Journal: bioRxiv

Article Title: CD47 as a potential biomarker for the early diagnosis of severe COVID-19

doi: 10.1101/2021.03.01.433404

Figure Lengend Snippet: SARS-CoV-2 infection is associated with increased CD47 levels. A) TF protein abundance in uninfected (control) and SARS-CoV-2-infected (virus) Caco-2 cells (data derived from . P-values were determined by two-sided Student’s t-test. B) CD47 and SARS-CoV-2 N protein levels and virus titres (genomic RNA determined by PCR) in SARS-CoV-2 strain FFM7 (MOI 1)-infected air-liquid interface cultures of primary human bronchial epithelial (HBE) cells and SARS-CoV-2 strain FFM7 (MOI 0.1)-infected Calu-3 cells. Uncropped blots are provided in Suppl. Figure 1. C) CD47 mRNA levels in post mortem samples from COVID-19 patients (data derived from ). P-values were determined by two-sided Student’s t-test.

Article Snippet: Detection occurred by using specific antibodies against CD47 (1:100 dilution, CD47 Antibody, anti-human, Biotin, REAfinityTM, # 130-101-343, Miltenyi Biotec), SARS-CoV-2 N (1:1000 dilution, SARS-CoV-2 Nucleocapsid Antibody, Rabbit MAb, #40143-R019, Sino Biological), and GAPDH (1:1000 dilution, Anti-G3PDH Human Polyclonal Antibody, #2275-PC-100, Trevigen).

Techniques: Infection, Quantitative Proteomics, Control, Virus, Derivative Assay

Journal: bioRxiv

Article Title: CD47 as a potential biomarker for the early diagnosis of severe COVID-19

doi: 10.1101/2021.03.01.433404

Figure Lengend Snippet: Results of the PubMed ( https://pubmed.ncbi.nlm.nih.gov ) literature search for “CD47 aging” (A) and “CD47 hypertension” (B). C) Overview figure of the data derived from the literature searches. Age-related increased CD47 levels may contribute to pathogenic conditions associated with severe COVID-19.

Article Snippet: Detection occurred by using specific antibodies against CD47 (1:100 dilution, CD47 Antibody, anti-human, Biotin, REAfinityTM, # 130-101-343, Miltenyi Biotec), SARS-CoV-2 N (1:1000 dilution, SARS-CoV-2 Nucleocapsid Antibody, Rabbit MAb, #40143-R019, Sino Biological), and GAPDH (1:1000 dilution, Anti-G3PDH Human Polyclonal Antibody, #2275-PC-100, Trevigen).

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: CD47 as a potential biomarker for the early diagnosis of severe COVID-19

doi: 10.1101/2021.03.01.433404

Figure Lengend Snippet: Results of the PubMed ( https://pubmed.ncbi.nlm.nih.gov ) literature search for “CD47 diabetes” (A). B) Overview figure of the data derived from the literature search. Hyperglycaemia- and diabetes-induced increased CD47 levels may contribute to immune escape of SARS-CoV-2-infected cells.

Article Snippet: Detection occurred by using specific antibodies against CD47 (1:100 dilution, CD47 Antibody, anti-human, Biotin, REAfinityTM, # 130-101-343, Miltenyi Biotec), SARS-CoV-2 N (1:1000 dilution, SARS-CoV-2 Nucleocapsid Antibody, Rabbit MAb, #40143-R019, Sino Biological), and GAPDH (1:1000 dilution, Anti-G3PDH Human Polyclonal Antibody, #2275-PC-100, Trevigen).

Techniques: Derivative Assay, Infection

Journal: Oncoimmunology

Article Title: IRE1α overexpression in malignant cells limits tumor progression by inducing an anti-cancer immune response

doi: 10.1080/2162402X.2022.2116844

Figure Lengend Snippet:

Article Snippet: PE-Vio770 anti-CD47 , REAfinity, Miltenyi Biotec , 130–103-105, RRID:AB_2659751.

Techniques:

Journal: Cancers

Article Title: Extracellular Vesicles Derived from Human Umbilical Cord Mesenchymal Stromal Cells as an Efficient Nanocarrier to Deliver siRNA or Drug to Pancreatic Cancer Cells

doi: 10.3390/cancers15112901

Figure Lengend Snippet: Phenotypical characterization of UC-MSCs by flow cytometry (unfilled histogram = CTRL/filled histogram = labeled cells). Histograms show positivity for the tetraspanins CD9, CD63 and CD81, as well as the MSC markers CD73, CD166, CD146, CD105, HLA-ABC, CD47 and CD200, and negativity for the hematopoietic markers HLA-DR and CD45.

Article Snippet: Cells were harvested after detachment with TrypLE Select, washed in PBS (Miltenyi Biotec, Bergisch Gladbach, Germany) and incubated for 30 min with the following monoclonal antibodies: CD105-FITC (Ancell corporation, Bayport, NY, USA), CD73-PE (Miltenyi Biotec, Bergisch Gladbach, Germany), CD146-PC5 (Beckman Coulter, Analis, Suarlée, Belgium), CD166-PE (BD Biosciences, Erembodegem, Belgium), CD45-PC7 (BD Biosciences, Erembodegem, Belgium), HLA-ABC- PC5 (BioLegend, San Diego, CA, USA), HLA-DR-PC5 (Beckman Coulter, Analis, Suarlée, Belgium), CD47-APC (Miltenyi Biotec, Bergisch Gladbach, Germany) and CD200-PC7 (BD).

Techniques: Flow Cytometry, Labeling

Journal: Cancers

Article Title: Extracellular Vesicles Derived from Human Umbilical Cord Mesenchymal Stromal Cells as an Efficient Nanocarrier to Deliver siRNA or Drug to Pancreatic Cancer Cells

doi: 10.3390/cancers15112901

Figure Lengend Snippet: Phenotypical characterization of EVs derived from hUC-MSCs by flow cytometry (black line = CTRL, red line = labeled cells). Histograms show positivity for the tetraspanins CD9, CD63 and CD81, as well as for the MSC markers CD73, CD166, CD146, CD105, HLA-ABC, CD47 and CD200, and negativity for the hematopoietic markers HLA-DR and CD45.

Article Snippet: Cells were harvested after detachment with TrypLE Select, washed in PBS (Miltenyi Biotec, Bergisch Gladbach, Germany) and incubated for 30 min with the following monoclonal antibodies: CD105-FITC (Ancell corporation, Bayport, NY, USA), CD73-PE (Miltenyi Biotec, Bergisch Gladbach, Germany), CD146-PC5 (Beckman Coulter, Analis, Suarlée, Belgium), CD166-PE (BD Biosciences, Erembodegem, Belgium), CD45-PC7 (BD Biosciences, Erembodegem, Belgium), HLA-ABC- PC5 (BioLegend, San Diego, CA, USA), HLA-DR-PC5 (Beckman Coulter, Analis, Suarlée, Belgium), CD47-APC (Miltenyi Biotec, Bergisch Gladbach, Germany) and CD200-PC7 (BD).

Techniques: Derivative Assay, Flow Cytometry, Labeling

Journal: Acta Pharmaceutica Sinica. B

Article Title: Multiparametric characterization of red blood cell physiology after hypotonic dialysis based drug encapsulation process

doi: 10.1016/j.apsb.2021.10.018

Figure Lengend Snippet: Comparison of the amount of each protein detected in RBCs, before and after encapsulation process, and with or without asparaginase (ASNase). The number of copies per cell of the proteins identified in pRBCs, proRBCs, eryaspase ( n = 4 per group) was compared between each pair of samples using a scatter plot. The Pearson correlation coefficient ( R ) was calculated for comparisons between all samples. Data for total and ghosts RBCs were separated. Some key RBCs proteins were displayed: hemoglobin proteins (HBB, HBA1), peroxiredoxin 2 (PRDX2), carbonic anhydrase (CA1, CA2), catalase (CAT), band 3 anion exchanger (SLC4A1), alpha and beta spectrin (SPTA1 and SPTB), ankyrin (ANK1), tropomyosin (TPM3), alpha and beta adducin (ADD1 and ADD2), calpain 1 catalytic subunit (CAPN1), glutathione- S -synthetase (GSS), actin (ACTB), glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), Glycophorin A (GYPA), CD47.

Article Snippet: Phosphatidylserine (PS) exposure at the outer membrane leaflet of RBCs and CD47 were assessed using Annexin V-PE (Miltenyi, 130-118-363) and anti-CD47-PE antibody (Miltenyi 130-101-348), respectively.

Techniques: Comparison, Encapsulation

Journal: Acta Pharmaceutica Sinica. B

Article Title: Multiparametric characterization of red blood cell physiology after hypotonic dialysis based drug encapsulation process

doi: 10.1016/j.apsb.2021.10.018

Figure Lengend Snippet: PS exposure, CD47 expression and RBCs-EVs release before and after encapsulation process.

Article Snippet: Phosphatidylserine (PS) exposure at the outer membrane leaflet of RBCs and CD47 were assessed using Annexin V-PE (Miltenyi, 130-118-363) and anti-CD47-PE antibody (Miltenyi 130-101-348), respectively.

Techniques: Expressing, Encapsulation

Journal: Journal of Nanobiotechnology

Article Title: Targeting and repolarizing M2-like tumor-associated macrophage-mediated MR imaging and tumor immunotherapy by biomimetic nanoparticles

doi: 10.1186/s12951-023-02122-8

Figure Lengend Snippet: ( A ) The expression of CD80 and CD206 on various macrophage phases detected by CLSM (scale bar: 20 μm). ( B ) Quantitative analysis of CD80 fluorescence intensity on various macrophage phases (***P < 0.001). ( C ) Quantitative analysis of CD206 fluorescence intensity on various macrophage phases (***P < 0.001). ( D ) Cell viabilities of M0 macrophage treated with HMMDN@PM or HMMDN-Met@PM. ( E ) Cell viabilities of M1 macrophage treated with HMMDN@PM or HMMDN-Met@PM. ( F ) Cell viabilities of M2 macrophage treated with HMMDN@PM or HMMDN-Met@PM

Article Snippet: Polyclonal antibodies CD47, CD80 and CD206 were obtained from Proteintech Group, Inc. (Wuhan, China).

Techniques: Expressing, Fluorescence

Journal: Journal of Nanobiotechnology

Article Title: Targeting and repolarizing M2-like tumor-associated macrophage-mediated MR imaging and tumor immunotherapy by biomimetic nanoparticles

doi: 10.1186/s12951-023-02122-8

Figure Lengend Snippet: ( A ) Fluorescence images observed the expression of CD80 and CD206 in vitro after M2-like TAMs were treated with HMMDN(I), HMMDN@PM(II), Met(III), HMMDN-Met(IV), HMMDN-Met@MM(V), HMMDN-Met@PM(VI) (scale bar: 20 μm). ( B ) Quantitative analysis of CD80 fluorescence intensity on M2-like TAMs treated with different nanoparticles; ( C ) Quantitative analysis of CD206 fluorescence intensity on M2-like TAMs after different treatments (***P < 0.001). ( D ) Flow cytometric analysis of the expression of CD80 in vitro after M2-like TAMs were treated with different nanoparticles. ( E ) Flow cytometric analysis of the expression of CD206 in vitro after M2-like TAMs were treated with different nanoparticles. ( F ) The levels of immune cytokines, including TNF-α, iNOS, Arg-1 and IL-10 in M2-like TAMs supernatant after different treatments (**P < 0.01, ***P < 0.001)

Article Snippet: Polyclonal antibodies CD47, CD80 and CD206 were obtained from Proteintech Group, Inc. (Wuhan, China).

Techniques: Fluorescence, Expressing, In Vitro

Journal: Journal of Nanobiotechnology

Article Title: Targeting and repolarizing M2-like tumor-associated macrophage-mediated MR imaging and tumor immunotherapy by biomimetic nanoparticles

doi: 10.1186/s12951-023-02122-8

Figure Lengend Snippet: ( A ) The levels of immune cytokines TNF-α, iNOS, Arg-1 and IL-10 in the serum of mice from indicated groups. *p < 0.05, **p < 0.01 and ***p < 0.001. ( B ) H&E and immunohistochemical staining for CD80, CD206 of tumor tissues harvested from different groups (scale bar: 100 μm)

Article Snippet: Polyclonal antibodies CD47, CD80 and CD206 were obtained from Proteintech Group, Inc. (Wuhan, China).

Techniques: Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: The Antitumor Activities of Anti-CD47 Antibodies Require Fc-FcγR interactions

doi: 10.1101/2023.06.29.547082

Figure Lengend Snippet: (A) Binding profiles to mouse FcγRs of Fc variants for anti-CD47 antibodies (MIAP301, chimeric-5F9 and chimeric-2D3), activating-to-inhibitory binding ratio (A/I) to mFcγRs is shown (B) Levels of expression of CD47 on different mouse cell lines, mean fluorescence intensity (MFI) expressed on the right. (C) Diagram showing the structure of the mouse antibody MIAP301 and binding of their Fc variants to recombinant mouse CD47 by ELISA (D) Diagram showing the structure of the chimeric antibody chimeric-5F9 and binding of their Fc variants to recombinant human CD47 by ELISA (E) Diagram showing the structure of non-bocking (Chimeric 2D3-mIgG2a) and blocking (Chimeric 5F9-mIgG2a) CD47 antibodies. Binding to recombinant human CD47 by ELISA (F) Competitive ELISA showing the capacity of blocking (mIgG2a Chimeric-5F9) and non-blocking (mIgG2a Chimeric-2D3) antibodies to disrupt the binding of recombinant SIRP⍺ to hCD47.

Article Snippet: Binding specificity of antibodies targeting mouse (MIAP301) and human (2D3 and 5F9) monoclonal antibodies were determined by ELISA using recombinant mouse (SinoBiological 57231-MNAH) and human (SinoBiological 12283-HCCH) CD47, respectively.

Techniques: Binding Assay, Expressing, Fluorescence, Recombinant, Enzyme-linked Immunosorbent Assay, Blocking Assay, Competitive ELISA

Journal: bioRxiv

Article Title: The Antitumor Activities of Anti-CD47 Antibodies Require Fc-FcγR interactions

doi: 10.1101/2023.06.29.547082

Figure Lengend Snippet: (A-C) Established MC38 tumors were analyzed by flow cytometry 72 hours after treatment with Fc variants for CD47 antibodies (MIAP301). Quantification of CD45 + , CD4 + and CD8 + cells from MC38 tumors 72h after treatment with Fc variants is shown. (D) Representative flow cytometry plots of macrophages, and (E) monocytes 72h after treatment with MIAP301 Fc variants. (F) Peripheral blood levels of monocytes 24 hours after treatment with anti-CCR2 ab (Clone MC-21-mIgG2a, 100 ug) or isotype control in WT mice. (G) Quantification of intratumoral macrophages from MC38 tumors pre-treated with control- or clodronate-liposomes intravenously (IV) or intratumorally (IT) (100 uL, d.-3, -1) by flow cytometry. (H) Quantification of intratumoral monocytes from MC38 tumors pre-treated with control- or clodronate-liposomes intravenously (IV) or intratumorally (IT) (100 uL, d.-3, -1) by flow cytometry.

Article Snippet: Binding specificity of antibodies targeting mouse (MIAP301) and human (2D3 and 5F9) monoclonal antibodies were determined by ELISA using recombinant mouse (SinoBiological 57231-MNAH) and human (SinoBiological 12283-HCCH) CD47, respectively.

Techniques: Flow Cytometry

Journal: bioRxiv

Article Title: The Antitumor Activities of Anti-CD47 Antibodies Require Fc-FcγR interactions

doi: 10.1101/2023.06.29.547082

Figure Lengend Snippet: (A) Representative flow cytometry plots of the percentage of phagocytosis of B16 cancer cells by BMDM from WT mice after treatment with control or Fc subclasses of MIAP301. (B) Phagocytosis of Lewis lung carcinoma (LLC) or HKP1 (Kras G12D p53 -/– ) lung adenocarcinoma after treatment with control or Fc subclasses of MIAP301. ( C) Representative flow cytometry plot and (D) phagocytosis of MC38 hCD47 KI or B16 hCD47 KI by BMDM from hCD47/hSIRP⍺ KI mice after treatment with control or Fc variants of Chimeric-5F9. (E) Phagocytosis of MC38 hCD47 KI cells by BMDM from hCD47/hSIRP⍺ KI mice pre-treated with different polarization conditions. (F) Phagocytosis of MC38 hCD47 KI or B16 hCD47 KI cells by BMDM from hCD47/hSIRP⍺ KI mice after treatment isotype control, blocking (5F9), non-blocking (2D3) antibodies targeting hCD47, both in mIgG2a Fc format (G) (Left) Representative flow cytometry plots and (Right) phagocytosis of B16 hCD47 KI mice by BMDM from hCD47/hSIRP⍺/hFcγR mice after treatment with Fc variants of fully humanized anti-CD47 ab (5F9). Macrophages were pre-treated with different polarization conditions as indicated. (H) (Left) representative flow cytometry plots and (right) phagocytosis of human cancer cell line Jurkat and (I) Raji by human macrophages derived from PBMCs after treatment with Fc variants of fully humanized 5F9.

Article Snippet: Binding specificity of antibodies targeting mouse (MIAP301) and human (2D3 and 5F9) monoclonal antibodies were determined by ELISA using recombinant mouse (SinoBiological 57231-MNAH) and human (SinoBiological 12283-HCCH) CD47, respectively.

Techniques: Flow Cytometry, Blocking Assay, Derivative Assay

Journal: bioRxiv

Article Title: The Antitumor Activities of Anti-CD47 Antibodies Require Fc-FcγR interactions

doi: 10.1101/2023.06.29.547082

Figure Lengend Snippet: (A) Levels of expression of CD47 on T cell subsets isolated from MC38 tumors, quantification (left) and representative flow cytometry plots (right) are shown. (B) Percentage of CD4 + FOXP3 + T regs of total CD4 + T cells, and (C) Percentage of tetramer + (KSPWFTTL) cells of total CD8 + T cells from MC38 tumors 72 hours after treatment with Fc variants of anti-mCD47 abs (MIAP301). (D) Average growth ± SEM of sq. MC38 tumors pre-treated with anti-CD8 ab (2.43) or isotype control (100 ug. d.7, 12, 17), then treatment with MIAP301-mIgG2a or isotype control was given (50 ug IT, d. 8, 10, 14 and 18). (E, left) MC38 tumors were treated with MIAP301-mIg2a (50 ug every 3 days) until rejection was achieved. Then mice were rechallenged with MC38 cells (10 million, d. 90). (E, right) Average growth ± SEM of sq. MC38 tumors in mice that achieved rejection 90 days before (rechallenged grouped) or in and mice without prior implantation of MC38 tumors (naïve group).

Article Snippet: Binding specificity of antibodies targeting mouse (MIAP301) and human (2D3 and 5F9) monoclonal antibodies were determined by ELISA using recombinant mouse (SinoBiological 57231-MNAH) and human (SinoBiological 12283-HCCH) CD47, respectively.

Techniques: Expressing, Isolation, Flow Cytometry

Journal: bioRxiv

Article Title: The Antitumor Activities of Anti-CD47 Antibodies Require Fc-FcγR interactions

doi: 10.1101/2023.06.29.547082

Figure Lengend Snippet: (A) Schematic drawing showing CRISPR/Cas9-mediated gene-targeting strategy to generate CD47 (top) and SIRP⍺ (bottom) KI mice. The double-strand break induced by the CRISPR/Cas9 in the flanking regions of the ECD of mCD47 and mSIRP⍺ was replaced by their human counterparts via non-homologous end joining. (B) Complete cell blood count of WT and hCD47/hSIRP⍺ KI mice (C) Right: Recombinant hSIRPα was incubated with RBCs from WT and hCD47/hSIRPα KI mice, binding was only detected in RBCs from the hCD47/hSIRPα mouse. Left: Recombinant hCD47 was incubated with leukocytes from WT and hCD47/hSIRPα KI mice, binding was primarily detected in leukocytes from the hCD47/hSIRPα mouse. (D) Flow cytometry analysis of expression of hCD47 and mCD47 in unstained (Black), MC38 WT (blue) and MC38 hCD47 KI cells (red). (E) Flow cytometry analysis of expression of hCD47 and mCD47 in unstained (Black), B16 WT (blue) and B16 hCD47 KI cells (red).

Article Snippet: Binding specificity of antibodies targeting mouse (MIAP301) and human (2D3 and 5F9) monoclonal antibodies were determined by ELISA using recombinant mouse (SinoBiological 57231-MNAH) and human (SinoBiological 12283-HCCH) CD47, respectively.

Techniques: CRISPR, Non-Homologous End Joining, Recombinant, Incubation, Binding Assay, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: The Antitumor Activities of Anti-CD47 Antibodies Require Fc-FcγR interactions

doi: 10.1101/2023.06.29.547082

Figure Lengend Snippet: (A) Flow cytometry analysis of hCD47 and hSIRP⍺ in red blood cells (RBC), platelets, CD3 + and CD11b + leucocytes isolated from peripheral blood of human (top) and hCD47/hSIRP⍺ KI mice (bottom). (B) Average growth ± SEM of sq. MC38 hCD47 KI tumors in hCD47/hSIRP⍺ KI mice, treated with chimeric anti-hCD47 ab Fc variants (chimeric-5F9) or isotype control (50 ug IT, d. 8,10,14 and 18). (C) Average of lung metastases ± SD of hCD47/hSIRP⍺ KI mice inoculated IV with B16 hCD47 KI tumor cells, treated with chimeric anti-hCD47 ab Fc variants (chimeric-5F9) or isotype control (20 mg/Kg IP, d. 1,4,7 and 11). (D) Schematic drawing showing the two ab formats proposed for effective antitumor activity of anti-CD47 antibodies. Left: An ab blocking the interaction between CD47 and SIRP⍺ AND an Fc domain engaging activating FcγRs. Right: A non-blocking anti-CD47 ab AND an Fc domain engaging activating FcγRs. (E) Average of lung metastases ± SD of hCD47/hSIRP⍺ KI mice inoculated IV with B16 hCD47 KI tumor cells, treated with blocking (5F9-mIgG2a) and non-blocking (2D3-mIgG2a) chimeric anti-hCD47 antibodies or isotype control (20 mg/Kg IP, d. 1,4,7 and 11).

Article Snippet: Binding specificity of antibodies targeting mouse (MIAP301) and human (2D3 and 5F9) monoclonal antibodies were determined by ELISA using recombinant mouse (SinoBiological 57231-MNAH) and human (SinoBiological 12283-HCCH) CD47, respectively.

Techniques: Flow Cytometry, Isolation, Activity Assay, Blocking Assay

Journal: bioRxiv

Article Title: The Antitumor Activities of Anti-CD47 Antibodies Require Fc-FcγR interactions

doi: 10.1101/2023.06.29.547082

Figure Lengend Snippet: (A) Characterization of FcγR profile on mice humanized for FcγRs (blue) and mice humanized for CD47, SIRP⍺ and FcγRs (yellow), representative flow cytometry plots and (B) Q uantification are shown.

Article Snippet: Binding specificity of antibodies targeting mouse (MIAP301) and human (2D3 and 5F9) monoclonal antibodies were determined by ELISA using recombinant mouse (SinoBiological 57231-MNAH) and human (SinoBiological 12283-HCCH) CD47, respectively.

Techniques: Flow Cytometry