Enrichment of high affinity subclasses and glycoforms from serum‐derived IgG using FcγRs as affinity ligands

Abstract

As antibodies continue to gain predominance in drug discovery and development pipelines, efforts to control and optimize their activity in vivo have matured to incorporate sophisticated abilities to manipulate engagement of specific Fc binding partners. Such efforts to promote diverse functional outcomes include modulating IgG-Fc affinity for FcγRs to alternatively potentiate or reduce effector functions, such as antibody-dependent cellular cytotoxicity and phagocytosis. While a number of natural and engineered Fc features capable of eliciting variable effector functions have been demonstrated in vitro and in vivo, elucidation of these important functional relationships has taken significant effort through use of diverse genetic, cellular and enzymatic techniques. As an orthogonal approach, we demonstrate use of FcγR as chromatographic affinity ligands to enrich and therefore simultaneously identify favored binding species from a complex mixture of serum-derived pooled polycloncal human IgG, a load material that contains the natural repertoire of Fc variants and post-translational modifications. The FcγR-enriched IgG was characterized for subclass and glycoform composition and the impact of this bioseparation step on antibody activity was measured in cell-based effector function assays including Natural Killer cell activation and monocyte phagocytosis. This work demonstrates a tractable means to rapidly distinguish complex functional relationships between two or more interacting biological agents by leveraging affinity chromatography followed by secondary analysis with high-resolution biophysical and functional assays and emphasizes a platform capable of surveying diverse natural post-translational modifications that may not be easily produced with high purity or easily accessible with recombinant expression techniques.

Hinge length contributes to the phagocytic activity of HIV-specific IgG1 and IgG3 antibodies

“…However, prior subclass switching work does not support a strong role for hinge modification in ADCC, as similar activity of IgG3 relative to IgG1 is typically observed [56,82]. We have previously observed similar induction of NK-degranulation by VRC01 IgG1 and IgG3 [14]. In contrast, a recent study found that hinge extension improved ADCC for one monoclonal antibody, but that this effect was not generalizable across specificities that recognized different epitopes on the same antigen [83].…”

“…While other factors such as antigen affinity and antigen density have also been associated with modulating ADCC activation [85], the most widely described means to enhanced ADCC activity is via variable fucosylation of the Fc domain. This effect is apparent in the potentiated ability of afucosylated VRC01 IgG1 to induce NK cell degranulation relative to fucosylated IgG1 or IgG3 types, whereas no activity was observed for aglycosylated IgG1 [14]. Antibody glycosylation status was not determined nor controlled for the majority of antibody variants evaluated in this study.…”

“…In contrast, as IgG2 and IgG4 antibodies have compromised effector function compared to IgG1 and IgG3, subclass switching has dramatic and generally predictable effects on the effector function of antiviral antibodies, as would be expected from their differing affinities for FcɣR [67]. Between IgG1 and IgG3, however, it has now been observed that the IgG3 subclass is associated with considerably enhanced phagocytic activity across multiple studies for multiple antibodies, including VRC01, 447-52D, CAP256, CH31, CH27, CH28, HG107, 7B2, 2158, and 1361 [12][13][14]89]. Here, these observations are extended to include two antibodies that recognize distinct viral epitopes and have differing phagocytic activities in their IgG1 forms.…”

“…Independent of the capture antigen, consistent activity patterns have been noted across hinge and subclass variants. For example, activity of VRC01 in IgG3 form has been reported to be similarly enhanced relative to IgG1 in the THP-1 phagocytosis assay when JRFL SOSIP target beads are used [14]. Further, the activity of myeloma-derived IgG3 relative to myeloma IgG1 shows the same enhancement in the THP-1 assay when an anti-IgG target bead is used [56].…”

“…Though modified by Fc domain glycosylation at a conserved motif, the effector functions of the limited number of human IgG subclasses are generally predictable, with IgG1 and IgG3 exhibiting higher affinity for FcɣR and consequently greater effector function than their IgG2 and IgG4 counterparts. Moving beyond these useful approximations, however, a number of recent studies aimed at defining the activity of subclass-switched HIV-specific antibodies have observed enhanced phagocytic activity of human IgG3 relative to IgG1 [12][13][14].…”