Generation of biparatopic antibody through two-step targeting of fragment antibodies on antigen using SpyTag and SpyCatcher

• Published in: Biotechnology reports (Amsterdam, Netherlands) Vol. 25; p. e00418
• Main Authors: Akiba, Hiroki, Takayanagi, Kensuke, Kusano-Arai, Osamu, Iwanari, Hiroko, Hamakubo, Takao, Tsumoto, Kouhei
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2020; Elsevier
• Subjects: Bio-layer interferometry; Biparatopic antibody; Bispecific antibody; Roundabout homolog 1 (Robo1); Single-Chain Fv (scFv); SpyCatcher; SpyTag; Single-Chain Fv (scFv); Bio-layer interferometry; Biparatopic antibody; SpyCatcher; Bispecific antibody; BpAb; AntiHis-AF488; B-STag; E-SCat; scFv; SpyTag; Roundabout homolog 1 (Robo1); BLI; AntiHis-AF488, anti-penta·His Alexa Fluor 488 conjugate; BLI, bio-layer interferometry; E-SCat, E2107 scFv fused with SpyCatcher; BpAb, biparatopic antibody; B-STag, B5209B scFv fused with SpyTag; scFv, single-chain variable fragment
• ISSN: 2215-017X; 2215-017X
• DOI: 10.1016/j.btre.2020.e00418

•Biparatopic antibody was generated on antigen using SpyTag/SpyCatcher pair.•Two-step targeting enabled slow dissociation and formation of a covalent bond.•Enhanced accumulation of biparatopic antibody was observed in a whole-cell model. Biparatopic fragment antibodies can overcome deficiencies in avidity of conventional antibody fragments. Here, we describe a technology for generating biparatopic antibodies through two-step targeting using a pair of polypeptides, SpyTag and SpyCatcher, that spontaneously react to form a covalent bond between antibody fragments. In this method, two antibody fragments, each targeting different epitopes of the antigen, are fused to SpyTag and to SpyCatcher. When the two polypeptides are serially added to the antigen, their proximity on the antigen results in covalent bond formation and generation of a biparatopic antibody. We validated the system with purified recombinant antigen. Results in antigen-overexpressing cells were promising although further optimization will be required. Because this strategy results in high-affinity targeting with a bipartite molecule that has considerably lower molecular weight than an antibody, this technology is potentially useful for diverse applications.