. (C) Quantitative analysis of the densities of immune markers between ARID1A loss CD47 high and ARID1A preserved CD47 low GC tissues. Error bars represent mean with SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: In brief, equal amounts of cells were harvested, were Fc-blocked, and were incubated with anti-CD47 antibody (PE) (cat. 12283-MM07-P; SinoBiological) or isotype control for 30 min at room temperature.

Techniques: Expressing, Multiplex Assay

Journal: Molecular Cancer Therapeutics

Article Title: Preclinical Development of SGN-CD47M: Protease-Activated Antibody Technology Enables Selective Tumor Targeting of the Innate Immune Checkpoint Receptor CD47

doi: 10.1158/1535-7163.MCT-24-0371

Figure Lengend Snippet: A protease-activated antibody approach using coiled-coil masking peptides improves the PK and tolerability of an antibody directed to mouse CD47. A, Masking impaired the binding of mIAP301 to mouse CD47 on HT-2 cells but could be restored upon cleavage with recombinant MMP. Symbols represent duplicate samples shown as mean ± SD. A, Decreased binding resulted in impaired RBC phagocytosis ( B ) in vitro . Representative data from three independent experiments. Symbols represent the mean ± SD from three biological replicates. *, P < 0.05; ***, P < 0.001 (one-way ANOVA followed by the Dunnett multiple comparisons test) compared with mIAP301. C, PK profiles of unmasked mIAP301 and Coil-MMP-mIAP301 in naïve BALB/c mice, which demonstrates strikingly different plasma exposure profiles because of avoidance of the large normal tissue sink for CD47. The dotted line denotes the level of quantitation in the experiment. It should be noted that the concentration of mIAP301 was only at or above the level of quantitation at the 15-minute time point. Symbols indicate n = 3 replicates and are shown as the mean ± SD. LOQ, level of quantitation. D , Antitumor activity of masked and unmasked mIAP301 antibodies was assessed in an A20 lymphoma model in BALB/c mice and showed similar antitumor activity (every two days for 8 cycles i.p. dosing). Error bars represent mean ± SD ( n = 6 mice per group). Statistical assessment for pairwise comparisons of tumor growth inhibition between treatment arms is provided in Supplementary Fig. S3. In tumor-bearing mice, masking decreased RBC depletion ( E ), and a comparison of bound antibody on RBCs vs. tumor cells provides evidence for tumor-specific mask activation ( F ). Data shown in E and F , are representative of two individual experiments. Symbols represent three biological replicates and are shown as the mean ± SD. *, P < 0.05; ***, P < 0.01; ***, P < 0.001 (one-way ANOVA followed by the Tukey multiple comparisons test) compared with mIAP301.

Article Snippet: Saturation binding ELISA experiments were performed using soluble recombinant human, mouse, or cyno CD47 (ACROBiosystems), which was diluted to an appropriate concentration in 50 mmol/L carbonate buffer at pH 9.6.

Techniques: Binding Assay, Recombinant, In Vitro, Clinical Proteomics, Quantitation Assay, Concentration Assay, Activity Assay, Inhibition, Comparison, Activation Assay

Journal: Molecular Cancer Therapeutics

Article Title: Preclinical Development of SGN-CD47M: Protease-Activated Antibody Technology Enables Selective Tumor Targeting of the Innate Immune Checkpoint Receptor CD47

doi: 10.1158/1535-7163.MCT-24-0371

Figure Lengend Snippet: SGN-CD47M has impaired binding and in vitro activity that can be restored upon mask cleavage with MMPs. Unmasked anti-CD47 antibody hB6H12.3 shows binding to SW780 pancreatic tumor cells ( A ) and human RBCs ( B ), whereas SGN-CD47M shows negligible binding. Representative data from three independent experiments. Symbols represent duplicate samples shown as mean ± SD. SGN-CD47M that had been pretreated with MMPs is able to bind at levels comparable with hB6H12.3. The ability of hB6H12.3, SGN-CD47M, and MMP-cleaved SGN-CD47M to elicit macrophage-mediated phagocytosis against Jurkat T-cell lymphoma cells and human RBCs was evaluated and demonstrated that masking can impair in vitro function that can be largely restored upon mask cleavage. Representative data from two independent experiments. Symbols indicate n = 6 biological replicates and are represented as the mean ± SD.

Article Snippet: Saturation binding ELISA experiments were performed using soluble recombinant human, mouse, or cyno CD47 (ACROBiosystems), which was diluted to an appropriate concentration in 50 mmol/L carbonate buffer at pH 9.6.

Techniques: Binding Assay, In Vitro, Activity Assay

Journal: Molecular Cancer Therapeutics

Article Title: Preclinical Development of SGN-CD47M: Protease-Activated Antibody Technology Enables Selective Tumor Targeting of the Innate Immune Checkpoint Receptor CD47

doi: 10.1158/1535-7163.MCT-24-0371

Figure Lengend Snippet: Intratumoral mask cleavage of SGN-CD47M leads to tumor cell binding and induction of tumor cell apoptosis in HT1080 xenograft tumors. A, SGN-CD47M mask cleavage was detected in plasma, spleen, and tumors of HT1080 tumor-bearing mice using Western blot analysis. Time-dependent preferential mask cleavage was observed in the tumor compared with plasma and spleen. Data are representative of three independent experiments. Intratumoral mask cleavage leads to tumor cell binding ( B ), as assessed by flow cytometry analysis of bound human antibody on dissociated tumor cells. C, Subsequent staining of a noncompeting anti-CD47 antibody demonstrates that hB6H12.3 and activated SGN-CD47M binding leads to a decrease in available CD47 receptors. D, Engagement of CD47 shows a trend for increased tumor cell apoptosis for hB6H12.3 and SGN-CD47M based on annexin V staining of dissociated tumor cells by flow cytometry. For B–D , the data are presented from a single independent experiment. For all panels, each experimental arm consisted of three biological replicates from individual mice and data presented are mean ± SEM. *, P < 0.05; ***, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (one-way ANOVA followed by the Tukey multiple comparisons test) compared with hB6H12.3. MFI, mean fluorescent index.

Article Snippet: Saturation binding ELISA experiments were performed using soluble recombinant human, mouse, or cyno CD47 (ACROBiosystems), which was diluted to an appropriate concentration in 50 mmol/L carbonate buffer at pH 9.6.

Techniques: Binding Assay, Clinical Proteomics, Western Blot, Flow Cytometry, Staining

Journal: Molecular Cancer Therapeutics

Article Title: Preclinical Development of SGN-CD47M: Protease-Activated Antibody Technology Enables Selective Tumor Targeting of the Innate Immune Checkpoint Receptor CD47

doi: 10.1158/1535-7163.MCT-24-0371

Figure Lengend Snippet: SGN-CD47M shows improved tolerability in nonhuman primates because of decreased systemic CD47 engagement. A, The extent of decrease in red cell mass (measured by hematocrit) was similar to animals receiving 20 mg/kg SGN-CD47M or 1 mg/kg hB6H12.3. Treatment at these doses resulted in about 65% decrease in red cell mass at the nadir that trended toward recovery by study day 22, and a dose of 10 mg/kg of SGN-CD47M resulted in about 44% decrease in red cell mass. Assessment of antibody concentrations in plasma ( B ) and blood pellets ( C ) provides supporting data indicating that SGN-CD47M remains masked in vivo . D, The production of cytokines such as MCP-1 for SGN-CD47M and hB6H12.3 was similar despite a 20-fold higher dose. For all panels, symbols represent two biological replicates from individual animals, shown as the mean ± SD. MCP-1, monocyte chemoattractant protein 1.

Article Snippet: Saturation binding ELISA experiments were performed using soluble recombinant human, mouse, or cyno CD47 (ACROBiosystems), which was diluted to an appropriate concentration in 50 mmol/L carbonate buffer at pH 9.6.

Techniques: Clinical Proteomics, In Vivo

Journal: Molecular Cancer Therapeutics

Article Title: Preclinical Development of SGN-CD47M: Protease-Activated Antibody Technology Enables Selective Tumor Targeting of the Innate Immune Checkpoint Receptor CD47

doi: 10.1158/1535-7163.MCT-24-0371

Figure Lengend Snippet: SGN-CD47M is stable in human cancer whole blood and plasma. Binding of FITC-labeled hB6H12.3 and SGN-CD47M to whole blood of human patients with cancer was assessed by flow cytometry after 20 hours incubation at 37°C ( A ). SGN-CD47M stability in plasma samples of human patients with cancer was assessed using a CD47-binding ELISA ( B ) after 96 hours of incubation at 37°C. At the concentration of antibody used in the experiment (20 μg/mL), SGN-CD47M does not bind to recombinant CD47 and is not detected by ELISA. Therefore, binding indicates that the masking domain of SGN-CD47M has been proteolyzed. Under these conditions, no binding of SGN-CD47M spiked into any of the 19 samples was detected, whereas spiked control samples of hB6H12.3 yielded quantitative detection of 20 μg/mL. All samples spiked with SGN-CD47M were quantified below 350 ng/mL, which equates to <2% binding of hB6H12.3. Symbols represent two biological replicates for each donor, shown as the mean ± SD. CRC, colorectal cancer; MFI, mean fluorescent index; NSCLC, non–small cell lung cancer.

Article Snippet: Saturation binding ELISA experiments were performed using soluble recombinant human, mouse, or cyno CD47 (ACROBiosystems), which was diluted to an appropriate concentration in 50 mmol/L carbonate buffer at pH 9.6.

Techniques: Clinical Proteomics, Binding Assay, Labeling, Flow Cytometry, Incubation, Enzyme-linked Immunosorbent Assay, Concentration Assay, Recombinant, Control