Antigen-binding activity of purified anti-KIFC1 scFvs

The antigen-binding activity of purified anti-KIFC1 scFvs in reducing or non-reducing conditions was analyzed by ELISA (Figure 18A). The KIFC1 1-50 and KIFC1 51-100 peptides, epitope of 6C407 and 2C281, respectively, were coated on the plate and the purified scFv proteins were treated; and the scFv antibodies bound to antigen were detected by rabbit IgG for Protein-A tag binding and AP-conjugated anti-rabbit IgG. In the results, 2C281 and 6C407 showed antigen-binding activity even in the absence of intra-chain disulfide bonds.

Further, in vitro staining was performed to confirm that the purified scFv binds not only to the peptide antigen fragment but also to the full-length KIFC1. KIFC1-GFP-expressing HeLa cells were fixed and then treated with anti-KIFC1 scFv, and the presence of KIFC1 in the nucleus was examined by confocal microscopy (Figure 18B). In the interphase cell, GFP-KIFC1 is in the nucleus, and the purified 2C281 and 6C407 scFvs were stained at the same position as KIFC1 (Figure 18C). These results indicate that the anti-KIFC1 scFv consisting only of the variable region of the anti-KIFC1 antibody maintained the ability of the antibody to bind both peptide and full-length protein antigen.

Figure 18. Antigen-binding activity of purified anti-KIFC1 scFvs. (A) Evaluation of antigen-binding activity by ELISA. Purified anti-KIFC1 scFv proteins were placed in wells coated with KIFC1 peptide#1 and #2, which are epitope of 2C281 and 6C407, respectively.

Bound scFvs to peptides were detected with rabbit IgG and AP-conjugated anti-rabbit IgG.

(B) Workflow of the assay. (C) HeLa cells stably expressing GFP-KIFC1 were fixed and permeabilized, followed by incubation with purified anti-KIFC1 scFv proteins. Bound proteins were detected by rabbit IgG and TRITC-conjugated anti-rabbit IgG. Stained cells on coverslips were mounted and observed by confocal microscopy.

59 K. Antigen-binding activity of cytosolic scFvs

It was confirmed whether the antigen-binding activity was maintained even when the antibody was expressed in scFv form in the cytosol. By removing the leader sequence, the scFv was expressed in the cytosol and the HA tag was added to the carboxy terminus for detection (Figure 19A). Antigen-antibody interaction was analyzed by confocal microscopy after cytosolic expression of anti-KIFC1 scFv (Figure 19B). Co-localization of KIFC1 and scFv was observed in mitotic HeLa cells expressing 6C407 and 2C281 scFv, but not HW6, an anti-DR5 antibody used as a negative control. To investigate antigen-binding activity of cytosolic scFv, the cell lysate obtained by disrupting the scFv-expressing HEK293T cells was treated to a peptide antigen-coated ELISA plate (Figure 20). ER-directed scFvs exhibited antigen-binding activity in the absence of DTT, whereas binding activity of 2C281 was decreased after DTT treatment. It is known that intra-chain disulfide bond in the scFv is reduced at 55 mM of DTT (Schouten et al., 2002). In the case of cytosolic scFv, 2C281 and 6C407 showed weak antigen-binding activity in the absence of DTT and no activity under DTT treatment. Interaction between endogenous KIFC1 and anti-KIFC1 scFv was also detected by IP (Figure 21). Myc-tagged scFv proteins were expressed in HeLa and precipitated by anti-myc antibody-conjugated agarose. The interaction between cytosolic anti-KIFC1 scFv and KIFC1 was confirmed by immunoblotting, and in particular, 2C281 was found to bind more KIFC1 protein than 6C407. KIFC1 was not detected when HW6 was precipitated, suggesting that KIFC1 protein is only recognized by anti-KIFC1 scFv. These results suggest that scFv antibodies expressed in the cytosol maintained their antigen-binding activity.

Figure 19. Confocal microscopic analysis to confirm antigen-binding activity of cytosolic scFvs. (A) Schematic diagram of plasmids construction to express scFv-HA in the ER or cytosol. (B) Co-localization of endogenous KIFC1 with cytosolic anti-KIFC1 scFvs were observed in the HeLa cells transiently transfected with plasmid encoding ΔLd-scFv-HA gene.

Cells in mitosis were collected through synchronization by double-thymidine method. After fixing and permeabilizing, cells were incubated with anti-HA IgG and anti-KIFC1 IgG, followed by TRITC-conjugated anti-mouse IgG and FITC-conjugated anti-rabbit IgG.

Stained cells were observed by confocal microscopy.

Figure 20. Evaluation of Antigen-binding activity of cytosolic anti-KIFC1 scFvs.

Antigen-binding activity of scFvs was evaluated by ELISA. Lysates of HEK293T cells transiently transfected with Ld- or ΔLd-scFvs were prepared in the absence (A) or presence (B) of 55 mM DTT at RT for 30 min, placed in wells coated with specific antigens. Bound scFvs were detected with rabbit anti-HA antibody and AP-conjugated goat anti-rabbit IgG (Fc specific). Data are presented as mean ± SEM (n = 3).

Figure 21. Investigation of binding activity of cytosolic scFvs to endogenous KIFC1 by IP. (A) Plasmid constructs for expression of Ld-anti-KIFC1-scFv-HA and ΔLd-anti-KIFC1-scFv-HA proteins. (B) Co-IP was performed to confirm binding of ΔLd-anti-KIFC1 scFv to endogenous KIFC1. Lysates of HeLa cells expressing ΔLd-anti-KIFC1 scFv-myc were immunoprecipitated using anti-myc antibody-conjugated agarose. Proteins in the eluate were detected with mouse anti-myc, rabbit anti-KIFC1, and rabbit anti-cytoskeletal actin antibody, followed by HRP-conjugated horse anti-mouse IgG, HRP-conjugated rabbit anti-goat IgG and HRP-conjugated goat anti-rabbit IgG, respectively. Input and elution samples were resolved by reducing SDS-PAGE. Input represents 10% of the total amount of lysate used for IP.

L. Increased multipolar spindle in anti-KIFC1 scFv expressing cells

It was investigated whether cytosolic scFvs can inhibit the function of antigen after antigen-antibody interaction. The function of KIFC1 is to cluster multiple centrosomes in cancer cells containing extra centrosomes, induce bipolar spindle formation and enable normal cell division. When the KIFC1 function is inhibited in mitosis, multiple centrosomes form their own spindle pole, resulting in multipolar spindle formation. We observed whether this phenomenon is caused by cytosolic scFvs expression. To observe spindle pole, gamma tubulin at the end of microtubule was stained and observed with a fluorescence microscope.

HeLa, MDA-MB-231, and RPE-1 (retina pigment epithelium) were selected for the analysis.

In MDA-MB-231 and HeLa, the centrosome amplification level of each cell is 44% and 11%, respectively (Kwon et al., 2008). RPE-1 is an immortalized normal cell that is used as a control. It was expected that more cell populations with a higher percentage of extra-centrosome will be more dependent on the clustering of KIFC1, resulting in more multipolar spindles when KIFC1 is suppressed in these cell lines. When 6C407 scFv was expressed in the cytosol, about 20% of MDA-MB-231 cells had multipolar spindle (Figure 22). This result is approximately 12-15% higher than 2C281 or HW6 expression, indicating the inhibitory effect of cytosolic 6C407 scFv on KIFC1. KIFC1 inhibitory activity of 6C407 scFv was higher than that of 2C281, indicating that there is a difference in intracellular activity depending on the antibody. Furthermore, unlike MDA-MB-231, the expression of 2C281 increased the multipolar spindle in HeLa, which indicates that the degree of cytosolic scFv activity varies from cell to cell.

When a multipolar spindle is formed, spindle assembly checkpoints inhibit anaphase onset until all chromosomes are properly located, increasing the time taken from metaphase

to the beginning of anaphase. Change in mitotic duration in the cells expressing cytosolic anti-KIFC1 scFv was observed by live cell imaging. In Figure 22B, the multipolar spindle was observed most frequently in MDA-MB-231 cells expressing 6C407 scFv in the cytosol, so we measured the mitotic duration under the same conditions. The mitotic duration of MDA-MB-231 cells was approximately 50 min in cytosolic HW6 scFv expression, but significantly increased to 71 min in cytosolic 6C407 scFv expression (Figure 23). Considering that mitotic duration of KIFC1 knock-downed MDA-MB-231 is only about 11 minutes longer than control (43 min for si-control and 54 min for si-KIFC1), the expression of cytosolic 6C407 scFv appears to have a significant effect on the mitotic duration of MDA-MB-231 cells.

The change in mitotic duration by cytosolic 6C407 scFv expression is further observed through the mitotic protein level. The levels of cyclin B1 and phospho-histone H3 in mitotic cells were monitored by immunoblotting. Expression level of cyclin B1 is increased in the G2/M progression and indicates that the cell has entered mitosis. The amount of cyclin B1 decreased as cells entered metaphase, and then phosphorylation was observed in ser10 of histone H3 (Juan et al., 1998). Since the level of phospho-histone H3 is maintained high until the end of mitosis, it is widely used as a mitotic marker. To observe the level of mitotic proteins, HeLa cells were transfected with ΔLd-scFv or si-RNA and harvested for every hour after 8 h release from thymidine block (Figure 24). If the mitotic duration is delayed, it is expected that cyclin B1 and phospho-histone H3 are maintained high until the cells enter anaphase. When the control HW6 scFv was expressed in the cytosol, the level of cyclin B1 peaked at 10 hours and rapidly decreased from 11 hours. When 6C407 scFv was expressed, the level of cyclin B1 maintained high at 11 hours. The amount of phospho-histone H3 began

to increase when cyclin B1 is decreased. In the cells expressing cytosolic 6C407 scFv, the expression level of phospho-histone H3 rapidly increased at 10 hours and maintained until 12 hours. The level of cyclin B1 and phospho-histone H3 in cells expressing cytosolic 6C407 scFv indicates delayed meta-to-anaphase transition. These results indicate that the cytosolic scFv that has antigen-binding activity could be used to inhibit the function of intracellular antigens.

Figure 22. Increased multipolar spindle formation in anti-KIFC1 scFv expressing cells.

(A) Immunoblotting to confirm the level of myc-conjugated anti-KIFC1 scFv expression and KIFC1 knockdown in HeLa, MDA-MB-231, and RPE-1 cells. Cell lysates were separated by SDS-PAGE and were detected by immunoblotting with mouse anti-myc, rabbit anti-KIFC1, and rabbit anti-actin antibody, followed by conjugated horse, anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG. (B) Multipolar spindle formation induced by anti-KIFC1 scFvs were demonstrated by counting the number of centrosomes. After expressing scFvs through transient DNA transfection, cells were synchronized at mitosis through one thymidine block followed by staining with mouse anti-γ-tubulin IgG and Alexa488-labeled rabbit anti-mouse IgG. Counting was performed at least three different regions and the number of counted cells was indicated in the graph. P values of paired t-tests (*p<0.05).

Figure 23. Prolonged mitotic duration of MDA-MB-231 cells expressing cytosolic 6C407 scFv. Mitotic duration during cells from round-up to anaphase onset was measured 24 h after DNA transfection by real-time microscopy in MDA-MB-231 cells for 48 h. Live cell images were analyzed by NIS-elements software and mitotic duration of cells was observed at least 5 different regions. Statistical significance is indicated in (A) and mitotic duration of individual cells were depicted in (B). P values of paired t-tests (*p<0.05).

Figure 24. Delayed meta-to-anaphase transition upon 6C407 scFv expression in the cytosol. (A) Cell synchronization process through one-thymidine block method. HeLa cells transfected with plasmids DNA encoding ΔLd-scFvs or siRNA were treated with 100 mM thymidine for 20 h. After replacing with thymidine-free medium and incubating for another 8 h to analyze mitotic cells, cells were harvested every hour. (B) SDS-PAGE and immunoblotting results to confirm the level of phospho-histone H3, cyclin B1, and GAPDH in the cell lysate. The expression level of scFv was detected by mouse anti-myc antibody and the knockdown level of KIFC1 by siRNA was detected by rabbit anti-KIFC1 antibody. (A:

asynchronized cells)

IV. 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

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.

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

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.

V. CONCLUSION

We found that variable regions of cytosolic IgG intrabodies determine H:L association and antigen-binding activity. Also, partial intra-chain disulfide bonds in the cytosolic IgG intrabodies were closely related to the integrity of constant region. Cytosolic antibody in a scFv form showed antigen-binding activity and could inhibited the function of target protein.

This is the first report to investigate structural properties of cytosolic IgG intrabody.

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