IMEX   05356
INSTITUTO DE MEDICINA EXPERIMENTAL
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
VHH antibodies against the B subunit of Shiga toxin 2 (Stx2B) as novel therapeutic tools against HUS. (Part I)
Autor/es:
HIRIART, Y; LAUCHÉ, CE; PARDO, RP; MEJIAS, MP; PALERMO, MS; GOLDBAUM, F; ZYLBERMAN, V
Reunión:
Congreso; 10th International symposium on Vero toxin-producing E. coli; 2015
Resumen:
Introduction: STEC infections are implicated in the development of life-threatening Hemolytic Uremic Syndrome (HUS), for which currently there is no specific therapy available. Neutralizing antibodies against the Shiga Toxin (Stx) should prevent the first step in the toxicity cascade. We generated a chimeric Stx2B-BLS immunogen, which raised high affinity and protective anti-Stx2B antibodies. The aim of this work was to use this immunogen to produce VHH antibodies (variable region of llama heavy chain only antibodies) against Stx2. Then generate by protein engineering an improved molecule with potential human therapeutic purposes. Methods: Two llamas (Lama glama) were immunized four times every 2 weeks intramuscularly with 0.1 mg of the BLS-Stx2B chimera. Two phagemid libraries of VHH fragments were generated and anti-Stx2B specific clones were selected by phage display system.Results and Discussion: The animals developed strong total IgG responses (>1/40,500). Two libraries of 7x108 and 6X108 clones were obtained and subjected to biopanning. From 400 clones screened by ELISA, 71 clones were selected considering their reactivity against BLS-Stx2B and Stx2B and grouped by CDR3 sequence similitude in 8 subfamilies. Only VHHs from subfamily 1 showed neutralizing activity in a Stx2 neutralization assay in Vero cells. Considering its neutralizing capacity, the clone 2vb27 was selected to generate bivalent and heterotrimeric antibody formats to improve its neutralizing capacity, avidity and half-life. The heterotrimeric molecule (two copies of anti-Stx2B VHH and one of anti-seroalbumin VHH) showed an extended circulating life in serum (14 days) compared to 2bv27 (1 day), and higher neutralizing capacity in Vero cells assays (mean of pmoles that neutralize 1CD50 Stx2 ±SD): 2vb27: 0.5±0.13, bivalent: 0.04±0.007*, heterotrimeric: 0.06±0.04* (*p