Expression of novel fusion proteins IL2/FU-MK-1-scFv in microorganism-host cells and its potential anti-tumor activities as a cytotoxic immunotherapy agent for FU-MK-1 expressing tumors

Abstract

MK-1, the target molecule of FU-MK-1, is encoded by the GA733-2 gene, which is currently being used as a target in clinical trials for gastric, intestinal, and biliary cancer treatment with monoclonal antibodies. Here, two different arrangement of heavy-chain and K light-chain variable fusion gene, IL2/FUscFv(VK-VH) or IL2/FUscFv(VWVK), were constructed. The efficiency of protein expression in prokaryotic host expression system, Escherichia coli strains BL21(DE3)pLysS and Rosetta-gami B was compared with eukaryotic host expression system, Pichia pastoris strains GS115 and KM71H, for their ability to produce fusion protein. It was found that the fusion proteins were expressed in E. coli strain Rosetta-gami B in reasonable yield with high percentage of soluble form (0.25 gil of IL2/FUscFv(Vw VK) with solubility 89.29% and 0.26 gil of IL2/FUscFv(VK-VH) with solubility 84.61 %) when cultivated at 25°C, pH 7, and induced with 0.05 mM IPTG for 10 h. In comparison, P. postoris produced the secreted fusion proteins. The highest production of the fusion protein at 0.258 ± 0.013 gil was obtained under pH 3, 30°C, and induction with 0.1 % (v/v) methanol for 96 h in shaken flask cultivation. Finally, fed-batch cultivation was performed in 5 l fermenter. The highest amount of secreted fusion protein was 0.425 ± 0.002 gil. Following purification, the fusion protein was examined the specific binding activity to CHO cell expressing MK-1. The fusion protein retained the specific binding activity to MK-1 antigen due to it significantly bound to MK-1 expressing CHO cell but not MK-1 non-expressing CHO cell when compared using Student's t test (p<0 .05).