Phage-displayed Peptide Library Research Articles

A peptide targeting outer membrane protein A of Acinetobacter baumannii exhibits antibacterial activity by reducing bacterial pathogenicity.

The World Health Organization has classified multidrug-resistant (MDR) Acinetobacter baumannii as a significant threat to human health, necessitating the urgent discovery of new antibacterial drugs to combat bacterial resistance. Outer membrane protein A of A. baumannii (AbOmpA) is an outer membrane-anchored β-barrel-shaped pore protein that plays a critical role in bacterial adhesion, invasion, and biofilm formation. Therefore, AbOmpA is considered a key virulence factor of A. baumannii. Herein, we screened three phage display peptide libraries targeting AbOmpA and identified several peptides. Among them, P92 (amino acid sequence: QMGFMTSPKHSV) exhibited the highest binding affinity with AbOmpA, with a KD value of 7.84 nM. In vitro studies demonstrated that although P92 did not directly inhibit bacterial growth, it significantly reduced the invasion and adhesion capabilities of multiple clinical isolates of MDR A. baumannii and concentration-dependently inhibited biofilm formation by acting on OmpA. Furthermore, the polymerase chain reaction results confirmed a significant positive correlation between the antibacterial effect of P92 and OmpA expression levels. Encouragingly, P92 also displayed remarkable therapeutic efficacy against A. baumannii infection in various models, including an in vitro cell infection model, a mouse skin infection model, and a mouse sepsis model. These results highlight P92 as a novel and highly effective antimicrobial molecule specifically targeting the virulence factor AbOmpA.

Discovery of broad-spectrum high-affinity peptide ligands of spike protein for the vaccine purification of SARS-CoV-2 and Omicron variants

To combat with emerging SARS-CoV-2 variants of concern (VOCs), we report the identification of a set of unique HWK-motif peptide ligands for the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein from a phage-displayed peptide library. These HWK-motif peptides exhibited nanomolar affinity for RBD. Among them, the peptide, HWKAVNWLKPWT (SP-HWK), had not only the highest affinities for RBD and trimer S protein, but also broad-spectrum affinities for RBDs from VOCs. Molecular dynamics simulations and competitive ELISA revealed a conserved pocket between the cryptic and the outer faces of RBD for SP-HWK binding, distinct from the human angiotensin-converting enzyme 2 receptor binding site. By coupling SP-HWK to agarose gel, the as-prepared affinity gel could efficiently capture RBD and trimer S from the ancestral strain and the Omicron variant, and the bound targets could be recovered by mild elution at pH 6.0. More importantly, the affinity gel presented excellent and stable chromatographic performance in the purification of inactivated SARS-CoV-2 and Omicron vaccines, affording high yields and purities, and strong HCP reduction. The results demonstrated the potential of SP-HWK as a broad-spectrum peptide ligand for developing a universal platform for the vaccine purification of SARS-CoV-2 and VOCs.

A novel delta opioid receptor specific peptide reduces craving in an animal model of cocaine seeking

Substance use disorder, and particularly cocaine use disorder, is a complex disease that affects societal, economic, and psychological factors. Endogenous β-endorphin released after prolonged cocaine withdrawal has been reported to activate the accumbal delta-opioid receptor (DOR), leading to attenuated cocaine seeking. However, using DOR β-endorphin activation to treat cocaine use disorder is impractical since β-endorphin does not cross the blood-brain barrier. Also, only activation of the sub-group DOR1 efficiently attenuates craving, as activation of DOR2 yields an opposite effect. Here, we isolated a specific peptide, PEP1, from a phage display peptide library with similar biological properties to β–endorphin, demonstrating specificity for DOR1 and functioning as full receptor agonists. Our pharmacodynamic results showed fast trafficking incorporation of DOR into the cell membrane, interpreted as superior rehabilitation of the receptor and its bioavailability compared to commercial agonists. We administered PEP1, either intrabrain or intranasal, to rats trained to self-administer cocaine. PEP1 induced a significant decrease in cocaine-craving behavior and reinstatement in three different animal models of addiction. Also, PEP1 did not exhibit rewarding properties and did not interfere with the natural reward system. ICP-OES analysis revealed that at least one hour post-administration, PEP1 was retained in the brain rather than in peripheral organs. These findings render PEP1 a potential novel regulator of cocaine craving, especially for being non-addictive. Hence, PEP1 should be further examined as a possible new therapy for substance use disorder.

Diversification of Phage-Displayed Peptide Libraries with Noncanonical Amino Acid Mutagenesis and Chemical Modification.

Sitting on the interface between biologics and small molecules, peptides represent an emerging class of therapeutics. Numerous techniques have been developed in the past 30 years to take advantage of biological methods to generate and screen peptide libraries for the identification of therapeutic compounds, with phage display being one of the most accessible techniques. Although traditional phage display can generate billions of peptides simultaneously, it is limited to expression of canonical amino acids. Recently, several groups have successfully undergone efforts to apply genetic code expansion to introduce noncanonical amino acids (ncAAs) with novel reactivities and chemistries into phage-displayed peptide libraries. In addition to biological methods, several different chemical approaches have also been used to install noncanonical motifs into phage libraries. This review focuses on these recent advances that have taken advantage of both biological and chemical means for diversification of phage libraries with ncAAs.

Predicting inhibitor development using a random peptide phage-display library approach in the SIPPET cohort

AbstractInhibitor development is the most severe complication of hemophilia A (HA) care and is associated with increased morbidity and mortality. This study aimed to use a novel immunoglobulin G epitope mapping method to explore the factor VIII (FVIII)–specific epitope profile in the SIPPET cohort population and to develop an epitope mapping–based inhibitor prediction model. The population consisted of 122 previously untreated patients with severe HA who were followed up for 50 days of exposure to FVIII or 3 years, whichever occurred first. Sampling was performed before FVIII treatment and at the end of the follow-up. The outcome was inhibitor development. The FVIII epitope repertoire was assessed by means of a novel random peptide phage-display assay. A least absolute shrinkage and selection operator (LASSO) regression model and a random forest model were fitted on posttreatment sample data and validated in pretreatment sample data. The predictive performance of these models was assessed by the C-statistic and a calibration plot. We identified 27 775 peptides putatively directed against FVIII, which were used as input for the statistical models. The C-statistic of the LASSO and random forest models were good at 0.78 (95% confidence interval [CI], 0.69-0.86) and 0.80 (95% CI, 0.72-0.89). Model calibration of both models was moderately good. Two statistical models, developed on data from a novel random peptide phage display assay, were used to predict inhibitor development before exposure to exogenous FVIII. These models can be used to set up diagnostic tests that predict the risk of inhibitor development before starting treatment with FVIII.

Development of New CD38 Targeted Peptides for Cancer Imaging

PurposeMultiple myeloma (MM) affects over 35,000 patients each year in the US. There remains a need for versatile Positron Emission Tomography (PET) tracers for the detection, accurate staging, and monitoring of treatment response of MM that have optimal specificity and translational attributes. CD38 is uniformly overexpressed in MM and thus represents an ideal target to develop CD38-targeted small molecule PET radiopharmaceuticals to address these challenges.ProceduresUsing phage display peptide libraries and pioneering algorithms, we identified novel CD38 specific peptides. Imaging bioconjugates were synthesized using solid phase peptide chemistry, and systematically analyzed in vitro and in vivo in relevant MM systems.ResultsThe CD38-targeted bioconjugates were radiolabeled with copper-64 (64Cu) with100% radiochemical purity and an average specific activity of 3.3 – 6.6 MBq/nmol. The analog NODAGA-PEG4-SL022-GGS (SL022: Thr-His-Tyr-Pro-Ile-Val-Ile) had a Kd of 7.55 ± 0.291 nM and was chosen as the lead candidate. 64Cu-NODAGA-PEG4-SL022-GGS demonstrated high binding affinity to CD38 expressing human myeloma MM.1S-CBR-GFP-WT cells, which was blocked by the non-radiolabeled version of the peptide analog and anti-CD38 clinical antibodies, daratumumab and isatuximab, by 58%, 73%, and 78%, respectively. The CD38 positive MM.1S-CBR-GFP-WT cells had > 68% enhanced cellular binding when compared to MM.1S-CBR-GFP-KO cells devoid of CD38. Furthermore, our new CD38-targeted radiopharmaceutical allowed visualization of tumors located in marrow rich bones, remaining there for up to 4 h. Clearance from non-target organs occurred within 60 min. Quantitative PET data from a murine disseminated tumor model showed significantly higher accumulation in the bones of tumor-bearing animals compared to tumor-naïve animals (SUVmax 2.06 ± 0.4 versus 1.24 ± 0.4, P = 0.02). Independently, tumor uptake of the target compound was significantly higher (P = 0.003) compared to the scrambled peptide, 64Cu-NODAGA-PEG4-SL041-GGS (SL041: Thr-Tyr-His-Ile-Pro-Ile-Val). The subcutaneous MM model demonstrated significantly higher accumulation in tumors compared to muscle at 1 and 4 h after tracer administration (SUVmax 0.8 ± 0.2 and 0.14 ± 0.04, P = 0.04 at 1 h; SUVmax 0.89 ± 0.01 and 0.09 ± 0.01, P = 0.0002 at 4 h).ConclusionsThe novel CD38-targeted, radiolabeled bioconjugates were specific and allowed visualization of MM, providing a starting point for the clinical translation of such tracers for the detection of MM.

Modification of a Selective NTRK2 Agonist and Confirmation of Activity in a Glaucoma-on-a-Chip Model.

RNYK is a selective agonist of the neurotrophic tyrosine kinase receptor type 2 (NTRK2) which has been screened from a phage-displayed peptide library. Its sequence is SGVYKVAYDWQH, similar to a native NTRK2 ligand, that is, brain-derived neurotrophic factor (BDNF). The current study was performed to recognize and confirm critical residues for RNYK activity in a glaucoma-on-a-chip model. We designed a modified RNYK (mRNYK) peptide based on hotspots of the RNYK sequence identified by alanine scanning. The critical residues consisted of tyrosine, valine, aspartic acid, and tryptophan (YVDW); however, lysine and glutamine were also maintained in the final sequence (YKVDWQ) for forming amide bonds and peptide dimerization. The affinity of mRNYK binding was confirmed by testing against NTRK2 receptors on the surface of ATRA-treated SH-SY5Y cells. The neuroprotective effect of mRNYK was also evaluated in cell culture after elevated pressure insult in a glaucoma-on-a-chip model. The primary amine on the lysine side-chain from one sequence (YKVDWQ) reacted with a γ-carboxamide group of glutamine from the other sequence, forming dimeric mRNYK. In silico, molecular dynamic simulations of the mRNYK-NTRK2 complex showed more stable and stronger interactions as compared to the RNYK-NTRK2 complex. In vitro, mRNYK demonstrated a neuroprotective effect on SH-SY5Y cells under normal and elevated pressure comparable to RNYK. The 50% effective concentration (logEC50) for mRNYK was 0.7009, which was better than RNYK with a logEC50 of 0.8318. The modified peptide studied herein showed improved stability over the original peptide (RNYK) and demonstrated potential for use as a BDNF agonist with neuroprotective properties for treatment of neurodegenerative disorders such as glaucoma.

Phage Display Identified Novel Leydig Cell Homing Peptides for Testicular Targeting.

Conventional drug delivery methods to treat testicular disorders face various challenges, which could be circumvented by using targeted drug delivery. Testicular cell targeting ligands, such as Leydig cell homing peptides, would be an excellent choice to achieve the targeted delivery of drugs to the testis. In this study, Leydig cell homing peptides (LCHPs), LCHP1 and LCHP2, were identified via in vitro, followed by in vivo biopanning of a phage display peptide library and next-generation sequencing. Both of the LCHPs were validated in vitro for their specific Leydig cell and in vivo testis targeting potential. Furthermore, molecular targets of the LCHP1 and LCHP2 were identified using affinity purification mass spectrometry (APMS). The LCHP1 and LCHP2 are able to specifically target Leydig cells of the testis and undergo cell internalization as well as target the testis at the in vivo level, hence providing an opportunity to be utilized as a potential ligand for drug delivery to the testis.

Mirror-Image Phage Display for the Selection of D-Amino Acid Peptide Ligands as Potential Therapeutics.

In neurodegenerative diseases like Alzheimer's disease (AD), endogenous proteins or peptides aggregate with themselves. These proteins may lose their function or aggregates and/or oligomers can obtain toxicity, causing injury or death to cells. Aggregation of two major proteins characterizes AD. Amyloid-β peptide (Aβ) is deposited in amyloid plaques within the extracellular space of the brain and Tau in so-called neurofibrillary tangles in neurons. Finding peptide ligands to halt protein aggregation is a promising therapeutical approach. Using mirror-image phage display with a commercially available, randomized 12-mer peptide library, we have selected D-amino acid peptides, which bind to the Tau protein and modulate its aggregation in vitro. Peptides can bind specifically and selectively to a target molecule, but natural L-amino acid peptides may have crucial disadvantages for in vivo applications, as they are sensitive to protease degradation and may elicit immune responses. One strategy to circumvent these disadvantages is the use of non-naturally occurring D-amino acid peptides as they exhibit increased protease resistance and generally do not activate the immune system. To perform mirror-image phage display, the target protein needs to be synthesized as D-amino acid version. If the target protein sequence is too long to be synthesized properly, smaller peptides derived from the full length protein can be used for the selection process. This also offers the possibility to influence the binding region of the selected D-peptides in the full-length target protein. Here we provide the protocols for mirror-image phage display selection on the PHF6* peptide of Tau, based on the commercially available Ph.D.™-12 Phage Display Peptide Library Kit, leading to D-peptides that also bind the full length Tau protein (Tau441), next to PHF6*. In addition, we provide protocols and data for the first characterization of those D-peptides that inhibit Tau aggregation in vitro. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Mirror image phage display selection against D-PHF6* fibrils Support Protocol 1: Single phage ELISA Basic Protocol 2: Sequencing and D-peptide generation Basic Protocol 3: Thioflavin-T (ThT) test to control inhibition of Tau aggregation Support Protocol 2: Purification of full-length Tau protein Basic Protocol 4: ELISA to demonstrate the binding of the generated D-peptides to PHF6* and full-length Tau fibrils.

A novel PDPN antagonist peptide CY12-RP2 inhibits melanoma growth via Wnt/β-catenin and modulates the immune cells.

Podoplanin (PDPN) is a highly conserved, mucin-type protein specific to the lymphatic system. Overexpression of PDPN is associated with the progression of various solid tumors, and plays an important roles in the tumor microenvironment by regulating the immune system. However, the role of PDPN-mediated signal activation in the progression of melanoma is still unknown. PDPN expression was first analyzed in 112 human melanoma tissue microarrays and melanoma cell lines. Functional experiments including proliferation, clone formation, migration, and metastasis were utilized to identify the suppressive effects of PDPN. The Ph.D.TM-12 Phage Display Peptide Library was used to obtain a PDPN antagonist peptide, named CY12-RP2. The immunofluorescence, SPR assay, and flow cytometry were used to identify the binding specificity of CY12-RP2 with PDPN in melanoma cells. Functional and mechanistic assays in vivo and in vitro were performed for discriminating the antitumor and immune activation effects of CY12-RP2. PDPN was overexpressed in melanoma tissue and cells, and inhibited melanoma cells proliferation, migration, and metastasis by blocking the EMT and Wnt/β-catenin pathway. PDPN antagonistic peptide, CY12-RP2, could specifically bind with PDPN, suppressing melanoma various functions inducing apoptosis in both melanoma cells and 3D spheroids. CY12-RP2 also enhanced the anti-tumor capacity of PBMC, and inhibited melanoma cells growth both in xenografts and allogeneic mice model. Moreover, CY12-RP2 could inhibit melanoma lung metastasis, and abrogated the immunosuppressive effects of PDPN by increasing the proportion of CD3 + CD4 + T cells, CD3 + CD8 + T cells, CD49b + Granzyme B + NK cells, and CD11b + CD86 + M1-like macrophages and the levels of IL-1β, TNF-α, and IFN-γ. This study has demonstrated the important role of PDPN in the progression of melanoma and formation of immunosuppressive environment, and provided a potential approach of treating melanoma using the novel CY12-RP2 peptide. In melanoma, PDPN is overexpressed in the cancer cells, and promotes melanoma cells growth and metastasis through activating the Wnt/β-catenin pathway. Treatment with the PDPN antagonistic peptide CY12-RP2 could not only inhibit the melanoma growth and metastasis both in vitro and in vivo through Wnt/β-catenin pathway blockade, but also abrogate the immunosuppressive effects of PDPN through modulating immune cells.

Protocols for Building and Producing High Diversity Peptide Phage Display Libraries.

Phage display is an important technology to study protein-protein interaction and protein evolution, with applications in basic science and applied biotechnology, such as drug discovery and the development of targeted therapies. However, in order to be successful during a phage display screening, it is paramount to have good phage libraries. Here, we described detailed procedures to generate peptide phage display libraries with high diversity and billions of transformants.

Identifying molecular tags selectively retained on the surface of brain endothelial cells to generate artificial targets for therapy delivery

Current strategies to identify ligands for brain delivery select candidates based on preferential binding to cell-membrane components (CMC) on brain endothelial cells (EC). However, such strategies generate ligands with inherent brain specificity limitations, as the CMC (e.g., the transferrin receptor TfR1) are also significantly expressed on peripheral EC. Therefore, novel strategies are required to identify molecules allowing increased specificity of therapy brain delivery. Here, we demonstrate that, while individual CMC are shared between brain EC and peripheral EC, their endocytic internalization rate is markedly different. Such differential endocytic rate may be harnessed to identify molecular tags for brain targeting based on their selective retention on the surface of brain EC, thereby generating ‘artificial’ targets specifically on the brain vasculature. By quantifying the retention of labelled proteins on the cell membrane, we measured the general endocytic rate of primary brain EC to be less than half that of primary peripheral (liver and lung) EC. In addition, through bio-panning of phage-displayed peptide libraries, we unbiasedly probed the endocytic rate of individual CMC of liver, lung and brain endothelial cells. We identified phage-displayed peptides which bind to CMC common to all three endothelia phenotypes, but which are preferentially endocytosed into peripheral EC, resulting in selective retention on the surface of brain EC. Furthermore, we demonstrate that the synthesized free-form peptides are capable of generating artificial cell-surface targets for the intracellular delivery of model proteins into brain EC with increasing specificity over time. The developed identification paradigm, therefore, demonstrates that the lower endocytic rate of individual CMC on brain EC can be harnessed to identify peptides capable of generating ‘artificial’ targets for the selective delivery of proteins into the brain vasculature. In addition, our approach identifies brain-targeting peptides which would have been overlooked by conventional identification strategies, thereby increasing the repertoire of candidates to achieve specific therapy brain delivery.

Combination of nanobody and peptidomimetic to develop novel immunoassay platforms for detecting ochratoxin A in cereals.

Mimotope-based immunoassays for mycotoxins eliminate the requirement for large amounts of mycotoxin standards for the chemosynthesis of artificial antigens. Herein, the nanobody-based magnetic beads were used to screen the mimotope (peptidomimetic) of ochratoxin A (OTA) from the phage-displayed peptide library. The interactions between nanobody and the most sensitive Y4 peptidomimetic were investigated by computer-assisted simulation and compared with those between nanobody and OTA. By combining the nanobody, the phage-displayed Y4 and alkaline phosphatase-tagged Y4 fusion protein as the competing antigens, were used to develop two novel immunoassay platforms (PN-ELISA and APN-ELISA). The two methods are advantageous in the use of nontoxic substitutes of OTA and avoiding the use of monoclonal antibodies. Moreover, good analytical performances of both methods were obtained and confirmed by liquid chromatography tandem mass spectrometry. Therefore, the proposed novel methods based on nanobody and peptidomimetic were demonstrated to be highly reliable for detecting OTA in food.

Clinical correlates of a subset of anti-fibroblast antibodies in systemic sclerosis

Anti-fibroblast antibodies (AFA) have been reported in systemic sclerosis (SSc) and are known to promote fibroblast activation. Aim of this study was to characterize the fine specificity of AFA and to analyze any correlations with clinical parameters associated to fibrosis. To this end, AFA were affinity-purified from a patient with diffuse cutaneous SSc (dcSSc) and interstitial lung disease (ILD). Panning of a phage display peptide library with purified AFA identified the motif <KxywxQ>. The peptide p121, bearing the AFA-specific motif, was used in ELISA to screen sera from 186 SSc patients and 81 healthy donors. Anti-p121 Ab serum levels were statistically higher in SSc than in healthy groups, and directly associated with dcSSc, reduced FVC (FVC < 70), and ILD. Given these clinical correlates, this study lays the groundwork for the identification of the antigen recognized by anti-p121 Ab, which might represent a novel therapeutic target for ILD.

A dual-targeting peptide for glioblastoma screened by phage display peptide library biopanning combined with affinity-adaptability analysis

The obstruction of blood–brain barrier (BBB) and the poor specific targeting are still the major obstacles and challenges of targeted nano-pharmaceutical therapy for glioblastoma (GBM) up to now. It is critical to find appropriate targeting ligands that can effectively mediate the nano-pharmaceuticals to penetrate brain capillary endothelial cells (BCECs) and then specifically bind to glioblastoma cells (GCs). Herein, a dual-targeting ligand for GBM was screened by the combination of phage display peptide library biopanning and affinity-adaptability analysis. Based on the acquisition of sub-library of peptide which exhibited the specific affinity to both BCECs and GCs, a comparison parameter of relative affinity was deliberately introduced to evaluate the relative affinity of candidate peptides to U251-MG cells and bEnd.3 cells. The optimized WTW peptide (sequenced as WTWEYTK) was provided with a high relative affinity (RU/B = 2.44), implying that its high affinity to U251-MG cells and moderate affinity to bEnd.3 cells might synergistically promote its receptor-mediated internalization and transport, the dissociation from bEnd.3, and the binding to U251-MG. The results of BBB model trials in vitro showed that the BBB penetration efficiency and GBM accumulation of WTW peptide were significantly higher than those of WSL peptide, GNH peptide, and REF peptide. Results of orthotopic GBM xenograft model assays in vivo also indicated that WTW peptide had successfully penetrated the BBB and improved accumulation in GBM. The screened WTW peptide might be the potential dual-targeting ligand to motivate the advancement of GBM targeted therapy.

Identification and epitope mapping of anti-p72 single-chain antibody against African swine fever virus based on phage display antibody library

African swine fever virus (ASFV) is a lethal pathogen that causes severe threats to the global swine industry and it has already had catastrophic socio-economic effects. To date, no licensed prophylactic vaccine exists. Limited knowledge exists about the major immunogens of ASFV and the epitope mapping of the key antigens. As such, there is a considerable requirement to understand the functional monoclonal antibodies (mAbs) and the epitope mapping may be of utmost importance in our understanding of immune responses and designing improved vaccines, therapeutics, and diagnostics. In this study, we generated an ASFV antibody phage-display library from ASFV convalescent swine PBMCs, further screened a specific ASFV major capsid protein (p72) single-chain antibody and fused with an IgG Fc fragment (scFv-83-Fc), which is a specific recognition antibody against ASFV Pig/HLJ/2018 strain. Using the scFv-83-Fc mAb, we selected a conserved epitope peptide (221MTGYKH226) of p72 retrieved from a phage-displayed random peptide library. Moreover, flow cytometry and cell uptake experiments demonstrated that the epitope peptide can significantly promote BMDCs maturation in vitro and could be effectively uptaken by DCs, which indicated its potential application in vaccine and diagnostic reagent development. Overall, this study provided a valuable platform for identifying targets for ASFV vaccine development, as well as to facilitate the optimization design of subunit vaccine and diagnostic reagents.

Identification of Eimeria tenella sporozoite immunodominant mimotopes by random phage-display peptide libraries-a proof of concept study.

Coccidiosis, caused by parasites of numerous Eimeria species, has long been recognized as an economically significant disease in the chicken industry worldwide. The rise of anti-coccidian resistance has driven a search for other parasite management techniques. Recombinant antigen vaccination presents a highly feasible alternative. Properly identifying antigens that might trigger a potent immune response is one of the major obstacles to creating a viable genetically modified vaccine. This study evaluated a reverse immunology approach for the identification of B-cell epitopes. Antisera from rabbits and hens inoculated with whole-sporozoites of E. tenella were used to identify Western blot antigens. The rabbit IgG fraction from the anti-sporozoite serum exhibited the highest reactogenicity; consequently, it was purified and utilized to screen two random Phage-display peptide libraries (12 mer and c7c mer). After three panning rounds, 20 clones from each library were randomly selected, their nucleotide sequences acquired, and their reactivity to anti-sporozoite E. tenella serum assessed. The selected peptide clones inferred amino acid sequences matched numerous E. tenella proteins. The extracellular domain of the epidermal growth factor-like (EGF-like) repeats, and the thrombospondin type-I (TSP-1) repeats of E. tenella micronemal protein 4 (EtMIC4) matched with the c7c mer selected clones CNTGSPYEC (2/20) and CMSTGLSSC (1/20) respectively. The clone CSISSLTHC that matched with a conserved hypothetical protein of E. tenella was widely selected (3/20). Selected clones from the 12-mer phage display library AGHTTQFNSKTT (7/20), GPNSAFWAGSER (2/20) and HFAYWWNGVRGP (8/20) showed similarities with a cullin homolog, elongation factor-2 and beta-dynein chain a putative E. tenella protein, respectively. Four immunodominant clones were previously selected and used to immunize rabbits. By ELISA and Western blot, all rabbit anti-clone serums detected E. tenella native antigens. Thus, selected phagotopes contained recombinant E. tenella antigen peptides. Using antibodies against E. tenella sporozoites, this study demonstrated the feasibility of screening Phage-display random peptide libraries for true immunotopes. In addition, this study looked at an approach for finding novel candidates that could be used as an E. tenella recombinant epitope-based vaccine.

Development a novel drug delivery formulation targeting to esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most popular cancers in digestive tract. Chemotherapy is still the primary treatment for ESCC patients with or without surgery. But the efficacy of the current chemotherapy for ESCC is poor with side effects because of none or weak ESCC targeting and multidrug resistance (MDR). To develop a novel targeting peptide for ESCC targeting diagnosis and therapy, a novel peptide, ESCP9 was screened from a 12-mer phage displayed peptide library. The satisfied sensitivity and specificity of ESCP9 binding to ESCC were characterized with cellular and tissue assays under fluorescence microscope, and by flow cytometry. By the assay of molecular docking, protein kinase C (PKC) was predicted as the target ESCP9 binding to. To develop a novel ESCC targeted and MDR inhibited doxorubicin (DOX) liposomal delivery formulation for ESCC targeting chemotherapy, the DOX delivery formulation (ESCP9-Lipo-DOX-miR101) was synthesized and characterized by using ESCP9 as ESCC directing fragment and miRNA101 as MDR suppressor. Finally, the therapeutic efficacy of the drug formulation for ESCC therapy was primarily evaluated by the therapeutic assay for the wild type and DOX resistant ESCC cells in vitro. The results demonstrated that the drug formulation enhanced cytotoxicity to Eca-109 cells and DOX-resistant Eca-109 cells (Eca-109/ADR) significantly. It is concluded that ESCP9 is a targeting peptide with high specificity and sensitivity, and the ESCP9 conjugated DOX liposomal formulation, ESCP9-Lipo-DOX-miR101, is of the potential to be further studied as an efficient drug formulation for ESCC and DOX resistant ESCC chemotherapy in future.

Propagation Capacity of Phage Display Peptide Libraries Is Affected by the Length and Conformation of Displayed Peptide.

The larger size and diversity of phage display peptide libraries enhance the probability of finding clinically valuable ligands. A simple way of increasing the throughput of selection is to mix multiple peptide libraries with different characteristics of displayed peptides and use it as biopanning input. In phage display, the peptide is genetically coupled with a biological entity (the phage), and the representation of peptides in the selection system is dependent on the propagation capacity of phages. Little is known about how the characteristics of displayed peptides affect the propagation capacity of the pooled library. In this work, next-generation sequencing (NGS) was used to investigate the amplification capacity of three widely used commercial phage display peptide libraries (Ph.D.™-7, Ph.D.™-12, and Ph.D.™-C7C from New England Biolabs). The three libraries were pooled and subjected to competitive propagation, and the proportion of each library in the pool was quantitated at two time points during propagation. The results of the inter-library competitive propagation assay led to the conclusion that the propagation capacity of phage libraries on a population level is decreased with increasing length and cyclic conformation of displayed peptides. Moreover, the enrichment factor (EF) analysis of the phage population revealed a higher propagation capacity of the Ph.D.TM-7 library. Our findings provide evidence for the contribution of the length and structural conformation of displayed peptides to the unequal propagation rates of phage display libraries and suggest that it is important to take peptide characteristics into account once pooling multiple combinatorial libraries for phage display selection through biopanning.

Screening epitope peptides based on a phage-displayed random peptide and peptide microarrays to contribute to improving the diagnostic efficiency of systemic lupus erythematosus

BackgroundSystemic lupus erythematosus (SLE) is one of the most common autoimmune diseases in China. At present, there are hundreds of autoantibodies in SLE patients; however, only a dozen of the autoantibodies can be routinely detected, and the available diagnostic antibodies are not sufficient for diagnosis or differential diagnosis of SLE patients with atypical clinical manifestations or other autoimmune diseases. Therefore, it is necessary to find new diagnostic markers to improve the diagnostic effect of SLE. MethodsThe displayed random peptide library and peptide microarray were combined to identify SLE-related epitope peptides. A case-control design was used. The IgG antibodies in the sera from SLE patients, healthy controls, and other autoimmune disease controls underwent a reaction with the phage-display random peptide library, respectively. Selected epitope peptides were used to construct a peptide chip. A total of 644 serum samples (including 296 SLE patients, 168 disease controls, and 180 healthy controls) were used for further screening and verification. Peptides with an area under the curve (AUC) > 0.650 were further verified by ELISA. Finally, 500 serum samples (including 200 SLE patients, 150 disease controls, and 150 healthy controls) were used to verify and evaluate the diagnostic and differential diagnostic efficacy of the selected peptides. ResultsAfter the previous screening, five epitope peptides (SLE_P19, SLE_P20, SLE_P27, SLE_P28, and SLE_P29) may have potential as SLE diagnostic markers. Additionally, SLE_P27 was superior to the other four peptides in the diagnosis and differential diagnosis of SLE and rheumatoid arthritis (RA). The AUC of SLE_P27 was 0.938, the sensitivity was 76.00%, the specificity was 92.70%, the positive likelihood ratio was 10.411, the negative likelihood ratio was 0.259, and the accuracy was 84.40%. The diagnostic efficacy of SLE can be increased by combining the five selected peptides with the anti-double stranded DNA antibody (anti-dsDNA) and anti-Smith antibodies (anti-Sm). ConclusionsIn this study, we identified five peptides that may serve as potential biomarkers for SLE diagnosis using the strategy of combining the displayed random peptide library with the peptide microarray. The combination of selected peptides and existing autoantibodies can significantly improve the diagnostic efficiency. These specific peptides are expected to be new diagnostic markers for SLE.