[Construction, screening and functional characterization of a mammalian cell-displayed antibody library targeting...

Objective T cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3), a critical regulator of innate and adaptive immune responses, has been considered as a promising new target for immunotherapy. Based on an existing laboratory murine-derived anti-human Tim-3 antibody, this study aims to employ computer-aided molecular design to construct a mammalian cell-displayed antibody library targeting human Tim-3 and to screen for antibodies with optimized function. Methods The parental Tim-3 sequence was designed and optimized via a computer-assisted molecular model construction platform and mammalian cell-displayed antibody library technology. The plasmid library encoding the Tim-3-targeted mammalian antibody library was obtained by molecular biology techniques including Overlap PCR, vector cleavage, enzyme ligation and transformation. Novel antibody sequences were identified through sequencing alignment, followed by ELISA-based screening to isolate high-expression and high-affinity candidates. The function of the optimized antibody was further verified by in vitro assays (RT-PCR and CCK-8). Results Utilizing computer-aided design platform, five key amino acid substitutions were designed and introduced into the light and heavy chains of the parental murine-derived anti-human Tim-3 antibody. Three mutant light chains (L-1, L-2 and L-3) and heavy chains (H-1, H-2 and H-3) were successfully generated by Overlap PCR. Subsequent ligation into vectors and transformation into TOP10 electrocompetent cells yielded a plasmid library with substantial sequence diversity. Sequence analysis and purification of new antibodies resulted in the identification of an antibody with higher affinity, named No.1. RT-PCR showed that after blockade with antibody No.1, transcription levels of IFN-β, TNF-α, IL-1β and IL-6 were significantly upregulated in the macrophage cell line RAW264.7 cells and microglia cell line BV2 cells, and a similar upregulation was observed in levels of IL-2 and IFN-γ in Jurkat^Tim-3-high cells. In these assays, antibody No.1 exhibited greater blocking efficacy than the parental Tim-3 antibody. CCK-8 assays demonstrated that antibody No.1 promoted Jurkat^Tim-3-high cells proliferation more effectively than the parental Tim-3 antibody. Conclusion A novel humanized Tim-3 antibody (No.1) with enhanced affinity and capacity to upregulate multiple cytokine expressions in both human and murine cell lines was obtained via a computer-aided molecular design platform and mammalian cell antibody display library technology.