Antibody Biotechnology

Introduction

Immune responses are highly complex, specific and dependent on biological recognition. Vertebrates’ distinction self from non-self-compounds can be induced by many different antigens (Ebioscience.com, 2015; Poljak et al., 1975). Differing from the innate immune system, the first lines of defence, the adaptive immune system drives a specific immunological memory through repeated interaction with the same agent. The adaptive response comprises a variety of cells and molecules performing a signalling cascade, in which lymphocytes and immunoglobulins (Ig) are the essential elements.  (Meulenbroek and Zeijlemaker, 1996).

Specific recognition is performed by adaptively synthesized antibody molecules capable of neutralizing antigens in complexes of high association due to closely complimentary interaction between the antigenic determinants and the active site of the antibody (Ebioscience.com, 2015; Poljak et al., 1975). In terms of structure, immunoglobulins are one of the most studied glycoproteins, composed of 82 to 96% protein and 4-18% oligosaccharide or polysaccharide (Meulenbroek and Zeijlemaker, 1996). Different techniques, such X-ray crystallography and diffraction, have allowed acquiring amino acid and nucleotid sequence data as well as three-dimensional structures (Padlan, 1994). Antibody molecules share a number of structural features which are a common characteristic of all classes of immunoglobulins (Poljak et al., 1975). Different immunoglobulin isotypes diverge in their biological function, structure, target specificity and distribution (Ebioscience.com, 2015).

The human Ig system is characterized by its diversity, composed of five main classes or isotypes (IgG, IgA, IgM, IgD and IgE) and subclasses within them, which differ in the primary structure, carbohydrate content and antigenic properties of their heavy chains. Normal human Immunoglobulin G (IgG) is the major class of the five classes of human beings and contains four subclasses: IgG1, IgG2, IgG3 and IgG4 (Merler, 1970; Nezlin, 1998; Vidarsson, Dekkers and Rispens, 2014).

The structure of an antibody determines its function. The chemical structure of antibodies determines the binding versatility, the binding specificity and the biological activity (Elgert, 2009). After a secondary immunization, B lymphocytes produce principally IgG molecules (Nezlin, 1998). IgG seems to accumulate with repeated exposure to antigen and is slowly catabolized; it is largely extravascular and is transported across the placenta (Merler, 1970).

Structure and function and potential antibody-based therapies

Basic immunoglobulin G molecules are produced by plasma cells (leucocytes) and composed of four polypeptide chains; two indistinguishable heavy chains (50-60 kDa each)  and two identical light chains (23 kDa each) connected by disulphide bridges (Nezlin, 1998). Intra-chain disulphide links are responsible for the establishment of loops, promoting the compacted, domain-like structure of the IgG (Meulenbroek and Zeijlemaker, 1996). Light chains can be either lambda (λ) or kappa (ϰ), depending on their primary structure and antigenic properties, frequently free of olygosaccharides.

Immunoglobulins G can be divided structurally into; variable domains, that connect antigens to the molecule and constant domains that lead effector functions such as activation of complement or binding to Fc receptors (B lymphocytes, follicular dendritic cells, etc.) (Schroeder and Cavacini, 2010).  As the amino acid sequence differs in the arms of the variable domain of every different IgG, each different antibody can bind specifically to only one epitope (Elgert, 2009).

Structurally, an immunoglobulin G is composed of three major fragments; the two Fab (Fragment antigen-binding), which are identical between them and each one contains the whole light chain and the first two domains of the four contained in heavy chain, and the Fc (Fragment crystallisable), which comprises the C-terminal constant domains of the two heavy chains (CH2 and CH3). The intermediate region between the Fab arms and the Fc is called hinge region, which differs in length and flexibility in the different antibody classes and isotypes (discussed below). The antigen binding active regions (paratopes) are placed at the tips of the Fab. Poljak et al. (1973) described the immunoglobulin fold as compact globular structures, whether the domains were variable or constant. Thus, the arms containing the Fab region confer the versatility and specificity of responses. On the other hand, the stem region of an antibody determines its biological activity and determines the immune response against the supposed pathogen (lysis, enhanced phagocytosis or allergy). These activities start once antibodies bind to antigen. The ratio Fab to Fc is 2:1. (Elgert, 2009). The overall structure is detailed in Fig. 1 and Fig. 2.[pic 1][pic 2]

Each domain consists of a constant arrangement of antiparallel strands with hydrogen bonds that form a bilayer structure, further stabilized by a disulfide bonds between the two layers. In the variable domains, the bilayer structure is formed by nine strands; in the constant domains, the bilayer is formed by seven strands. Bends of different sizes and conformations connect the strands. The predominant secondary structure in immunoglobulins G is the anti-parallel beta sheet, with short stretches of r-helix found in certain bends (Padlan, 1994).

An IgG immunoglobulin consists of two γ “heavy” (H) polypeptide chains rounding the 50,000 g/mol of molecular weight (50 kDa) and two “light” (L) chains of molecular mass 25,000 g/mol (25 kDa). The H and L chains can be partitioned into “homology” regions; VL and CL, compounding the L chain and VH, CH1, CH2 and CH3 forming the H chain. Each of those regions contains about 110 amino acids residues. The amino acid sequences of CL, CH1, CH2 and CH3 regions are constant and highly homologous between them (Poljak et al., 1975). VL and VH amino acid sequences act as a critical part of the adaptive immune response by precisely identifying and binding to specific antigens to aid their removal (Ebioscience.com, 2015). A schematic representation of all its regions can be seen in Fig. 1. In fact, each IgG contains about 3% carbohydrate, while the carbohydrate content in other immunoglobulins is usually greater (8-12%) (Nezlin, 1998). IgG contains a biantennary complex N-linked carbohydrate attached to CH2, where binding sites for Fc are located. The oligosaccharides can influence their biological and functional properties by affecting the polymerization and effector consequences such as complement activation and FcγR binding (Coloma et al., 2000). [pic 3][pic 4]