Human Anti-Histone 3.3 Antibodies as Potential Biotherapeutics for Chronic Obstructive Pulmonary Disease (COPD)

Abstract

Chronic Obstructive Pulmonary Disease (COPD), which is characterized by limitation of pulmonary air flow, is now the third major cause of death worldwide. Barrero et al. have reported that the elevation of extracellular hyperacetylated histone H3.3 in the lungs of COPD patients is associated with cytotoxicity and disease progression. They found that extracellular hyperacetylated H3.3 was cytotoxic to lung structural cells and resistant to proteasomal degradation, and that mouse antibodies to either the C- or N- termini of H3.3 could partially reverse H3.3 toxicity in vitro. Thus, we hypothesize that human antibodies directed against H3.3 may be effective biotherapeutics useful to control progression of COPD in vivo. The discovery and development of human monoclonal antibodies (mAbs) is a fast growing field of biotherapeutics. In addition to full length mAbs, antibody fragments also have been used in antibody discovery research. We have used phage display technology in this project to discover human anti-H3.3 antibody Fab fragments. This technology utilizes genetically engineered phage particles containing genes encoding diverse Fab fragments displayed on the particles. The “Ylanthia” library from MorphoSys AG, a synthetic fully human Fab antibody phage display library with 1.3 x 1011 independent clones, was panned against purified recombinant human H3.3 immobilized on 96-well plates. Seven H3.3-binding Fab fragments with unique DNA sequences were isolated after four rounds of panning. Following their expression in E.coli and purification, Fab purities and electrophoretic mobilities were evaluated on SDS-PAGE. The concentration-dependent binding activities of all seven Fabs to human H3.3 were tested by ELISA. All seven Fabs were shown by ELISA to bind H3.3 but not histones 2A, 2B or 4. Since H3.3 is localized to the nucleus, western blotting was used to demonstrate that seven Fabs recognize purified, recombinant H3.3 and denatured natural histone(s) from nuclear extracts of human 293T cells. In order to characterize these molecules further, biological activity assays will be done to test their potential to reverse the toxic effects of H3.3 in cell culture. If these Fabs prove active in cell culture, they will be converted to IgGs and tested in animal models as potential biotherapeutics for COPD.