Agonistic anti-CD40 single-chain antibody enhances anti-tumor effect of mouse NK cells / 中华微生物学和免疫学杂志

ABSTRACT

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 microen-vironment were detected by ELISA. Changes in the percentages of tumor-infiltrating NK cells and the expres-sion of NKG2D protein(natural-killer group 2,member D) were detected by flow cytometry and immunohis-tochemistry,respectively. Results The recombinant plasmid CD40 scFv-pET28a was confirmed to be con-structed correctly. Results of SDS-page and His-tag Western blot revealed that anti-CD40 scFv could be ex-pressed 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 signifi-cantly higher than those in NS group (P<0.05). Conclusion Agonistic anti-mouse CD40 scFv could en-hance the anti-tumor ability of NK cells by activating DCs.