Antibody Structure

mAbs, based upon the immunoglobulin (Ig) “Y” shape, are tetrameric glycoprotein therapeutics with two HC and two LC constituents. These four chains form the basis of the homoheterodimer structure and are linked via a flexible stretch of polypeptide chain referred to as the hinge region [21]. In mammals there are five different isoforms consisting of one or more replicas of the “Y”-shaped unit: gamma (IgG), mu (IgM), alpha (IgA), delta (IgD), and epsilon (IgE) [22]. Therapeutic templates however have been based primarily upon the IgG1 class (though therapeutics based on IgG2 and IgG4 are also used) as it is considered to be the most active within the immune system, resulting from its ability to efficiently engage with cytotoxic cell receptors (FcyR) and its ability to destroy target cells through activation of the complement [23]. The IgG form also accounts for 75% of total serum Ig [24]. A schematic representation of an IgG1 molecule is shown in Figure 10.1. The heavy chain (HC) and light chain (LC) are made up of different regions that each contain approximately 110 amino acids, which fold into discrete compact regions referred to as domains [25]. Each of the four chains has a variable domain (VH and VL) at the N-terminus, which is responsible for antigen binding. The nomenclature of the Ig isoforms is based upon the antigen binding specificity that is determined by the amino acid sequence in these variable domains. The remaining domains are referred to as “constant” as their amino acid composition is highly conserved across the different Ig isoforms. The antibody molecule can be further divided into two functionally distinct regions: the Fab and Fc subunits. The Fab corresponds to the two identical antibody “arms,” which consist of the entire LC coupled with VH and CH1, and is hence responsible for antigen recognition and binding [26]. The Fc interacts with cell surface receptors and complement proteins [27].

The IgGs are split into additional subclasses (IgG1-4), which differ from one another most significantly with the location and quantity of disulfide bonds

The immunoglobulin G antibody structure consists of a variable region in the heavy and light chains

Figure 10.1 The immunoglobulin G antibody structure consists of a variable region in the heavy and light chains (VH and VL, respectively), a constant region in light chain (CL), and three constant regions in the heavy chain (CH1, CH2, and CH3). The complementarity determining region (CDR) consists of three loops connecting the variable domains at which antigen binding and selectivity occur. The Fab region recognizes and binds antigens, while the Fc region interacts with effector cells. N-glycosylation in IgG1 mAbs, such as trastuzumab, occurs in the Fc CH2 domain.

within the hinge region [28-30]. In each case a single disulfide bridge connects the HC and LC, with two disulfide bonds connecting the two HC strands in IgG1 and IgG4, four in IgG2, and eleven in IgG3 at the flexible hinge region [30, 31]. Contributing to the overall 3D structure of the mAbs are 12 additional intramolecular disulfide bridges (common to all IgGs) and PTMs such as N-glycosylation, O-glycosylation, galactosylation, and mannosylation, to name a few - all of which need to be characterized during the drug development process [32]. It is the N-termini of the variable domains that facilitate antigen binding through six sites in the complementarity determining region (CDR): three from variable HC and three from variable LC (VH and VL, respectively). The CDR expresses loops formed from р-pleated sheets with antigen binding affinities that are based upon sequence structure in the variable domains [33], whereas the constant domains host the most abundant PTMs [34]. The majority of approved therapeutic mAbs are produced using Chinese hamster ovary (CHO) cells [35, 36]; however other expression platforms such as E. coli [37] and mouse myeloma cells [38] have also been employed. The majority of approved mAbs for therapeutic use are subclasses or derivatives of humanized IgG1, although IgG2 and IgG4 therapeutics have also been reported [39, 40]. IgG3 structures, however, are not currently used in therapeutics as they have been reported to have a significantly shorter half-life and faster clearance rates than the other isotypes [21, 38, 40].

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