MimoDB 2.0: a mimotope database and beyond

Currently, the phage display technology is probably one of the most mature animal-free alternatives to the hybridoma technology for developing antibodies. However, the performances of these two technologies have been rarely compared. To address this question, hybridoma and phage display approaches were used to generate antibodies against Zika virus (ZIKV) non-structural protein 1 (NS1), a biomarker of the acute phase of flavivirus infection that displays high sequence homology among flaviviruses. Phage display-derived antibodies showed enhanced specificity towards ZIKV recombinant NS1 (rNS1). Conversely, hybridoma-derived antibodies cross-reacted with other flavivirus rNS1. This likely reflects differences in selection strategies rather than in the intrinsic properties of the technologies. Despite the moderate binding affinity of paired antibodies obtained by phage display, a limit of detection of 25ng/mL was achieved in human serum spiked with ZIKV rNS1. This sensitivity dropped to 1ng/mL with antibodies obtained by mice immunization. Then, the high specificity of phage display-derived antibodies and sensitivity of hybridoma-derived antibodies were combined in a hybrid antibody format immunoassay. In this assay, a very specific phage display-derived antibody was used for capture and a hybridoma-derived antibody with a low dissociation rate constant (koff) was used for detection, maintaining sensitivity as low as 1ng/mL. This hybrid assay format also preserved high specificity when tested using serum samples from patients with acute Dengue fever that contain high levels of Dengue virus NS1. Our findings highlight the complementary strengths of both antibody generation strategies for the sensitive and specific detection of ZIKV NS1.

Ribosome display presents an innovative in vitro technology for the rapid isolation and evolution of high-affinity peptides or proteins. Displayed proteins are bound to and recovered from target molecules in multiple rounds of selection in order to enrich for specific binding proteins. No transformation step is necessary, which could lead to a loss of library diversity. A cycle of display and selection can be performed in one day, enabling the existing gene repertoire to be rapidly scanned. Proteins isolated from the panning rounds can be further modified through random or directed molecular evolution for affinity maturation, as well as selected for characteristics such as protein stability, folding and functional activity. Recently, the field of display technologies has become more prominent due to the generation of new scaffolds for ribosome display, isolation of high-affinity human antibodies by phage display, and their implementation in the discovery of novel protein–protein interactions. Applications for this technology extend into the broad field of antibody engineering, proteomics, and synthetic enzymes for diagnostics and therapeutics in cancer, autoimmune and infectious diseases, neurodegenerative diseases and inflammatory disorders. This review highlights the role of ribosome display in drug discovery, discusses advantages and disadvantages of the system, and attempts to predict the future impact of ribosome display technology on the development of novel engineered biopharmaceutical products for biological therapies.

Introduction Nanobodies (Nb) are single‐domain antibodies derived from camelids and are one‐tenth the size of normal antibodies. Various methods have been developed to obtain potent Nb using phage display and yeast display, but the processes of introducing mutations and evaluating Nb mutants are separated, making Nb acquisition a relatively time‐consuming and costly process. Recently, in vivo continuous evolution, in which genes are diversified within an organism, has been focused on as a means to accelerate the improvement of protein function. In in vivo continuous evolution, mutagenesis enzymes accumulate mutations in the target gene, and the target gene is continuously diversified simply by culturing the organism. Although in vivo continuous evolution is useful, it is not compatible enough with display technologies. For example, in yeast display, Nb is expressed as a fusion protein with a scaffold protein to be immobilized on the yeast cell wall. If the scaffold protein is mutated, the display itself does not occur. In this study, we have developed a method for binding scaffold proteins expressed from different gene cassettes to Nb in yeast and presenting them on the yeast surface using SpyTag/SpyCatcher [1] protein ligation, which irreversibly forms isopeptide bonds. This method can be integrated with in vivo continuous evolution because Nb can be expressed separately from the scaffold proteins required for the surface display system, allowing selective directed evolution of Nb alone. Methods As a model, we generated a yeast strain which produced anti‐lysozyme Nb fused with SpyTag (ST, 16‐residue) and a 649‐stalk scaffold protein fused with an epitope tag and SpyCatcher (SC, 113‐residue). To investigate potential crosstalk between yeast cells, two yeast strains, which produced different Nbs and epitope tag, were co‐cultured and stained with anti‐epitope antibody and fluorescently labeled lysozyme for microscopic observation. To confirm that our display method can be used for library screening, we attempted to enrich the target yeast cells using a flow cytometer. We generated a mixture sample of yeast cells displaying anti‐lysozyme Nb or Nb that does not bind lysozyme. The sample was mixed with fluorescently labeled lysozyme, and fluorescence‐positive cells were sorted. Results and Discussion Yeast cells producing anti‐lysozyme Nb fused with SpyTag (ST) and 649‐stalk scaffold protein fused with an epitope tag and SpyCatcher (SC) were stained with anti‐HA antibody and fluorescently labeled lysozyme, and we confirmed that 93% of the yeast cells successfully displayed anti‐lysozyme Nb on the yeast cell surface. We observed no crosstalk between yeast cells. In the enrichment experiment, we successfully confirmed that the fluorescence‐positive cells were enriched to about 94% by using a flow cytometer. In conclusion, our technique is expected to be an efficient cell surface display platform which can be integrated with in vivo continuous evolution[2]. [1] Anthony. et al. PNAS , Dec 2019, 116, 26523. [2] Kajiwara et al. submitted.

Phage display technology has become an important research tool in biological research, fundamentally changing the traditional monoclonal antibody preparation process, and has been widely used in the establishment of antigen-antibody libraries, drug design, vaccine research, pathogen detection, gene therapy, antigenic epitope research, and cellular signal transduction research.The phage display is a powerful platform for technology development. Using phage display technology, single chain fragment variable (scFv) can be screened, replacing the disadvantage of the large size of traditional antibodies. Phage display single chain antibody libraries have significant biological implications. Here we describe the types of antibodies, including chimeric antibodies, bispecific antibodies, and scFvs. In addition, we describe the phage display system, phage display single chain antibody libraries, screening of specific antibodies by phage libraries and the application of phage libraries.

Specific molecular markers for various normal and pathogenic cell states and cell types provide knowledge of basic biological systems and have a direct application in targeted therapy. We describe a proteomic method based on the combination of new and improved phage display antibody technologies and mass spectrometry that allows identification of cell type-specific protein markers. The most important features of the method are (i) reduction of experimental noise originating from background binding of phage particles and (ii) isolation of affinity binders after a single round of selection, which assures a high diversity of binders. The method demonstrates, for the first time, the ability to detect, identify, and analyze both secreted and membrane-associated extracellular proteins as well as a variety of different cellular structures including proteins and carbohydrates. The optimized phage display method was applied to analysis of human skin keratinocytes resulting in the isolation of a panel of antibodies. Fourteen of these antibodies were further characterized, half of which predominantly recognized keratinocytes in a screen of a range of different cell types. Three cognate keratinocyte antigens were subsequently identified by mass spectrometry as laminin-5, plectin, and fibronectin. The combination of phage display technology with mass spectrometry methods for protein identification is a general and promising approach for proteomic analysis of cell surface complexity.

In-vitro protein evolution by ribosome display and mRNA display

The antibody display technology (ADT) such as phage display (PD) has substantially improved the production of monoclonal antibodies (mAbs) and Ab fragments through bypassing several limitations associated with the traditional approach of hybridoma technology. In the current study, we capitalized on the PD technology to produce high affinity single chain variable fragment (scFv) against tumor necrosis factor-alpha (TNF- α), which is a potent pro-inflammatory cytokine and plays important role in various inflammatory diseases and malignancies. To pursue production of scFv antibody fragments against human TNF- α, we performed five rounds of biopanning using stepwise decreased amount of TNF-α (1 to 0.1 μ g), a semi-synthetic phage antibody library (Tomlinson I + J) and TG1 cells. Antibody clones were isolated and selected through enzyme-linked immunosorbent assay (ELISA) screening. The selected scFv antibody fragments were further characterized by means of ELISA, PCR, restriction fragment length polymorphism (RFLP) and Western blot analyses as well as fluorescence microscopy and flow cytometry. Based upon binding affinity to TNF-α , 15 clones were selected out of 50 positive clones enriched from PD in vitro selection. The selected scFvs displayed high specificity and binding affinity with Kd values at nm range to human TNF-α . The immunofluorescence analysis revealed significant binding of the selected scFv antibody fragments to the Raji B lymphoblasts. The effectiveness of the selected scFv fragments was further validated by flow cytometry analysis in the lipopolysaccharide (LPS) treated mouse fibroblast L929 cells. Based upon these findings, we propose the selected fully human anti-TNF-α scFv antibody fragments as potential immunotherapy agents that may be translated into preclinical/clinical applications.

We combined cell-free ribosome display and cell-based yeast display selection to build specific protein binders to the extracellular domain of the human interleukin 9 receptor alpha (IL-9Rα). The target, IL-9Rα, is the receptor involved in the signalling pathway of IL-9, a pro-inflammatory cytokine medically important for its involvement in respiratory diseases. The successive use of modified protocols of ribosome and yeast displays allowed us to combine their strengths-the virtually infinite selection power of ribosome display and the production of (mostly) properly folded and soluble proteins in yeast display. The described experimental protocol is optimized to produce binders highly specific to the target, including selectivity to common proteins such as BSA, and proteins potentially competing for the binder such as receptors of other cytokines. The binders were trained from DNA libraries of two protein scaffolds called 57aBi and 57bBi developed in our laboratory. We show that the described unconventional combination of ribosome and yeast displays is effective in developing selective small protein binders to the medically relevant molecular target.

Mimotopes are peptides mimicking an epitope. They can readily be selected out from phage-displayed random peptides libraries and have been widely used, especially in epitope mapping. There are now several computational tools available for the mimotope based epitope mapping. Most of these tools implicitly take peptides acquired through phage display as mimotopes in an extended conformation and map the sequential neighbor residue pair of the mimotopes to the spatial neighbor residue pair on the antigen structure. In this study, we evaluated the implicit assumptions above. The results show that mimotopes have many conformations and the indirect sequential neighbors separated by one amino acid should also map to spatial neighbors, indicating there is information loss with these tools. Furthermore, all the existing tools do not have a noise sifting procedure, though peptides acquired through phage display can be target unrelated peptides also. Thus, the risk of noise inclusion may exist. Our results may be helpful for developing new tools with improved performance on mimotope mapping.

Phage display has been shown to facilitate greatly the selection of polypeptides with desired properties by establishing a direct link between the polypeptide and the gene that encodes it. However, selection for catalytic activities displayed on phage remains a challenge, since reaction products diffuse away from the enzyme and make it difficult to recover catalytically active phage-enzymes. We have recently described a selection methodology in which the reaction substrate (and eventually the reaction product) is anchored on calmodulin-tagged phage-enzymes by means of a calmodulin binding peptide. Phage displaying a catalytic activity are physically isolated by means of affinity reagents specific for the product of reaction. In this study, we investigated the efficiency of selection for catalysis by phage display, using a ligase (the Escherichia coli biotin ligase BirA) and an endopeptidase (the rat trypsin His57--> Ala mutant) as model enzymes. These enzymes could be displayed on phage as fusion proteins with calmodulin and the minor coat protein pIII. Both the display of functional enzyme and the efficiency of selection for catalysis were significantly improved by using phage vectors, rather than phagemid vectors. In model selection experiments, phage displaying BirA were consistently enriched (between 4-fold and 800-fold) per round of panning, relative to negative controls. Phage displaying the trypsin His57-->Ala mutant, a relatively inefficient endopeptidase which cleaves a specific dipeptide sequence, were enriched (between 15-fold and 2000-fold), relative to negative controls. In order to improve the catalytic properties of the trypsin His57-->Ala mutant, we constructed a combinatorial phage display library of trypsin mutants. Selection of catalytically active phage-enzymes was evidentiated by increasing phage titres at the different rounds of panning relative to negative control selections, but mutants with catalytic properties superior to those of trypsin His57-->Ala mutant could not be isolated. The results obtained provide evidence that catalytic activities can be recovered using phage display technology, but stress the importance of both library design and stringent biopanning conditions for the recovery of novel enzymes.

Phage display technology uses the interaction between nanomaterials and phage display polypeptides, playing an important role in the selection of peptides on the surface of nanomaterials.

Although yeast two-hybrid assay and biochemical methods combined with mass spectrometry have been successfully employed for the analyses of protein-protein interactions in the field of proteomics, these methods encounter various difficulties arising from the usage of living cells, including inability to analyze toxic proteins and restriction of testable interaction conditions. Totally in vitro display technologies such as ribosome display and mRNA display are expected to circumvent these difficulties. In this study, we applied an mRNA display technique to screening for interactions of a basic leucine zipper domain of Jun protein in a mouse brain cDNA library. By performing iterative affinity selection and sequence analyses, we selected 16 novel Jun-associated protein candidates in addition to four known interactors. By means of real-time PCR and pull-down assay, 10 of the 16 newly discovered candidates were confirmed to be direct interactors with Jun in vitro. Furthermore, interaction of 6 of the 10 proteins with Jun was observed in cultured cells by means of co-immunoprecipitation and observation of subcellular localization. These results demonstrate that this in vitro display technology is effective for the discovery of novel protein-protein interactions and can contribute to the comprehensive mapping of protein-protein interactions.

Camelids produce functional antibodies devoid of light chains of which the single N-terminal domain is fully capable of antigen binding. These single-domain antibody fragments (VHHs or Nanobodies®) have several advantages for biotechnological applications. They are well expressed in microorganisms and have a high stability and solubility. Furthermore, they are well suited for construction of larger molecules and selection systems such as phage, yeast, or ribosome display. This minireview offers an overview of (1) their properties as compared to conventional antibodies, (2) their production in microorganisms, with a focus on yeasts, and (3) their therapeutic applications.

Ribsosome display is a PCR-based in vitro display technology that it well suited for the selection and evolution of high-affinity antibodies. In particular, ribosome display lends itself to the evolution of functional characteristics, such as potency, and thereby facilitates the production of therapeutic antibodies from lead candidates. In this chapter, we describe how to mature large phage display antibody populations (>10(7)) by performing increasingly stringent selections with decreasing antigen concentration. This process takes advantage of ribosome display's intrinsic ability to evolve sequence during selection. Ribosome display can also be used as a complementary tool to phage display for isolating high-affinity antibodies from naïve libraries. Ultimately, maturation of large antibody populations by ribosome display will help to speed up the process of generating antibody therapeutics.

Nanobodies (Nbs) can be successfully retrieved following phage, bacterial, yeast, or ribosome display of immune, synthetic, or naïve libraries. However, after panning, multiple individual Nb clones need to be screened and assessed for solubility, antigen specificity, affinity, and potential biological function. Therefore, it is highly desirable to have a convenient expression strategy to obtain sufficient protein for in-depth characterization of the Nbs. The presence of a purification and detection tag, as well as a chemically reactive group to enable simple generation of Nb derivatives, would be of great help in this regard. Here, we provide a general protocol for high yield cytoplasmic expression and purification of formylglycine generating enzyme (FGE)-tagged Nbs. The cysteine within the FGE tag is easily converted to formylglycine by passing the FGE-tag containing Nb over a continuous-flow bio-catalysis system. The aldehyde group within the formylglycine side chain at the C-terminal end of the Nb is suitably located for subsequent bio-orthogonal reactions to fluorescent dyes, biotin, polyethylene glycol, or chromatography resins. We also include methods for production of high yield recombinant FGE, as well as conditions for its immobilization on Sepharose to produce the continuous-flow bio-catalysis system.