The Fc (fragment crystallizable) region of the antibody is responsible for activating pathways that eliminate pathogens and abnormal cells by binding to Fc gamma (Fcγ) receptors on innate immune cells and complement proteins in plasma. However, in autoimmune diseases, the loss of tolerance to self allows antibodies to incorrectly activate these damaging pathways against the body's own tissues.
Technological advances have revealed the intricate molecular determinants regulating antibody interactions with the innate immune system. Innovations in x-ray crystallography, cryogenic electron microscopy, yeast surface display and next generation sequencing have unlocked high-resolution understanding of Fc binding dynamics.
Notably, the recent review from Damelang et al. (2024) consolidates the latest findings on both natural and engineered structural modifications of IgG antibodies. This comprehensive review encompasses diverse aspects such as allotypic variation, glycosylation patterns, Fc engineering, and optimization of Fc gamma receptor binding. By pinpointing and mutating key residues participating at the different antibody-effector interfaces, antibodies are engineered to selectively enhance or silence effector functions.1
Subclass Distinctions Underlie Effector Hierarchy
Humans express five isotype classes of antibodies—IgM, IgD, IgG, IgA and IgE—which share similar structures but fulfill distinct roles. IgG constitutes the predominant circulating immunoglobulin, engaged in most effector responses. The four IgG subclasses—IgG1, IgG2, IgG3 and IgG4— exhibit fine differences in their capacity to interact with distinct FcγR subtypes on innate immune cells like natural killer cells, neutrophils, monocytes and macrophages.1
The IgG subclasses diversify through allotypic variations encoded by non-synonymous single nucleotide polymorphisms in the IgG constant heavy chain loci directly impacting FcγR and complement binding sites. 1 Differential patterns of N-linked glycan addition at conserved residues also contribute to subclass distinctions.2 The variable occupancy with fucose, bisection, galactose and sialic acid alters adherence to different FcγR isoforms. Meanwhile, the flexibility imparted by variable hinge lengths controls spatial factors influencing hexamerization and Fc-engaging molecule complement component (C1q) engagement.3
Neonatal Fc Receptor Regulates Serum Persistence
In addition to activating immune cells, IgG requires protection from catabolism to ensure sustained antibody-mediated surveillance and response.1 The major histocompatibility complex class I related neonatal Fc receptor (FcRn) interacts with the Cγ2-Cγ3 interface of IgG at acidic pH, salvaging endocytosed antibodies from lysosomal degradation in vascular endothelia and recycling them back to circulation.4 Loss of FcRn protection results in dramatically shortened serum half-life of IgG antibodies. 5 Overall, FcRn binding rescues IgG from breakdown, maintaining antibody levels for sustained immune responses against pathogens.
The interaction depends on histidine 310, histidine 435 and isoleucine 253. 6,7 Mutating all three residues to alanine abrogates IgG binding. However, increased affinity selectively within the acidic pH of the endosome leads to improved protection and longer persistence. 1 The M252Y/S254T/T256E (YTE) variant exhibits stronger adherence to FcRn at the optimal pH < 6.5 without increasing binding requisite for efficient release back into circulation.1
Antigen Binding Induces Effector System Activation
In contrast to constitutive IgG recycling by FcRn, interaction with FcγRs and complement strictly relies on antigen-mediated hexamerization of the antibodies. The IgG molecules first bind soluble antigen through their Fab variable region composition or recognize surface antigens on pathogens, infected cells or cancerous cells.1 Soluble immune complexes cluster into ordered hexamers with the Fc regions facing outward. Meanwhile, densely clustered membrane antigens can directly promote Fc-Fc interactions between neighboring antibodies.8
The dynamic process of avidity-enhanced multimerization depends on multiple molecular factors including antibody subclass flexibility, epitope binding angle, binding affinity, target size and antigen mobility. Optimal orientations allow the hexameric structures to recruit effector interactions. The flexible hinge regions permit free rotation of the Fab and Fc which facilitates hexamer assembly.9
Fcγ Receptors Bind Distinct IgG Residues
The interaction epitopes between human IgG and FcγRI, FcγRII or FcγRIII receptors rely on distinct combinations of amino acids, allowing subclass-specific adherences amenable to selective engineering.1 Binding strongly depends on the lower hinge region, where the IgG1 glycine 236 and glycine 237 residues constitute key interacting contacts. Simply adding a glycine residue at position 236 of human IgG2 allows it to gain binding to FcγRI and FcγRIIIa. Conversely, mutating glycine 236 in IgG1 abrogates binding to all FcγR.10
The alternative Leu234Ala/Leu235Ala double substitution reduces adherence to FcγRI, FcγRIIa and FcγRIIIa. Further introduction of Pro331Ser or Pro329Gly eliminates residual binding. Dissociating the conserved N297 glycan by substituting alanine similarly annuls FcγR interaction. Other mutant combinations can selectively increase or decrease affinity to certain stimulating or inhibitory receptors. For example, the triple Gly236Ala/Ser239Asp/Ile332Glu variant shows augmented binding to activating FcγR subtypes.11
Conclusion
Comprehensively optimizing antibody function requires assimilating the molecular participation of subclass genetics, allotypic variation, hinge flexibility, glycan occupancy, binding valency and epitope orientation in differential FcγR activation. While structure-guided mutations offer promising avenues for enhancing therapeutic efficacy, anticipated modifications must still undergo rigorous preclinical analysis in humanized FcγR mice tracking cytotoxicity, phagocytosis and bioavailability.1
Future innovations might attempt to leverage the unique properties of the less studied IgG3 subclass and its allotypic portfolio conferring maximal effector potency. Alternatively, variable domain engineering to specify target epitope selection allows tuning of hexamer geometries. Screening platforms assessing antibody clustering dynamics offer another avenue for custom engineering. Ultimately, the accumulating structural insights set the stage for an exciting new generation of tailored immunotherapies. But realization requires interdisciplinary expertise traversing structural biology, bioinformatics and immune monitoring.
