Sviluppo ed umanizzazione di un anticorpo in formato scFv contro β-1,3 glucano

INTRODUCTION Fungal diseases caused by opportunistic pathogenic species of fungi, are increasingly impacting on health of populations, particularly in patients under immunosuppressive chemotherapy and hospitalized. Classic therapies are often unable to control these infections and may be associated with severe side effects and the emergence of resistant fungal strains. There is increasing interest in the development of new biological tools, such as monoclonal antibodies, to fight these opportunistic fungi. These molecules may be used to replace or integrate the classical antifungal therapy. Our work aimed to develop and humanize a single chain Fv (scFv) derived from the murine antibody 2G8, which specifically recognizes β1,3-glucan, a major cell wall polysaccharide crucial for growth and survival of the most common pathogenic species of fungi, such as Candida Albicans, Aspergillus fumigatus and Criptococcus neoformans. METHODS: The murine framework (FRW 1, 2, 3, 4) regions of 2G8 were humanized using bioinformatics tools by choosing the human variable domains with high sequence homology. The humanized scFv 2G8 was obtained by CDR-graft method of the murine CDRs into humanized framework regions. scFv 2G8 was expressed in E. Coli as periplasmic-secreted protein, and purified through 6 His-tag, using immobilized metal chelate affinity chromatography (IMAC) with Ni-Sepharose column. Biological activity of the molecule was tested by ELISA and in vitro growth inhibition analysis. RESULTS: Recombinant protein was purified from insoluble fraction in denatured conditions. The purified scFv showed a specific binding activity for β1,3-glucans as determined by ELISA test. scFv 2G8 at the concentration of 100 µg/ml showed a growth inhibition of 33% on Candida Albicans cells with germ tubes. CONCLUSIONS: Future studies will concern: site-directed mutagenesis, new in silico modeling studies, new in vitro studies and VL-linker-VH orientation.