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Antibody Phage Display Overview

What is Antibody Phage Display

Antibody phage display (APD) is a technique based on genetic engineering of bacteriophages and repeated rounds of antigen-guided selection and phage propagation. It allows in vitro selection and production of recombinant monoclonal antibodies of high specificity and affinity.

In 1985, Smith was first to discover that foreign DNA fragments can be fused to the gene encoded for pIII coat protein of a nonlytic filamentous phage and expressed as a fusion protein on the virion surface without disturbing the infectivity of the phage. Winter flipped the process of phage display by using the phage to display antibodies (rather than proteins) then fishing out the desired antibodies that bind to molecules or even cells. The invention of antibody phage display has revolutionised monoclonal antibody drug discovery.

To establish which monoclonal antibody technique is appropriate, researchers should weigh the pros and the cons of each method, summarized in the table below:

  Phage display Hybridoma
Pros Large scale production
• Fast process
• Great control over the selection process
• Easy to screen a large diversity of clones
• Possible to directly screen human libraries
• Possible to screen toxic antigens
• No immunogenicity issue (for naïve libraries)
• No clone viability issues
• Direct access to sequence
• No animal use (for naïve libraries)
• Large scale production
• High antibody yield
• High specificity
• High antibody sensitivity
• Lower cost
Cons • More expensive
• Binders may have lower affinity
• Technically more difficult
• Long generation time
• Incomplete epitope identification
• Often requires humanization

Types of Combinatorial Antibody Libraries

According to the source of library sequences, there are four types of antibody libraries: naïve, immune, semi-synthetic and synthetic libraries.

Naïve libraries

Naïve libraries are amplified from a natural source, such as primary B-cells of non immunized donors. Naturally rearranged variable region genes have been used to construct large repertoires of up to 1011 members. In contrast to immune libraries, one single library can be used for the generation of antibodies against a wide variety of antigens, including toxins and self-antigens.

The CAT1.0 library (Cambridge Antibody Technology, now part of MedImmune/AstraZeneca) is the first large naïve antibody phage display library. Rearranged antibody V genes for heavy and light chain genes were amplified from B-cells of 43 human donors and randomly combined into scFvs resulting in a library with a size of 1.4×1010. The CAT2.0 library was a further extension of CAT1.0. the total library size has increased to 1.29×1011.

Sino Biological has constructed a naïve fully human scFv library with a size of 1.56×1011. We can offer you tailored antibody phage display services and get your monoclonal antibody generation as soon as possible. The variety of our libraries allows us to propose a wide range of formats to fit a wide range of applications.

Immune libraries

Immune libraries are generated from B-cell derived antibody repertoire of immunized or immune donors and are predisposed for a limited panel of antigens. Due to this predisposition, they are usually comparatively small in size. In contrast to naïve libraries, however, immune libraries are not well suited for the identification of antibody fragments against a large panel of antigens, especially self-antigens.

Semi-synthetic libraries

Semi-synthetic libraries comprise both CDRs from natural sources as well as in silico design of defined parts. The first semi-synthetic libraries used a variety of different framework genes to keep diversity high. In 1992 Hoogenboom and Winter described semi-synthetic scFv-antibody phage display libraries, comprising 49 germline VH sequences and a single V_lambda 3 light chain sequence. Five or eight residues in the CDR-H3 were randomized in a PCR-based approach to generate libraries with a size of 1×107.

Synthetic libraries

Synthetic libraries are based on computational in silico design and gene synthesis. CDR design and composition is precisely defined and controlled. HuCAL (human combinatorial antibody library) PLATINUM is a synthetic library of human Fab antibodies with a size of 4.5×1010.

Four types of antibody libraries
Fig 1. Four types of antibody libraries

Construction and Production of Phage Display Antibody

Phage display is a process in which phage DNA is manipulated to produce a fusion of a protein or peptide to one of the phage coat proteins. The most commonly used phage for phage display are members of the Ff family (M13, Fd, f1) and the most commonly used fusion partners are coat proteins of the parental phage, such as protein III of M13. For M13, both the N and C termini of the five coat proteins have been used as fusion proteins for library display.

A bacteriophage highlighting the genotype-phenotype coupling that is fundamental to phage display technology. The gene of interest (pink) is cloned into the gene 3 protein (g3p) of phage DNA, which results in the display of the pink protein product (antibody, peptide) on the surface of the phage as a polypeptide fusion.

The selection process of phage display is as follows: (1) A phage library containing 106-1011 clones is incubated with immobilized antigen. (2) Unbound phage are removed by washing. (3) Bound phage are eluted. (4) E.coli. are infected with eluted phage with or without helper phage to amplify eluted candidates. (5) Cells are plated onto selective plates and amplified. Process is reiterated 2–3 times resulting in enriched population of antibody/peptide fragments for the antigen of interest. Additional site directed mutagenesis or depletion approaches can be used to further tune desired antibody properties.

Phage display and selection
Fig 2. Phage display and selection

Applications of Phage Display Antibody

Antibody phage display has played a key role in the quest to generate monoclonal antibodies for basic research, diagnostic and therapeutic applications.

For diagnostic applications, recombinant antibodies are used due to its binding specificity and affinity. There are many platforms, such as lateral‐flow assay (LFA), ELISA and cell imaging available in the market today, which are rapid and accurate in identifying the target antigens found in sample. Most of the platforms make use of either the antigen‐capture assay or the antibody‐capture assay to diagnose the presence of certain diseases.

The first clinically approved therapeutic antibody obtained with the help of phage display was adalimumab (marketed as Humira®), which neutralizes tumor necrosis factor and is mainly used against rheumatoid arthritis. Phage display technology is very helpful for high throughput screening for human therapeutic antibody candidates.

Table 1. Phage display derived drugs approved by FDA

Generic name Brand name Target Launch year Indications
Durvalumab Imfinzi programmed death ligand-1 (PD-L1) 2017 metastatic urothelial carcinoma, unresectable non-small cell lung cancer
Avelumab Bavencio programmed death ligand-1 (PD-L1) 2017 metastatic Merkel-cell carcinoma (MCC)
Necitumumab Portrazza epidermal growth factor receptor (EGFR) 2015 metastatic squamous non-small cell lung cancer
Ramucirumab Cyramza vascular endothelial growth factor receptor 2 (VEGFR2) 2014 advanced gastric cancer or gastro-esophageal junction (GEJ) adenocarcinoma
Raxibacumab ABthrax Protective antigen (PA) component of anthrax (Bacillus anthracis) 2012 Prophylaxis and treatment of anthrax
Belimumab Benlysta B-lymphocyte stimulator (BLyS) 2011 Autoantibody-positive, systemic lupus
Ranibizumab Lucentis Vascular endothelial growth factor A (VEGF-A) 2006 Neovascular (wet) age-related macular degeneration
Adalimumab Humira Tumor necrosis factor-α (TNF) 2002 Rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, crohn's disease, ulcerative colitis, plaque psoriasis

References

1. Hammers, C. M., & Stanley, J. R. (2014). Antibody phage display: technique and applications. The Journal of investigative dermatology, 134(2), e17.
2. Ponsel, D., Neugebauer, J., Ladetzki-Baehs, K., & Tissot, K. (2011). High affinity, developability and functional size: the holy grail of combinatorial antibody library generation. Molecules, 16(5), 3675-3700.
3. Burkovitz, A., & Ofran, Y. (2016, February). Understanding differences between synthetic and natural antibodies can help improve antibody engineering. In MAbs (Vol. 8, No. 2, pp. 278-287). Taylor & Francis.
4. Bahara, N. H. H., Tye, G. J., Choong, Y. S., Ong, E. B. B., Ismail, A., & Lim, T. S. (2013). Phage display antibodies for diagnostic applications. Biologicals, 41(4), 209-216.
5. Ch'ng, A. C. W., Choong, Y. S., & Lim, T. S. (2016). Phage display-derived antibodies: application of recombinant antibodies for diagnostics. Proof and concepts in rapid diagnostic tests and technologies. InTech, London, 107-135.

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