We summarized the results of ELISA of Fig. 7, 8, 9, and 11 (Table 4). Clone 6C407 and 8C346 against Glu43 - Arg54 and Met1 - Lys12 of HSET, bound all kinds of antigen and were expected to have high binding activity. Clone 2C280 and 2C281, these bound to whole HSET protein, but not to yeast displayed protein. There are two clones that could bind peptide and whole HSET protein but yeast displayed protein, clone 9C352 and 9C353 which have epitope between Lys12 - Lys23. Because of different targeting region of clone 1C274 and 3C290 (Pro123 - Arg134 and Gln115 - Ala126, respectively), they were not available using Met1 - Lys50 peptide and Met1 - Ala100 yeast displayed protein as antigens to perform ELISA.
Nevertheless, clone 1C274 and 3C290 showed comparatively high binding to whole HSET protein. All of these clones were targeting N-terminus of HSET, not C-terminus. The reason could be explained when amino acid sequence of human HSET and mouse KIFC1, were aligned.
Because C-terminus region of HSET and KIFC1 were almost similar sequence, it is thought that C-terminus of HSET could not induce immune response in mouse (Fig 12).
27 Table 4. Summary of anti-HSET mAbs
MAbs in ascitic fluids Purified mAbs Peptide divided by negative control value (1-1.5: +, 1.5-2: ++, 2-2.5: +++, 2.5-3:
++++, >3: +++++).
28
Figure 12. Sequence comparison between human HSET and mouse KIFC1. Human HSET and its mouse homolog, KIFC1, have higher homology of amino acid sequence at C-terminus than N-terminus.
29 E. Sequence analysis of anti-HSET mAbs
Sequence of mAbs were analyzed for the generation or enginieering of recombinant Ab.
DNA sequences of all mAbs were analyzed regardless of their binding activities. It is because that even mAbs with low binding activity against the prepared HSET could strongly bind to the intracellularly expressed HSET. Variable region genes were amplified and read by the primers that hybridize to immunoglobulin genes (Table 1). In case of clone 5C303 and 6C406, Vĸ genes were not able to be identified because those genes were amplified by MKV2 primer which also annealed to pseudo Vĸ gene from myeloma fusion partner cell. In case of clone 8C342, VH gene was not amplified by any MHV primers for unknown reason.
Comparing the sequences of mAbs, mAbs that target same region of HSET had similar variable region sequence (Table 5). Variable heavy chain genes (VH) of 3C288, 3C289, 3C290, and 3C292 that target Gln115 - Ala126 (peptide 3) were 100% identical. In particular, 3C288 and 3C289 had 100% identical variable kappa chain (Vĸ). Clone 6C404 and 6C406 that target Glu43 - Arg54 (peptide 6) had 100% identical VH sequence, although their Vĸ sequences could not be compared. Clone 4C295 and 4C298 that recognize Ser33 - Asp44 (peptide 4) also had 100% identical Vĸ and were different in only one amino acid on VH.
30
Table 5. Amino acids sequence of variable regions in anti-HSET mAbs
Variable heavy chain
31 Variable kappa chain
32
IV. DISCUSSION
It is well known that several cancer cells have amplified centrosomes compare with normal cells. There were many attempts to target centrosome bundling mechanism as a development of therapeutic molecules, including antifungal drug griseofulvin (Rebacz et al., 2007), kinesin spindle protein inhibitor, CENP-E inhibitor (Huszar et al., 2009), and phenanthrene-derived PARP inhibitor (Castiel et al., 2011). In recent studies, some small molecules that have been developed to block the centrosome clustering function of HSET induced the death of cancer cells, suggesting that HSET would be a promising target molecule for cancer therapy (Watts et al., 2013; Wu et al., 2013).
Generally, comparing to small molecule inhibitors which can cause side effects from the lack of specificity, mAbs are known to have high specificity against target antigen (Huang et al., 2004). Moreover, the half-lives of mAbs are 4-fold longer than small molecule inhibitors (Dancey and Sausville, 2003).
In this study, we successfully produced mAbs against N-terminus region of HSET molecules using a hybridoma technique, with the expectation to inhibit the function of HSET in the cancer cells in which function of HSET is indispensable for their cell division. HSET sequences were analyzed by BLAST search to design antigen for immunization. Some regions of HSET which show relatively hydrophilic and low homology with other proteins were synthesized as 12 amino acid-length peptides. The advantage of short length antigens, instead of immunization with full length protein, is that the production of anti-HSET mAbs which show cross-reactivity with other kinesin could be avoided. Though several mAbs to peptide or whole protein form of HSET were obtained, but the binding activity against endogenous HSET
33
molecule through intracelluar expression of these mAbs should be confirmed by the method of immunoprecipitation and immunofluorescence.
Binding activity of mAbs was different, depending on the types of antigen. It may be two reasons as follows. First, the glycosylation pattern may affect the binding site of mAbs. HSET fragment displayed on the yeast surface can be glycosylated in unpredicted regions of protein.
Different glycosylation pattern can lead to the change of protein conformation. Second, mAbs that had been derived from the immunization with HSET peptide antigens could not recognize the secondary/tertiary structure of intact HSET protein.
In fact, we tried simultaneously a hybridoma technique and a phage display technique to obtain anti-HSET mAbs. Unfortunately, however, none of the mAbs was obtained from phage display technique in which phage surface-displayed human Ab (single chain variable fragments) library was panned against the peptide antigens (corresponding to Met1 - Ala100 and Gln625 - Lys673 of HSET, respectively) as well as yeast surface-displayed HSET fragment (Met1 - Ala100).
All most of IgG mAbs of 150 kDa cannot enter cells. To target intracellular molecules using an Ab, internalization of Ab should be allowed. As a matter of fact, many researchers pay attention to intracellular targeting Ab and a variety of attempts are being tried to penetrate Ab into the cells (Kaiser et al., 2014). HSET is localized in nucleus and cytosol during interphase and mitosis (Cai et al., 2009), respectively. It implies that anti-HSET Ab should enter across the cellular membrane to target HSET molecule. Therefore, to inhibit HSET function by the treatment of anti-HSET mAbs to cancer cells, the conversion of non-internalizing anti-HSET mAbs to the internalizing Ab (intrabody) by Ab engineering should be followed.
34
V. CONCLUSION
We generated mouse mAbs against HSET protein involved in centrosome clustering essential for division of the cancer cells with extra-centrosome, and analyzed the binding activity of the mAbs to HSET.
35
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표면에 발현시킨 HSET과의 결합을 확인했을 때 클론 8C346이 높은 결합력을 보였고, 하이브리도마 배양액으로부터 정제된 항체의 HSET 단백질과의 결합력은 클론 1C274, 2C280, 2C281, 6C407, 9C352, 9C353에서 상대적으로 높게 나타났다. 이들 항체가 HSET의 기능을 억제하여 암세포 특이적인 사멸을 유도할 것으로 기대된다.
핵심어 : HSET, 중심체 군집, 항-HSET 항체, 하이브리도마, 암