Asymmetrical Fc engineering greatly enhances ADCC effector function and stability of the modified antibodies [Protein Structure and Folding]

December 5th, 2013 by Liu, Z., Gunasekaran, K., Wang, W., Razinkov, V., Sekirov, L., Leng, E., Sweet, H., Foltz, I., Howard, M., Rousseau, A.-M., Kozlosky, C., Fanslow, W., Yan, W.

Antibody Dependent Cellular Cytotoxicity (ADCC) is mediated through the engagement of Fc segment of antibodies with Fc gamma receptors (FcγRs) on immune cells upon binding of tumor or viral antigen. Co-crystal structure of FcγRIII in complex with Fc revealed that Fc binds to FcγRIII asymmetrically with two Fc chains contacting separate regions of the FcγRIII by utilizing different residues. To fully explore this asymmetrical nature of Fc:FcγR interaction, we screened more than 9,000 individual clones in Fc heterodimer format in which different mutations were introduced at the same position of two Fc chains using a high throughput competition AlphaLISA assay. To this end, we have identified a panel of novel Fc variants with significant binding improvement to FcγRIIIA (both F158 and V158 allotypes), increased ADCC activity in vitro and strong tumor growth inhibition in mice xenograft human tumor models. Compared to previously identified Fc variants in conventional IgG format, Fc heterodimers with asymmetrical mutations can achieve similar or superior potency in ADCC-mediated tumor cell killing and demonstrate improved stability in CH2 domain. Fc heterodimers also allow more selectivity toward activating FcγRIIA than inhibitory FcγRIIB. Afucosylation of Fc variants further increases the affinity of Fc to FcγRIIIA, leading to much higher ADCC activity. The discovery of these Fc variants will potentially open up new opportunities in building the next generation of therapeutic antibodies with enhanced ADCC effector function for the treatment of cancers and infectious diseases.