Agonistic anti-CD40 single-chain antibody enhances anti-tumor effect of mouse NK cells

Objective To obtain a mouse CD40-specific single-chain antibody (scFv) with high purity and to investigate its in vivo and in vitro agonistic activity on natural killer (NK) cells against tumor. Methods Agonistic anti-mouse CD40 scFv with high purity was obtained by genetic engineering. Mouse dendritic cells (DCs) were treated with different strategies including anti-CD40 monoclonal antibody (McAb), anti-CD40 scFv and negative control. Expression of IL-12 in the supernatants of DC cultures was measured by ELISA. Then DCs and NK cells were co-cultured to obtain activated NK cells, which were co-cultured with T6-17 cells. WST-8 was used to test the cytotoxic effects of NK cells on T6-17 cells. A mouse tumor model was established by injecting BALB/c nude mice with T6-17 cells. Anti-CD40 scFv was injected into tumor to evaluate its inhibitory effect on tumor growth. Levels of IL-12 and IFN-γ in the tumor microenvironment were detected by ELISA. Changes in the percentages of tumor-infiltrating NK cells and the expression of NKG2D protein (natural-killer group 2, member D) were detected by flow cytometry and immunohistochemistry, respectively. Results The recombinant plasmid CD40 scFv-pET28a was confirmed to be constructed correctly. Results of SDS-page and His-tag Western blot revealed that anti-CD40 scFv could be expressed successfully with a relative molecular mass of 27×103. The level of IL-12 in the supernatant of DC culture of anti-CD40 scFv group was (555.86±40.48) pg/ml, which was significantly higher than that of negative control group (P<0.05). The killing ability of NK cells in anti-CD40 scFv group was (72.23±3.99)%, which was significantly higher than that in negative control group (P<0.05). Anti-CD40 scFv significantly inhibited the tumor growth in BALB/c nude mice as compared with negative control group (P<0.05). The levels of IL-12 and IFN-γ in tumor microenvironment of anti-CD40 scFv group were (188.801±32.718) pg/ml and (121.428±30.994) pg/ml, respectively, which were significantly higher than those in normal saline (NS) group (P<0.05). The positive rate of NKG2D protein and the percentage of CD3-DX5+ cells in anti-CD40 scFv group were respectively (8.18±2.01)% and (19.15±2.24)%, which were significantly higher than those in NS group (P<0.05). Conclusion Agonistic anti-mouse CD40 scFv could enhance the anti-tumor ability of NK cells by activating DCs. Key words: Agonistic anti-CD40 single-chain antibody; Dendritic cell; Natural killer cell; T6-17 cell