ANTIBODY PRODUCTION THROUGH PHAGE DISPLAY TECHNOLOGY

Phage Display

Phage display technology is based on the linkage between phenotype and genotype and on the replicative capacity of filamentous bacteriophage. Foreign DNA fragments can be inserted into filamentous phage genes coding for phage coat protein to create a fusion protein that is displayed in the phage surface. Billions of antibody fragment (Fab or scFv) sequences can be displayed on phage and presented to different type of proteins in various possible conformations to select specific monoclonal antibodies (mAbs), as shown by pioneer and co-inventor John McCafferty. Due to the flexibility of the phage display-based selection process, this technology enables the identification of monoclonal antibodies with very specific characteristics, representing an alternative to the classical hybridoma technology.

The phage display technology, first developed by George Smith in the 1980s for peptide libraries, and later adapted to the fast screening of antibodies, has earned the Nobel Prize in Chemistry in 2018. Since then, at least 10 antibodies derived from the phage display technology were granted approval by the FDA and EMA for clinical use.

The unparalleled benefits of using the phage display technology for antibody discovery include:

• Fast lead times (only a few weeks are needed to obtain antigen-specific binders by panning antibody libraries via phage display)
• Adapted to the use of toxic or non-immunogenic antigens
• Possibility to tailor an antibody’s cross-reactivity properties according to each project’s needs
• Minimization of animal use in antibody generation

Beyond these unique advantages, phage display technologies continue to push the boundaries of antibody engineering, especially in improving the production of bispecific antibodies. These antibodies, especially promising in the clinical context, are hard to produce in vitro, which has hindered their further development.

Bispecific antibodies are remarkable for their ability to bind two different epitopes on the same or different antigens. In this way, they can engage the immune system and direct the immune response to specific markers. This has proven to be particularly useful in the treatment of cancer where bispecifics engage T cells and direct them towards specific cell surface markers overexpressed on the surface of cancerous cells, which would normally evade the patient’s immune system.

HOW ARE BISPECIFIC ANTIBODIES DEVELOPED?

Bispecific antibodies are built by merging two antibodies with affinities to different epitopes/antigens into a single bispecific molecule. But producing these different antibody-encoding genes in a single cell line often leads to chain misassembly, co-expression of undesired antibody pairs, and, subsequently, reduced purity.

One strategy that has been successfully used to overcome this constraint and help bispecific antibodies maintain a native and stable structure is the “knobs-into-holes” amino acid changes. To ensure a correct chain assembly between the two variants, the “knobs-into-holes” strategy bases itself on the rational design of the CH3 (constant heavy chain 3) region to create a “knob” into one of the variants and a hole in the other. This configuration ensures that the bispecific configuration is favored during antibody production and thus increases the purity and yield of the desired bispecific antibody.

However, this strategy does not solve another important challenge in bispecific production – heavy and light chain mispairing between the two variants of the bispecific molecule.

Several strategies have been developed to minimize this issue such as constant domain fusions, chemical ligation, reshaped Fab interfaces, and the use of common light or heavy chains. But these strategies introduce many changes into conserved antibody regions or impose a heavy restriction in chain diversity. These changes are prone to cause an increase in immunogenicity and to limit epitope coverage or antibody affinity.

FUTURE PERSPECTIVES FOR BISPECIFIC ANTIBODY DEVELOPMENT VIA PHAGE DISPLAY TECHNOLOGIES

A better method for bispecific antibody development via the phage display technology was recently devised by Daniel Christ and his team at the Garvan Institute of Medical Research (Sydney, Australia). In their recent study, they make use of the phage display technology as well as negative and positive selection strategies to boost the final purity and yield of an IgG bispecific antibody. The elegant solution consists of boosting the affinity between a VH domain and its natural VL pair via random mutagenesis and phage display selection in the presence of a competitor molecule.

For drug research on Monoclonal Antibodies In India, phage display-based monoclonal antibody manufacturing is an alternative to hybridoma technology.

Smith is credited with inventing the antibody phage display technology in 1985, with a gene coding for a specific antibody incorporated into the DNA sequence of a filamentous bacteriophage, allowing it to be expressed on the bacteriophage capsid’s surface. This distinction creates a relationship between the genotype and the phenotype. The phage infects Escherichia coli and uses its replication machinery to display new phage constantly without harming the host cell, as this permits large-scale antibody manufacturing in a short amount of time.

Screening procedures can be used to create a library of naive or immune phages that can be utilized to detect an antigen-antibody interaction of interest. Libraries can be made from any animal, including humans, allowing for direct screening of human antibodies. Because the genotype and phenotype are linked, it’s also simple to get immediate access to the sequence, making additional engineering or recombinant protein manufacturing easier for the Phage Display Antibody Library.

Advantages of Phage Display

• Large scale production
• Fast process
• Great control over the selection process
• It’s easy to screen a large number of different 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)

• Phage displayis a rapidly evolving technology that has been used to support a range of applications depending on the nature of the phage display library. These include epitope mapping, receptor and ligand identification, protein-protein interaction studies, recombinant antibody production, directed evolution of proteins, and drug discovery. The widespread utility of this technology is backed by a growing number of citations, highlighting its importance to accelerate research.

Epitope Mapping

• When an antibody recognises a specific antigen, the binding region is referred to as the epitope. Knowledge of antigenic epitopes is valuable for the development of effective antibody reagents for research use and therapeutics. Moreover, epitope mapping has significant utility in vaccine development as it can help elucidate immune responses. Consequently, knowledge of epitope mapping has allow the construction of peptide vaccines based on epitopes specificity.

Receptor and Ligand Identification

• Phage display is an efficient method to determine which proteins or peptides bind specifically to predefined targets. This facilitates the study of receptor-ligand interactions. Furthermore, it sheds new light on cellular signalling pathways through the identification of key inhibitors, agonists and antagonists. In situations where a receptor interacts with multiple ligands, this technology can also help to classify those ligands with the highest binding affinity. This allow further downstream research on receptor and multiple ligands interaction.

Protein-Protein Interaction Studies

• Virtually all biological processes are mediated by protein-protein interactions. Phage display provide means of evaluating complex inter-protein relationship. It also allows the identification of known and novel protein binding partners to evaluate the specificity of these associations. As an example, the use of phage display to present multiple phosphorylated and unmodified peptides to a target protein can inform the phospho-specificity of a protein-protein interaction.

Recombinant Antibody Production

• Antibody phage display is regarded by many as the gold standard in recombinant antibody production. By screening large numbers of antibody-displaying phages against a target antigen, phages which expresses antibody specific to the target can be identified quickly. One method of producing a library of antibody-displaying phage is to immunise an animal, isolate B-cells, extract mRNA and synthesise cDNA encoding antibody single-chain variable regions (scFv) for cloning into phagemid vectors. It is also possible to purchase pre-synthesised libraries consisting of many different antibody scFv genes.

Directed Evolution of Proteins

• Directed evolution introduces desirable features into an existing protein, typically through the acquisition of mutations. In addition, this can also change the function of the protein and alter the specificity for a binding partner. Using a phage display library composed of multiple protein variants, researchers can identify those mutations with the required characteristics.

  Drug Discovery

• Phage display has widespread utility within drug discovery. Not only can it facilitate the identification of peptide ligands for therapeutic targets, it can also provide a launch point for drug discovery efforts, and is pivotal to understand how these biomolecules may cross-react with other proteins. Therefore, research groups are focused on developing antibody drugs using phage display to investigate antibody selectivity. On top of that, phage technology is also use to identify scFv fragments suitable for targeted delivery of cytotoxic agents to tumours.

Principle & Methodology

Amber non-suppressor host, such as WK6 cells transformed with VHH-pHEN6c DNA allow IPTG inducible expression of soluble dAbs with 6xHis tag. For expression, WK6 cells grown to mid-log phase in terrific broth/ampicillin/Mg++ is induced with IPTG for expression of soluble dAb-6xHis products at 28°C for 16-18 hrs with shaking. The expressed products are transported in the periplasmic space due to the presence of pelB leader sequence in the vector, which is cleaved from the finished product in the periplasmic contents. The periplasmic contents are released from the bacterial pellet suspension in hypotonic tris-EDTA-sucrose solution. Yield of the expressed clones may range between 1–15 mg/litre culture.

Materials, reagents & equipment

1. WK6 colonies on LB/ampicillin/glucose, freshly grown by plating VHH-pHEN6c positive transformants 2. Terrific broth/ampicillin (100μg/ml)/0.1% glucose/2mM MgCl2, one litre for each clone 3. 1M IPTG stock solution 4. TES buffer (10 mM tris.HCl, pH 8.0- 0.5 mM EDTA- 20 mM sucrose) & TES/4 (TES diluted 1:4 in sterile deionized water) 5. One litre conical flasks, sterilized 6. 250 ml bottles for centrifugation of cultures 7. 50 ml sterile culture/centrifuge tubes 8. Biophotometer/spectrophotometer for OD600 measurement 9. Shaker water bath 10. Cooling centrifuge 11. Pipettes, disposable, 2 ml & 10 ml

Procedure

1. Inoculate each colony in 10 ml TB/amp/glucose/MgCl2broth medium in a 50 ml sterile culture tube and incubate at 37ºC in a shaking water bath until the OD600 reached between 0.6-0.9. 2. Then, add one ml of this mid-log phase growth in 330 ml TBampglucose/MgCl2 broth in each of three one litre conical flasks and incubate at 37ºC in a shaking water bath until the OD600 reached between 0.6-0.9. 3. Add IPTG to a final concentration of 1 mM in the flask and further incubation it at 28ºC for 16-18 hrs. 4. Distribute the culture in four 250 ml bottles and centrifuge at 4000 rpm for 15 min. at 4ºC.
2. Decant the bottles completely and resuspend the pellet of each in 2 ml TES buffer to release the bacterial periplasmic contents. Pool the contents in one 50 ml sterile tube and store on ice for 60 min. 6. Then, add 12 ml of TES/4 solution to the contents of the tube and further incubate for 60 min. on ice. 7. Centrifuge the tube at 13,000 rpm for 30 min. at 4ºC and collect the supernatant as periplasmic contents having expressed dAbs in fresh 50 ml tube. These antibody fragments expressed from pHEN6c vector have dAbs with hexa-histidine epitope in their C-terminal end. Designate them as dAb.6xHis. 8. Use the preparations for indirect ELISA, immunoblotting, purification by Ni chelate chromatography, etc

Compiled  & Shared by- Team, LITD (Livestock Institute of Training & Development)

Image-Courtesy-Google

Reference-On Request.

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