DIFFERENT FORMATS OF RECOMBINANT ANTIBODIES FOR STEC DIAGNOSIS AND THERAPY

PIAZZA ROXANE MP; LOZZARDO FERREIRA RAISSA; AMARAL MARÍA MARTA; SACERDOTI FLAVIA; GAVIAO PRADO LUAN; HENRIQUE CAMILA; SOUSA MELO BRUNO; ANDRADA SHIGA EMERSON; GUTH BEATRIZ; BERNAL ALAN MAURO; MORO ANA MARÍA; QUINTILLO WAGNER; MACEDO HENRIQUE IZABELLA; PALERMO MARINA; IBARRA CRISTINA; CHEN GANG; SIDHU SACHDEV S; LUZ DANIELA

Congreso; Reunión de Sociedades de Biociencias.; 2021

Sociedad Argentina de Investigación Clínica

Antibodies innovative recombinant DNA technologieshave enhanced the murine mAb clinical eficacy and, inthe preceding decades, have led to regulatory approv-als for immunoglobulin and classic monovalent antibodyfragment (Fab) molecules, either for therapy or diagnosis. Single chain fragment variable (scFv) is the formatwhere the VH and VL are joined by a exible peptidelinker to prevent their dissociation. Besides the variablechains, Fab fragments have one constant region in eachchain. Both fragments retain the parental IgG specific antigen-binding af nity. Recombinant antibodies fragmentstargeting Stx1 and/or Stx2 were produced in bacteriaand have been comprehensively studied. The scFvStx1and scFvStx2 genes were constructed on the basis ofmurine hybridoma (mAb 3E2) secreting Stx1 or (mAb2E11) Stx2 IgG monoclonal antibodies. Both scFv werecoupled to latex nanoparticles and provide a toxin assaywith a competitive Stx detection limit of 30 ng/mL for Stx1and 10 ng/mL for Stx2, which has low cost and can beperformed in a short-term time. For the therapeutic ap-proach, four Fab fragments were selected against Stx1and Stx2 from human phage display library, and showedthe following dissociation constants analyzed by surfaceplasmon resonance: B6 4X10-8 M and C8 1X10-8 M, bothspeci c against Stx1, F8 1X10-8 M speci c against Stx2and the C11 which cross-recognizes both toxins with anaf nity of 7X10-9 M to Stx2 toxin and 3X10-8 M to Stx1.The cross-reaction of FabC11 is due to the binding epitope GKIEFSKYNEDDTF, localized on subunit B of bothtoxins. The Fabs neutralizing ability was tested eitheremploying puri ed toxins or bacterial supernatants inrenal (Vero and HK-2) and glomerular cells (HGEC) as-says. Considering different ranges of neutralization, theFabF8 and FabC11 neutralized the cytotoxicity in 90 and100% of the Stx2 producing strains, respectively. Also,the FabB6 and FabC8 were able to neutralize the cy-totoxicity in 50 and 85% of the Stx1 producing strains,respectively. Moreover, the FabC11 was able to preventStx2 toxicity to human kidney cells and in mice. This neu-tralizing capacity seems to involve receptor binding siteblocking, preventing the translocation of effective subunitA into the target cells. Taken together, our results indicatethe recombinant fragments are promising molecules tobe used therapeutically against Stx1 and Stx2 intoxica-tion, as well as for rapid screening detection of Stx pro-ducing strains.