•Antigen presentation strategy is important for the antibody selection campaign.•Phage display selection strategy can be tailored to individual selection campaigns.•Specialized libraries can be ...constructed to benefit specific discovery projects.•The selection strategy determines the characteristics of the discovered antibodies.
Phage display technology can be used for the discovery of antibodies for research, diagnostic, and therapeutic purposes. In this review, we present and discuss key parameters that can be optimized when performing phage display selection campaigns, including the use of different antibody formats and advanced strategies for antigen presentation, such as immobilization, liposomes, nanodiscs, virus-like particles, and whole cells. Furthermore, we provide insights into selection strategies that can be used for the discovery of antibodies with complex binding requirements, such as targeting a specific epitope, cross-reactivity, or pH-dependent binding. Lastly, we provide a description of specialized phage display libraries for the discovery of bispecific antibodies and pH-sensitive antibodies. Together, these methods can be used to improve antibody discovery campaigns against all types of antigens.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Phage display enables to screen probes such as peptide, antibody and nanobody with high specificity and affinity to the target, providing excellent probes for various analytical or biosensing ...platforms. Furthermore, by employing techniques like as genetic engineering and chemical modification, it is possible to conveniently functionalize these ligands for analytical chemistry with selectivity and sensitivity. This review initially presents the fundamental aspects of phage biology that are commonly employed in phage display vectors, elaborates the construction process of phage display peptide library and phage display antibody library, and evaluates different biopanning strategies. Then from the viewpoint of analytes, this paper summarizes phage display based biosensing or bioanalysis of a wide range of target molecules. This paper presents knowledge insights and research foundations for the identification of additional phage display probes and their subsequent application in biosensing or bioanalysis with excellent performance.
•An overview of phage-display to screen peptide, antibody and nanobody.•Phage display derived probes in biosensing are summarized.•Challenges and perspectives in phage display and biosensing application envisioned.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The low response rate and adaptive resistance of PD‐1/PD‐L1 blockade demands the studies on novel therapeutic targets for cancer immunotherapy. We discovered that a novel immune checkpoint TIGIT ...expressed higher than PD‐1 in many tumors especially anti‐PD‐1 resistant tumors. Here, mirror‐image phage display bio‐panning was performed using the d‐enantiomer of TIGIT synthesized by hydrazide‐based native chemical ligation. d‐peptide DTBP‐3 was identified, which could occupy the binding interface and effectively block the interaction of TIGIT with its ligand PVR. DTBP‐3 showed proteolytic resistance, tumor tissue penetrating ability, and significant tumor suppressing effects in a CD8+ T cell dependent manner. More importantly, DTBP‐3 could inhibit tumor growth and metastasis in anti‐PD‐1 resistant tumor model. This is the first d‐peptide targeting TIGIT, which could serve as a potential candidate for cancer immunotherapy.
The d‐peptide DTBP‐3 was identified, which could effectively block TIGIT/PVR interaction. DTBP‐3 could inhibit tumor growth and metastasis in anti‐PD‐1 resistant tumor model and could serve as a potential candidate for cancer immunotherapy.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Nanobodies that are small and thermally stable, as well as have high expression level, are leading alternative to produce anti-idiotypic antibodies. These antibodies have the advantage of replacing ...mycotoxins and their conjugates for immunoassays. In this work, anti-fumonisin B1 (FB1) monoclonal antibody (mAb) was used as the target for biopanning from a naïve alpaca nanobody (Nb) phage display library. After three cycles of panning, one anti-idiotypic nanobody (Ab2β Nb) was isolated and subjected to a Nb-ELISA for the detection of FB1. Surface plasmon resonance was used to analyze the reaction kinetics between the Ab2β Nb and anti-FB1 mAb. The developed assay showed a half inhibitory concentration (IC50) of 0.95±0.12ng/mL, a limit of detection of 0.15ng/mL, a linear range of 0.27–5.92ng/mL, and a low cross-reactivity toward FB2 of 4.93%. The sensitivity was enhanced approximately 20-fold compared with that of the chemosynthetic FB1–BSA conjugates. The equilibrium dissociation constant (KD) measured for Ab2β Nb: anti-FB1 mAb was 164.6nM. The assay was compared with conventional ELISA (the commercial ELISA kit), and the results indicated the reliability of Ab2β Nb replacing the antigen–carrier protein conjugates. The use of biotechnology in developing the surrogate is an ideal strategy for replacing conventional synthesized antigens.
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•We evidenced that Ab2β Nbs can be used as a substitute for hapten-conjugates.•We developed and optimized a green and rapid Nb-ELISA for the determination of FB1.•The sensitivity of Nb-ELISA was improved 20-fold than that of the hapten-conjugate.•Ab2β Nb showed lower affinity than the hapten-conjugates to mAb.•Ab2β Nb was more fit for working as coating antigen than the hapten-conjugates.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The unique class of heavy chain-only antibodies, present in Camelidae, can be shrunk to just the variable region of the heavy chain to yield VHHs, also called nanobodies. About one-tenth the size of ...their full-size counterparts, nanobodies can serve in applications similar to those for conventional antibodies, but they come with a number of signature advantages that find increasing application in biology. They not only function as crystallization chaperones but also can be expressed inside cells as such, or fused to other proteins to perturb the function of their targets, for example, by enforcing their localization or degradation. Their small size also affords advantages when applied in vivo, for example, in imaging applications. Here we review such applications, with particular emphasis on those areas where conventional antibodies would face a more challenging environment.
Glucose in biological cells is metabolized and exists as glucose-6-phosphate (G6P). A G6P biosensor was fabricated using a combination of phage display and autodisplay technologies for the detection ...of G6P. Active G6P dehydrogenase (G6PDH) was expressed and anchored to the outer membrane of Escherichia coli (E. coli) cells using autodisplay technology. Autodisplayed G6PDH exhibited high activity as confirmed by OM protein analysis. A conductive nanomesh layer was formed using a phage display to enhance the direct electron transfer of the enzyme. G6PDH autodisplaying E. coli cells were then immobilized on the layer using a polyelectrolyte interlayer. The nicotinamide adenine dinucleotide phosphate redox pairs in the fabricated electrode of the autodisplayed G6PDH exhibited a redox reaction, resulting in a significant increase in the peak currents in the presence of G6P. The G6P biosensor exhibited a wide dynamic range with high sensitivity, exhibiting linear responses in the ranges of 0.1 fM – 10 nM and 10 nM – 100 μM. The biosensor detected G6P without interference from other sugar molecules. The combination of phage display and autodisplay technologies is a promising approach for the fabrication of highly sensitive and specific biosensors.
•First biosensor utilizing two surface displays, phage display and autodisplay.•Phage display based nanomesh enhanced the direct electron transfer of the enzyme.•Autodisplay utilized for simultaneous expression and immobilization of enzyme.•Sensitive glucose-6-phosphate detection with wide linear range.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Most antibodies used in immunohistochemistry (IHC) have been developed by animal immunization. We wanted to explore naive antibody repertoires displayed on filamentous phages as a source of ...full-length antibodies for IHC on Formalin-Fixed and Paraffin-Embedded (FFPE) tissues. We used two isogenic mouse fibroblast cell lines that express or not human HER2 to generate positive and negative FFPE pseudo-tissue respectively. Using these pseudo-tissues and previously described approaches based on differential panning, we isolated very efficient antibody clones, but not against HER2. To optimize HER2 targeting and tissue specificity, we first performed 3-4 rounds of in vitro panning using recombinant HER2 extracellular domain (ECD) to enrich the phage library in HER2 binders, followed by one panning round using the two FFPE pseudo-tissues to retain binders for IHC conditions. We then analyzed the bound phages using next-generation sequencing to identify antibody sequences specifically associated with the HER2-positive pseudo-tissue. Using this approach, the top-ranked clone identified by sequencing was specific to the HER2-positive pseudo-tissue and showed a staining pattern similar to that of the antibody used for the clinical diagnosis of HER2-positive breast cancer. However, we could not optimize staining on other tissues, showing that specificity was restricted to the tissue used for selection and screening. Therefore, future optimized protocols must consider tissue diversity early during the selection by panning using a wide collection of tissue types.Most antibodies used in immunohistochemistry (IHC) have been developed by animal immunization. We wanted to explore naive antibody repertoires displayed on filamentous phages as a source of full-length antibodies for IHC on Formalin-Fixed and Paraffin-Embedded (FFPE) tissues. We used two isogenic mouse fibroblast cell lines that express or not human HER2 to generate positive and negative FFPE pseudo-tissue respectively. Using these pseudo-tissues and previously described approaches based on differential panning, we isolated very efficient antibody clones, but not against HER2. To optimize HER2 targeting and tissue specificity, we first performed 3-4 rounds of in vitro panning using recombinant HER2 extracellular domain (ECD) to enrich the phage library in HER2 binders, followed by one panning round using the two FFPE pseudo-tissues to retain binders for IHC conditions. We then analyzed the bound phages using next-generation sequencing to identify antibody sequences specifically associated with the HER2-positive pseudo-tissue. Using this approach, the top-ranked clone identified by sequencing was specific to the HER2-positive pseudo-tissue and showed a staining pattern similar to that of the antibody used for the clinical diagnosis of HER2-positive breast cancer. However, we could not optimize staining on other tissues, showing that specificity was restricted to the tissue used for selection and screening. Therefore, future optimized protocols must consider tissue diversity early during the selection by panning using a wide collection of tissue types.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Bacterial quorum sensing (QS) is a well-known behavior for bacterial communication and plays an important role in various bacterial biological functions. Several approaches have been developed to ...interrupt the QS mechanism in bacterial pathogens. This study developed a novel, high-throughput screening method combined with phage peptide display libraries and a QS biosensor system (PDQBS) to identify quorum-sensing inhibitors (QSIs). We screened 11 hit peptide candidates for QSI activity, which inhibited the C4-AHL mediated quorum-sensing system. Although the synthetic peptides showed moderate inhibitory effects and six candidate peptides ligated with a cell-penetrating peptide (CPP) dramatically inhibited QS signals. The protective efficacies of candidate DNA expression vector ligated with CPP-QSIs were tested against Aeromonas hydrophila infection in the zebrafish model. The obtained result revealed that at least two peptides (CPP21 and CPP23) exhibited an increased protective ability against A. hydrophila infection with RPSs of >50%. Furthermore, the three candidates CPP-QSIs (CPP21, CPP23, and CPP24), also showed high protective abilities with RPSs of >50% against A. hydrophila infection in the mice model. The qPCR analysis further confirmed that these selected CPP-QSIs might stimulate host immunity and inhibit the virulence of A. hydrophila. Finally, the results of dot blot and ELISA assays indicated that these CPP-QSI peptides could bind to the QS receptor protein, AhyR/CviR. Overall, this study provides a promising approach to screening novel QSI candidates for controlling QS-mediated bacterial infections.
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•A novel PDQSR method is designed to screen QSI peptides.•CPP-QSI peptides can inhibit violacein production of CV260.•CPP-QSI peptides display anti-infection abilities in zebrafish and mice.•CPP-QSI peptides bind with the AhyR protein of A. hydrophila.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
