Design,expression And Activity Of Nanobodies-based Bispecific Neutralizer For Shiga Toxin-producing E.coli

Shiga toxin-producing Escherichia coli（STEC）is an infectious enteric pathogen that becomes a great threat to human health worldwide.Non-specific strategies,such as antibiotics and hemodialysis are mainly used to treat STEC infection,which resulting in poor clinical efficacy and antibiotic resistance.Currently,no specific preventions and therapeutics against STEC infection are available for clinical use and to the best of our knowledge,there is no reported biotherapeutic both targeting to Shiga toxin（Stx）and STEC adhesion.Nanobody,derived from the variable region of heavy-chain-only antibody in Camelidae spp.and Carcharhinidae spp.,is the smallest fragment with excellent antigen-binding capability.It has the advantages of high affinity,strong stability,high solubility and strong tissue permeability.It has been shown to have great prospects in the development of anti-infection therapeutics.In this study,based on the anti-Stx2 B subunit（Stx2B）nanobody Nb113 and the anti-intimin nanobody IB10,we constructed a nanobodies-based bispecific neutralizer Nb113-IB10,targeting both to Stx and intimin to neutralize the toxins as well as block STEC colonization.The recombinant protein Nb113-IB10 was expressed by E.coli expression system,followed by binding activity and neutralization protective activity evaluation using enzyme-linked immunosorbent assay（ELISA）,co-immunoprecipitation（Co-IP）and flow cytometry（FCM）.Finally,the stability of Nb113-IB10 under simulated intestinal extreme p H,different temperatures and repeat freeze-thaw conditions were evaluated.The main conclusions are as follows:（1）A recombinant protein Nb113-（G₄S）₃-IB10-6His was constructed.The protein was successfully expressed in E.coli expression system with 4.5 mg·L^-1 yield with 1 m M IPTG induction at 16℃.（2）Antigen binding evaluations showed that the EC₅₀ of Nb113-IB10 to Stx2B and intimin were 296 n M and 46 n M,respectively,which were 8.6 times and 2.7 times higher than that of the corresponding monovalent nanobody.Besides,Nb113-IB10 could bind Stx2B and intimin simultaneously,indicating that it has bilateral antigen binding activity.（3）Antigen neutralization evaluations showed that the IC₅₀ of Nb113-IB10 to block Stx2B binding to Gb₃ receptor was 134 n M and that of intimin binding to translocated intimin receptor（Tir）was 475 n M.Besides,Nb113-IB10 has bilateral antigen neutralization activity.Cell-based neutralization evaluations showed that Nb113-IB10 could effectively inhibit the binding of Stx2B to Gb₃ receptor in HT-29 cell line.（4）Nb113-IB10 had excellent protein stability and maintained high STEC antigen neutralization activity under different temperatures,repeat freeze-thaw conditions and simulated intestinal extreme p H conditions.It has the potential to be developed as therapeutics for oral and inhalable administration.