Directed evolution is a proven approach to fine tune or modify biomolecules for various applications ranging from research to industry. The process of evolution requires methods that are capable of not only generating genetic diversity but also to distinguish the variants of desired characteristics. One method that is synonymous with directed evolution of proteins is phage display. Here, we present a protocol describing the application of magnetic nanoparticles coupled with a processor to carry out the identification of monoclonal antibodies (mAbs) from a diverse antibody library via phage display. Target antigens are coupled to magnetic nanoparticles as the solid phase for the isolation of the binding mAbs via affinity. A gradual enrichment in clones would result in increasing ELISA readouts with increasing rounds of panning. During monoclonal level analysis, positivity can be deduced with comparison to background and controls. The biopanning process can also be adopted for the directed evolution of enzymes, scaffold proteins or even peptides.
Bio-panning is a common process involved in recombinant antibody selection against defined targets. The biopanning process aims to isolate specific antibodies against an antigen via affinity selection from a phage display library. In general, antigens are immobilized on solid surfaces such as polystyrene plastic, magnetic beads, and nitrocellulose. For high-throughput selection, semi-automated panning selection allows simultaneous panning against multiple target antigens adapting automated particle processing systems such as the KingFisher Flex. The system setup allows for minimal human intervention for pre- and post-panning steps such as antigen immobilization, phage rescue, and amplification. In addition, the platform is also adaptable to perform polyclonal and monoclonal ELISA for the evaluation process. This chapter will detail the protocols involved from the selection stage until the monoclonal ELISA evaluation with important notes attached at the end of this chapter for optimization and troubleshooting purposes.
Panning is a common process used for antibody selection from phage antibody libraries. There are several methods developed for a similar purpose, namely streptavidin mass spectrometry immunoassay (MSIA™) Disposable Automation Research Tips, magnetic beads, polystyrene immunotubes, and microtiter plate. The advantage of using a magnetic particle processor system is the ability to carry out phage display panning against multiple target antigens simultaneously in parallel. The system carries out the panning procedure using magnetic nanoparticles in microtiter plates. The entire incubation, wash, and elution process is then automated in this setup. The system also allows customization for the introduction of different panning stringencies. The nature of the biopanning process coupled with the limitation of the system means that minimal human intervention is required for the infection and phage packaging stage. However, the process still allows for rapid and reproducible antibody generation to be carried out.
The application of human TCR in cancer immunotherapy has gained momentum with developments in tumor killing strategies using endogenous adaptive immune responses. The successful coverage of a diverse TCR repertoire is mainly attributed to the primer design of the human TCR V genes. Here, we present a refined primer design strategy of the human TCR V gene by clustering V gene sequence homolog for degenerate primer design based on the data from IMGT. The primers designed were analyzed and the PCR efficiency of each primer set was optimized. A total of 112 alpha and 160 beta sequences were aligned and clustered using a phylogram yielding 32 and 27 V gene primers for the alpha and beta family. The new primer set was able to provide 93.75% and 95.63% coverage for the alpha and beta family, respectively. A semi-qualitative approach using the designed primer set was able to provide a relative view of the TCR V gene diversity in different populations. Taken together, the new primers provide a more comprehensive coverage of the TCR gene diversity for improved TCR library generation and TCR V gene analysis studies.
The immune system is tasked to keep our body unharmed and healthy. In the immune system, B- and T-lymphocytes are the two main components working together to stop and eliminate invading threats like virus particles, bacteria, fungi and parasite from attacking our healthy cells. The function of antibodies is relatively more direct in target recognition as compared to T-cell receptors (TCR) which recognizes antigenic peptides being presented on the major histocompatibility complex (MHC). Although phage display has been widely applied for antibody presentation, this is the opposite in the case of TCR. The cell surface TCR is a relatively large and complex molecule, making presentation on phage surfaces challenging. Even so, recombinant versions and modifications have been introduced to allow the growing development of TCR in phage display. In addition, the increasing application of TCR for immunotherapy has made it an important binding motif to be developed by phage display. This review will emphasize on the application of phage display for TCR discovery as well as the engineering aspect of TCR for improved characteristics.
The application of recombinant human antibodies is growing rapidly mainly in the field of diagnostics and therapeutics. To identify antibodies against a specific antigen, panning selection is carried out using different display technologies. Phage display technology remains the preferred platform due to its robustness and efficiency in biopanning experiments. There are both manual and semi-automated panning selections using polystyrene plastic, magnetic beads, and nitrocellulose as the immobilizing solid surface. Magnetic nanoparticles allow for improved antigen binding due to their large surface area. The Kingfisher Flex magnetic particle processing system was originally designed to aid in RNA, DNA, and protein extraction using magnetic beads. However, the system can be programmed for antibody phage display panning. The automation allows for a reduction in human error and improves reproducibility in between selections with the preprogrammed movements. The system requires minimum human intervention to operate; however, human intervention is needed for post-panning steps like phage rescue. In addition, polyclonal and monoclonal ELISA can be performed using the semi-automated platform to evaluate the selected antibody clones. This chapter will summarize the suggested protocol from the panning stage till the monoclonal ELISA evaluation. Other than this, important notes on the possible optimization and troubleshooting are also included at the end of this chapter.
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.
Phage display was first described by George P. Smith when it was shown that virus particles were capable of presenting foreign proteins on their surface. The technology has paved the way for the evolution of various biomolecules presentation and diverse selection strategies. This unique feature has been applied as a versatile platform for numerous applications in drug discovery, protein engineering, diagnostics, and vaccine development. Over the decades, the limits of biomolecules displayed on phage particles have expanded from peptides to proteomes and even alternative scaffolds. This has allowed phage display to be viewed as a versatile display platform to accommodate various biomolecules ranging from small peptides to larger proteomes which has significantly impacted advancements in the biomedical industry. This review will explore the vast array of biomolecules that have been successfully employed in phage display technology in biomedical research.
Pulmonary tuberculosis, caused by Mycobacterium tuberculosis, is one of the most persistent diseases leading to death in humans. As one of the key targets during the latent/dormant stage of M. tuberculosis, isocitrate lyase (ICL) has been a subject of interest for new tuberculosis therapeutics. In this work, the cleavage of the isocitrate by M. tuberculosis ICL was studied using quantum mechanics/molecular mechanics method at M06-2X/6-31+G(d,p): AMBER level of theory. The electronic embedding approach was applied to provide a better depiction of electrostatic interactions between MM and QM regions. Two possible pathways (pathway I that involves Asp108 and pathway II that involves Glu182) that could lead to the metabolism of isocitrate was studied in this study. The results suggested that the core residues involved in isocitrate catalytic cleavage mechanism are Asp108, Cys191 and Arg228. A water molecule bonded to Mg2+ acts as the catalytic base for the deprotonation of isocitrate C(2)-OH group, while Cys191 acts as the catalytic acid. Our observation suggests that the shuttle proton from isocitrate hydroxyl group C(2) atom is favourably transferred to Asp108 instead of Glu182 with a lower activation energy of 6.2 kcal/mol. Natural bond analysis also demonstrated that pathway I involving the transfer of proton to Asp108 has a higher intermolecular interaction and charge transfer that were associated with higher stabilization energy. The QM/MM transition state stepwise catalytic mechanism of ICL agrees with the in vitro enzymatic assay whereby Asp108Ala and Cys191Ser ICL mutants lost their isocitrate cleavage activities.
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.
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.