Development of chimeric humanized monoclonal antibodies against HIV-1

Development of chimeric humanized monoclonal antibodies against HIV-1 Dalila Astone, Pierpaolo Racchiolli, Serena Ziglio, Donato Zipeto Laboratory of Molecular Virology, Section of Biology and Genetics, Department of Life and Reproduction Sciences, University of Verona, Strada le Grazie 8, 37134 Verona, Italy Introduction: Fusion complexes were used to elicit HIV-1 neutralizing antibodies. To avoid antibody production loss in hybridoma clones, CHO cells expressing neutralizing antibodies were obtained by co-infection with lentiviral vectors expressing the murine variable heavy and light chains (Hv, Lv) and the human constant chains (Hc, Lc). Methods: IgG variable regions were obtained from hybridomas producing neutralizing antibodies and cloned into lentiviral vectors bearing respectively Hc and Lc. The neutralization activity of humanized antibodies was tested using both TzmBl cells and TzmBl variants expressing FcγR. Results: Following cell passage and amplification, antibody production and neutralizing activity of selected hybridomas decreased, requiring the stabilization of antibodies expression. Hv and Lv of IgG from selected clones were inserted into lentiviral vectors expressing human Hc and Lc. After lentiviral infection of CHO cells, 16% of CHO clones showed evidence of production of humanized chimeric IgG. Clones with higher IgG production were analyzed for neutralization activity against some reference Env pseudoviruses. Preliminary results showed an 80% average neutralizing activity against tested pseudovirues. In addition, chimeric humanized antibodies showed neutralizing activity using TzmBl FcγR cells, on which the original murine mAbs did not neutralize. Conclusions: Monoclonal antibodies obtained by immunizing with fusion complexes showed broad spectrum neutralizing activity against several pseudoviruses, but unstable antibody production. Following humanization, antibodies exploit both classic and FcγR-mediated neutralization. Fusion complexes represent potential new immunogens that can induce neutralizing antibodies with activity against a wide panel of HIV-1 isolates. Humanization of antibodies may widen their neutralization activity through different mechanisms