IgG homeostasis: (see right hand panel)

IgG participates in defending against viral antigens, whereas IgM is predominantly active in antibacterial and antitoxin immune responses (US2013/0052208). 

Antibodies belong to a family of globular proteins called immunoglobulins. IgG is the most common. Eight percent of all the antibodies present in the blood are IgG. IgG is a relatively large molecule (about 150 kDa). It has four subclasses, IgG1, IgG2, IgG3 and IgG4. (see below). Chakrabarti, “separation of monoclonal antibodies by analytical size exclusion chromatography” Antibody engineeering, Cahpt 7, 2018).

Which Isotype to choose?

The human IgG compartment consists of four distinct subclasses, designated IgG1 (6.98 mg/ml in serum) , IgG2 (3.80 mg/ml) , IgG3(0.51 mg/ml) and IgG4 (0.56 mg/ml). The principal biological activities of IgGs are related to their effector functions, including activation of complement and binding Fcgamma receptors to mediate antibody-dependent cellular cytotoxicity. Although their heavy chains are about 95% similar in sequence homology, IgG subclasses express unique effector activities. In general, protein antigens characteristically elicit IgG1 and IgG3 responses, and these isotypes are capable of activating all Fc receptors and the C1 component of complement. Induction of IgG4 subclass appears to be characteristic of chronic anitgen stimulation, such as observed in autoimmune disease. IgG4 exhibits restricted Fc receptor activation and does not activate C1q. The IgG2 sublcass often predominantes in response to carbohydrate antigens and also exhibits restricted Fc receptor and C1q activation. (Taylor, Gynecologic Oncology 116, (2010) 213-221). 

Different IgG isotypes, such as IgG1, 2, 3, and 4, demonstrate unique recognition and activation profiles when interacting with favrious FcyRs. IgG1 has proved to be most effective in complement dependent (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). This corresponds with the fucntion of this highest cirulating serum IgG subclass for binding exogenous pathogens and for effective destrcution and clearance of antigens via activation of various effector mechanisms. IgG3 has been shown to be as effective as IgG1 in complement activation and cell mediated toxicity, whereas IgG2 and IgG4 isotypes are relatively inactive in eliciting effector functions. (Drug Discov. Today, 2006 Jan; 11(1-2):81-8).

A recent paper comparing all four human isotypes of an anti-GXM antibody in a Crytococcus neoformans infeciton model is evidence that there are no general rules for istype slection. Contrary to expectations that an active istype might have superior bactericidal function, IgG2 or IgG4 were optimal, whereas IgG1 was determiental. (Salfeld “isotype selection in antibody engineering” Nautre Biotechnology, 25,(12), 2007).

Specific Isotypes

Structurally, the human IgG subclasses are remarkably similar with an overall >95% sequence identity. This implies that relatively few amino acid differences unerlie functional variation between for example IgG1 and IgG. (Rispens “Mechanisms of Immunoglobulin G4 Fab-arm Exchange” J Am Chem Soc 2011, 10302-10311). 

IgG1: The most abundant isotype of human serum antiboides is IgG1. IgG1 is a glycoprotein whose N-linked carbohydrate is located in the CH2 domain of its H chain. Mainy studies show that thsi carbohydrate is critical in maintaning the structure and funciton of IgG1. Dorai (Hybridoma, 10(2), 211-217 (1991)) show that aglycosylated IgG1 failed to exhibit any ADCC activity but a significant level of CDC activity was retained. Accordingly, some, but not all, effector functions of a human IgG1 Ab are affected by aglycosylation.  

IgG1 has been the most popular choice for antibody isotype choice and has been approved for over 13 chimeric, humanized or fully human antibodies compared with only two instances for IgG4 and one fo IgG2. (Salfeld “isotype selection in antibody engineering” Nautre Biotechnology, 25,(12), 2007).

IgG2: The IgG2 sublcass often predominates in response to carbohydrate antigens and also exhibits restricted Fc receptor and C1 q activation. For example, in acute lymphocytic leukemia (ALL), elevated levels of 9-O-acetylated sialic acid specific IgG2 were induced, which were unable to trigger activaiton of Fcy receptors, the complement cascade and cell mediated cytotoxicity, although its glycotope binding ability was unaffected. The subclass switching of anti-9-OAcSa to IgG2 in ALl was linked with alterations in the tumor cell’s glycosylation profile. (Taylor, Gynecologic Oncology 116, (2010) 213-221).

Human IgG2 antibodies have been shown to consist of three major disulfide structural isoforms IgG2-A, -B and -A/B. This structural heterogeneity is due to different light chain to heavy chain connectivity in each isoform. It has been shown that the individual isoforms can have unique and different structural and functional properites, including differences in potency or other quality attributes including Fcy receptor binding, viscosity, stability, and particle formation. (Dillon, US14/363735)

The natural IgG2 response is typically directed twoard carbohydrate antgiens, which are arrayed in repetitive patterns on bacterial surfaces. Dimers of these often low affinity IgG2s can enhance protection and provide an evolutionary advantage. (Salfeld “isotype selection in antibody engineering” Nautre Biotechnology, 25,(12), 2007).

Panitumumab is a human IgG2k mAb that binds specifically to EGFR with high affinity, and not to the other ErbB family members. Treatment with panitumumab has been associated with low immunogenicity and low risk of hypersensitivity reacitons. The fully human IgG2 mAb, panitumumab is devoic of natural killer (NK) cell mediated classical ADCC. The IgG2 isotype was chosen to minimiz potential toxicity to EGFR expressing normal tissues form ADCC and CDC. A novel anti-EGFR mAb, denoted as Pan-P, has been developed with the goal of enhancing ADCC and target selective activity and using probody technology where an epitope blocking peptide was tethered to Pan via a tumor specific protease selective linker. Pan-P showed almost 40 fold weaker affinity compared with Pan, but funcitonal activity was restored to a similar extent as Pan when Pan-P was selectively activated by urakinase-type plasminogen activator (uPA)  (Yang, “Generation and characterization of a target-selectively activated antibody against epidermal growth factor receptor with enhanced anti-tumor potency” mAbs (2015). 

IgG3: Antibodies desigend for slective eradication of cancerl ells typically require an active isotype that permits complement activaiton and effector mediated cell killing by antibody dependent cell mediated cytotoxicity. Although IgG1 and IgG3 both meet these criteria, IgG3 has not been used for therpaeutic antibody development probably because of a shomewhat shorter half life susceptibility of the longer hinger region to proteolysis and extensive allotypic polymorphism. (Salfeld “isotype selection in antibody engineering” Nautre Biotechnology, 25,(12), 2007).

IgG4:

IgG4s possess several properties that make them attractive therapeutic candidates. For example, IgG4s have long serum half-lives and low Fc function and/or effector function. However, IgG4 antibodies also have unusual properties which are undesirable in vivo: they are unstable, dynamic molecules which engage in Fab arm exchange. An administered therapeutic IgG4 antiboy may exchange with endogenous IgG4 antibodies with undesired specificites. (Brower, US 14/668820, published as US 2016/0108127). 

–Structure of IgG4:

For the CH1 domain, the IgG4 has a Cys at position 151, which is used for linking to the light chain. In contrast, IgG1 has a Ser at this position, lining to the light chain via Cys220 in the hinge, which is one of the three extra amino acids in the IgG1 hinge compared to IgG4. At positions 137-138 both IgG2 and IgG4 have Glu-Ser, whreas IgG1 has Gly-Gly. IgG4 also has Lys at position 196, which is Gln in IgG1 as wella s in IgG2. The IgG4 hinge 9as well as the IgG2 hinge) is three amino acids shorter than the hinge of IgG1. Similar to IgG1, IgG4 has two cysteins that are available for the covalent interaciton between the H chains. The CH2 domain of IgG4 has its most marked ifferences with IgG1 in a surface exposed patch of the C temrinal part of the doamin which in 3D structure is clsoe to the hinge. Starting at position 337, the IgG4 sequences reades GLPSS, that of IgG1: ALPAP. The IgG4 patch is clearly much less hydrophobic than the IgG1 patch. (Shuurman, Immunology, 2002, 105, 9-19). 

–Fab Arm Exchange (IgG4 shuffling):

IgG4 antibodies are known to undergo a process called Fab arm exchange, also known as IgG4 shuffling in which the heavy chains separate and randomly re-assocaite to produce a mixed population of IgG4 molecules with randomized heavy-chain and light-chain pairs. 

It has been demonstrated that a Serine to Proline mutation at position 228 using the EU index in the hinge region of human IgG4 results in the reduction of IgG4 “half-antibody” molecules and reduced heterogeneity/shuffling of IgG4 molecules. 

Relative to the Cys-Pro-Pro-Cys motif in IgG1, the IgG4 sequence appararently increases hinge flexibility, allowing justaposition of the two intrachain cysteines and bringing intra and interchain disulfide bonds into equilibrium. When the interchain bonds of IgG4 break and the intrachain bonds are created, new interchain bonds can be formed. The relative concentrations of different IgG4 molecuels (and their derived half molecules) determine whether these new interchain disulfide bonds are formed between two identical or two different IgG4 half molecules, leading to bivalent moelcules with mono or bispecificity, respectively. To address this problem, antibody engineers introduced a serine to proline substitution that made the middle hinge region of IgG4 more like that found in IgA which gave a more homeogenous monospecific product but also extended serum half-life and improved tissue distribution. (Salfeld “isotype selection in antibody engineering” Nautre Biotechnology, 25,(12), 2007).

Comapred to IgG1, two structural features of IgG4 are important for the exchagne reaction. First, the CH3 domain of IgG4 is necessary for exchange. The (noncovalent) interheavy chain interactions in the CH3 domains are most likely significantly weaker, thereby allowing dissociation and inevitable step in the exchange process. Second, a serine instead of proline at position 228 in the core hinge further facilitates exhcange, probably because the hinge is more easily reduced, since the exchange reaction can be observed at higher concentrations of GSH also if this feature is absent. Introducing boht features in IgG1 results in a mutant antibody that is able to exchange half-molecules similar to IgG4. (Rispens “Mechanisms of Immunoglobulin G4 Fab-arm Exchange” J Am Chem Soc 2011, 10302-10311).