Fig. 3

Bioactivity analysis of bispecific nanobodies in vitro. a Jurkat cell chemotaxis was inhibited by BsNb PX4 and Nb CXCR4. b The half-maximal inhibitory concentrations of BsNb PX4 and Nb CXCR4. c The levels of IFN-γ were measured by ELISA. d Inhibition of cancer cell proliferation induced by BsNb PX4. e Inhibition of AsPC-1 cell proliferation induced by BsNb PX4 in the presence of CXCL12. f Cytotoxicity of PBMCs against tumour cells treated with BsNb PX4 and IL-2 in vitro. Control group: hPBMCs incubated with tumour cells for 96 h; IL-2 group: 100 IU/mL IL-2 was added to wells after 24 h of hPBMC and tumour cell coculture, then the mixture was cultured for 72 h; IL-2 + BsNb (or IL-2 + Nb PD-L1) group: hPBMCs were precultured with tumour cells for 24 h, followed by incubation with IL-2 for 24 h, and then the mixture was cultured for 48 h in the presence of BsNb PX4 (or Nb PD-L1). g Changes in PD-L1 expression levels in AsPC-1 cells were analysed by flow cytometry. (a-f) The data are shown as the mean ± S.D. (n = 3). *P < 0.05, ** P < 0.01, ***P < 0.001