Fig. 1

Screening specific nanobodies, and identification their functional characterization. (A) The transcript expression levels of BCMA and CD47 in MM patients (n = 170) was significantly higher than control (n = 6) (P < 0.001) from MMRF-CoMMpass data. (B) The correlation of expression between BCMA and CD47 is 0.15, indicating no significant correlation. (C) Schematic presentation of library construction and screening nanobodies by phage display technology. (D) Amplification of VHH gene (400 bp) by nest-PCR from PBMCs and estimation the VHH gene insert rate of nanobodies library. The VHH-CH1-CH2 (900 bp), VHH-CH2 (700 bp) gene of camel IgG were amplified after first round PCR (i), and VHH gene (400 bp) was amplified after second round PCR (ii). After amplification with MP57 and GIII primers, the PCR product containing the VHH gene is 700 bp, whereas the un-inserted product is 357 bp (iii). (E) The candidate nanobodies Nb303, Nb381, Nb388, Nb394 and Nb396 against BCMA could specifically binding to BCMA protein according to iELISA. (F) Real-time binding profile of anti-BCMA humanized nanobody hu388 with BCMA protein by SPR. The binding affinity between hu388 and BCMA protein was 3.87 × 10^− 11M. (G) Specificity identification of anti-BCMA humanized nanobodies by flow cytometry. Humanized nanobodies hu303, hu381, hu388, hu394 and hu396 could specifically bind to RPMI-8226 and MM.1S cells, and not with MM.1S-BCMA^KO, K562 and THP-1 cells. (H-I) Anti-BCMA humanized nanobody hu388 showed strong binding ability with BCMA protein on the BCMA-GFP-Hela cells (H), RPMI-8226 and MM.1S cells (I), 11D5.3 was positive control antibody against BCMA. Alexa Fluor® 594 rabbit anti-human IgG as the secondary antibody for detection. Scale bar 5 μm. Results were expressed as mean ± SD, statistical analyses were performed using t-test (A) and two-way ANOVA test with Dunnett’s multiple comparisons test (E). *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001; ns, no significance