A modified single-domain antibody candidate for the treatment of botulism caused by botulinum toxin type A

INTRODUCTION. Currently, the primary treatment method for botulism is the use of botulinum antitoxin, which causes a number of side effects, including allergic reactions. The development of medicinal products based on monoclonal antibodies (mAbs), in particular, single-domain mAbs fused to the human IgG1 Fc fragment, holds promise for the treatment of botulinum toxin poisoning. AIM. This study aimed to optimise the technology for laboratory-scale production of a single-domain mAb fused to the human IgG1 Fc fragment (B11-Fc) for botulism treatment and post-exposure prophylaxis and to conduct a preclinical efficacy study of this mAb. MATERIALS AND METHODS. The study used CHO cells. B7, a stable clone producing the B11-Fc single-domain mAb, was cultured in Erlenmeyer flasks using commercially available media and feeds. The B11-Fc mAb was purified using multistep chromatography (including affinity, anion exchange, and multimodal chromatography steps), virus elimination, and tangential flow filtration. The purity of the B11-Fc mAb was assessed by high-performance liquid chromatography (HPLC) and electrophoresis. The glycan profile was established by HPLC. Bio-layer interferometry was used to measure the mAb concentration in the culture fluid and to determine the equilibrium dissociation constants for the mAb and various Fc receptors. Botulinum toxin type A (BoNT/A) was produced by culturing the Clostridium botulinum A98 strain and purified by chromatography. In vivo experiments involved intraperitoneal and intragastric administration of BoNT/A to female BALB/c mice, with a subsequent assessment of the severity of toxic signs. The B11-Fc mAb was administered intramuscularly or intravenously (to study the pharmacokinetics). The efficacy of the B11-Fc mAb (in terms of mouse survival) was studied using various toxicity models and the prophylactic and therapeutic modes of administration. RESULTS. The study optimised culture conditions for the B11-Fc mAb producer clone and developed a mAb purification technology that ensured a high yield (0.5 g/L) and a purity of over 99%. The average particle size in the mAb preparation was 7.85 nm. The study characterised the glycan profile of the B11-Fc mAb and determined the equilibrium dissociation constants for the mAb and human Fc receptors. Poisoning with BoNT/A was modelled in mice. The intramuscular administration of the B11-Fc mAb at a dose of 0.6 mg/kg provided 100% protection from poisoning with BoNT/A that was simultaneously administered at a dose of 20 LD 50 . The study determined the main pharmacokinetic parameters of the B11-Fc mAb. The experiments demonstrated that prophylactic administration of the B11-Fc mAb for 21 days had a protective effect against BoNT/A administered intraperitoneally at a dose of 5 LD 50 , and therapeutic administration of the mAb 14 h after intragastric administration of the toxin at a dose of 12,000 intraperitoneal LD 50 provided 100% protection. CONCLUSIONS. The authors optimised the technology for laboratory-scale production of the candidate modified single-domain mAb. In vivo experiments conducted using BoNT/A toxicity models demonstrated that the B11-Fc mAb is highly effective in botulism prevention and treatment. On the basis of preclinical data, phase I clinical trials have been initiated to study B11-Fc in healthy volunteers.