Immunoglobulin M (IgM) is the major circulating Ig isotype in teleost fish. Unique features in crucial parts of the IgM molecule have been uncovered in Antarctic fish species that have experienced a special evolutionary history. The most striking structural characteristic is an extraordinary long hinge region, located between the second and third heavy chain constant domains. It can be viewed as a result of adaptive evolution to enhance the functionality of the molecule under very extreme environmental conditions. This finding prompted the idea to modify the heavy chain constant region (IgH) of a murine monoclonal antibody (mAb) by replacing its hinge with that from Antarctic fish IgM by using the CRISPR-Cas9 system. This technology has been recently proposed as an RNA-guided DNA targeting platform and widely used as a powerful tool for precise gene editing. Given its simplicity and flexibility, the CRISPR-Cas9 system has been successfully used also in the field of immunology to edit mouse and human Ig genes. A stepwise approach was chosen for targeted genome editing of a hybridoma cell line secreting IgG mAb. The first step was the creation of a targeted DNA double-stranded break at the hybridoma IgH gene locus to be modified. Homology-directed repair was then used to insert the "Antarctic" hinge sequence through recombination of a DNA donor template with the target locus. The correct sequence insertion was assessed by using a fluorescent protein as selection marker. A preliminary characterization of the antigen binding activity of the engineered mAb was performed by the Localized Surface Plasmon Resonance. The association constant k of the engineered mAb was found to be three-fold higher than that of the murine counterpart, suggesting an enhanced ability of the "antartized" mAb to recognize its target antigen, when immobilized on a rigid substrate. Overall, these results may open a new frontier in the field of antibody engineering by using an innovative and versatile CRISPR-based method.
Investigation of the IgM heavy chain gene from Antarctic fish inspired a novel engineered monoclonal antibody
A Ametrano;B Miranda;L De Stefano;U Oreste;MR Coscia
2023
Abstract
Immunoglobulin M (IgM) is the major circulating Ig isotype in teleost fish. Unique features in crucial parts of the IgM molecule have been uncovered in Antarctic fish species that have experienced a special evolutionary history. The most striking structural characteristic is an extraordinary long hinge region, located between the second and third heavy chain constant domains. It can be viewed as a result of adaptive evolution to enhance the functionality of the molecule under very extreme environmental conditions. This finding prompted the idea to modify the heavy chain constant region (IgH) of a murine monoclonal antibody (mAb) by replacing its hinge with that from Antarctic fish IgM by using the CRISPR-Cas9 system. This technology has been recently proposed as an RNA-guided DNA targeting platform and widely used as a powerful tool for precise gene editing. Given its simplicity and flexibility, the CRISPR-Cas9 system has been successfully used also in the field of immunology to edit mouse and human Ig genes. A stepwise approach was chosen for targeted genome editing of a hybridoma cell line secreting IgG mAb. The first step was the creation of a targeted DNA double-stranded break at the hybridoma IgH gene locus to be modified. Homology-directed repair was then used to insert the "Antarctic" hinge sequence through recombination of a DNA donor template with the target locus. The correct sequence insertion was assessed by using a fluorescent protein as selection marker. A preliminary characterization of the antigen binding activity of the engineered mAb was performed by the Localized Surface Plasmon Resonance. The association constant k of the engineered mAb was found to be three-fold higher than that of the murine counterpart, suggesting an enhanced ability of the "antartized" mAb to recognize its target antigen, when immobilized on a rigid substrate. Overall, these results may open a new frontier in the field of antibody engineering by using an innovative and versatile CRISPR-based method.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
