Antibody Biotechnology

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Variations in molecular structure and functionality of the different subclasses of IgG are detailed in Table 1. Even though there is more than 90% of similarity between them, each subclass has a exclusive profile according to antigen binding (due to the paratopes of the Fab arms), immune complex formation, complement activation, activating of effector cells, half-life and placental transport. IgG, as previously stated, can be additionally classified in four different subclasses, listed according their relative abundance in a decreasing order; IgG1, IgG2, IgG3 and IgG4 (Vidarsson, Dekkers and Rispens, 2014).

The general structures of those subclasses are very similar, even though there are critical differences between them that affect directly their binding to antigen and effector promoters, changing their functionality, as seen in Fig. 3 and Table 1. Those differences are not placed arbitrarily, many of this variations are concentrated in the hinge region and N-terminal CH2 domain, while less changes in amino acid sequences are found in other regions. Functional consequences due to structural variations in CH1 (if there is any) are not known, as a difference from structural differences in CH2 and CH3 domains, which form the Fc tail, that indeed are quite known.

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The hinge region constitutes a flexible linkage between the Fab arms and the Fc part in IgG. The differences between the hinge region of the diverse IgG subclasses can be seen in Fig. 3. In fact, this affects the conformation of the Fab arms between them and relative to Fc. The residues next to the hinge region in the CH2 domain fragment crystallisable are determining the effector reactions and further impacts and functions of immunoglobulins as they contain the fundamentally overlapping binding site for C1q (complement) and IgG-Fc receptors (FcγR) on effector cells of the innate immune system. The contact with Fc receptors induces a specific effector reaction and promotes the destruction of possibly damaging agents recognized by immunoglobulins G.

Taking into account that the hinge exon of IgG1 comprises 15 amino acids, it results in a quite flexible, while IgG2 has a shorter hinge than IgG1 because it is formed by 12 amino acids. The distal hinge region of IgG2 (comprised in the CH2 domain) also lacking one amino acid (deletion of one of the double glycines found at position 235-6), causing the smallest hinge region of all the immunoglobulin G subclasses. Moreover, this region of IgG2 is stiffer due to a poly-proline helix, stabilized by up to four extra inter-heavy sequence disulfide links, increasing the rigidity of the IgG2 molecule. Likewise IgG2, the hinge region of IgG4 is also made of 12 Aa and is consequently smaller than IgG1 hinge region. It appears to have a medium rigidity between IgG1 and IgG2. Different to IgG2, it encodes for the CH2 glycines 235-6 in the proximal hinge. IgG3 has the longest hinge region of all the IgG human isotype, comprising up to 62 amino acids (21 prolines and 11 cysteines), establishing a poly-proline helix with restricted flexibility (as in IgG2). The hinge length of this last subclass gives the molecule a great flexibility, as a result of duplications of a hinge exon. It is also responsible for its greater molecular weight in comparison to other subclasses. Binding sites for C1q and/or FcγR can be incompletely or entirely guarded by Fab arms, varying the binding capacity and immune complex formation (Vidarsson, Dekkers and Rispens, 2014). The data shows that the rank order (most to least flexible) of the IgG subclasses for hinge-folding mode of rigidity between Fab arms is IgG3 > IgG1 > IgG4 > IgG2 (Roux et al., 1997).

The four IgG subclasses also vary in quantity of inter-heavy chain disulphide links, affecting its rigidity. In addition, another structural difference is found in the association of the light and heavy chain through a disulphide bond. In IgG1, this bond links the carboxy-terminal cysteine of the light chain to the cysteine located in the position 220 of the heavy chain, while IgG2, IgG3 and IgG4 are linked by a cysteine residue in the position 113 of the heavy chain. Both residues 220 and 131 are in close proximity in the three dimensional globule, and the heavy chains with both variants of the heavy-light disulphide linkage have the same general structure (Nezlin, 1998).

Another structural difference between IgG subclasses are the hinge isomers in IgG2 and IgG4. It has been found that there are different isomers of those subclasses as a result of alternative formation of disulphide bonds between the cysteines in the hinge region connecting the heavy with the light chain. However, FcRn (neonatal Fc receptor) binding does not seem to be affected by this difference in the linkage formation (Vidarsson, Dekkers and Rispens, 2014).

Furthermore, half molecules of IgG4 can recombine arbitrarily with other half-particles of IgG4, resulting in monovalent-bispecific antibodies (in vivo).The unique S228 in the hinge of IgG4 allows an intra-chain isomer, and R409 (rather than the equivalent lysine in IgG1) results in weaker CH3-CH3 interactions and promotes Fab arm exchange. The functional significance is that the resulting IgG4 cannot successfully crosslink the

target antigen. Moreover, IgG4 has a reduced attraction to activate FcγR while holding high affinity when still a high role in inhibiting FcγRIIb (Table 1). As IgG4 tends to dominate after repeated antigen exposure, therefore has been described as a “blocking antibody”, especially in the framework of allergy, where it competes with IgE for antigen linking (Vidarsson, Dekkers and Rispens, 2014).

Improvements and developments in the knowledge of molecular biology permitted in the early 90s to clone the genes of IgG molecules in eukaryotic expression vectors. As a consequence, production issues provoked by the instability of hybridoma lines could be fixed. This was the first step towards the manipulation or modification of antibodies to get antibody-based therapies. Development of therapeutic antibodies requires a precise understanding of the structure and functionality of the immunoglobulin as well as its subclass’ characteristics (Chames et al., 2009). However, therapeutic antibodies are biologically functional through two mechanisms; they can block the interactions between the receptors and their ligands due to the high specificity of their variable region or epitope (as previously mentioned), the called antagonist antibodies, or agonist antibodies, which can generate powerful biological responses as cell proliferation or apoptosis once they are linked to the surface of molecules; and they