Humanized VHH-hFc Fusion Proteins Targeting the L-HN Fragment of Tetanus Toxin Provided Protection In Vivo

Abstract

Background: Tetanus toxin, produced by Clostridium tetani, is the second deadliest known toxin. Antibodies capable of neutralizing tetanus toxin (TeNT) are vital for preventing and treating tetanus disease.

Methods: Herein, we screened thirty-six single variable domains on a heavy chain (VHHs) binding to the light chain (L) and the translocation domain (HN) (L-HN) fragment of TeNT from a phage-display library. Then, the L-HN-specific clones were identified, humanized, and fused with a human fragment crystallizable region (hFc) to form humanized VHH-hFc fusion proteins.

Results: The humanized VHH-hFc fusion proteins TL-16-h1-hFc, TL-25-h1-hFc, and TL-34-h1-hFc possessed potent efficacy with high binding affinity, specificity, and neutralizing activity. Only 0.3125 μg was required for TL-16-h1-hFc or TL-25-h1-hFc, and 0.625 μg was required for TL-34-h1-hFc to provide full protection against 10 × Lethal Dose 50 (LD₅₀) TeNT. In the prophylactic setting, 125 μg/kg of TL-16-h1-hFc or TL-25-h1-hFc provided full protection even when they were injected 12 days before exposure to 10 × LD₅₀ TeNT, while TL-34-h1-hFc was less effective. In the therapeutic setting, 25 μg/kg of TL-16-h1-hFc or TL-25-h1-hFc could provide complete protection when administered 24 h after exposure to 5 × LD₅₀ TeNT, while TL-34-h1-hFc required 50 μg/kg.

Conclusion: Our results suggest that TL-16-h1-hFc, TL-25-h1-hFc, and TL-34-h1-hFc provide a bright future for the development of anti-TeNT preventive or therapeutic drugs.

Keywords: TL-HN; VHH; humanized VHH-hFc fusion protein; neutralization; phage-display library; tetanus toxin.

Figure 1

Evaluation of the VHH-hFc fusion proteins using SDS-PAGE. (left) SDS-PAGE under reducing conditions. Lane 1, TL-16-hFc; lane 2, TL-25-hFc; lane 3, TL-34-hFc. (right) SDS-PAGE under non-reducing conditions. Lane 4, TL-16-hFc; lane 5, TL-25-hFc; lane 6, TL-34-hFc; M, protein markers.

Figure 2

Evaluation of the basic properties of the VHH-hFc fusion proteins. (a) Binding assay of the VHH-hFc fusion proteins to rTeNT or TL-HN proteins using ELISA. The concentration of the antigens was 2 μg/mL, and that of VHH-hFc fusion proteins was initially 0.2 μg/mL, followed by 2-fold dilutions for a total of 14 gradients. (b) The specificity of TL-16-hFc, TL-25-hFc, and TL-34-hFc. The binding between the VHH-hFc fusion proteins and different antigens. The concentrations of the antigens were all 2 μg/mL, and the concentration of the VHH-hFc fusion proteins was 0.1 μg/mL.

Figure 3

Competitive binding analysis of three VHH-hFc fusion proteins using BLI. (a–c) Kinetic processes of the three antibodies TL-16-hFc, TL-25-hFc, and TL-34-hFc binding to the TL-HN protein in different orders; in the legend box, samples separated by a slash “/” represent the “Loading,” “Association,” and “Re-association” stages of the sample molecules. TE-7-hFc and T23-hFc serve as control groups. (d) Structural diagram of TeNT and the mapped epitopes of TL-16-hFc, TL-25-hFc, and TL-34-hFc on the TL-HN fragment in the competitive binding experiment.

Figure 4

Evaluation of the neutralization efficiency of the VHH-hFc fusion proteins. The VHH-hFc fusion proteins were diluted into five different doses and mixed with 10 × LD₅₀ of TeNT. Then, the mixture was injected intraperitoneally into mice, with four mice in each group. (a–c) The time of death and the number of surviving mice were observed once a day, and the percentage of surviving mice was plotted for each VHH-hFc fusion protein.

Figure 5

ELISA binding assay of the humanized VHH-hFc fusion proteins to rTeNT or TL-HN proteins. The concentration of rTeNT or TL-HN was 2 μg/mL, and that of the VHH-hFc fusion proteins was initially 0.2 μg/mL, followed by 2-fold dilutions for a total of 14 gradients.

Figure 6

Evaluation of the neutralization efficiency of VHH-hFc fusion proteins. (a–c) Survival curves for TL-16-hFc, TL-25-hFc, and TL-34-hFc fusion proteins, respectively. (d) The three VHH-hFc fusion proteins were diluted to 1.25 μg, 0.625 μg, 0.3125 μg, 0.156 μg, and 0.078 μg and then mixed with 10 × LD₅₀ TeNT3.8. Prophylactic and Therapeutic Efficacy of Humanized VHH-hFc Fusion Proteins In Vivo.