Journal: Nature
Article Title: Engineered CD47 protects T cells for enhanced antitumour immunity
doi: 10.1038/s41586-024-07443-8
Figure Lengend Snippet: (a) Engineered CD47 (47 E ) mechanism: αCD47 antibodies bind tumour cells, but not 47 E -T cells, triggering tumour-specific phagocytosis. The diagram was created using BioRender. (b) Cartoon of yeast displayed CD47 Ig-like domain using the pCTCON2 vector. CD47 is displayed as an N-terminal fusion. (c) Binding of 100 nM B6H12 to yeast displayed CD47 in pCTCON2 by flow cytometry. Representative of n = 3 independent experiments. (d) Binding curve of B6H12 to yeast displayed CD47 in pCTCON2, measured over multiple concentrations by flow cytometry. MFI of n = 1 experiment. (e) Binding of 300 nM human (top) and mouse (bottom) SIRPα to yeast displayed CD47 in pCTCON2 by flow cytometry. Data are representative of n = 3 independent experiments. (f) Binding of 500 nM CV-1 to yeast displayed CD47 in pCTCON2 by flow cytometry. Data are representative of n = 3 independent experiments. (g) Flow cytometry sorting plots of all six sorts of the CD47 library, indicating negative sorts to B6H12 and positive sorts to CV-1. Collected population indicated by the black box in each plot. (h) Binding of 500 nM B6H12 or 100 nM CV-1 to the yeast displayed CD47 library population collected after sort 6 or yeast displayed WT CD47. Data are representative of n = 2 independent experiments. (i) Binding of 1 μM B6H12 or 100 nM CV-1 to CD47 variants displayed on yeast using the pCTCON2 vector. Mean ± SD of n = 3 individual yeast clones, normalized to MFI from binding to 47 WT . (j) Cartoon of yeast-displayed CD47 Ig-like domain using the pFreeNTerm (pFNT) vector. CD47 is displayed as a C-terminal fusion, along with GFP to monitor protein expression. (k) Binding of 100 nM human and mouse SIRPα to yeast displayed CD47 in pFNT by flow cytometry. Representative of n = 3 independent experiments. (l) Crystal structure of CD47 (yellow) binding SIRPα (orange) [PDB: 2JJS], identifying the CD47 BC loop (green), containing CD47 residues T26 – Q31. (m) Crystal structures of CD47 (red) binding SIRPα (dark pink, left) [PDB: 2JJS] and B6H12 (light blue, right) [PDB: 5TZU], identifying residues A30 (gold) and Q31 (blue), and the BC and FG loops of CD47. Structures are enlargements of the boxed regions in the full structures shown in Fig. . (n) Binding of 100 nM B6H12, TJC4, Hu5F9, and hSIRPα to yeast displayed 47 T26A , 47 N27A , and 47 M28A variants. Mean ± SD of n = 3 individual yeast clones, normalized to MFI from binding to 47 WT . [(i), (n)] Two-way analysis of variance (ANOVA) test with Tukey’s multiple comparison test. ns = not significant. Comparison is between indicated group and binding to CD47 WT expressing cells.
Article Snippet: A DNA sequence encoding the CD47 Ig-like domain (Gln19–Ser135) was cloned into the pCTCON2 yeast-surface display vector (Addgene) using the NheI and BamHI sites.
Techniques: Plasmid Preparation, Binding Assay, Flow Cytometry, Clone Assay, Expressing, Comparison
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![(a) Engineered CD47 (47 E ) mechanism: αCD47 antibodies bind tumour cells, but not 47 E -T cells, triggering tumour-specific phagocytosis. The diagram was created using BioRender. (b) Cartoon of yeast displayed CD47 Ig-like domain using the <t>pCTCON2</t> vector. CD47 is displayed as an N-terminal fusion. (c) Binding of 100 nM B6H12 to yeast displayed CD47 in pCTCON2 by flow cytometry. Representative of n = 3 independent experiments. (d) Binding curve of B6H12 to yeast displayed CD47 in pCTCON2, measured over multiple concentrations by flow cytometry. MFI of n = 1 experiment. (e) Binding of 300 nM human (top) and mouse (bottom) SIRPα to yeast displayed CD47 in pCTCON2 by flow cytometry. Data are representative of n = 3 independent experiments. (f) Binding of 500 nM CV-1 to yeast displayed CD47 in pCTCON2 by flow cytometry. Data are representative of n = 3 independent experiments. (g) Flow cytometry sorting plots of all six sorts of the CD47 library, indicating negative sorts to B6H12 and positive sorts to CV-1. Collected population indicated by the black box in each plot. (h) Binding of 500 nM B6H12 or 100 nM CV-1 to the yeast displayed CD47 library population collected after sort 6 or yeast displayed WT CD47. Data are representative of n = 2 independent experiments. (i) Binding of 1 μM B6H12 or 100 nM CV-1 to CD47 variants displayed on yeast using the pCTCON2 vector. Mean ± SD of n = 3 individual yeast clones, normalized to MFI from binding to 47 WT . (j) Cartoon of yeast-displayed CD47 Ig-like domain using the pFreeNTerm (pFNT) vector. CD47 is displayed as a C-terminal fusion, along with GFP to monitor protein expression. (k) Binding of 100 nM human and mouse SIRPα to yeast displayed CD47 in pFNT by flow cytometry. Representative of n = 3 independent experiments. (l) Crystal structure of CD47 (yellow) binding SIRPα (orange) [PDB: 2JJS], identifying the CD47 BC loop (green), containing CD47 residues T26 – Q31. (m) Crystal structures of CD47 (red) binding SIRPα (dark pink, left) [PDB: 2JJS] and B6H12 (light blue, right) [PDB: 5TZU], identifying residues A30 (gold) and Q31 (blue), and the BC and FG loops of CD47. Structures are enlargements of the boxed regions in the full structures shown in Fig. . (n) Binding of 100 nM B6H12, TJC4, Hu5F9, and hSIRPα to yeast displayed 47 T26A , 47 N27A , and 47 M28A variants. Mean ± SD of n = 3 individual yeast clones, normalized to MFI from binding to 47 WT . [(i), (n)] Two-way analysis of variance (ANOVA) test with Tukey’s multiple comparison test. ns = not significant. Comparison is between indicated group and binding to CD47 WT expressing cells.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8929/pmc11168929/pmc11168929__41586_2024_7443_Fig11_ESM.jpg)