Use of bacteriophage for discovery of therapeutically relevant antibodies against infectious diseases

Scientists George P Smith and Gregory Winter were recently awarded half of the 2018 Nobel Prize for Chemistry for developing a technology to display exogenous peptides and proteins on the surface of bacteriophage. ‘Phage display’ has revolutionised the development of monoclonal antibodies, allowing fully human-derived antibodies to be isolated from large antibody libraries. It has been used for the discovery of many blockbuster drugs, including Humira (adalimumab), the highest selling drug yearly since 2012, with US$18.4b in sales globally in 2017¹. Phage display can be used to isolate antibodies to almost any antigen for a wide range of applications including clinical use (for cancer, inflammatory conditions and infectious diseases), diagnostic use or as research tools. The technology is accessible to any laboratory equipped for molecular biology and bacteria culture.

Phage display technology was first demonstrated by Smith in 1985, who showed that DNA encoding peptides could be inserted into the bacteriophage gene III resulting in the expression and display of the corresponding peptides on the surface of the virion as a fusion to the coat protein pIII². Winter then showed that this technology could be used to display antibody fragments on the surface of bacteriophage³. His group also showed that highly specific antibodies could be fished out of large libraries of antibody gene sequences cloned into phage expression vectors^4,5. This now allowed the isolation of fully human antibodies, from cloned human antibody gene repertoires, reducing the impact of immunogenicity of mouse-derived therapeutic antibodies.

The bacteriophage biology that allows the display of peptides and proteins is well reviewed by Russel et al.⁶. The most commonly used phage display system uses phagemid vectors, where the antibody-pIII gene fusion is cloned into a bacterial expression vector containing a periplasmic leader sequence, an ampicillin resistance gene and an f1 viral origin of replication. When the phagemid is transformed into Escherichia coli, and grown in the presence of ampicillin and M13-derived filamentous helper phage (usually M13K07), the antibody-pIII fusion protein is expressed and incorporated into the newly synthesised phage particles, and the phagemid is replicated as single-stranded DNA and preferentially packaged into the particle (Figure 1). Phage particles are released into the culture media and are purified by precipitation with high salt and polyethylene glycol.

Figure 1.  Left: A phagemid cloning vector containing an f1 origin of replication (f1 ori), and antibody variable region genes (Heavy chain (orange) and Light chain (blue)), assembled as a single chain variable fragment (scFv), cloned in frame with the gene for the bacteriophage p3 coat protein (green). Right: A bacteriophage particle containing a phagemid vector inside the particle, and the scFv antibody fragment displayed on its surface as a fusion to the p3 protein.

Phage antibody libraries can either be ‘naïve’ or ‘immunised’. Naïve libraries are usually human derived, and are created by collecting peripheral blood samples from a large group of healthy donors from a general population, with no bias towards any particular disease or condition. Naïve libraries can be used indefinitely to isolate antibodies to almost any target presented to the library. For this reason, naïve libraries are also termed ‘single-pot’ libraries since the same library can be used for any antigen⁷. Immunised libraries are focussed on the isolation of particular antibodies, with blood samples collected from individuals with a defined condition or from mice immunised with an antigen-of-interest⁸. Immunised libraries increase the likelihood of obtaining highly specific and high affinity antibodies, but also limits their use towards a single antigen.

The process of isolating specific antibodies from a phage antibody library is termed ‘biopanning’, and is summarised in Figure 2. Biopanning involves incubating the library of phage particles with immobilised antigen, washing away non-binding phage, and then eluting the bound phage using a buffer that breaks the antibody-antigen interaction. After enriching the library for binding phage, individual clones can be isolated, characterised and further developed as either laboratory tools, or as commercial diagnostic and therapeutic antibodies.

Figure 2.  Summary of the biopanning process. The phage particles are depicted in blue with the scFv-p3 fusion protein on their tips. (1) The phage particles displaying a library of scFv is incubated with immobilised antigen (depicted in red), which could be purified proteins, or whole cells or viruses. (2) The surface is washed to remove any non-binding phage. (3) Bound phage are eluted using a low pH, high pH or high salt buffer. (4) The eluted phage are infected into Escherichia coli to amplify these phage, enriching the library for specific binders. This process is then repeated with the newly amplified, enriched pool 3–5 times with increasing stringency at step 2 to further enrich the library for strong binders.

As of December, 2018, there were 82 antibodies approved in the US and/or EU for therapeutic use in humans, and 10 of these were isolated using phage display^9–12 (Table 1). The majority of therapeutic antibodies target endogenous antigens such as proteins involved in the inflammatory response, or cell-surface or circulating proteins overexpressed in cancers.

Table 1.  FDA approved therapeutic antibodies isolated using phage display technology. Information was obtained from the ImMunoGeneTics antibody database (IMGT/mAb-DB)^11,13, and the numbers following each drug name indicate the IMGT database entry number.

Therapeutic antibodies can also target infectious agents, including bacteria and viruses; examples include bezlotoxumab, which targets the B toxin of Clostridium difficile, obiltoxaximab and raxibacumab, which target the anthrax toxin, and palivizumab, which targets the F protein of respiratory syncytial virus. These are currently the only antibodies approved for therapy against infectious agents, and only raxibacumab was isolated using phage display. The others were isolated from mice using traditional hybridoma technology followed by humanisation, or using transgenic mice with humanised immune repertoires.

However, phage display, using immunised human antibody libraries created from individuals who have survived viral infections or from vaccinated individuals, offers a unique advantage for the isolation of neutralising antibodies to infectious agents. Antibodies have been isolated using such techniques from several viruses including Enterovirus 71¹⁴, Ebola virus¹⁵, HIV¹⁶, West Nile Virus¹⁷ and Rabies virus¹⁸. Neutralising antibodies can also be isolated from naïve human libraries using phage display. m102.4 antibody neutralises Hendra and Nipah viruses, and was isolated by panning a naïve library against the G-protein of Hendra virus¹⁹. This antibody has recently completed Phase I clinical trials in Australia²⁰ and has been used as passive immunotherapy in several individuals exposed to Hendra virus²¹.

Biopanning strategies for isolation of antibodies to microbial targets requires a source of antigen for incubation with the phage library. The antigen can be a highly purified preparation of the target, for example viral proteins^22–24or purified bacterial toxins^25,26, or crude preparations such as whole bacterial cells^27,28 or virus particles^29,30.

Phage display offers several advantages over mouse immunisation strategies for antibody discovery, especially for targets that are either toxic or non-immunogenic in a mouse host, or where precision over epitope targeting is required³¹. Guidance towards particular epitopes can be incorporated into the biopanning strategy, by competing with a ligand, or alternating between mouse and human equivalent antigens, or depleting the library to binders that are cross-reactive to similar antigens. For example, antibodies specific for each of the four serotypes of Dengue virus (DENV) NS1 were isolated from a human naïve phage library³². Serotype specificity was achieved by first exposing the library to the other three DENV NS1 serotypes to deplete cross-reactive binders. Such antibodies may be useful in serotyping assays.

Phage display is a simple but powerful tool for antibody discovery, either for therapeutic use or for research tools. It is accessible to any laboratory equipped for standard culturing and molecular biology. Libraries can be created in-house, obtained commercially (Source Bioscience, Creative Biolabs) or shared from other researchers through material transfer agreements. Within Australia, the National Biologics Facility (NBF) at the University of Queensland offers phage display services and access to their naïve human library, and has experience in isolating antibodies against infectious targets including Dengue virus³² and the malaria parasite³³. Isolation of viral neutralising antibodies using phage display of libraries generated from immunised or recovered patients is an emerging field in infectious disease therapy.

Martina Jones is Operations Manager of the Queensland node of the National Biologics Facility, which offers phage display services to industry and academic groups.