Screening And Functional Study Of Humanized Anti-tetanus Toxin Monoclonal Antibody

Tetanus is caused by Clostridium tetani,a bacterium widely present in the soil environment,whose spores enter the body through improperly treated skin or mucosal wounds,recover and multiply under anaerobic conditions,and secrete large amounts of tetanus toxin（TT）,which travels retrograde to the central nervous system,blocking neurotransmitter transmission and causing generalized muscle spasms or rigidity in patients,which can be life-threatening in severe cases.Tetanus remains a serious public health problem,especially in low-income areas with high mortality rates.Tetanus prevention and treatment consists mainly of active immunization with inactivated toxin（toxoid）tetanus vaccine and passive immunization based on equine tetanus antitoxin（TAT）and human immunoglobulin tetanus（HTIG）.TTV is primarily used for long-term prophylaxis,especially in immunocompromised children and the elderly.TAT for short-term tetanus prophylaxis is manufactured from horse plasma and carries a high potential risk of allergic reactions and other infections.HTIG is expensive because there are few sources of raw materials for mass production,and because it is a human blood product,there is a potential risk of infection with human viruses such as hepatitis,AIDS,and other infectious diseases.Currently,there is a huge demand for TAT and HTIG in the medical market.In addition,tetanus infections can easily occur and threaten the lives of patients during severe natural disasters and exceptional war trauma.Therefore,there is an urgent need for a new antibody against tetanus infections that is affordable,efficient,and safe.Tetanus toxin consists of a light chain（L,50 kDa）and a heavy chain（H,100 kDa）linked by disulfide bonds.The light chain（fragment A）is a zinc metalloprotease that blocks the release of inhibitory neurotransmitters.In contrast,the heavy chain consists of fragment B（50 kDa）,which functions as a toxin transporter,and fragment C（50 kDa）,which is a receptor binding domain necessary for neuronal cells to bind toxins and endocytose them into vesicles.Currently,there is great potential to develop antibodies against fragments A and C.In addition,tetanus toxin fragment C retains many properties such as intact binding to gangliosides,comparable immunogenicity to natural toxins,low toxicity and hypo allergenicity.Therefore,the focus of this study was to prepare humanized monoclonal antibodies by immunization with tetanus toxin fragment C to address the difficulties faced in the fight against tetanus infection.In this study,based on the humanized monoclonal antibody development platform,the tetanus toxin C fragment（TT-HC）,fragment A and fragment B were expressed by the prokaryotic system E.coli receptor state BL21（DE3）,and the optimal conditions for induction of expression（0.1 mM IPTG,16°C,16h）were explored.Mice were immunized with TT-HC protein as an immune antigen and their splenocytes were fused with P3X63Ag myeloma cells to obtain hybridoma cells that could proliferate indefinitely and produce antibodies.TT-HC was used as the detection agent and HTIG was used as the positive control,screening was performed from4608 cell counting plate wells by indirect ELISA,then the eligible cell lines were subcloned for the first time,19 monoclonal antibody cell lines with high affinity were successfully isolated,and 5 monoclonal cell lines with high affinity were obtained by subcloning again,further mRNA was extracted,and the amino acid sequence of the monoclonal antibody was obtained by reverse The amino acid sequences of the monoclonal antibodies were obtained by reverse transcription,ligation,transformation and sequencing,and the optimized sequences were used to synthesize plasmids and transfected into Expi293 cells for purification and affinity verification,and the best monoclonal antibodies were selected.The humanized monoclonal antibodies were obtained by replacing part of the mouse-derived amino acid sequences with human-derived sequences through biogenetic engineering and further transfected with eukaryotic cells for expression,which reduced the immunogenicity and improved the safety of clinical treatment while maintaining the high affinity of the antibodies.These monoclonal antibodies and humanized monoclonal antibodies were subjected to in vitro experiments and in vivo neutralization experiments in mice to verify their potency.The study finally resulted in 4 stable and high-affinity monoclonal antibodies（9E10,12C6,14C7 and 20G9）,of which the highest affinity 20G9was further humanized as H-20G9 and tested in vitro and neutralized in vivo.In a lethality protection assay with tetanus toxin in mice,a single injection of 50μg·kg^-1 of H-20G9 completely protected mice from surviving a 20LD50dose of the toxin.Approximately 2.5 mg of H-20G9 antibody was equally effective against 1 commercially available human immunoglobulin（250 IU）.In summary,we obtained a humanized monoclonal antibody H-20G9with high affinity for neutralizing tetanus toxin,which has great potential against tetanus infection.