Basics of Antibody Phage Display Technology

Abstract

Antibody discovery has become increasingly important in almost all areas of modern medicine. Different antibody discovery approaches exist, but one that has gained increasing interest in the field of toxinology and antivenom research is phage display technology. In this review, the lifecycle of the M13 phage and the basics of phage display technology are presented together with important factors influencing the success rates of phage display experiments. Moreover, the pros and cons of different antigen display methods and the use of naïve versus immunized phage display antibody libraries is discussed, and selected examples from the field of antivenom research are highlighted. This review thus provides in-depth knowledge on the principles and use of phage display technology with a special focus on discovery of antibodies that target animal toxins.

Keywords: M13 phage; antibody discovery; phage display; recombinant antivenom; toxinology.

Figure 1

Schematic representation of the M13 bacteriophage, which is a filamentous phage carrying a single-stranded DNA (ssDNA) chromosome. The genome contains nine genes, which encode 11 proteins. Five of these proteins are coat proteins (G3P, G6P, G7P, G8P and G9P), while the remaining six proteins are used for replication of the genome, assembly of the phage, and phage extrusion.

Figure 2

Infection of Escherichia coli by the M13 phage. The phage G3P binds to the tip of the F pilus on E. coli. Normal disassembly of the F pilus transports the phage to the surface of the bacterium, where it interacts with the TolA receptor, mediating uptake of the phage genome.

Figure 3

Infection cycle of the M13 phage. (A) Upon infection, the single stranded (+) chromosome of the M13 phage is converted into the double-stranded replicative form (RF) (B) After proper accumulation, the G2P nicks the (+) strand in the RF and binds covalently to the 5′-end (C) The genome is then replicated from the 3′ end of the nick, using the (−) strand as a template. The original G2P-bound (+) strand is physically displaced by the Rep helicase throughout the elongation process (D) The old (+) strand is re-circularized by the bound G2P after dissociation, ready to be converted into an RF or to be packaged into new M13 phages (D) Genome replication continues until the concentration of G5P has accumulated to sufficient levels to sequester the ssDNA (E) When sufficient G5P is accumulated, G5Ps will bind the ssDNA in a back-to-back dimeric conformation, causing the more rod-shaped appearance of the ssDNA (F) A pore is formed in the membrane, and the phage genome is translocated through this pore, while the phage coat is assembled.

Figure 4

Engineering of the M13 phage for phage display experiments. The G3P is genetically fused to a human single-chain variable fragment (scFv) with a trypsin cleavage site shown in yellow as part of a peptide linker connecting the two proteins.

Figure 5

Gene III product from helper phage and gene III-scFv product from phagemid. The helper phage carries the gene encoding a trypsin-sensitive ‘bald’ G3P. The phagemid carries the gene encoding the G3P-scFv fusion protein. As M13 phages will be assembled with three to five copies of G3P, the number of assembled phages containing more than one G3P-scFv fusion protein will be too low to influence the outcome of selections, because more ‘bald’ G3P than G3P-scFv fusion proteins will be expressed. Thereby, the majority of the scFv-displaying phagemids will only be carrying one scFv (monovalent display).

Figure 6

Trypsin treatment of G3P and G3P-scFv fusion proteins on the M13 phage. G3P encoded by the helper phage is trypsin-sensitive and can thus be cleaved by trypsin, rendering the phage non-infective. In contrast, the G3P-scFv fusion protein encoded by the phagemid contains a myc-tag in the linker between the G3P and the scFv, which will be cleaved during trypsin treatment. This leaves a G3P on the phage, rendering it infective.

Figure 7

The five steps in a phage display selection experiment. Addition of phage library (A) refers to the addition of phages to an antigen-coated vial. The phages displaying the highest affinity scFvs will bind (B) the antigen, while unspecific binders will be removed during the washing step (C).After washing, the antigen-specific phages can be eluted (D) using trypsin digestion (or other means of elution) Then, phages are amplified (E) in E. coli and a new panning round can be initiated to further accumulate phages displaying high affinity antibody fragments. Binding can be evaluated using both ELISA and plate tests.

Figure 8

Antigen immobilization strategies. Direct coating can result in denaturation of the antigen or distortion of its conformation, whereas immobilization through a streptavidin-biotin capture system, involving biotinylation of the antigen, can give rise to over-biotinylation resulting in low availability of binding sites.

Figure 9

Diversification methods used for in vitro affinity maturation of antibody fragments. Chain shuffling (A) is where one heavy or light chain is paired with chains of the opposite type from a naïve library. The principle of both light and heavy chain shuffling is presented in the figure. Random mutagenesis (B) is used to introduce mutations (red lines) in the entire variable regions of the antibody fragment. Site-directed mutagenesis (C) is used to specifically introduce mutations in one or more of the complementarity-determining regions (CDR) regions of the antibody fragment.