4. Discussion
IL-18 has been proposed as a candidate for cancer treatment for a long
time. Many studies have revealed the role of this cytokine as inducing
factor for IFN-γ, which plays an important role in anti-cancer immunity
(Castro et al., 2018). However, the limitation of using this cytokine
was demonstrated by the low impact of this cytokine in clinical trials
(Robertson et al., 2006). This may be due to the presence of natural
inhibitor, IL-18 binding protein, that increases in several types of
cancers (Srivastava et al., 2010). Interestingly, many research tried to
improve both efficiency and stability of this protein, mainly by protein
engineering. The popular technique that has been applied to modify this
protein is site-directed mutagenesis based on the knowledge of binding
mode of IL-18 to its receptor (Kato et al., 2003; S. H. Kim et al.,
2001; S.-H. Kim et al., 2002; Saetang et al., 2016; Yamamoto et al.,
2004). Most of studies showed the successful development of engineered
IL-18 with enhancing activity and bioavailability, especially the
development of the decoy-resistant IL-18 (DR-18) that displayed the low
affinity to IL-18 binding protein while maintaining the signaling
cascade when binding to its receptor (T. Zhou et al., 2020). These all
emphasized the role of this cytokine in cancer immunotherapy.
In this work, we tried to improve the aggregation phenomenon of our
modified IL-18 found at the high concentration (data not shown).
Although the improvement of efficiency of our IL-18 could enhance the
satisfactory result from the study, the aggregation has still been the
problem because of the unpredictable activity and immunogenicity
results. This was revealed by several types of FDA-approved
alpha-helical cytokines, such as IL-2 (Fatima et al., 2012), IFN-β-1b
(Lipiäinen et al., 2015) or granulocyte colony stimulating factor
(G-CMS) (Krishnan et al., 2002) that were found to form multimer
precipitation in some condition, especially physiological condition. The
aggregation may result in loss of activity or induction of unfavorable
immune response. For IL-18, Yamamoto and colleagues reported that the
free surface cysteines, including C38, C68, C76 and C127 may contribute
to IL-18 aggregation based on the computational structure prediction
(Yamamoto et al., 2004). After the replacement of these cysteine by
serine and the exposure of oxidative stress, the C-to-S mutations tend
to form aggregation lower than wild-type IL-18, especially when all four
surface cysteines were replaced (Yamamoto et al., 2004). This is in
accordance with our study that when applied all these modifications to
IL-18 DM, the fluorescent signal corresponding to protein aggregation
was lower compared to IL-18 DM and wild-type. This suggested the
contribution of surface cysteine on intermolecular bonding which also
found in other cytokines (Karpusas et al., 1997; Lipiäinen et al.,
2015). For example, the cysteine to serine substitution was performed
for IFN-β-1b, which has been commercially used to treat relapsing forms
of multiple sclerosis (MS) to increase the stability of this cytokine
(Lin, 1998). The aggregation due to cysteine residue was also found in
IL-31 which the formation of disulfide bond occurs at the intracellular
levels (Shen et al., 2011). The replacement of these cysteine residues
by serine also helped to improve the aggregation phenomenon (Shen et
al., 2011). Interestingly, human α-synuclein is another case that
emphasized the role of cysteine in protein stability. The aggregation of
α-synuclein in Lewy bodies in midbrain dopamine neurons is usually
associated with Parkinson’s disease. Importantly, amino acid replacement
of tyrosine by cysteine showed the increasing aggregation rate under the
oxidative stress led to the cellular toxicity which may be associated
with Parkinson’s disease (W. Zhou & Freed, 2004).
In addition to the decrease of aggregation rate under oxidative
condition, IL-18 DM1234 showed the higher ability to induce IFN-γ from
NK-92MI cell compared to IL-18 DM. This may be caused by the structural
changes of IL-18 DM1234 that led to the more suitable form for receptor
binding. As our MD simulation revealed, D35 of IL-18 DM1234 changed its
direction closer to V125 and S127 of IL-18 receptor compared to IL-18 WT
and IL-18 DM. This probably led to the formation of stronger hydrogen
bonds between these molecules. Importantly, this interaction was
reported to played an important role for stabilizing α-helix structure
of IL-18 that mediated the interaction between IL-18 and IL-18 receptor
α (Tsutsumi et al., 2014). One another explanation is that the
replacement of cysteine by serine may increase the surface polarity
since serine showed that less hydrophilic index compared to cysteine
(Kyte & Doolittle, 1982) and this scenario may improve IL-18-receptor
binding. The reduction of aggregation rate might be another factor that
improve the efficacy of this cytokine since it may increase the active
form of IL-18 DM1234 in the system.