Page 12 - Tyrosine-Based Bioconjugations - Jorick Bruins

Chapter 1
1.1. Role and anatomy of monoclonal antibodies
Antibodies, or immunoglobulins, are Y-shaped proteins released by B cells of the adaptive immune system to neutralize pathogens.1 Five main isotypes of immunoglobulins (Ig) are known, immunoglobulin A (IgA), immunoglobulin D (IgD), immunoglobulin E (IgE), immunoglobulin G (IgG), immunoglobulin M (IgM), each with different structural properties (i.e. glycosylation patterns, oligomeric complexes) and functions (i.e. toxin neutralization, pathogen binding).1 The full extent of antibodies and their functions are well reviewed and beyond the scope of this thesis.2, 3 Here, we will only focus on a single isotype, immunoglobulin G1 (or IgG1), which is the most common antibody found in blood circulation1 and in pharmaceutical applications as biological drugs. We will also only focus on monoclonal antibodies, which are antibodies that come from a single B cell lineage and bind a unique epitope on a specific target antigen, versus polyclonal antibodies that can bind multiple epitopes on the same target.4 For clarity, we will refer to monoclonal IgG1 as antibodies (or mAbs).
The structure of an IgG1 antibody consist of two identical light chains of about 25 kDa, and two identical heavy chains of about 50 kDa (Figure 1A, C).4 There are four interchain disulfide bridges that connect the light and heavy chains together (Figure 1A, B, depicted in red), as well as 12 intrachain disulfide bridges that assist in stabilizing the structure (not depicted).5 The overall structure can also be divided into six domains, two variable domains (VL and VH) and four constant domains (CL and CH1, CH2, and CH3). Whereas the constant domains remain largely identical between antibodies, the variable domains contain the binding sites of the target, or antigen, and vary significantly from antibody to antibody depending on the target antigen. More specifically, several strands of β-loops in both the VL and VH domains are responsible for the antigen-binding site (also called epitope), these strands are called the complementary determining regions (CDR).1, 4 Finally, the antibody can also be divided into three 50 kDa fragments; the two Fab regions (fragment, antigen-binding) consisting of the VL, VH, CL, and CH1 domains, and the Fc region (fragment, crystallizable) consisting of the CH2 and CH3 domains. The Fab regions have the antigen-binding site, whereas the Fc regions that can interact with cell surface receptors (Fc receptors) that can activate the immune system1 and have a conserved glycan-residue at N297 essential for Fc receptor-mediated activity.6
While different glycans do not have any effect on antigen binding, they do have an effect on biological mechanisms.6 For example: the absence of a fucose on the penultimate GlcNAc improves binding to human FcγRIII, which translates into improved antibody-dependent cellular toxicity (ADCC).7 Similarly, addition or removal of sugars such as sialic acid, or even the removal of the total carbohydrate can have profound effects on functional activities.8 This is even true between the two most common glycoforms, G0F and G1F (Figure 1D), where a single galactose can increase the complement-dependent cytotoxicity by a factor two.6 These facts illustrate the importance of understanding the importance of detailed analysis of the antibody structure.
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