28. Characterization of the Epitope for Anti Human Respiratory Syncytial Virus F Protein Monoclonal Antibody 101F.

MARIAN KRUSZYNSKI, GABRIELA CANZIANI, ET AL.

Radnor, PA, USA

Jornada; Annual Scientific Meeting, Centocor R&D; 2006

CENTOCOR, INC

Human respiratory syncytial virus (HRSV) is a membrane-enveloped virus that encodes two major surface glycoproteins (G and F). Antibodies that interact with RSV F protein reduced infectivity by blocking viral and cell membranes fusion. The lead RSV mAb,101F, is a potent RSV neutralizing that recognize the antigenic region IV/V/VI region of the F protein. The specific aa recognition by mAb 101F to the F protein in this region was recently identified as amino acid K433 by the characterization of the RSV that escaped from the mAb neutralization in cell culture. We characterized 101F epitope binding by a systematic analysis of peptides covering the aa 422-436, encompass the antigenic region of IV/V/VI of the F protein. We showed that the peptide (422-436) [CTASNKNRGIIKTFS] bound to 101F with the highest affinity in ELISA assays. As demonstrated by the substitution analysis in ELISA assays, R429 and K433 were identified as critical for binding of 101F, while another basic residue, K427 showed a minor contribution to the binding. These results were further confirmed by ELISA binding of 101F to the recombinant F protein mutants which incorporated a single amino acid substitutions at positions 427, 429 and 433 expressed in cells. The 101F binding was eliminated in mutants K433L, K433N, and K433T, and reduced at K433Q, K433R and K433S. Binding for 101F at K433D was drastically reduced. Both, the peptides and HRSV F protein recombinant mutants ELISA indicated the K433 is critical for the binding of 101F. The epitope recognition by the humanized 101F mAb, B21M was characterized by SPR analysis. Specific binding of B21M to the peptide 422-436 was confirmed, indicating the humanization conserved the epitope recognition by the parental murine mAb. From the equilibrium binding data, the free energies of binding of different sequence lengths as well as that of single residue substitutions indicated that the positive charged residues of the RSV F protein epitope are critical for antibody recognition. These data confirmed the ELISA results and corroborated the role of basic residues (in particular, Arg429 and Lys433) in steering binding as predicted from recombinant escape mutant (R429S and K233T) data.