DIFFERENT FORMATS OF RECOMBINANT ANTIBODIES FOR STEC DIAGNOSIS AND THERAPY

PIAZZA FONTES, ROXANE MARIA; FERREIRA, RAISSA LOZZARDO; AMARAL, MARÍA M.; SACERDOTI, FLAVIA; PRADO, LUAN GAVIAO; HENRIQUE, CAMILA; MELO, BRUNA S.; SHIGA, EMERSON ANDRADE; CABILIO GUTH, BEATRIZ ERNESTINA; BERNAL, ALAN MAURO; MORO, ANA MARIA; QUINTILIO, WAGNER; HENRIQUE, IZABELLA DE MACEDO; PALERMO, MARINA; IBARRA, CRISTINA; CHEN, GANG; SIDHU, SACHDEV S.; LUZ, DANIELA

Congreso; Reunión conjunta SAIC-SAI-AAFE-NANOMED.ar; 2021

Antibodies innovative recombinant DNA technologies have enhanced themurine mAb clinical efficacy and, in the preceding decades, have led toregulatory approvals for immunoglobulin and classic monovalent antibodyfragment (Fab) molecules, either for therapy or diagnosis. Single chain fragmentvariable (scFv) is the format where the VH and VL are joined by a flexiblepeptide linker to prevent their dissociation. Besides the variable chains, Fabfragments have one constant region in each chain. Both fragments retain theparental IgG specific antigen-binding affinity. Recombinant antibodies fragmentstargeting Stx1 and/or Stx2 were produced in bacteria and have beencomprehensively studied. The scFvStx1 and scFvStx2 genes were constructedon the basis of murine hybridoma (mAb 3E2) secreting Stx1 or (mAb 2E11)Stx2 IgG monoclonal antibodies. Both scFv were coupled to latex nanoparticlesand provide a toxin assay with a competitive Stx detection limit of 30 ng/mL forStx1 and 10 ng/mL for Stx2, which has low cost and can be performed in a short-term time. For the therapeutic approach, four Fab fragments wereselected against Stx1 and Stx2 from human phage display library, and showedthe following dissociation constants analyzed by surface plasmon resonance:B6 4X10 -8 M and C8 1X10 -8 M, both specific against Stx1, F8 1X10 -8 M specificagainst Stx2 and the C11 which cross-recognizes both toxins with an affinity of7X10 -9 M to Stx2 toxin and 3X10 -8 M to Stx1. The cross-reaction of FabC11 isdue to the binding epitope GKIEFSKYNEDDTF, localized on subunit B of bothtoxins. The Fabs neutralizing ability was tested either employing purified toxinsor bacterial supernatants in renal (Vero and HK-2) and glomerular cells (HGEC)assays. Considering different ranges of neutralization, the FabF8 and FabC11neutralized the cytotoxicity in 90 and 100% of the Stx2 producing strains,respectively. Also, the FabB6 and FabC8 were able to neutralize the cytotoxicityin 50 and 85% of the Stx1 producing strains, respectively. Moreover, theFabC11 was able to prevent Stx2 toxicity to human kidney cells and in mice.This neutralizing capacity seems to involve receptor binding site blocking,preventing the translocation of effective subunit A into the target cells. Takentogether, our results indicate the recombinant fragments are promisingmolecules to be used therapeutically against Stx1 and Stx2 intoxication, as wellas for rapid screening detection of Stx producing strains.