DISCUSSION

In this study, structural and functional characteristics of anti-KIFC1 antibodies after cytosolic expression in the form of IgG or scFv were analyzed. First, the variable region of the mouse hybridoma antibody specific to the KIFC1 protein was fused with the constant region of human IgG1. Mouse-human chimeric anti-KIFC1 IgGs that were purified from mammalian cells maintained their binding activity. To determine whether antigen-binding activity was maintained when chimeric anti-KIFC1 IgG was expressed in the cytosol of mammalian cells, ELISA, IP, and confocal microscopy were performed. Of the three antibodies, 2C281 and 6C407 retained their ability to bind to antigen, but 10C358 did not. In order to investigate the factors that determine the difference of antigen-binding activity between each cytosolic IgG, the association of heavy and light chains was analyzed.

Assembly between heavy and light chains was observed in 2C281 and 6C407, which showed antigen-binding activity, but not in 10C358, which lost antigen-binding activity. In other words, if H:L association did not occur, and the antigen-binding site was not formed properly, leading to loss of the antigen-binding ability. However, the investigation of the hybrid 2C281 IgG showed that even if the H:L assembly occurs, antigen-binding activity may not be exhibited if the heavy and the light chains are not matched. Therefore, it was found that the coordination between heavy and the light chains plays an important role in forming antigen-binding activity. To analyze the characteristics of cytosolic IgG differentiated from ER-directed IgG, disulfide bonds were analyzed. In the results, it was found that disulfide bonds partially exist in the cytosolic IgG, and the intra-chain disulfide bond in the constant region

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plays an essential role in H:L association. The presence of disulfide bonds was also confirmed by PEGylation of cysteine residues in cytosolic IgG. To investigate whether antibodies consisting of only the variable region in absence of constant region retain their function, scFv form of anti-KIFC1 antibodies were purified or cytosolically expressed to be analyzed. The anti-KIFC1 scFv purified in E. coli had antigen-binding activity, which was maintained even when expressed in the cytosol of mammalian cells. In addition, cytosolic expression of anti-KIFC1 scFv inhibited the function of anti-KIFC1, resulting in an increase in multipolar spindle formation and mitotic duration in cells containing extra-centrosomes. These results suggest that antibodies expressed in the cytosol in the form of IgG and scFv can effectively target intracellular antigens.

The most commonly used forms of antibodies for cytosol expression are scFvs because disulfide bonds connecting the chains that make up the antigen-binding site are not properly formed in the cytosolic reducing environment (Biocca et al., 1995; Saaranen and Ruddock, 2013). Various methods have been attempted to obtain functional antibodies when expressed in scFv form in the cytosol (Schiefner et al., 2011; Mazuc et al., 2014; Waraho-Zhmayev et al., 2014). Methods for selecting antibodies to act in cells (Visintin et al., 2002; Lee et al., 2006) or methods for fusion of proteins to existing antibodies have been devised (Visintin et al., 2002; Zheng et al., 2003; Shaki-Loewenstein et al., 2005). I have expressed antibodies in the cytosol in the form of IgG in this study. From a broader perspective, it could be believed that CH is fused to VH and CL to VL. However, the fact that the interaction that is formed between constant regions plays a crucial role in determining the characteristics of the antibody shows that the presence of a constant region is different from the role of proteins that have simply been previously fused to scFv.

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The way in which cytosolic IgG is assembled in the cytosol is different from what is known.

In general, it is known that cytosol is a reducing environment and disulfide bonds are hardly formed. The results confirm the existence of intra-chain disulfide bond of cytosol-expressed IgG. However, even when inter-chain disulfide bond was not formed, the binding between heavy and light chains was observed. It was an interesting finding that ER-directed IgG and cytosolic IgG had different properties depending on where they were expressed, even though the amino acid sequences were identical. Further study of these features may increase understanding of cytosolic IgG.

Studies on cytosolic scFv were associated with the inhibition of antigen function. Because the target molecule, KIFC1, is present in the nucleus rather than the cytosol, the antigen-antibody interaction can occur only briefly during mitosis. However, cytosol expression of anti-KIFC1 scFv showed suppression of antigen function despite its short action during mitosis. These results suggest that cytosolic antibodies can be used to target not only cytosolic antigens, but also nuclear antigens without addition of nuclear localization signal.

One of the further studies that can be performed is to find a more effective way to express intracellular antibodies through studies comparing cytosolically expressed IgG and scFv. IgG is known to have a high stability and a long duration due to the constant region. In contrast, it is known that scFv proteins expressed in the cytosol have lower expression level compare to those of its secretory counterparts (Biocca et al., 1995). It can be confirmed how much it differs from the cytosolic expression of IgG proteins comparing to scFvs. It is also known that the cytosolic Fc receptor present in the cytosol recognizes and degrades purified IgG delivered from outside the cell bound to the antigen (Clift et al., 2017). If this phenomenon is also observed in cytosolic expression IgG, the intracellular action of cytosolic IgG can be

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improved more efficiently by applying a strategy such as avoiding recognition of cytosolic Fc receptor.

This study has improved the understanding of the structural and functional properties of cytosolic antibodies and provides the information needed for using various types of cytosolic antibodies.

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