Human IgG Fc domain engineering enhances antitoxin neutralizing antibody activity

Abstract

The effector activity of antibodies is dependent on engagement with Fcγ receptors (FcγRs) and activation of the associated intracellular signaling pathways. Preclinical evaluation of therapeutic humanized or chimeric mAbs to study the interactions of their Fc regions with FcγRs is hampered by substantial structural and functional FcγR diversity among species. In this report, we used mice expressing only human FcγRs to evaluate the contribution of FcγR-mediated pathways to the neutralizing activity of an anti-anthrax toxin chimeric mAb. We observed that the protective activity of this mAb was highly dependent upon FcγR engagement, with minimal protection against anthrax toxin observed in FcγR-deficient mice following mAb administration. We generated anti-anthrax toxin mAbs with specific Fc domain variants with selectively enhanced affinity for particular human FcγRs and assessed their activity in FcγR-humanized mice. We determined that Fc domain variants that were capable of selectively engaging activating FcγRs substantially enhanced the in vitro and in vivo activity of anthrax toxin-neutralizing antibodies. These findings indicate that the application of Fc domain engineering is a feasible strategy to enhance toxin-neutralizing activity and suggest that engineered antitoxin antibodies will have improved therapeutic efficacy.

Broadly Neutralizing Anti-HIV-1 Antibodies Require Fc Effector Functions for In Vivo Activity

“…To overcome this problem, we have recently developed a mouse model, in which the mouse FcγRs were deleted and all the human FcγRs were expressed as transgenes, faithfully recapitulating the human-specific FcγR expression pattern and diversity (Smith et al, 2012). These mice have been previously characterized and successfully used for the study of human IgG Fc effector activity in vivo (Bournazos et al, 2014; DiLillo et al, 2014; Smith et al, 2012). Luciferase reporter mice were crossed to FcγR humanized mice and the activity of anti-HIV-1 human IgG1 bNAbs and Fc domain variants was evaluated.…”

“…As the amino acid backbone of the Fc domain is the main determinant for Fc-FcγR interaction, introduction of certain mutations in the Fc region that participates in the FcγR binding interface modulates the capacity of an IgG molecule to interact with FcγRs (Lazar et al, 2006; Shields et al, 2001). This approach has been previously employed by several groups to successfully generate antibodies with improved Fc effector functions, including ADCC and opsonization through enhancement of Fc-FcγR interactions (Bournazos et al, 2014; DiLillo et al, 2014; Heider et al, 2011; Horton et al, 2010; Lazar et al, 2006; Smith et al, 2012). Similarly, in our study, Fc domain variants (GASDALIE) of anti-HIV-1 bNAbs that exhibited enhanced binding capacity for activating human FcγRs, such as FcγRIIa and FcγRIIIa (Figure 4A–B) also presented augmented in vivo protective and therapeutic activity compared to wild-type or FcγR null binding variants (GRLR).…”

“…While the neutralizing activity of an antibody has been commonly considered to be the result of Fab-antigen interactions that block viral entry, substantial evidence suggests that the in vivo activity of an antibody is highly dependent upon interactions of the IgG Fc domain with its cognate receptors, Fcγ receptors (FcγR) expressed on the surface of effector leukocytes (Abboud et al, 2010; Bournazos et al, 2014; Corti et al, 2011; DiLillo et al, 2014; Li and Ravetch, 2011; Nimmerjahn and Ravetch, 2005; Smith et al, 2012). The FcγR system represents a balance of activating and inhibitory receptors that transduce immunostimulatory or immunomodulatory signals following engagement with IgG immune complexes, determining thereby the outcome of IgG-mediated inflammation and immunity (Pincetic et al, 2014).…”

“…Indeed, selective engagement of activating or inhibitory FcγRs by IgG has been shown to determine the in vivo outcome of antibody treatment, suggesting that Fc-mediated pathways play a key role in modulating the effector activity of a mAb (Li and Ravetch, 2011; Nimmerjahn and Ravetch, 2005). For example, the neutralizing activity of anti-toxin and anti-viral antibodies has been demonstrated to require selective FcγR engagement to mediate their in vivo protective activities (Akiyoshi et al, 2010; Bournazos et al, 2014; Corti et al, 2011; DiLillo et al, 2014). …”

LILR-B1 blocks activating FcγR signaling to allow antibody dependent enhancement of dengue virus infection

“…Although the process of virus neutralization was considered to be independent of the recruitment of effector pathways afforded by the innate immune system, recent evidence suggests that binding of IgG-virus immune complexes to innate immune effector cells via Fcγ-receptors (FcγR) is of great importance. Thus, neutralizing antibody dependent inhibition of HIV or influenza infection, as well as blocking the activity of bacterial toxins by passive immunotherapy, was shown to be critically dependent on activating FcγRs in vivo (5)(6)(7)(8).…”