A review of in vitro stochastic and non-stochastic affinity maturation strategies for phage display derived monoclonal antibodies.

Monoclonal antibodies identified using display technologies like phage display occasionally suffers from a lack of affinity making it unsuitable for application. This drawback is circumvented with the application of affinity maturation. Affinity maturation is an essential step in the natural evolution of antibodies in the immune system. The evolution of molecular based methods has seen the development of various mutagenesis approaches. This allows for the natural evolutionary process during somatic hypermutation to be replicated in the laboratories for affinity maturation to fine-tune the affinity and selectivity of antibodies. In this review, we will discuss affinity maturation strategies for mAbs generated through phage display systems. The review will highlight various in vitro stochastic and non-stochastic affinity maturation approaches that includes but are not limited to random mutagenesis, site-directed mutagenesis, and gene synthesis.

Generation of a Naïve Human scFv Phage Display Library and Panning Selection.

Phage display antibody libraries have been successfully used as the essential tool to produce monoclonal antibodies against a plethora of targets ranging from diseases to native biologically important proteins as well as small molecules. It is well documented that diverse antibody genes are the major genetic source for the construction of a high-quality antibody library and selection of high-affinity antibodies. Naïve antibody libraries are derived using the IgM repertoire of healthy donors obtained from B-cells isolated from human peripheral blood mononuclear cell (PBMC). Single-chain fragment variable (scFv) is a routinely used format due to its smaller size and preference for phage display. The process involves the use of a two-step cloning method for library construction. The protocol also covers the biopanning process for target positive clone selection.

Construction of Naïve and Immune Human Fab Phage Display Library.

Phage display has been applied successfully for the rapid isolation of monoclonal antibodies against various targets including infectious diseases, autoantigens, cancer markers, and even small molecules. The main component in any phage display experiment is the availability of an antibody library to carry out the selection process of target-specific antibodies through an iterative process termed as biopanning. To generate human antibody libraries, the antibody repertoire can be obtained from human peripheral blood mononuclear cell (PBMC) or directly from cell-sorted B-cell populations. The choice of antibody isotype is dictated by the nature of the library. Naïve libraries would utilize IgM repertoires, whereas the IgG repertoire is commonly used for immune libraries. Antibody genes are amplified through polymerase chain reaction (PCR) and paired in a combinatorial fashion to expand the diversity of the cloned library repertoire. The protocol here describes the use of a two-step cloning method that can be applied for the construction of either a naïve or immune human antibody library in Fab format followed by the subsequent panning.

Antibody Phage Display.

The application of antibodies has transcended across many areas of work but mainly as a research tool, for diagnostic and for therapeutic applications. Antibodies are immunoproteins from vertebrates that have the unique property of specifically binding foreign molecules and distinguish target antigens. This property allows antibodies to effectively protect the host from infections. Apart from the hybridoma technology using transgenic animals, antibody phage display is commonly considered the gold standard technique for the isolation of human monoclonal antibodies. The concept of antibody phage display surrounds the ability to display antibody fragments on the surface of M13 bacteriophage particles with the corresponding gene packaged within the particle. A repetitive in vitro affinity based selection process permits the enrichment of target specific binders. This process of recombinant human monoclonal antibody generation also enables additional engineering for various applications. This makes phage display an indispensable technique for antibody development and engineering activities.

Streptavidin-Coated Solid-Phase Extraction (SPE) Tips for Antibody Phage Display Biopanning.

Phage display is a technique that allows the presentation of unique proteins on the surface of bacteriophages. The phage particles are usually screened via repetitive rounds of antigen-guided selection and phage amplification. The main advantage of this approach lies in the physical linkage between phenotype and genotype. This feature allows the isolation of single unique clones from a panning campaign consisting of a highly diverse population of clones. Due to the high-throughput nature of this technique, different approaches have been developed to assist phage display selections. One of which involves utilizing a streptavidin-coated solid-phase extraction (SPE) tip that is mounted to an electronically controlled motorized multichannel pipette. In this chapter, we will entail the procedures involved in the adaptation of a commercial SPE tip (MSIA™ streptavidin D.A.R.T's®) as the solid phase. This protocol is an updated version of a previous protocol with some minor refinements.

Subtractive panning for the isolation of monoclonal PEPITEM peptide antibody by phage display.

Antibody phage display is a key tool for the development of monoclonal antibodies against various targets. However, the development of anti-peptide antibodies is a challenging process due to the small size of peptides for binding. This makes anchoring of peptides a preferred approach for panning experiments. A common approach is by using streptavidin as the anchor protein to present biotinylated peptides for panning. Here, we propose the use of recombinant expression of the target peptide and an immunogenic protein as a fusion for panning. The peptide inhibitor of trans-endothelial migration (PEPITEM) peptide sequence was fused to the Mycobacterium tuberculosis (Mtb) α-crystalline (AC) as an anchor protein. The panning process was carried out by subtractive selection of the antibody library against the AC protein first, followed by binding to the library to PEPITEM fused AC (PEPI-AC). A unique monoclonal scFv antibodies with good specificity were identified. In conclusion, the use of an alternative anchor protein to present the peptide sequence coupled with subtractive panning allows for the identification of unique monoclonal antibodies against a peptide target.

Naïve antibody library derived monoclonal antibody against VP35 of Ebola virus.

Ebola virus is notorious for causing severe and even deadly haemorrhagic fever in infected humans and non-human primates. The high fatality rate of Ebola virus disease (EVD) has highlighted the need for effective diagnosis and treatment. Two monoclonal antibodies (mAbs) have been approved by USFDA for treatment of EVD. Virus surface glycoprotein is the common target for diagnostic and therapy including vaccines. Even so, VP35, a viral RNA polymerase cofactor and interferon inhibitor could be a potential target to curb EVD. The present work describes the isolation of three mAb clones from a phage-displayed human naïve scFv library against recombinant VP35. The clones showed binding against rVP35 in vitro and inhibition of VP35 in luciferase reporter gene assay. Structural modelling analysis was also carried out to identify the binding interactions involved in the antibody-antigen interaction model. This allows some insight into the "fitness" of the binding pocket between the paratope and target epitope which would be useful for the design of new mAbs through in silico means in the future. In conclusion, the information obtained from the 3 isolated mAbs could be potentially useful in the quest to improve VP35 targeting for therapeutic development in the future.

A semi-rational mutagenesis approach for improved substrate activity of microbial transglutaminase.

A higher specific activity of microbial transglutaminase (mTGase) is desirable for a broad range of applications ranging from food industry to biotechnology. Three-dimensional docking simulation of mTGase revealed that residues V65, W69, and Y75 were critical for substrate recognition. A semi-rational mutagenesis approach was applied to each residue to generate three separate mini mutant libraries. A high-throughput screening process identified five mutants that demonstrated improved specific activities than the wild type (WT) mTGase were isolated from the Y75 mini mutant library. Mutant Y75L showed approximately 60% increment in specific activity and improved substrate specificity. Conjugation of two heterologous single-chain fragment variable clones to generate a diabody with mutant Y75L was successfully performed and validated. This work demonstrates the successful application of semi-rational mutagenesis coupled with a high-throughput screening approach to identify mTGase mutants with improved specific activities and specificities which are beneficial for protein-protein conjugation.

Potential of Phage Display Antibody Technology for Cardiovascular Disease Immunotherapy.

Cardiovascular disease (CVD) is one of the leading causes of death worldwide. CVD includes coronary artery diseases such as angina, myocardial infarction, and stroke. "Lipid hypothesis" which is also known as the cholesterol hypothesis proposes the linkage of plasma cholesterol level with the risk of developing CVD. Conventional management involves the use of statins to reduce the serum cholesterol levels as means for CVD prevention or treatment. The regulation of serum cholesterol levels can potentially be regulated with biological interventions like monoclonal antibodies. Phage display is a powerful tool for the development of therapeutic antibodies with successes over the recent decade. Although mainly for oncology, the application of monoclonal antibodies as immunotherapeutic agents could potentially be expanded to CVD. This review focuses on the concept of phage display for antibody development and discusses the potential target antigens that could potentially be beneficial for serum cholesterol management.

One-step synthesis of M13 phage-based nanoparticles and their fluorescence properties.

Fluorescent carbon nanoparticles have been gaining more attention in recent years for their excellent fluorescence properties and simple synthesis routes. Different carbon sources have been reported for fluorescent carbon nanoparticle synthesis but the use of virus particles as a carbon source is scarce. Herein, we report the utilization of M13 bacteriophage particles as the carbon source to synthesize phage-based nanoparticles through facile, one-step microwave heating. M13 bacteriophage is a nanosized filamentous virus particle with a single-stranded DNA genome encapsulated by a large number of coat proteins. These amino acid rich building blocks provide a substantial amount of carbon source for the synthesis of fluorescent nanoparticles. The resulting nanoparticles from M13 bacteriophage showed good water solubility and exhibited bright blue luminescence. The selectivity and sensitivity of the phage-based nanoparticles towards Fe(iii) ions showed a quenching effect with a linear correlation and a detection limit of 8.0 µM. This process highlights the potential application of virus particles as a source for the synthesis of fluorescent carbon nanoparticles and the sensing application.

Applicability of Brugia malayi immune antibody library for the isolation of a human recombinant monoclonal antibody to Echinococcus granulosus antigen B.

Echinococcus granulosus is a worldwide zoonotic infection that causes human cystic echinococcosis (CE) or hydatid disease. The present study describes the isolation and production of a monoclonal antibody against recombinant AgB protein using the developed Human AntibodY Disease ENhanced (HAYDEN)-Filariasis library. The DNA sequences of the isolated clones were analyzed, followed by gene analysis and binding assays. Clone E1 showed a full-length sequence and represents the IgHV5-LV3 antibody gene family. The antibody protein yield was satisfactory, and it reacted specifically against rAgB. The novel E1 protein is potentially useful for the development of an antigen detection assay for CE. The ability of the Brugia malayi immune antibody library to isolate antibodies against Echinococcus granulosus antigens highlights the broad coverage of immune antibody libraries.

Infectious disease antibodies for biomedical applications: A mini review of immune antibody phage library repertoire.

Antibody phage display is regarded as a critical tool for the development of monoclonal antibodies for infectious diseases. The different classes of antibody libraries are classified based on the source of repertoire used to generate the libraries. Immune antibody libraries are generated from disease infected host or immunization against an infectious agent. Antibodies derived from immune libraries are distinct from those derived from naïve libraries as the host's in vivo immune mechanisms shape the antibody repertoire to yield high affinity antibodies. As the immune system is constantly evolving in accordance to the health state of an individual, immune libraries can offer more than just infection-specific antibodies but also antibodies derived from the memory B-cells much like naïve libraries. The combinatorial nature of the gene cloning process would give rise to a combination of natural and un-natural antibody gene pairings in the immune library. These factors have a profound impact on the coverage of immune antibody libraries to target both disease-specific and non-disease specific antigens. This review looks at the diverse nature of antibody responses for immune library generation and discusses the extended potential of a disease-specified immune library in the context of phage display.

Potential application of Leishmania tarentolae as an alternative platform for antibody expression.

Through the discovery of monoclonal antibody (mAb) technology, profound successes in medical treatment against a wide range of diseases have been achieved. This has led antibodies to emerge as a new class of biodrugs. As the "rising star" in the pharmaceutical market, extensive research and development in antibody production has been carried out in various expression systems including bacteria, insects, plants, yeasts, and mammalian cell lines. The major benefit of eukaryotic expression systems is the ability to carry out posttranslational modifications of the antibody. Glycosylation of therapeutic antibodies is one of these important modifications, due to its influence on antibody structure, stability, serum half-life, and complement recruitment. In recent years, the protozoan parasite Leishmania tarentolae has been introduced as a new eukaryotic expression system. L. tarentolae is rich in glycoproteins with oligosaccharide structures that are very similar to humans. Therefore, it is touted as a potential alternative to mammalian expression systems for therapeutic antibody production. Here, we present a comparative review on the features of the L. tarentolae expression system with other expression platforms such as bacteria, insect cells, yeasts, transgenic plants, and mammalian cells with a focus on mAb production.

Cassette hybridization for vector assembly application in antibody chain shuffling.

Gene assembly methods are an integral part of molecular cloning experiments. The majority of existing vector assembly methods stipulate a need for exonucleases, endonucleases and/or the use of single-stranded DNA as starting materials. Here, we introduced a vector assembly method that employs conventional PCR to amplify stable double-stranded DNA fragments and assembles them into functional vectors specifically for antibody chain shuffling. We successfully formed vectors using cassettes amplified from different templates and assembled an array of single chain fragment variable clones of fixed variable heavy chain, with different variable light chains - a chain shuffling process for antibody maturation. The method provides an easy alternative to the conventional cloning process.

Delineation of B-cell Epitopes of Salmonella enterica serovar Typhi Hemolysin E: Potential antibody therapeutic target.

Hemolysin E (HlyE) is an immunogenic novel pore-forming toxin involved in the pathogenesis of typhoid fever. Thus, mapping of B-cell epitopes of Salmonella enterica serovar Typhi (S. Typhi) is critical to identify key immunogenic regions of HlyE. A random 20-mer peptide library was used for biopanning with enriched anti-HlyE polyclonal antibodies from typhoid patient sera. Bioinformatic tools were used to refine, analyze and map the enriched peptide sequences against the protein to identify the epitopes. The analysis identified both linear and conformational epitopes on the HlyE protein. The predicted linear GAAAGIVAG and conformational epitope PYSQESVLSADSQNQK were further validated against the pooled sera. The identified epitopes were then used to isolate epitope specific monoclonal antibodies by antibody phage display. Monoclonal scFv antibodies were enriched for both linear and conformational epitopes. Molecular docking was performed to elucidate the antigen-antibody interaction of the monoclonal antibodies against the epitopes on the HlyE monomer and oligomer structure. An in-depth view of the mechanistic and positional characteristics of the antibodies and epitope for HlyE was successfully accomplished by a combination of phage display and bioinformatic analysis. The predicted function and structure of the antibodies highlights the possibility of utilizing the antibodies as neutralizing agents for typhoid fever.

Naïve Human Antibody Libraries for Infectious Diseases.

Many countries are facing an uphill battle in combating the spread of infectious diseases. The constant evolution of microorganisms magnifies the problem as it facilitates the re-emergence of old infectious diseases as well as promote the introduction of new and more deadly variants. Evidently, infectious diseases have contributed to an alarming rate of mortality worldwide making it a growing concern. Historically, antibodies have been used successfully to prevent and treat infectious diseases since the nineteenth century using antisera collected from immunized animals. The inherent ability of antibodies to trigger effector mechanisms aids the immune system to fight off pathogens that invades the host. Immune libraries have always been an important source of antibodies for infectious diseases due to the skewed repertoire generated post infection. Even so, the role and ability of naïve antibody libraries should not be underestimated. The naïve repertoire has its own unique advantages in generating antibodies against target antigens. This chapter will highlight the concept, advantages and application of human naïve libraries as a source to isolate antibodies against infectious disease target antigens.