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

Abstract

Background: IL-15 is a potential activator of T and NK cells and a promising candidate for vaccine adjuvant and immunotherapy including cancer. IL-15 has drawn attention of researchers to explore its therapeutic potential. However, its short half-life (~1 hr) and poor bioavailability limit its therapeutic use. Need of higher amount and multiple infusions to achieve the optimum therapeutic response increase the risk of side effects and overall cost of therapy. Various approaches have been adopted to address these issues but the efficacy of modified IL- 15 is questionable, particularly for its ability to generate focused T cell response. In the present study we have designed chimeric IL-15 with an objective of concentrating its effect/influence on T cells to augment memory response. Methods: The in silico evaluation of chimeric IL-15 including design, prediction, structural assessment and docking was performed. In wet lab, IL-15 and IgG2 genes from mouse and human sources were amplified and separately cloned into PCR2.1 TOPO vector. The K322A mutations into IgG2 Fc and N72D mutation into IL-15 were introduced by site directed mutagenesis. IL-2 signal peptide (SP) was inserted in pcDNA3.1 vector downstream of CMV promoter by using GeneArt Service, Thermo Fisher Scientific. Sequentially IL-15 CDS and IgHg2 genes were inserted in IL-2-pcDNA3.1 vector to make IL-2SP-N72DIL-15CDSK322AIgHg2- pcDNA3.1 construct (chimeric IL-15 constructs). All cloning, mutagenesis and fusion constructs were confirmed by Sanger sequencing. CHO cells were transfected with pEGFP-n1 plasmid (positive control), Chimeric IL-15 construct or pcDNA3.1 vector by using Lipofectamine LTX plus reagents (Thermo Fisher Scientific). Secretion of chimeric IL-15 was measured by sandwich ELISA. Affinity chromatography was used to purify/concentrate the chimeric IL-15 protein. The purified chimeric IL-15 was loaded on SDS-PAGE under non-reducing or reducing conditions to assess the formation of dimers. The coomassie dye was used to stain the protein bands. Densitometry analysis was performed to compare the percentage of dimer vs. monomer. The chimeric IL-15 was also confirmed by western blot analysis. Pharmacokinetic evaluation including determination of Half-life, stability assessment and shelf-life determination was performed. Biological activity of chimeric IL-15 was determined by various in vitro and in vivo experiments. Results and Conclusion: In the current study, we have addressed the above limitations with our novel design and systematically improved the stability and half-life of IL-15 with an objective to make it a T cell centric adjuvant. The in silico evaluation suggest that our model for chimeric IL-15 not only fulfils the requirements of homodimer formation but also maintains the native structure and functionality of both partner proteins. In wet lab experiments (1) we covalently conjugated human or mouse-IL-15 with IgG2/2a heavy constant chain (IgHg2/2a) with an intention of targeting specifically to APC thereby presenting IL-15 to T-cells along with MHC/p complex, (2) we have fused both the molecules via a linker, which would provide flexibility to the molecule, (3) we have introduced K322A mutation in the immunoglobulin heavy constant chain to delay the immune clearance. The above engineering made IL-15 more stable (Tm or Tmax=78.26ᵒC), longer bio-available i.e., >40 folds increment in half-life. (4) We have also introduced N72D mutation in the IL-15 distal region to improve its binding affinity with the IL-15Rß present on T cells. Our results show that the chimeric IL-15 is predominantly expressed as dimer, very stable whose half life is >40 hr. The preliminary in vitro and in vivo studies demonstrate that the molecule is bioactive and has ability to activate IL-15 pathway as well as to modulate CD8+ T cell responses. In the current study, we have not only mimicked the typical trans-presentation of IL-15 action, but also concentrated its effect predominantly on CD8+ T cells by fusing it with IgG2/2a base (chimeric IL-15). The current strategy presents the novel design to improve the stability, half-life of IL-15 and to make it longer bioavailable. In this study we have developed stable chimeric IL-15 which has improved half-life and longer bioavailability. Our chimeric IL-15 induces IL-15 pathway, anti-apoptotic factors and gene required for conversion of naive/effector T cells to memory. Currently we are exploring is suitability for vaccine adjuvant and for immunotherapy including cancer therapy.