(719b) Design and Characterization of Peptide-Tagged T-Cell Immunoglobulin and Mucin (TIM) for Efficient Recovery of Exosomal Vesicles

In recent years, micro RNA (miRNA) enpacsulated in exosomal vesicles which were secreted from a variety of mammalian cells has been recognized as a next generation of biomarker in clinical diagnosis research area, and therefore, it has been requisite to separate and recover the intact exosomal vesicles efficiently from the serum as well as culture supernatant. Among the separation methods that have been proposed, affinity-based separation of exosomes using solid-materials immobilized with affinity ligand proteins such as antibodies have a possibility to use in the actual fields, because simple, efficient and rapid recovery of exosomes with high purity is requisite. Here, we demonstrate use of T-cell Immunoglobulin and Mucin (TIM) as a ligand for affinity separation of exosome, and TIM fragments that have an ability to capture intact exosomes were successfully produced by HEK293T cells. In this study, both Avi-tag as well as PMMA-binding peptide (PMMA-tag) were adopted as an affinity peptide tag, and were genetically-introduced at the C-terminus of TIM fragments for site-directed immobilization and maintenance of their binding activity in adsorption states.

Avi-tagged TIM fragments (TIM-VM) as well as their Fc-fusion protein (TIM-VM-Fc) were expressed in culture supernatant, and they were purified by Ni-NTA column. Site-specific biotinylation to Avi-tag-fused TIM-VM and TIM-VM-Fc were confirmed by Westernblot analysis and sandwich ELISA. Site-specific immobilization of Avi-tag-fused TIM-VM and TIM-VM-Fc was confirmed, and biotinylation yields were increased with expression levels of biotin ligase (BirA). According to the results of sandwich ELISA using streptavidin-coated plate and HRP-labeled streptavidin, it was found that Avi-tag-fused TIM fragments were immobilized site-specifically, and their orientation is controlled preferentially. Consequently, binding activity of both TIM-VM and TIM-VM-Fc were highly maintained, and therefore, 10-times lower concentration of exosomes were detectable in sandwich ELISA that TIM fragments were immobilized on a surface of streptavidin-coated plate.

On the other hand, PMMA-tagged TIM-VM and TIM-VM-Fc were also prepared by the same method as described above. Both fusion proteins were successfully produced and purified. In order to identify the optimal adsorption conditions for each TIM fragment, 96 different conditions that pH and NaCl concentrations were comprehensively changed, were tested. Tim-VM-PM showed the highest binding activity against exosomal vesicles, when it was immobilized at pH 6.0. Similar tendency was observed when Tim-VM-Fc-PM was tested. Then, exosomal vesicles in culture supernatant including FBS, were detected by TIM-VM-PM-coated PMMA plate with 35-times higher signal intensity.

We further investigated site-directed immobilization of single-chain Fv (scFv) in a same manner as PMMA-tagged TIM. Anti-CD9 scFvs with high antigen-binding activities were isolated from scFv-displayed phage library, and binding activities of scFv clones in an adsorption state were compared. According to the results of sandwich ELISA that scFvs were immobilized, the clone 4-6 showed the highest binding ability against the antigen among 6 positive clones that were selected from phage library. PMMA-tag-fused scFv was successfully produced by cultivation of recombinant E. coli. Therefore, it was immobilized on the surface of latex beads, and showed higher antigen-binding activity compared with the tagless scFv. According to the results of flow cytometry (FCM) as well as fluorescence microscopy, we confirmed that scFv-PM developed in this study have a potential to capture exosomal vesicles more efficiently than tagless scFv in immobilization state.

Thus, the fusion proteins developed in this study, namely tagged TIM fragments and scFvs would be significantly useful for rapid, efficient and quantitative detection of intact exosomes, and it might be applicable in actual clinical diagnosis fields.