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Chen, Yilun

University of British Columbia

Nanobodies (nAbs) are emerging as potent alternatives over traditional antibodies, offering advantages in production efficiency, cost and reactivity. Despite their promising features, the application of nAbs can be limited by insufficient affinities. Traditional affinity maturation processes are costly and time-consuming; consequently, efforts have been directed toward developing engineering strategies to transform nAbs into multimeric formats for affinity enhancement via avidity effect. Additionally, nAbs are engineered into multispecific formats to broaden their functionality. Although these strategies have shown potential, a universal and easily implementable in vitro platform for producing the engineered nAbs remains underdeveloped. In this thesis, we develop a peptidisc-based approach for in vitro assembly of nAbs into multimeric and multispecific formats, leveraging on thermodynamically favorable hydrophobic effect, hereafter termed "polybody". Utilizing a nAb targeting the green fluorescent protein (GFP), we showcase the successful production of a GFP polybody and the introduction of avidity upon multimerization. The advantage of this enhanced affinity is demonstrated through the reformatting of a suboptimal-affinity human serum albumin (HSA) nAb into an HSA polybody in ELISA assays. Extending the in vitro self-assembly principle, we also produce bispecific and fluorescent polybodies, thereby validating our approach as a universal platform for generating multispecific and multifunctional entities. This peptidisc-based protein engineering design aims to expand the range of nAb applications in life sciences.

Text

2024-09-25

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/89300

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/