Development of Chimeric IL 15 to augment T cell response and memory

Abstract

IL-15 is a potential activator of T and NK cells and a promising candidate for vaccine newlineadjuvant and immunotherapy including cancer. IL-15 has drawn attention of researchers to newlineexplore its therapeutic potential. However, its short half-life (~1 hr) and poor bioavailability newlinelimit its therapeutic use. Need of higher amount and multiple infusions to achieve the newlineoptimum therapeutic response increase the risk of side effects and overall cost of therapy. newlineVarious approaches have been adopted to address these issues but the efficacy of modified IL- newline15 is questionable, particularly for its ability to generate focused T cell response. In the newlinepresent study we have designed chimeric IL-15 with an objective of concentrating its newlineeffect/influence on T cells to augment memory response. newlineMethods: newlineThe in silico evaluation of chimeric IL-15 including design, prediction, structural assessment newlineand docking was performed. In wet lab, IL-15 and IgG2 genes from mouse and human newlinesources were amplified and separately cloned into PCR2.1 TOPO vector. The K322A newlinemutations into IgG2 Fc and N72D mutation into IL-15 were introduced by site directed newlinemutagenesis. IL-2 signal peptide (SP) was inserted in pcDNA3.1 vector downstream of CMV newlinepromoter by using GeneArt Service, Thermo Fisher Scientific. Sequentially IL-15 CDS and newlineIgHg2 genes were inserted in IL-2-pcDNA3.1 vector to make IL-2SP-N72DIL-15CDSK322AIgHg2- newlinepcDNA3.1 construct (chimeric IL-15 constructs). All cloning, mutagenesis and newlinefusion constructs were confirmed by Sanger sequencing. CHO cells were transfected with newlinepEGFP-n1 plasmid (positive control), Chimeric IL-15 construct or pcDNA3.1 vector by using newlineLipofectamine LTX plus reagents (Thermo Fisher Scientific). Secretion of chimeric IL-15 newlinewas measured by sandwich ELISA. Affinity chromatography was used to purify/concentrate newlinethe chimeric IL-15 protein. The purified chimeric IL-15 was loaded on SDS-PAGE under newlinenon-reducing or reducing conditions to assess the formation of dimers. The coomassie dye newlinewas used to stain the prot