Mimotopes for Mycotoxins Diagnosis Based on Random Peptides or Recombinant Antibodies from Phage Library

Mycotoxins, the small size secondary metabolites of fungi, have posed a threat to the safety of medicine, food and public health. Therefore, it is essential to create sensitive and effective determination of mycotoxins. Based on the special affinity between antibody and antigen, immunoassay has been proved to be a powerful technology for the detection of small analytes. However, the tedious preparation and instability of conventional antibodies restrict its application on easy and fast mycotoxins detection. By virtue of simplicity, ease of use, and lower cost, phage display library provides novel choices for antibodies or hapten conjugates, and lead random peptide or recombinant antibody to becoming the promising and environmental friendly immune-reagents in the next generation of immunoassays. This review briefly describes the latest developments on mycotoxins detection using M13 phage display, mainly focusing on the recent applications of phage display technology employed in mycotoxins detection, including the introduction of phage and phage display, the types of phage displayed peptide/recombinant antibody library, random peptides/recombinant antibodies-based immunoassays, as well as simultaneous determination of multiple mycotoxins.

Search and Characterization of ITM2A as a New Potential Target for Brain Delivery

Background Integral membrane protein 2A (ITM2A) is a transmembrane protein whose function is not well described. This target was identified as highly enriched in human brain vs peripheral endothelial cells by transcriptomic and proteomic studies during the European Collaboration on the Optimization of Macromolecular Pharmaceutical Innovative Medicines Initiative (COMPACT IMI) consortium. The object of the present paper is to report the work we have undertaken to characterize this protein as a potential brain delivery target. Methods A series of ITM2A constructs, cell lines and specific anti-human and mouse ITM2A antibodies were generated. Binding and internalization in Human Embryonic Kidney 293 (HEK293) cells overexpressing ITM2A and in brain microvascular endothelial cells from mouse and non-human primate (NHP) were performed with these tools. The best antibody was evaluated in an in vitro human blood brain barrier (BBB) model and in a mouse in vivo pharmacokinetic study to validate blood brain barrier crossing. Results Antibodies specifically recognizing extracellular parts of ITM2A or tags inserted in its extracellular domain showed selective binding and uptake in ITM2A-expressing cells. However, despite high RNA expression in mouse and human microvessels, this protein, is rapidly downregulated upon endothelial cells culture, probably explaining why transcytosis could not be evidenced in vitro. An attempt to directly demonstrate in vivo transcytosis in mice turned out unconvincing, using a cross reactive anti ITM2A antibody on one hand and in vivo phage panning of an anti ITM2A phage library on the other hand. Conclusions The present article describes the work we have undertaken to explore the potential of ITM2A as target to mediate transcytosis through BBB. This work highlights the multiple challenges linked to the identification of new brain delivery targets.

Reproducible Discovery of Cell-Binding Peptides "Lost" in Bulk Amplification via Emulsion Amplification in Phage Display Panning

Many pharmaceutically-relevant cell surface receptors are functional only in the context of intact cells. Phage display, while being a powerful method for the discovery of ligands for purified proteins often fails to identify a diverse set of ligands to receptors on a cell membrane mosaic. To understand this deficiency, we examined growth bias in naive phage display libraries and observed that it fundamentally changes selection outcomes: The presence of growth-biased (parasite) phage clones in a phage library is detrimental to selection and cell-based panning of such biased libraries is poised to yield ligands from within a small parasite population. Importantly, amplification of phage libraries in water-oil emulsions suppressed the amplification of parasites and steered the selection of biased phage libraries away from parasite population. Attenuation of the growth bias through the use of emulsion amplification reproducibly discovers the ligands for cell-surface receptors that cannot be identified in screen that use conventional "bulk" amplification.

A human antibody against human endothelin receptor type A that exhibits antitumor potency

Endothelin receptor A (ETA), a class A G-protein-coupled receptor (GPCR), is involved in the progression and metastasis of colorectal, breast, lung, ovarian, and prostate cancer. We overexpressed and purified human endothelin receptor type A in Escherichia coli and reconstituted it with lipid and membrane scaffold proteins to prepare an ETA nanodisc as a functional antigen with a structure similar to that of native GPCR. By screening a human naive immune single-chain variable fragment phage library constructed in-house, we successfully isolated a human anti-ETA antibody (AG8) exhibiting high specificity for ETA in the β-arrestin Tango assay and effective inhibitory activity against the ET-1-induced signaling cascade via ETA using either a CHO-K1 cell line stably expressing human ETA or HT-29 colorectal cancer cells, in which AG8 exhibited IC50 values of 56 and 51 nM, respectively. In addition, AG8 treatment repressed the transcription of inhibin βA and reduced the ETA-induced phosphorylation of protein kinase B and extracellular regulated kinase. Furthermore, tumor growth was effectively inhibited by AG8 in a colorectal cancer mouse xenograft model. The human anti-ETA antibody isolated in this study could be used as a potential therapeutic for cancers, including colorectal cancer.

Filamentous Bacteriophage—A Powerful Carrier for Glioma Therapy

Glioma is a life-threatening malignant tumor. Resistance to traditional treatments and tumor recurrence present major challenges in treating and managing this disease, consequently, new therapeutic strategies must be developed. Crossing the blood-brain barrier (BBB) is another challenge for most drug vectors and therapy medications. Filamentous bacteriophage can enter the brain across the BBB. Compared to traditional drug vectors, phage-based drugs offer thermodynamic stability, biocompatibility, homogeneity, high carrying capacity, self-assembly, scalability, and low toxicity. Tumor-targeting peptides from phage library and phages displaying targeting peptides are ideal drug delivery agents. This review summarized recent studies on phage-based glioma therapy and shed light on the developing therapeutics phage in the personalized treatment of glioma.

Anti-Cancer Effects of Cyclic Peptide ALOS4 in a Human Melanoma Mouse Model

We examined the effects of ALOS4, a cyclic peptide discovered previously by phage library selection against integrin αvβ3, on a human melanoma (A375) xenograft model to determine its abilities as a potential anti-cancer agent. We found that ALOS4 promoted healthy weight gain in A375-engrafted nude mice and reduced melanoma tumor mass and volume. Despite these positive changes, examination of the tumor tissue did not indicate any significant effects on proliferation, mitotic index, tissue vascularization, or reduction of αSMA or Ki-67 tumor markers. Modulation in overall expression of critical downstream αvβ3 integrin factors, such as FAK and Src, as well as reductions in gene expression of c-Fos and c-Jun transcription factors, indirectly confirmed our suspicions that ALOS4 is likely acting through an integrin-mediated pathway. Further, we found no overt formulation issues with ALOS4 regarding interaction with standard inert laboratory materials (polypropylene, borosilicate glass) or with pH and temperature stability under prolonged storage. Collectively, ALOS4 appears to be safe, chemically stable, and produces anti-cancer effects in a human xenograft model of melanoma. We believe these results suggest a role for ALOS4 in an integrin-mediated pathway in exerting its anti-cancer effects possibly through immune response modulation.

Preparation and Directed Evolution of Anti-Ciprofloxacin ScFv for Immunoassay in Animal-Derived Food

An immunized mouse phage display scFv library with a capacity of 3.34 × 109 CFU/mL was constructed and used for screening of recombinant anti-ciprofloxacin single-chain antibody for the detection of ciprofloxacin (CIP) in animal-derived food. After four rounds of bio-panning, 25 positives were isolated and identified successfully. The highest positive scFv-22 was expressed in E. coli BL21. Then, its recognition mechanisms were studied using the molecular docking method. The result showed the amino acid residue Val160 was the key residue for the binding of scFv to CIP. Based on the results of virtual mutation, the scFv antibody was evolved by directional mutagenesis of contact amino acid residue Val160 to Ser. After the expression and purification, an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) based on the parental and mutant scFv was established for CIP, respectively. The IC50 value of the assay established with the ScFv mutant was 1.58 ng/mL, while the parental scFv was 26.23 ng/mL; this result showed highly increased affinity, with up to 16.6-fold improved sensitivity. The mean recovery for CIP ranged from 73.80% to 123.35%, with 10.46% relative standard deviation between the intra-assay and the inter-assay. The RSD values ranged between 1.49% and 9.81%. The results indicate that we obtained a highly sensitive anti-CIP scFv by the phage library construction and directional evolution, and the scFv-based IC-ELISA is suitable for the detection of CIP residue in animal-derived edible tissues.

Isolation and characterization of broad host-range of bacteriophages infecting Cronobacter sakazakii and its biocontrol potential in dairy products

Cronobacter sakazakii (C. sakazakii) is an important pathogen contaminating dairy products (e.g., milk pow-der) and causes high mortality in infants. Bacteriophage as a potential biocontrol agent is a good alternative method for the control of this pathogen in dairy production and its environment. Thus, it is important to complete the C. sakazakii phage library by isolating and characterizing the broad host range of bacteriophage against C. sakazakii for control use. In this study, C. sakazakii strains from different sources were used as hosts to isolate and purify phages from human stool and sewage samples by double-layer plates. The biological characteristics, antibacterial properties, and genomes of these phages were then studied. Finally, ten virulent phages (EspYZU01–EspYZU10) infecting C. sakazakii were isolated and identified as belonging to the Myoviridae, Podoviridae, Tectivirus, and Stylovinidae families. Phage EspYZU08 presented the broadest host range and could infect all the five host strains of C. sakazakii. All 10 phages retained their infectivity at 50°C and pH 5–9. Both genomes of EspYZU05 and EspYZU08 were double-stranded DNAs with sizes of 41723 bp and 145582 bp, G+C contents of 55.69% and 46.75%, and open reading frames of 47 and 103, respectively. No toxins and antibiotic resistance genes were detected in both EspYZU05 and EspYZU08. Phage EspYZU08 and phage cocktail-3 (EspYZU01 + EspYZU03 + EspYZU08 + EspYZU09 + EspYZU10) presented excellent antibacterial efficacy for C. sakazakii in liquid broth and milk at 4°C, 25°C, and 37°C, suggesting that the phages in this study have great potential for the development of biocontrol agents against C. sakazakii in dairy and its processing environment.