Characterization of a novel format scFv×VHH single-chain biparatopic antibody against metal binding protein MtsA

Abstract

Biparatopic antibodies (bpAbs) are engineered antibodies that bind to multiple different epitopes within the same antigens. bpAbs comprise diverse formats, including fragment-based formats, and choosing the appropriate molecular format for a desired function against a target molecule is a challenging task. Moreover, optimizing the design of constructs requires selecting appropriate antibody modalities and adjusting linker length for individual bpAbs. Therefore, it is crucial to understand the characteristics of bpAbs at the molecular level. In this study, we first obtained single-chain variable fragments and camelid heavy-chain variable domains targeting distinct epitopes of the metal binding protein MtsA and then developed a novel format single-chain bpAb connecting these fragment antibodies with various linkers. The physicochemical properties, binding activities, complex formation states with antigen, and functions of the bpAb were analyzed using multiple approaches. Notably, we found that the assembly state of the complexes was controlled by a linker and that longer linkers tended to form more compact complexes. These observations provide detailed molecular information that should be considered in the design of bpAbs.

Keywords: VHH; biparatopic antibody; complex formation; interaction; metal binding; nanobody; physicochemical analysis; protein engineering; scFv.

FIGURE 1

Constructs of the bpAb in this study. Schematic representation of the scFv‐VHH bpAb. scFv and VHH are fused by a single amino acid linker.

FIGURE 2

Binding of scFv13 and VHH43 to MtsA. (a) SPR data corresponding to the binding of scFv13 and VHH43 to MtsA. Raw sensorgrams of scFv13 and VHH43 and fitted sensorgrams are shown in yellow, green, and black lines, respectively. (b) ITC data corresponding to the binding of scFv13 and VHH43 to MtsA. Representative results are shown.

FIGURE 3

Crystal structure of the complex of scFv13 with MtsA. (a) Structure of scFv13 bound to MtsA. scFv13 is colored in blue, and MtsA is in pink. (PDB ID: 8YJ7) (b) Side view of the scFv13‐bound MtsA (c) Superposition of scFv13‐Zn‐bound MtsA with Zn‐bound MtsA. Zn‐bound MtsA is colored in sky blue, scFv13 is in grayish blue, and scFv13‐Zn‐bound MtsA is in pink. (d) Superposition of the metal binding site. Zn‐bound MtsA is colored in sky blue, scFv13‐Zn‐bound MtsA is in pink, and Zn is in red. Protein structures were visualized with UCSF Chimera (Pettersen et al., 2004).

FIGURE 4

Crystal structure of the complex of VHH43 with MtsA. (a) Structure of VHH43‐metal‐unbound MtsA. VHH43 is colored in light blue, and metal‐unbound MtsA is in orange. (PDB ID: 8YJ5) (b) Side view of the VHH43‐metal‐unbound MtsA. (c) Close‐up view of the metal binding site. (d) Structure of VHH43‐Zn‐bound MtsA. VHH43 is colored in light blue, and Zn‐bound MtsA is in green. (PDB ID: 8YJ8) (e) Side view of the VHH43‐Zn‐bound MtsA. (f) Close‐up view of the metal binding site. Zn is colored in red. (g) Superposition of VHH43‐metal‐unbound MtsA with Zn‐bound MtsA. Zn‐bound MtsA is colored in sky blue, VHH43 is in light blue, VHH43‐metal‐unbound MtsA is in orange, and Zn is in red. (h) Close‐up view of the metal binding site. (i) Superposition of VHH43‐Zn‐bound MtsA with Zn‐bound MtsA. Zn‐bound MtsA is colored in sky blue, VHH43‐Zn‐bound MtsA is in green, and Zn is in red. (j) Close‐up view of the metal binding site. Protein structures were visualized with UCSF Chimera (Pettersen et al., 2004).

FIGURE 5

Design of the bpAb based on crystal structure. (a) Superposition of scFv13‐bound MtsA with VHH43‐metal‐unbound MtsA. The dotted lines show the distance between Cα atoms of the C‐terminus residue of scFv13 and the N‐terminus residue of VHH43. scFv13 is shown in gold, the CDR of scFv13 is in blue, VHH43 is in green, the CDR of VHH43 is in yellow, the N‐lobe of MtsA is in pink, the C‐lobe of MtsA is in purple, and the backbone of MtsA is in magenta. Protein structures were visualized with UCSF Chimera (Pettersen et al., 2004). (b) Constructs of scFv‐VHH bpAb. scFv13 is linked to VHH43 by the amino acid linker composed of four glycines and one serine repeated six times [(G₄S)x6] at both the N‐ or C‐terminus.

FIGURE 6

Physicochemical properties of the GS30 bpAb. (a) CD spectra of the antibodies. scFv13 is shown in yellow, VHH43 is in green, the sum of scFv13 and VHH43 is in blue, and the GS30 bpAb is in orange. (b) Thermal stability of the antibodies determined by DSC. scFv13 is in yellow, VHH43 is in green; and the GS30 bpAb is in orange. Representative results are shown.

FIGURE 7

Binding of the GS30 bpAb to MtsA. (a) SPR data corresponding to the binding of the GS30bpAb to MtsA. Raw sensorgrams and fitted curves are shown in orange and black lines, respectively. (b) ITC data corresponding to the binding of the GS30 bpAb to MtsA. Representative results are shown.

FIGURE 8

Complex formation of the GS30 bpAb with MtsA. (a) SEC‐MALS of the GS30 bpAb and MtsA complex. Orange lines represent relative value of absorbance at 280 nm. Red lines represent molecular weight. (b) Representative nsTEM micrograph of the GS30bpAb:MtsA complex. (c) Close‐up view of a representative GS30bpAb:MtsA ring‐shaped complex. (d) Model structure of the 3:3 complex. The scale bar represents 15 nm. Protein structures were visualized with UCSF Chimera (Pettersen et al., 2004). (e) 17 representative 2D nsTEM class averages of the ring‐shaped GS30 bpAb:MtsA complex. The scale bar represents 10 nm.

FIGURE 9

Complex formation of the bpAb linked by various linkers. SEC‐MALS of bpAbs with various linkers and the MtsA complex. Relative value of absorbance at 280 nm and molar mass are represented by thin and bold lines, respectively. GS5 is shown in dark orange, GS20 is in blue, GS30 is in orange, and PAS60 is in purple.

FIGURE 10

Equilibrium between bpAb and MtsA complexes. Analytical SEC of fractioned the bpAb and MtsA complex. Ultraviolet absorbance at 280 nm is represented. (a) Reinjection of the 3:3 complex of GS30 bpAb and MtsA (orange: day 0, brown: day 7). (b) Reinjection of the 2:2 complex of GS30 bpAb and MtsA (orange: day 0, brown: day 7). (c) Reinjection of the 2:2 complex of PAS60 bpAb and MtsA (light purple: day 0, purple: day 7). (d) Reinjection of the 1:1 complex of PAS60 bpAb and MtsA (light purple: day 0, purple: day 7).

FIGURE 11

Metal binding inhibition of the bpAb. Titration curve of the ITC analysis of the interaction between Mn²⁺ and MtsA in the presence of 2M equivalent antibodies: (a) buffer, (b) scFv13, (c) VHH43, (d) scFv13 and VHH43, (e) GS30 bpAb (scFv13‐GS30‐VHH43), and (f) PAS60 bpAb (scFv13‐PAS60‐VHH43). Representative results are shown.