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

Vladimir Razinkov,Zhi Liu,Laura Sekirov,Esther Leng,Carl Kozlosky,Wei Wang,William Fanslow,Ian Foltz,Monique Howard,Kannan Gunasekaran,Anne-Marie Rousseau,Wei Yan,Heather Sweet

doi:10.1074/jbc.m113.513366

Vladimir Razinkov, Zhi Liu + Show 11 more

Open Access

https://doi.org/10.1074/jbc.m113.513366

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Abstract

Antibody-dependent cellular cytotoxicity (ADCC) is mediated through the engagement of the Fc segment of antibodies with Fcγ receptors (FcγRs) on immune cells upon binding of tumor or viral antigen. The 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 the 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 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 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 of building the next generation of therapeutic antibodies with enhanced ADCC effector function for the treatment of cancers and infectious diseases.

Highlights

Co-crystal structure of Fc-Fc␥RIII complex revealed that Fc binds to Fc␥RIII asymmetrically
Heterodimeric IgG1 Antibodies Containing Asymmetrical Fc Mutants Enhance Antibody-dependent cellular cytotoxicity (ADCC) Effector Function—The crystal structure studies revealed that the interaction of human Fc␥RIII with IgG1 Fc region is asymmetric, i.e. Fc␥RIII comes into contact with different amino acid residues on the two Fc polypeptide chains that make up the Fc region (15)
Based on the nature of asymmetric interaction between the Fc region of IgG and Fc␥RIII (15), we hypothesized that an optimal way to improve Fc binding to Fc␥RIII is to make asymmetrical changes in the two Fc chains so that each Fc chain can be individually optimized to interact with the Fc␥RIII and

Summary

Background

Co-crystal structure of Fc-Fc␥RIII complex revealed that Fc binds to Fc␥RIII asymmetrically. The co-crystal structure of Fc␥RIII in complex with Fc revealed that Fc binds to Fc␥RIII asymmetrically with two Fc chains contacting separate regions of the Fc␥RIII by utilizing different residues To fully explore this asymmetrical nature of the Fc-Fc␥R 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 Fc␥RIIIA (both Phe-158 and Val-158 allotypes), increased ADCC activity in vitro, and strong tumor growth inhibition in mice xenograft human tumor models. This leads to an hypothesis that the IgG subclass with a higher activating to inhibitory ratio, i.e. IgG with increased binding to Fc␥RIIIA or Fc␥RIIA but decreased/unchanged binding to Fc␥RIIB, could translate into significantly enhanced in vivo activity (9)

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