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

Abstract

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.

Keywords: AntiHis-AF488, anti-penta·His Alexa Fluor 488 conjugate; B-STag, B5209B scFv fused with SpyTag; BLI, bio-layer interferometry; Bio-layer interferometry; Biparatopic antibody; Bispecific antibody; BpAb, biparatopic antibody; E-SCat, E2107 scFv fused with SpyCatcher; Roundabout homolog 1 (Robo1); Single-Chain Fv (scFv); SpyCatcher; SpyTag; scFv, single-chain variable fragment.

Fig. 1

A) Scheme of the sequential targeting protocol. First, two antibody fragments targeting different epitopes of an antigen molecule are each fused with one of a pair of covalent-bond-forming units. Second, the two fragments are serially added to the antigen. Third, a covalent bond spontaneously forms between the units, resulting in a biparatopic antibody (BpAb) fragment bound to antigen. B) The covalent-bond-forming units SpyCatcher and SpyTag. Ribbon diagrams produced from PDB ID 2X5P. C) Robo1 and the regions bound by the two mAbs used here. mAb E2107 binds to an Ig domain (red), and B2212A binds to an Fn domain (blue). The C-termini of scFvs produced from E2107 and B2212A were fused to SpyCatcher and SpyTag, respectively. TM, transmembrane domain. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

A) Graphic representations of the antibody fragments. B, C) Changes in bio-layer thickness upon addition of the first antibody fragment, the second antibody fragment, and buffer. Responses were compared with one-step targeting of B-STag (orange), E-SCat (green), or pre-formed BpAb (blue) at 10 nM. In panel B, B-STag was as the first antibody fragment and E-SCat was the second. In panel C, E-SCat was first antibody fragment and B-STag was the second. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

A) Covalent bond formation between B-STag (2 μM) and E-SCat (2 μM) in the presence (left) or absence (right) of recombinant antigen sRobo1 (2 μM). Samples were analyzed at indicated times by SDS-PAGE. B) Intensity of the band corresponding to the BpAb at 71 kDa versus time in the presence of sRobo1 (blue) and in the absence of sRobo1 (red). Data are means ± S.D. of three independent experiments. *P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Histograms of the areas of AlexaFluor 488 fluorescence. Robo1-CHO cells were incubated with antiHis-AF488 only (green); B-STag and antiHis-AF488 (cyan); E-SCat and antiHis-AF488 (orange); and B-STag, E-SCat, and antiHis-AF488 added sequentially (red). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)