"Antarctized antibody": a new format of monoclonal antibody, engineered by the crispr/cas9 system

The CRISPR/Cas9 system, a prokaryotic adaptive defense, has been recently suggested as an RNA-guided DNA targeting platform and widely used as a powerful tool for genome editing. Over the past few years, given its simplicity and flexibility, the CRISPR-Cas9 system has been successfully applied also in the field of immunology to edit mouse and human Ig genes. Immunoglobulin M (IgM) is the most expressed Ig isotype in teleost fish and, in particular, it has been shown to possess unique features in Antarctic fish species, e.g., an extraordinary long hinge region, crucial for the flexibility of the Ig molecule. This structural peculiarity can be viewed as a result of adaptive evolution to improve the performance of the antibody under very cold environmental conditions. This finding prompted the idea to engineer the heavy chain (IgH) carboxy-terminal region of a murine monoclonal antibody (mAb) by replacing its hinge with that from Antarctic Ig using the CRISPR-Cas9 system. A stepwise approach was chosen for targeted genome editing of a hybridoma cell line secreting IgG mAbs. The first step was the creation of a DNA double-stranded break at the hybridoma IgH constant region gene locus. The Antarctic hinge sequence was inserted in the target locus by homologous recombination through a DNA donor template. The correct sequence insertion was evaluated by using a fluorescent protein (mCherry) as selection marker. The "antarctized" mAb was successfully produced by the engineered hybridoma cell line. A preliminary biochemical and functional characterization in comparison to the wild type counterpart was performed. Overall, these results could offer new perspectives on further structural modifications of mAbs for different purposes by applying an innovative and versatile technology.