1. Introduction
Interleukin-18, IL-18, is a cytokine that plays a role in many aspects of human immunological system. This cytokine was previously named as interferon-γ-inducing factor that reflected its function in human physiology (Okamura et al., 1995). Nowadays, this cytokine has been recognized as one of the cytokine that promote type 1 helper T cell response which is essential for anti-tumor immunity (Fabbi et al., 2015; Nakamura et al., 2018). Moreover, IL-18 also polarizes human NK cells to develop a distinct helper differentiation phenotype (CD83+CCR7+CD25+) and stimulate cytotoxic lymphocytes to enhance IFN-γ secretion, granule-mediated cytotoxicity and Fas ligand expression (Mailliard et al., 2005; Srivastava et al., 2010). From these reasons, the using of IL-18 in cytokine therapy for cancer has been established and developed in both animal and human studies (Robertson et al., 2006, 2008). However, although cancer patients were good tolerated to this cytokine, the efficacy was still limited due to its high-affinity antagonist IL-18-binding protein (IL-18BP) which is increased in many cancers (Srivastava et al., 2010). Moreover, the precipitation or aggregation which is commonly found in recombinant cytokine therapeutics is another problem that may cause the loss of activity and increase the risk of immunogenicity (Lipiäinen et al., 2015).
To overcome this immunological inhibition of IL-18BP, many studies tried to apply the knowledge of molecular engineering to this protein by site directed mutagenesis and protein engineering. A study showed that the substitution of amino acid of IL-18 from glutamic acid to alanine at position 6 (E6A) and lysine to alanine at position 53 (K53A) could enhance the activity of IL-18 and reduce the affinity to IL-18BP (S. H. Kim et al., 2001). In addition, they also changed the type of mutation of E6 from E6A to E6K and found that this mutation increased the activity of IL-18 to induce IFN-γ about 3-8 times compared to wild-type (S.-H. Kim et al., 2002). Another research highlighted the change of intramolecular core threonine at position 63 to alanine enhanced the activity of IL-18 approximately 3 times compared to native IL-18 (Swencki-Underwood et al., 2006). Recently, decoy-resistant IL-18 has been developed for applying to cancer immunotherapy. This modified IL-18 was reported to be less sensitive to IL-18BP than native IL-18 and promoted the polarization and stimulation of effector cytotoxic T cells and natural killer cells, respectively (T. Zhou et al., 2020). While most of studies focused on IL-18 activity improvement, some works tried to develop the low aggregating IL-18. For example, the surface cysteine of IL-18 was found to be associated with protein aggregation and instability and this led to the replacement by serine at position 38, 68, 76 and 127, which caused IL-18 to be tolerated to oxidative stress and decreased the aggregation (Yamamoto et al., 2004).
In previous work, we have developed the modified IL-18 with the high activity about 16 times more than wild-type protein (Saetang et al., 2016). This engineered IL-18 was found to be a potent inducer for IFN-γ induction from NK-92MI cell. Moreover, it showed the anti-tumor activity that promoted type 1 helper T cell and cytotoxic T lymphocyte in CT26-WT colon cancer animal model (Saetang et al., 2020). However, according to the stability testing from the other works, IL-18 tend to form multimer which led to the inactive IL-18 aggregates (Kato et al., 2003; Li et al., 2003; Yamamoto et al., 2004). This problem was also found in our work when the high concentration of modified IL-18 was prepared. Therefore, to improve the quality of modified form of IL-18 to be more stable and less aggregated, we tried to rely on the method that changed the surface cysteine by serine to our IL-18.
This study described the generation of double modified IL-18 with the increasing of both activity and stability. Interestingly, the replacement of cysteine by serine also improved the activity of engineered IL-18 indicating the less aggregation led to the higher activity.