The Preparation Of Human Neutralizing Antibodies Against Botulinum Neurotoxin Serotype A

The botulinum neurotoxins (BoNT) produced by Clostridium botulinum are the most poisonous protein substance known, which include seven serotypes(A-G). Due to their extreme potency and lethality, ease of production and transport, BoNT are used as weapons or for bioterrorism by some countries and organizations. As a result of these threats, specific pharmaceutical agents are needed for prevention and treatment of intoxication. Small quantities of equine antitoxin are currently used to treat adult and infant botulism. Unfortunately, this product is associated with a high incidence of side-effects, including serum sickness and anaphylactic shock.As an alternative, genetic monoclonal antibody (mAb)-based antitoxins against botulinum neurotoxin can effectively prevent and cure the toxicosis, with large quantities and without any side-effect to human bodies. In this research, we focus on screening specific neutralizing antibodies against botulinum neurotoxin serotype A from fully synthetic human antibody libraries.Gene encoding protective antigen Hc fragment of butulinum neurotoxin serotype A (BoNT/A-Hc) was synthesized, optimized and reconstructed Hc proteins were expressed in E.coli BL21(DE3) in soluble form which covered 36-53% total proteins in the supernatant of sonicated bacteria. When purified with Ni column, Hc covered 95% total proteins and can be achieved as 30mg/L. There was no report about higher expression before.Purified BoNT/A-Hc as antigens, the specific antibodies were screened from fully synthetic human antibody libraries, and more than 1000 clones were identified of which 70% are positive clones. About 73% positive clones can specifically bind to BoNT/A-Hc. After sequencing of 18 positive clones, 5 unique scFv antibodies were acquired, with all VL genes belonging toλ3 family and VH genes belonging to H5 family. The results of competitive ELISA showed that the 5 scFv antibodies had the same epitope.One scFv antibody named C10 was selected from the 5 antibodies for its high affinity (KD=7.8×10⁽-9(M) to BoNT/A-Hc and was reconstructed to a Diabody for stable form. C10 scFv Diabody was expressed in E.coli KS1000(DE3) and was purified from culture medium supernatants. Its affinity constant KD was 3.57×10^-10M and 20 times higher than C10 scFv through BiaCore determination. Furthermore, an IgG4 formed full antibody of C10 was constructed by linking the variable regions of high and light chain domains with their constant regions. The full antibody of C10 was transiently expressed in FreeStyleTM293-F cells. After purified the full antibodies with protein A column, a more stable antibody was acquired and no changes in activity was observed upon storage at 4℃for a month. Detected by BiaCore, the affinity constant KD of C10 full antibodies were 6×10^-11M and 100 times higher than scFv antibodies of C10.Botulinum neurotoxin had more than one receptor epitopes when binding to its receptor cells, several antibodies with different epitopes must be prepared and combined for more satisfied block to epitopes and neutralizing activities. In this study, two methods of sandwich selection and solid competitive selection were used to screen antibodies with different epitopes from C10. After 2 and 3 periods of selections, 3 monoclonal antibodies with unique sequence were obtained. Only two antibodies named 1B6 and 2G4 still kept their binding activity to BoNT/A-Hc after being constructed to a full antibody. According to the result of a competitive ELISA, the 1B6 and 2G4 monoclonal antibodies have the different antigen epitopes. The affinity constant KD of 1B6 full antibody was 2.38×10-8M determined by uncompetitive ELISA.For analyzing the mapping epitopes of two monoclonal antibodies-C10 and 1B6, 5 epitope fragments of BoNT/A-Hc were designed and their binding to C10 and 1B6 were identified. The results showed that C10 antibodies mapped to the new epitope of 1200-1223Aa of BoNT/A-Hc, while 1B6 had the different epitope from C10.Cellular immunofluorescence assay was used to detect the in vitro activities of C10 ,1B6 and 2G4 full antibodies. All the antibodies can effectively inhibit the binding of BoNT/A-Hc to differentiated PC-12 cells, showing C10, 1B6 and 2G4 had neutralizing activities in vitro.In vivo toxin neutralization was studied using a mouse assay in which toxin and 50μg Ab are premixed and injected i.p., and time to death and number of surviving mice determined. Fifty micrograms of 1B6 and C10 single mAb prolonged the time to death but failed to protect mice challenged with 20 LD50s. In contrast, any pair of mAbs completely protected mice challenged with 20 LD50s of toxin and a combination of three mAbs (C10, 1B6 and 2G4) neutralized at least 100 LD50 doses of BoNT/A and was determined to be 2000 LD50 / mg Ab, suggesting their high neutralizing potency in vivo. All the three antibodies are all human species and can be used directly to human bodies.In conclusion, we obtained three human monoclonal antibodies C10, 1B6 and 2G4 against botulinum neurotoxin serotype A with different epitopes and effectively neutralizing activities in vitro. All the results would set light on the further production of neutralizing antibody drugs against botulinum neurotoxin serotype A.