Construction And Charaterization Of Dual-Variable-Domain Antibody Against Ricin

Ricin is a kind of plant toxin produced in the seeds of the castor oil. The toxin consists of twosubunits (A and B) linked by a disulfide bridge. The B-chain (RTB) is a galactose-specific lectin whichbinds to the cell surface. These binding toxins depend on host cell machinery for internalization,retrograde trafficking from endosomes to the ER. In endoplasmic Reticulum, A-chain (RTA) can’trelease a steric active site unless the interchain disulfide bonds are reduced by Proteindisulphide-isomerase(PDI).when RTA is translocated to the cytosol, it’s activity of N-glycosidasewhich recognize the universally conserved α-sarcin loop of large rRNAs inactivates elongation ofpolypeptides and leads to cell death. Because of its high lethality, relative ease of dissemination andavailability. RICIN is considered as a potential biological warfare agent. Although several types oftherapy for this threat are under development, special cure antibody still remains one of the mosteffective therapies, immediately active and very specific.People have reported that the cocktail of two or more mAbs have a promoted efficacy in protectionand similar phenomenons had been found in mAbs against ricn. In previous work, we have producedseveral mAbs against RTA and RTB chains of ricin. A better efficacy is observed when two of them arenamed6C2and3E1against RTA or RTB respectively are combined in ricin protecting in vitro andvivo.However, as the knowledge of traditional antibodies has matured, people was trying to developeseveral ways towards developing next-generation antibodies in last decade. The variety of newgeneration antibodies was developed and bispecific antibody is the most noted one. A key strength istheir potential of simultaneously neutralizing two targets like ligands, receptors or cytokines with onemolecular. Beyond this, the bispecific approach may also enable increased antigen specificity andprotective efficacy by targeting different epitopes at one antigen.In our study, we develope a bispecific antibody consists of variable domains of6C2,3E1, and ahuman IgG1Fc domain that is able to possess two antigen-binding sites in one arm. This new DVDantibody is confirmed that conserved all the properties and functions derive from parental mAbs:theability of recognizing both RTA and RTB, inhibition of RTA’s ribosome-inactivating enzymatic activityand RTB’s binding activity with glycoproteins and glycolipids in cell surface.Data of cytotoxicity andmouse protection assay show that there is a obviously promoted efficacy for new DVD molecular, atleast10folds better than its parent mAbs, even approximately3folds better than combination ofchimeric6C2and3E1which consists of variable domains of6C2or3E1, and a human IgG1Fc. Theefficacy of antibodies have regularly connected with affinity between antibody and antigen.Nevertheless, opposite to our suppose, we found there is no big distinction among kinetic parameters of 6C2,3E1and6C2-3E1-DVD antibody.The equilibrium dissociation constants and other kineticparameters like Kon or Koff among three antibodies were extraordinarily similar. So,we reasoned thatthere must be some unknown mechanism inside cells, mediated by the special characterization of DVD.The largest distinction between the DVD and two traditional mAbs is that our DVD antibody is ableto grip both RTA and RTB at the same time and same arm.We also confirm that DVD antibody leads toremaining of interchain disulfide bond in non-cell system and the same phenomenon did not appear inother antibody-antigen combination when the western blot and SRP analyse was carried out.Beside,there is a obviously evidence that DVD antibody is responsible for RTA lingering and otherantibodies including cocktail of two mAbs6C2and3E1can’t cause the same result. Based on thesedata, we consider that when DVD and holotoxin was uptaking into ER where interchain disulfide bondwas reduced and toxic subunit is separated from holotoxin，reduction of interchain disulfide bondimplemented by PDI was interfered by DVD.This could be the reasonable reason why DVD antibodyhave more powerful efficacy than other antibodies even though they have similar kinetic parameters.