Asymmetrical Fc Engineering Greatly Enhances Antibody-dependent Cellular Cytotoxicity (ADCC) Effector Function and Stability of the Modified Antibodies

Background: Co-crystal structure of Fc-FcRIII complex revealed that Fc binds to FcRIII asymmetrically. Results: We identified a panel of novel Fc heterodimers with enhanced ADCC activity. Conclusion: Asymmetrical Fc engineering is an efficient approach for enhancing ADCC activity and stability of engineered antibodies. Significance: The discovery could be applied in therapeutic antibodies for the treatment of cancers and infectious diseases. Antibody-dependent cellular cytotoxicity (ADCC) is mediated through the engagement of the Fc segment of antibodies with Fc receptors (FcRs) on immune cells upon binding of tumor or viral antigen. The co-crystal structure of FcRIII in complex with Fc revealed that Fc binds to FcRIII asymmetrically with two Fc chains contacting separate regions of the FcRIII by utilizing different residues. To fully explore this asymmetrical nature of the Fc-FcR 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 (R) assay. To this end, we have identified a panel of novel Fc variants with significant binding improvement to FcRIIIA (both Phe-158 and Val-158 allotypes), increased ADCC activity in vitro, and strong tumor growth inhibition in mice xenograft human tumor models. Compared with 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 the C(H)2 domain. Fc heterodimers also allow more selectivity toward activating FcRIIA than inhibitory FcRIIB. Afucosylation of Fc variants further increases the affinity of Fc to FcRIIIA, leading to much higher ADCC activity. The discovery of these Fc variants will potentially open up new opportunities of building the next generation of therapeutic antibodies with enhanced ADCC effector function for the treatment of cancers and infectious diseases.