IgG is generated through isotype switching and makes up a majority of circulating antibody. IgGs are generally higher affinity than the primary IgM antibodies and make up a vast majority of the memory response. IgGs have four major effector functions: 1) neutralization, 2) opsonization, 3) complement fixation, and 4) antibody dependent cell-mediated cytotoxicity (ADCC). Each of the IgG subclasses has slightly different structures that dictate their ability to perform the effector functions previously listed. In past “Biology of Antibodies” blog articles we have examined the development of antibodies (B Cell development), antibody structure, as well as immunoglobulin genetics. With all of that as background, let’s dive a little deeper into the general subject of antibody structure and function, by focusing on IgGs.
IgG Structural Variation
There are four IgG subclasses in both human (IgG1, IgG2, IgG3, & IgG4) and mice (IgG1, IgG2a, IgG2b, & IgG3). Within each of these two species, the IgG subclasses are 95% identical at the amino acid level. The relatively minor differences have rather important functional differences. Without walking through all of the individual changes, the differences are mainly in the size and configuration of the hinge region, glycosylation sites, and structures, as well as a few key amino acid changes that impact the ability to interact with complement and Fc receptors (Figure 1).
These changes, particularly the size of the hinge region, have an impact on the flexibility of the antibody at the hinge. Another factor that influences the flexibility of the hinge region is the number and the location of interchain disulfide bonds between the two IgG heavy chain strands. Each of these variations, as well as the glycosylation pattern, will define the effector function of the IgG.
Human IgGs also have some interesting features that distinguish the valency. IgG1 and IgG3 are monomeric (2 heavy chains & 2 light chains) and bivalent (2 variable regions). IgG2 has a distinct disulfide bond pattern which allows for two monomeric IgG2 antibodies to form a dimeric (and tetravalent) structure through unique inter-molecule disulfide bonds. IgG4 has an even more unique structure (again dictated by the heavy chain intrachain disulfide bond). The intrachain disulfide bonds (there are two) can be reduced, which generates a monovalent structure. In addition, the monovalent structures can reform the disulfide bonds, but may not be the same IgG4 monovalent chain; meaning the resulting IgG4 will be a bivalent monomer but will have two different variable regions.
IgG Fc Receptor
IgG receptor, known as Fc𝝲Rs, have three distinct functions: cellular activation, cellular inhibition, and recycling and transport. There are several Fc𝝲Rs in both humans and mice. Fc𝝲RIIB is the inhibitory receptor in both humans and mice and acts as a feedback loop to prevents the development of new antibody responses to antigens that the host is already producing IgG antibodies. In humans, this inhibitory receptor is almost exclusively expressed on B cells and dendritic cells, however in mice there is a much broader expression profile across several leukocyte populations. The FcRn is involved in transport across tissue barriers (placenta, gut, and others) as well as recycling IgG from acidic endosomes and releasing it from the cell.
Activating Fc𝝲Rs (I, IIA, IIC, IIIA, & IIIB for human and I, III, IV in mice) all have different cellular expression patterns and affinities for total IgG and each of the sub-classes. Therefore, the individual IgG isotype (G1, G2, etc) will lead to different cellular outcomes based on the Fc𝝲R expression pattern and the abundance of specific IgG produced.
IgG Functional Variation
The overall effector function of antibodies is to increase the efficiency of detection and clearance of pathogens and toxins. Toxins need to be neutralized and therefore, antibodies that can bind to the toxin will prevent the toxin from performing its biological function. While neutralizing the toxin is important it must also clear the toxin from the system in order to protect the host cells and tissues. The IgG-toxin complex will be bound by Fc𝝲Rs activating phagocytosis and degradation of the toxin. The IgG may be saved from degradation in the endosome by FcRn. A similar process is undertaken for microbial and viral pathogens. As these are cells or complex particles, many antibodies may coat the surface of the virus or microbial pathogen, a process referred to as opsonization. There can be two outcomes of this process, complement fixation or phagocytosis. For viruses, the Fc𝝲Rs will recognize IgG-virus complex and induce phagocytosis and degrade the virus in the endosome. A pathogenic microbial cell can be removed in this fashion or the IgG can “fix” complement on the cell surface which increases phagocytosis activation and induce complement to induce cell killing (by essentially punching holes in the membrane). Note the complement-induced cell killing will not be effective on viruses as they do not have a cell membrane to target. The final type of antibody effector function is ADCC, which directs (predominantly NK cells) to a pathogen infected target cell and activates the NK cell to kill the target cell.
In all of the described mechanisms, the function of the antibody is to specifically recognize the antigen (toxin or pathogen component) and induce the clearance and removal. The specificity is mediated by the variable region and the function is mediated by the constant region, the Fc specifically. Each of the subtypes (G1, G2, etc.) have different abilities to be recognized by Fc𝝲Rs or complement and therefore result in different effector function outcomes.
The type of immunogen (allergen/toxin/pathogen), route of exposure, and duration defines the immunological response, including the isotype of antibody produced, which is extremely important for appropriate protection of the host. It has also become important in therapeutic antibody development to match the isotype with the desired effector function. A vast majority of therapeutic antibodies are IgG1, although IgG4 and IgG2 are more recently becoming important. More recently, genetic manipulation of the IgG Fc region is occurring to increase or decrease ADCC, complement fixation, glycosylation patterns, and FcRn binding to manipulate the effector functions as well as stability and antigenicity. For mouse hybridoma development the subclass (G1, G2a, G2b, etc) can be manipulated to some extent by adjusting immunization strategies to drive responses that result in the specific generation of a particular IgG subclass.