Humanization of murine neutralizing monoclonal antibodies against HIV-1 through a new approach based on lentiviral vectors

More than 25 years after the discovery of HIV, AIDS still represents a pandemic emergency. Antiretroviral therapies are becoming more and more effective, but no real cure has yet been found to eradicate it. In order to develop a preventive vaccine, the isolation of new immunogens, capable to stimulate humoral immune response towards highly conserved viral epitopes, is pivotal. In a past work performed by our lab, the fusion between HIV-1 envelope and cell membrane was exploited to produce fusion intermediates exposing viral conserved epitopes. Such fusion complexes were used to immunize mice to elicit antibody production, and to produce hybridomas population expressing antibodies against HIV-1. During the course of the present work of thesis, hybridoma populations were screened for antibody production and neutralization activity. Cells were gradually subcloned down to single cell population in order to identify broad-spectrum monoclonal antibodies. In order to stabilize antibody expression, and to obtain antibodies supernatants close to human antibody, IgG mRNA sequences of the selected monoclonal population were cloned to be humanized. Viral vectors expressing humanized light and heavy IgG chains were produced by matching human constant regions with murine variable regions. Such viral vectors were used to co-infect CHO cells in order to obtain a cell line stably expressing humanized antibodies. Transgenes integration was confirmed by amplification of genomic DNA and humanized antibody production was quantified by ELISA assay. Obtained results show that viral vectors can integrate transgenes inside target cells and, more precisely, that our method works to produce humanized antibodies. Moreover, preliminary functional tests show that the produced antibody is working. Nevertheless, such protocol needs to be devised, as antibody expression remained at low concentration values to perform functionality assays. Optimization of this method and tests of antibody functionality will allow to the release of a rapid and easy protocol to produce cell line stably expressing the antibody of interest. Antibody characterization and analysis could be favored by a stable production source.