Immunoglobulin G (IgG) is the most abundant antibody class in human serum, accounting for approximately 70%–85% of total serum immunoglobulins, and plays a central role in adaptive immune responses. The IgG molecule features a characteristic Y-shaped structure composed of two identical heavy chains and two identical light chains linked by disulfide bonds. The amino-terminal regions of both heavy and light chains exhibit highly variable sequences, designated as variable regions (V regions, VH and VL). The remaining segments possess genetically conserved sequences and are referred to as constant regions (C regions, CH and CL). The heavy chain constant region of IgG is divided into three domains: CH1, CH2 and CH3. Structurally, IgG can be further split into the antigen-binding fragment (Fab) and the crystallizable fragment (Fc). The Fab region specifically recognizes and binds antigens, while the Fc region mediates a variety of immune effector functions. A highly conserved glycosylation site, Asn297, is located within the Fc region, carrying glycan moieties including mannose, N-acetylglucosamine, galactose, sialic acid and fucose.
IgG is categorized into four subtypes: IgG1, IgG2, IgG3 and IgG4. Their constant regions share high sequence homology except for the CH2 domain. The hinge region acts as a flexible linker between the Fab arms and the Fc domain, governing the conformational flexibility of Fab relative to Fc and between individual Fab arms. Distinct structural differences exist in the hinge regions across subtypes, particularly in the number and position of disulfide bonds: IgG1 and IgG4 contain 2 interchain hinge disulfide bonds, IgG2 has 4, and IgG3 possesses as many as 11. Additionally, the linkage site between light chains and heavy chains varies by subtype: in IgG1, the linkage occurs at the 5th cysteine residue of the heavy chain, whereas in IgG2, IgG3 and IgG4, it is located at the 3rd cysteine. These structural disparities lead to divergent glycosylation patterns, molecular flexibility and receptor binding affinities among subtypes, ultimately resulting in distinct effector functions.
The four IgG subtypes display unique properties during immune responses. IgG1 is predominantly induced upon exposure to soluble protein antigens and membrane protein antigens, often accompanied by low levels of other subtypes, especially IgG3 and IgG4. Immune responses targeting bacterial capsular polysaccharide antigens mainly trigger the production of IgG2. As a potent pro-inflammatory antibody, IgG3 efficiently activates downstream immune effector cascades but has a relatively short half-life, which may help prevent excessive inflammatory reactions. IgG4 generally predominates under conditions of persistent or repeated antigen exposure without infection, such as chronic allergies and certain autoimmune disorders.
Effector functions represent the core biological activities of antibodies, functioning to neutralize and eliminate pathogens and their products. IgG mediates two primary effector mechanisms: complement activation and Fcγ receptor (FcγR)-dependent opsonization and cytotoxicity. Notably, the glycan structure at the conserved Fc glycosylation site Asn297 critically regulates the binding of IgG to various FcγRs and complement component C1q, thereby modulating key immune effector pathways including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC).
Complement activation is initiated by C1q binding. Key residues within the CH2 domain responsible for C1q interaction include L235, D270, K322, P329 and P331. The impaired C1q binding of IgG2 is primarily attributed to the A235 residue, which is a leucine in other subtypes. In IgG4, the P331 residue largely accounts for its diminished or abolished C1q binding capacity. The extended hinge region of IgG3 renders C1q binding sites more accessible, enabling robust complement activation. In contrast, IgG2 and IgG4 have short hinge regions consisting of only 12 amino acid residues; their Fab arms may sterically hinder C1q access to binding sites. Collectively, IgG1 and IgG3 potently trigger the classical complement pathway, while IgG2 and IgG4 exhibit markedly low activity in this regard.
The interaction between the Fc region and FcγRs mediates diverse immune reactions, and IgG subtypes differ substantially in their FcγR binding profiles. FcγRI binds all human IgG subtypes except IgG2, with IgG1 and IgG3 showing stronger affinity than IgG4. Mutations in the lower hinge region of IgG1 at positions corresponding to those in IgG2, particularly E233P, L235A and G236, disrupt FcγRI binding. The substitutions P331S and L234F are responsible for the reduced FcγRI affinity of IgG4 compared with IgG3. All IgG subtypes bind weakly to FcγRIIb/IIc, with the preference ranking: IgG3 = IgG1 = IgG4 > IgG2. FcγRIII selectively interacts with IgG1 and IgG3, but not IgG2 or IgG4, and IgG3 demonstrates superior binding to FcγRIII relative to IgG1. Overall, IgG1 and IgG3 exhibit far stronger FcγR binding capabilities than IgG2 and IgG4.
The neonatal Fc receptor (FcRn) binds to the Fc domain of IgG to protect antibodies from degradation and extend their in vivo half-life. FcRn does not bind its ligands at physiological pH (7.4). Binding only occurs in the acidic environment of endocytic vesicles (pH < 6.5), where histidine residues on IgG become protonated. Histidine residues located at the CH2-CH3 interface of the IgG Fc region are essential for high-affinity binding to β2-microglobulin (β2M) and the α-chain of FcRn. Residue H435 sits at the core of this interaction interface. IgG3 carries an arginine at position 435 instead of histidine, which reduces its affinity for FcRn, explaining its shorter half-life and decreased transplacental transport.
Nearly all currently marketed therapeutic antibodies are built on IgG scaffolds. IgG1 stands as the most widely adopted subtype due to its robust effector functions, while IgG2 and IgG4 are utilized in selected drug candidates. Despite its superior complement activation and cellular cytotoxicity, IgG3 is rarely employed for therapeutic antibody development, owing to its short half-life, extensive allotypic polymorphisms and potential immunogenicity. In practical antibody drug design, IgG subtypes are rationally selected or engineered according to therapeutic goals and mechanisms of action, as detailed below:
1. Scenarios requiring potent effector functions (anti-cancer and anti-infective drugs)
With high affinity for FcγRs and C1q, IgG1 effectively mediates ADCC, ADCP and CDC, making it the first choice for such applications. Although IgG3 delivers stronger effector activities, its suboptimal pharmacokinetic properties render it a secondary option. Fc region point mutations can further enhance effector functions: mutations such as S239D/I332E (DE) and G236A/S239D/I332E (GASDIE) strengthen FcγR binding, while E345R/E430G mutations promote antibody hexamerization and dramatically boost complement activation.
2. Scenarios requiring minimal effector functions (anti-inflammatory and signal-blocking antibodies)
IgG2 possesses relatively weak effector functions, making it suitable for treating certain autoimmune and infectious diseases. IgG4 binds FcγRs poorly and barely activates the complement system, which is ideal for blocking antibodies where Fc-mediated off-target cytotoxicity must be avoided. To completely ablate Fc effector functions, mutations including L234A/L235A (LALA) and P329G (LALAPG) can be introduced to weaken binding to FcγRs and C1q. Alternatively, the N297A/Q mutation eliminates Fc glycosylation and fully abolishes effector activities.
3. Scenarios requiring extended half-life
All IgG subtypes can be engineered at the FcRn binding interface to prolong serum half-life. Common strategies include introducing point mutations such as M252Y/S254T/T256E (YTE) and M428L/N434S (LS). These variants enhance pH-dependent FcRn binding, reduce lysosomal degradation and extend antibody circulation time.
Furthermore, IgG4 is capable of Fab-arm exchange (FAE), which enables the formation of functionally monovalent bispecific antibodies in vivo and provides a unique approach for developing naturally formatted bispecific antibodies. Characterized by low effector activity, IgG4 is regarded as a non-inflammatory subtype and fits well with multiple therapeutic indications. Currently, bispecific antibodies based on the IgG4 scaffold have become a major hotspot in antibody drug research and development.
In conclusion, rational selection and protein engineering of IgG subtypes are critical strategies for precise modulation of therapeutic antibody functions. With advancing understanding of antibody structure-function relationships and the maturation of protein engineering technologies, a growing number of antibody drugs featuring enhanced effector functions, prolonged half-life, low immunogenicity and bispecific binding capabilities will be developed for broad applications in oncology, autoimmune diseases, infectious diseases and other therapeutic areas.
