Fig. 2
From: BCMA/CD47-directed universal CAR-T cells exhibit excellent antitumor activity in multiple myeloma
Anti-CD47 nanobodies hu404 has strong binding ability with CD47 protein, and showed high safety for human RBCs. (A-B) Immunofluorescence assay showed anti-CD47 nanobody hu404 has strong binding ability with CD47 protein on the CD47-GFP-Hela cells (A), RPMI-8226 and MM.1S cell lines (B). Scale bar 5 μm. (C) Real-time binding profile showed the binding affinity between hu404 and CD47 was 5.49 × 10− 10 M. (D) The tandem linked of hu388 and hu404 with flexibility linker (3×G4S) or rigidity linker (3×EAAAK) were obtained after purification with Ni-NTA agarose. M: Marker; lane 1,3,5,7 and lane 2,4,6,8: non-reducing and reducing of hu388-hFc, hu404-hFc, h31-hFc and h32-hFc. (E) Anti-CD47 nanobodies hu404-hFc, h31-hFc and h32-hFc fusions did not bind to human RBCs from healthy donors. (F) Anti-CD47 nanobodies hu404-hFc, h31-hFc and h32-hFc fusions did not agglutinate human RBCs. (G) Anti-CD47 nanobody hu404-hFc, h31-hFc and h32-hFc fusions did not bind to human RBCs, but still bind to MM.1S cells. (H) iELISA revealed the binding affinity of bispecific antibodies (h31-hFc, h32-hFc) were significantly higher than monospecific antibodies at 0.1 and 0.01 nM (P < 0.01), and h32-hFc shows the highest affinity (P < 0.0001). (I) The humanized nanobodies hu404-hfc, hu388-hFc and h32-hFc fusions showed strong binding activity with primary MM cells from multiple myeloma patients. Results were expressed as mean ± SD, statistical analyses were performed using a two-way ANOVA test with Bonferroni post-test. *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001; ns, no significance