NDLI: A novel synthetic trivalent single chain variable fragment (tri-scFv) construction platform based on the SpyTag/SpyCatcher protein ligase system

Content Provider	Springer Nature : BioMed Central
Author	Alam, Md. Kausar Brabant, Michelle Viswas, Raja Solomon Barreto, Kris Fonge, Humphrey Ronald Geyer, C.
Abstract	Background Advances in antibody engineering provide strategies to construct recombinant antibody-like molecules with modified pharmacokinetic properties. Multermerization is one strategy that has been used to produce antibody-like molecules with two or more antigen binding sites. Multimerization enhances the functional affinity (avidity) and can be used to optimize size and pharmacokinetic properties. Most multimerization strategies involve genetically fusing or non-covalently linking antibody fragments using oligomerization domains. Recent studies have defined guidelines for producing antibody-like molecules with optimal tumor targeting properties, which require intermediates size (70–120 kDa) and bi- or tri-valency. Results We described a highly modular antibody-engineering platform for rapidly constructing synthetic, trivalent single chain variable fragments (Tri-scFv) using the SpyCatcher/SpyTag protein ligase system. We used this platform to construct an anti-human epidermal growth factor receptor 3 (HER3) Tri-scFv. We generated the anti-HER3 Tri-scFv by genetically fusing a SpyCatcher to the C-terminus of an anti-HER3 scFv and ligating it to a synthetic Tri-SpyTag peptide. The anti-HER3 Tri-scFv bound recombinant HER3 with an apparent KD of 2.67 nM, which is approximately 12 times lower than the KD of monomeric anti-HER3 scFv (31.2 nM). Anti-HER3 Tri-scFv also bound endogenous cell surface expressed HER3 stronger than the monomer anti-HER3 scFv. Conclusion We used the SpyTag/SpyCatcher protein ligase system to ligate anti-HER3 scFv fused to a SpyCatcher at its C-termini to a Tri-SpyTag to construct Tr-scFv. This system allowed the construction of a Tri-scFv with all the scFv antigen-binding sites pointed outwards. The anti-HER3 Tri-scFv bound recombinant and endogenously expressed HER3 with higher functional affinity (avidity) than the monomeric anti-HER3 scFv. The Tri-scFv had the size, valency, and functional affinity that are desired for therapeutic and imaging applications. Use of the SpyTag/SpyCatcher protein ligase system allows Tri-scFvs to be rapidly constructed in a simple, modular manner, which can be easily applied to scFvs or other antibody fragments targeting other antigens.
Related Links	https://bmcbiotechnol.biomedcentral.com/counter/pdf/10.1186/s12896-018-0466-6.pdf
Ending Page	8
Page Count	8
Starting Page	1
File Format	HTM / HTML
ISSN	14726750
DOI	10.1186/s12896-018-0466-6
Journal	BMC Biotechnology
Issue Number	1
Volume Number	18
Language	English
Publisher	BioMed Central
Publisher Date	2018-09-10
Access Restriction	Open
Subject Keyword	Applied Microbiology Biotechnology Biochemical Engineering Genetic Engineering Plant Breeding Biomedical Engineering SpyTag/SpyCatcher Multimerization scFv Tri-scFv Avidity HER3 Plant Breeding/Biotechnology Biomedical Engineering/Biotechnology
Content Type	Text
Resource Type	Article
Subject	Biotechnology
Journal Impact Factor	3.5/2023
5-Year Journal Impact Factor	3.2/2023

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