Abstract
Interleukin-15 (IL-15) is a cytokine previously suggested as a potential immunotherapy for cancer treatment. IL-15 can effectively reduce tumor growth in many preclinical tumor models including prostate cancer. This is due to its ability to expand and activate immune cells, such as CD8+ T cells and natural killer cells. To increase the potency of IL-15, we have engineered a protein variant that can be modified to localize and be retained in tissues where it is administered. However, the production of recombinant IL-15, the purity, and correct refolding of the final protein is not always ideal. In the current study, we aimed to optimize the methodology for production and purification of a modified recombinant human IL-15 and investigate the efficacy of the produced protein in the treatment of prostate tumors. Human IL-15 with its polypeptide sequence modified at the C-terminus to enable thiol conjugation with membrane localizing peptides, was produced in E. coli and purified using mild denaturing conditions (2M urea) from a washing step or from solubilization of inclusion bodies. The purified protein from the wash fraction was conjugated to a myristoylated peptide to form a membrane-localizing IL-15 (cyto-IL-15). The efficacy of cyto-IL-15 was investigated in subcutaneous TRAMP-C2 prostate tumors in mice and compared with cyto-IL-15 derived from protein purified from inclusion bodies (cyto-IL-15 Gen). When mild denaturing conditions were used for purification, the largest amount of IL-15 was collected from the wash fraction and a smaller amount from inclusion bodies. The protein from the wash fraction was mainly present as a monomer, whereas the one from inclusion bodies formed homodimers and higher complexes. After cytotopic modification, the purified IL-showed great efficacy in delaying prostate tumor growth (∼50%) and increased mice survival by ∼1.8-fold compared with vehicle. This study demonstrates an alternative, inexpensive and efficient method to produce and purify a modified version of IL-15 using mild denaturing conditions. This IL-15, when cytotopically modified, showed great efficacy as a monotherapy in prostate tumors in mice further highlighting the potential of IL-15 as a cancer immunotherapy.
Highlights
Interleukin-15 (IL-15), a protein with a molecular weight of 14–15 kDa, is a member of the four α-helix bundle family of cytokines that is involved in the proliferation and activation of natural killer (NK) and T cells (Waldmann, 2006)
Recombinant IL-15 was obtained from inclusion bodies, resulting in low yields of protein recovered after refolding and most of the purified protein being in the form of complexes
In this study, decreasing the temperature did not lead to the production of soluble IL-15 since, after disruption of E. coli, no IL-15 was present in the soluble fraction
Summary
Interleukin-15 (IL-15), a protein with a molecular weight of 14–15 kDa, is a member of the four α-helix bundle family of cytokines that is involved in the proliferation and activation of natural killer (NK) and T cells (Waldmann, 2006). IL-15 has been considered one of the most promising cytokines in the treatment of cancer due to its ability to enhance the anti-tumoral response of CD8+ T and natural killer (NK) cells in pre-clinical studies (Klebanoff et al, 2004; Teague et al, 2006). Our laboratory has shown that IL-15 is the only protein among a panel of several cytokines that was able to expand and activate immune cells in vitro; this effect was increased when prostate cancer cells were present (Sakellariou et al, 2020). In in vivo studies, we have shown induction of cell death and improved mice survival when a membranelocalizing cytotopically modified IL-15 was injected directly into prostate tumors (Papaevangelou et al, 2020)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
