Antitumor effect of agonistic CD40 antibody via the regulation of Th1/Th2 balance in the tumor microenvironment / 中国肿瘤临床

ABSTRACT

Objective:To explore the agonistic CD40 antibody(CD40ScFv and CD40mAb)-mediated suppression of mouse breast cancer cell growth and change in tumor tissue Th1/Th2 balance,as well as the mechanism underlying its antitumor effect.Methods:The re-combinant plasmid containing the CD40ScFv gene fragment was transformed into the Rosetta strain of Escherichia coli to express and purify the recombinant functional CD40ScFv protein.The 4T1 mouse breast cancer cells were cultured in vitro.Balb/C tumor model mice were divided into CD40ScFv,CD40mAb agonist,and saline(NS group)groups,which were administered CD40ScFv,CD40mAb ago-nist,and saline,respectively,to observe the change in tumor volume.The tumor tissues were removed and enzyme-linked immunosor-bent assay(ELISA)was used to determine IL-12 concentration in the tumor tissue supernatants.The tumor infiltrating lymphocytes (TILs)were extracted by enzymatic digestion.The proportion of Th1 and Th2 cells in TILs was determined by flow cytometry.Results:The CD40ScFv protein was successfully identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot.The molecular size and concentration of the purified protein were 27kDa and 1.12 mg/mL,respectively.The tumor sizes of the CD40ScFv and CD40mAb groups were(3.044±0.239)cm3and(2.749±0.261)cm3,respectively,which were significantly smaller(P<0.05)than that of the NS group(3.933±0.326)cm3.The tumor IL-12 concentration(determined by ELISA)in the CD40ScFv group(396.27±48.13)pg/mL and the CD40mAb agonist group(457.63±58.37)pg/mL were significantly higher(P<0.05)than that of the NS group(79.51±14.97) pg/mL.The results of flow cytometry showed that the excited Th1/Th2 cell ratios were 6.32±0.87 and 5.54±0.71 for the CD40ScFv and CD40mAb groups,respectively,which were significantly higher(P<0.05)than that of the NS group(1.79±0.38).Conclusions:The agonis-tic CD40 antibody inhibited tumor growth by regulating the Th1/Th2 ratio and IL-12 secretion via promotion of DC activation,which is one of the important mechanisms affecting Th1/Th2 balance in the tumor microenvironment