RECOMBINANT ANTIBODIES FOR STEC DIAGNOSIS AND THERAPY

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

Modalidad Virtual

Congreso; Reunión de Sociedades de Biociencias 2021; 2021

SAIC. SAI. AAFE. NANOMED

Antibodies innovative recombinant DNA technologies have enhanced the murine mAb clinical efficacy and, in the preceding decades, have led to regulatory approvals for immunoglobulin and classic monovalent antibody fragment (Fab) molecules, either for therapy or diagnosis. Single chain fragment variable (scFv) is the format where the VH and VL are joined by a flexible peptide linker to prevent their dissociation. Besides the variable chains, Fab fragments have one constant region in each chain. Both fragments retain the parental IgG specific antigen-binding affinity. Recombinant antibodies fragments targeting Stx1 and/or Stx2 were produced in bacteria and have been comprehensively studied. The scFvStx1 and scFvStx2 genes were constructed on the basis of murine hybridoma (mAb 3E2) secreting Stx1 or (mAb 2E11) Stx2 IgG monoclonal antibodies. Both scFv were coupled to latex nanoparticles and provide a toxin assay with a competitive Stx detection limit of 30 ng/mL for Stx1 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 were selected against Stx1 and Stx2 from human phage display library, and showed the 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 specific against Stx2 and the C11 which cross-recognizes both toxins with an affinity 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 both toxins. The Fabs neutralizing ability was tested either employing purified toxins or bacterial supernatants in renal (Vero and HK-2) and glomerular cells (HGEC) assays. Considering different ranges of neutralization, the FabF8 and FabC11 neutralized the cytotoxicity in 90 and 100% of the Stx2 producing strains, respectively. Also, the FabB6 and FabC8 were able to neutralize the cytotoxicity in 50 and 85% of the Stx1 producing strains, respectively. Moreover, the FabC11 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. Taken together, our results indicate the recombinant fragments are promising molecules to be used therapeutically against Stx1 and Stx2 intoxication, as well as for rapid screening detection of Stx producing strains.