Design and characterization of novel dual Fc antibody with enhanced avidity for Fc receptors

Abstract

Monoclonal antibodies (mAbs) have become an important class of therapeutics, particularly in the realm of anticancer immunotherapy. While the two antigen‐binding fragments (Fabs) of an mAb allow for high‐avidity binding to molecular targets, the crystallizable fragment (Fc) engages immune effector elements. mAbs of the IgG class are used for the treatment of autoimmune diseases and can elicit antitumor immune functions not only by several mechanisms including direct antigen engagement via their Fab arms but also by Fab binding to tumors combined with Fc engagement of complement component C1q and Fcγ receptors. Additionally, IgG binding to the neonatal Fc receptor (FcRn) allows for endosomal recycling and prolonged serum half‐life. To augment the effector functions or half‐life of an IgG1 mAb, we constructed a novel “2Fc” mAb containing two Fc domains in addition to the normal two Fab domains. Structural and functional characterization of this 2Fc mAb demonstrated that it exists in a tetrahedral‐like geometry and retains binding capacity via the Fab domains. Furthermore, duplication of the Fc region significantly enhanced avidity for Fc receptors FcγRI, FcγRIIIa, and FcRn, which manifested as a decrease in complex dissociation rate that was more pronounced at higher densities of receptor. At intermediate receptor density, the dissociation rate for Fc receptors was decreased 6‐ to 130‐fold, resulting in apparent affinity increases of 7‐ to 42‐fold. Stoichiometric analysis confirmed that each 2Fc mAb may simultaneously bind two molecules of FcγRI or four molecules of FcRn, which is double the stoichiometry of a wild‐type mAb. In summary, duplication of the IgG Fc region allows for increased avidity to Fc receptors that could translate into clinically relevant enhancement of effector functions or pharmacokinetics.

Avidity in antibody effector functions and biotherapeutic drug design

“…Over the past few years, engineering strategies for enhancing antibody clustering to improve antibody function have primarily aimed at increasing antibody valency by replicating antibody binding or Fc domains in fragments and IgG-like or IgM-like structures. Indeed, several studies have reported on the design of antibody formats containing multiple Fc domains to enhance FcγR crosslinking and ADCC 143 – 146 . In a recent study, Miller and colleagues adapted the tetramerization domain of the tumour suppressor p53 and fused it to different antigen binding domains to create octavalent monospecific and bispecific antibody variants with increased functional activity, known as quads 147 .…”

Antibody Fc-chimerism and effector functions: When IgG takes advantage of IgA

“…Knowing that effector functions rely mainly on the Fc domains of Ab’s, the insertion of additional Fc domains into the design of Abs could result in chimeric antibodies with enhanced FcR binding and downstream Fc effector functions and therapeutic activities. To this end, from the parental Abs harboring one Fcγ ( Figure 4A ), chimeric Abs against respiratory syncytial virus (RSV) were designed with two or three Fcγ domains, resulting in a chimeric 2-Fc Ab with a tetrahedral-like geometry ( 121 ), as shown in Figure 4B . Surface plasmon resonance experiments showed that each 2-Fc chimera molecule binds to two FcγR molecules, thereby improving the Fc avidity of the Ab while maintaining the Fab antigen specificity of the original Ab.…”

In Translation: FcRn across the Therapeutic Spectrum

“…The investigators attribute the diminished half-life of these Fc tandem molecules to FcγR binding, which causes a sink effect by promoting endocytosis and degradation. In another interesting study, a group of investigators chose to attach an additional Fc to the light chain of a mAb [ 152 ]. This resulted in a 35-fold increase in FcRn affinity at pH 6.0, although pharmacokinetic data were not presented.…”