Why recombinant antibodies - benefits and applications

Abstract

Antibodies (Abs) are ubiquitous reagents for biological and biochemical research and are rapidly expanding into new therapeutic areas. They are one of the most important probes for determining how proteins function under normal and pathophysiological conditions. Abs are required for the quantification of targets, detection of temporal and spatial patterns of protein expression in cells and tissues, and identification of interacting partners and their biological activities. Their remarkable specificity and unique binding properties can facilitate three-dimensional structure determination using X-ray crystallography and electron cryomicroscopy. While hybridoma technology that involves animal immunization is often productive, many antigen targets do not generate useful Abs. This is particularly true if unique states of the target or critical non-immunogenic target sequences need to be recognized by the Abs. By using the methods of recombinant antibody generation, identification, and engineering, these 'hybridoma-refractory' antigens can be readily targeted. Specific, reproducible, and renewable recombinant Abs are proving to be invaluable reagents in applications ranging from biological discovery to structure determination of challenging macromolecules.

Figure 1:. Antigen presentation in hybridoma antibody generation versus recombinant antibody display.

Target antigen (red) follows hybridoma immunoglobulin G generation path (top) or recombinant antibody (rAb) generation path (bottom). In top path, the pink cell is a major histocompatibility complex (MHC) presenting cell, the purple cell is a T-cell, the blue cell is a B-cell, the black receptor is a MHC, and the grey receptor is a T-cell receptor (TCR). In bottom path, the grey oval particles are display library members (yeast or phage) with colored displayed rAb and the yellow cell is Escherichia coli.

Figure 2:. Different formats and important applications of recombinant antibodies.

Various antibody formats are shown in the center of the circle: immunoglobulin G (IgG), fragment of antigen-binding domain (Fab), single-chain variable fragment (scFv), and nanobody (Nb). IgGs are composed of two unique polypeptide chains, a heavy chain (H) and a light chain (L) based on their molecular weights. Each chain has one variable region (V_H, V_L). The light chain has one constant region (C_L) and the heavy chain has three constant regions (C_H1-3). Two heavy chains and two light chains come together to make an IgG. A Fab is composed of V_H, V_L, C_H1 and C_L. An scFv is a single polypeptide chain composed of both V_H and V_L connected with a flexible linker. A Nb consists of only a V_H. Heavy chains are colored in dark blue, light chains are colored in cyan, and antibody targets are colored in red.