Two-dimensional immune profiles improve antigen microarray-based characterization of humoral immunity

Antigen arrays are becoming widely used tools for the characterization of the complexity of humoral immune responses. Current antibody profiling techniques provide modest and indirect information about the effector functions of the antibodies that bind to particular antigens. Here we introduce an antigen array-based approach for obtaining immune profiles reflecting antibody functionality. This technology relies on the parallel measurement of antibody binding and complement activation by features of the array. By comparing sera from animals immunized against the same antigen under different conditions, we show that identifying the position of an antigen in a 2-D space, derived from antibody binding and complement deposition, permits distinction between immune profiles characterized by diverse antibody isotype distributions. Additionally, the technology provides a biologically interpretable graphical representation of the relationship between antigen and host. Our data suggest that 2-D immune profiling could enrich the data obtained from proteomic scale serum profiling studies.     

In vitro panning of a targeting peptide to hepatocarcinoma from a phage display peptide library

Phage display technology has been used as a powerful tool in the discovery of ligands specific to receptor(s) on the surface of a cancer cell and could also impact clinical issues including functional diagnosis and cell-specific drug delivery. After three rounds of in vitro panning and two rounds of reverse absorption, a group of phages capable of addressing BEL-7402 enormously were obtained for further analysis. Through a cell-based ELISA, immunofluorescence, FACS, and in vivo binding study, WP05 (sequence TACHQHVRMVRP) was demonstrated to be the most effective peptide in targeting four kinds of liver cancer cell lines (BEL-7402, BEL-7404, SMMC-7721, and HepG2), but not the normal liver cell line HL-7702. In conclusion, the peptide WP05 which was screened by in vitro phage display technology was proved to be a targeting peptide to several common hepatocellular carcinoma cell lines.     

The making of the minibody: An engineered β-protein for the display of conformationally constrained peptides

Conformationally constraining selectable peptides onto a suitable scaffold that enables their conformation to be predicted or readily determined by experimental techniques would considerably boost drug discovery process by reducing the gap between the discovery of a peptide lead and the design of a peptidomimetic with a more desirable pharmacological profile. With this in mind, we designed the minibody, a 61-residue β-protein aimed at retaining some desirable features of immunogloblin variable domains, such as tolerance to sequence variability in selected regions of the protein and predictability of main chain conformation of the same regions, based on the ‘canonical structures’ model. To test the ability of the minibody scaffold to support functional sites we also designed a metal binding version of the protein by suitably choosing the sequences of its loops. The minibody was produced both by chemical syntyhesis and expression in E. coli and charactgerized by size exclusion chromatography, UV CD (circular dichroism) spectroscopy and metal binding activity. All our data supported the model, but a more detailed structural characterization of the molecule was impaired by its low soubility. We were able to overcome this problem both by further; mutagenesis of the framework and by addition of a solublizing motif. The minibody is being used to select constrained human IL-6 peptidic ligands from a library displayed on the surface of the f1 bacteriophage.     

Selection of peptides that bind to the core oligosaccharide of R-form LPS from a phage-displayed heptapeptide library

To characterize common sites within the core oligosaccharide of the R-form lipopolysaccharide (LPS), we screened peptides from a phage-displayed heptapeptide library by using the most truncated form of R-LPS, Re-LPS (S. Typhimurium SL1165) as a ligand. After three rounds of biopanning/amplification and subsequent screening by phagemid enzyme-linked immunosorbent assay (ELISA), we selected three distinct clones that bind to the ligand LPS. We characterized the binding sites of the three clones by ELISA and thin-layer chromatography immunostaining and found that the three clones bind the two Re-LPSs (SL1165 and S. Minnesota Re595) and Rb2-LPS. In addition, one of the clones also bound to S-form LPS (S. Enteritidis). Current data show that those clones bind to common carbohydrate structure(s) expressed in the core oligosaccharides of those LPS samples.     

Efficient construction of a highly useful phage-displayed human antibody repertoire

We have constructed a highly useful phage-displayed human antibody repertoire with limited cloning efforts. Our strategy was to maximize diversity during the first steps of library construction through the use of various lymphoid sources from several donors, inclusion of different immunoglobulin isotypes, and performance of multiple separate amplification reactions with all possible combinations within a complex primer set. The resulting variable region collections were cloned to form a moderate size library, composed by 4.25x10(8) single chain antibody fragments. This repertoire was successfully used to retrieve binders to seven model antigens: six proteins and one 12 aa peptide. Binding affinities reached nanomolar and even subnanomolar range. Sequence diversity and V-gene usage variability among binders were proven. Our approach was not focused on absolute library size, but on a high quality sampling of variable regions from the human antibody repertoire.     

Development of a bacteriophage-based system for the selection of structured peptides

Short structured peptides can provide scaffolds for protease-resistant peptide therapeutics, serve as useful building blocks in biomedical and biotechnological applications, and shed light on the role of secondary structure elements in protein folding. It is well known that directed evolution is a powerful method for creating proteins and peptides with novel properties, and a system for the selection of short peptides based on structure from a randomized library would be an important advancement. In this study, phage particles monovalently displaying a short peptide and an N-terminal 6×His tag on their P3 coat protein were bound to nickel agarose resin and were subsequently challenged with a protease that specifically cleaves at a site within the peptide. The extent to which phage is proteolytically released from the resin was found to be dependent on the structural properties of the inserted peptide sequences. As proofs-of-concept, a structured peptide has been isolated from a pool of flexible peptides using a trypsin selection, and a flexible peptide has been isolated from a pool of structured peptides using a chymotrypsin selection. This selection system will be a strong technological platform for the creation of short peptides with interesting structural properties using directed evolution.     

Seek & Destroy, use of targeting peptides for cancer detection and drug delivery

Accounting for 16 million new cases and 9 million deaths annually, cancer leaves a great number of patients helpless. It is a complex disease and still a major challenge for the scientific and medical communities. The efficacy of conventional chemotherapies is often poor and patients suffer from off-target effects. Each neoplasm exhibits molecular signatures – sometimes in a patient specific manner – that may completely differ from the organ of origin, may be expressed in markedly higher amounts and/or in different location compared to the normal tissue. Although adding layers of complexity in the understanding of cancer biology, this cancer-specific signature provides an opportunity to develop targeting agents for early detection, diagnosis, and therapeutics. Chimeric antibodies, recombinant proteins or synthetic polypeptides have emerged as excellent candidates for specific homing to peripheral and central nervous system cancers. Specifically, peptide ligands benefit from their small size, easy and affordable production, high specificity, and remarkable flexibility regarding their sequence and conjugation possibilities. Coupled to imaging agents, chemotherapies and/or nanocarriers they have shown to increase the on-site delivery, thus allowing better tumor mass contouring in imaging and increased efficacy of the chemotherapies associated with reduced adverse effects. Therefore, some of the peptides alone or in combination have been tested in clinical trials to treat patients. Peptides have been well-tolerated and shown absence of toxicity. This review aims to offer a view on tumor targeting peptides that are either derived from natural peptide ligands or identified using phage display screening. We also include examples of peptides targeting the high-grade malignant tumors of the central nervous system as an example of the complex therapeutic management due to the tumor’s location. Peptide vaccines are outside of the scope of this review.     

利用噬菌体表面展示技术筛选转铁蛋白黏附肽的研究

为了筛选转铁蛋白黏附肽,应用噬菌体表面展示技术经过三轮生物淘选,成功地从随机七肽库中得到黏附转铁蛋白的重组噬菌体克隆,经过相对亲和力常数测定和DNA测序得到4个转铁蛋白黏附肽的序列。实验中以回收率和选择比为操作参数,对淘选进行了优化,并发展了一种基于噬菌体滴度的相对亲和力常数测定方法。转铁蛋白受体是一种有效的肿瘤标记物,利用转铁蛋白为载体可以实现药物靶向运输,因此转铁蛋白黏附肽将是重组蛋白质药物连接转铁蛋白的有用标签。     

Identification of anti-TNFalpha peptides with consensus sequence

Phage displayed peptide library was used to select tumor necrosis factor alpha (TNFalpha) binding peptides. After three sequential rounds of biopanning, some linear TNFalpha-binding peptides were identified from a 12-mer peptide library. A consensus sequence (L/M)HEL(Y/F)(L/M)X(W/Y/F), where X might be variable residue, was deduced from sequences of these peptides. The phages bearing these peptides showed specific binding to immobilized TNFalpha, with over 80% of phages bound being competitively eluted by free TNFalpha. To confirm the binding activity and to explore further functional properties, three peptides with typical structure were selected and expressed as GST-fused protein. These recombinant peptides effectively competed for [125I]TNFalpha binding to TNFR1 in a dose-dependent manner, with IC(50) from 10 to 160 microM. Furthermore, the GST-fused derivatives showed inhibitory effects on TNFalpha-induced cytotoxicity. Taken together, these data demonstrate that the TNFalpha-binding peptides are effective antagonists of TNFalpha and the deduced motif might be useful in development of novel low molecular weight anti-TNFalpha drugs.     

Selection of trkB-binding peptides from a phage-displayed random peptide library

Brain-derived neurotrophic factor (BDNF) shows potential in the treatment of neurodegenerative diseases, but the therapeutic application of BDNF has been greatly limited because it is too large in molecular size to permeate blood-brain barrier. To develop low-molecular-weight BDNF-like peptides, we selected a phage-displayed random peptide library using trkB expressed on NIH 3T3 cells as target in the study. With the strategy of peptide library incubation with NIH 3T3 cells and competitive elution with 1 μg/mL of BDNF in the last round of selection, the specific phages able to bind to the natural conformation of trkB and antagonize BDNF binding to trkB were enriched effectively. Five trkB-binding peptides were obtained, in which a core sequence of CRA/TXΦXXΦXXC (X represents the random amino acids, Φ represents T, L or I) was identified. The BDNF-like activity of these five peptides displayed on phages was not observed, though all of them antagonized the activity of BDNF in a dose-dependent manner. Similar results were obtained with the synthetic peptide of C1 clone, indicating that the 5 phage-derived peptides were trkB antagonists. These low-molecular-weight antagonists of trkB may be of potential application in the treatment of neuroblastoma and chronic pain. Meanwhile, the obtained core sequence also could be used as the base to construct the secondary phage-displayed peptide library for further development of small peptides mimicking BDNF activity.     

Selection of trkB-binding peptides from a phage-displayed random peptide library

Brain-derivedneurotrophicfactor(BDNF),originallypurifiedfrompigbrainbyBarderetal.[1]in1982,belongstothefamilyofneurotrophins(NTs)aswellasnervegrowthfactor(NGF),neurotrophin-3(NT-3),NT-4/5.Itisabletopromotesurvivalanddifferentiationofseveralpopu-lationsofneurons,includingmesencephalicdopaminergicneurons,motorneurons,andcholiner-gicneurons,andtoprotectthemagainstneurotoxicityandischemia.BDNFplaysanimportantroleinregulatingneuronsurvivalanddifferentiationduringdevelopmentandinmaintainingthe…     

Biotechnology techniques for the development of new tumor specific peptides

Peptides, proteins and antibodies are promising candidates as carriers for radionuclides in endoradiotherapy. This novel class of pharmaceuticals offers a great potential for the targeted therapy of cancer. The fact that some receptors are overexpressed in several tumor types and can be targeted by small peptides, proteins or antibodies conjugated to radionuclides has been used in the past for the development of peptide endoradiotherapeutic agents such as ⁹⁰Y-DOTATOC or radioimmunotherapy of lymphomas with Zevalin. These procedures have been shown to be powerful options for the treatment of cancer patients.Design of new peptide libraries and scaffolds combined with biopanning techniques like phage and ribosome display may lead to the discovery of new specific ligands for target structures overexpressed in malignant tumors. Display methods are high throughput systems which select for high affinity binders. These methods allow the screening of a vast amount of potential binding motifs which may be exposed to either cells overexpressing the target structures or in a cell-free system to the protein itself. Labelling these binders with radionuclides creates new potential tracers for application in diagnosis and endoradiotherapy. This review highlights the advantages and problems of phage and ribosome display for the identification and evaluation of new tumor specific peptides.     

Selection of phage-displayed peptides for the detection of imidacloprid in water and soil

Imidacloprid is the most widely used neonicotinoid insecticide in the world and shows widespread environment and human exposures. A phage clone designated L7-1 that selectively binds to imidacloprid was selected from a commercial phage display library containing linear 7-mer randomized amino acid residues. Using the clone L7-1, a competitive enzyme-linked immunosorbent assay (ELISA) for imidacloprid was developed. The half-maximum signal inhibition concentration (IC₅₀) and the limit of detection (LOD) of the phage ELISA for imidacloprid were 96 and 2.3 ng ml⁻¹, respectively. This phage ELISA showed relatively low cross-reactivity with all of the tested compounds structurally similar to imidacloprid, less than 2% with the exception of 6-chloronicotinic acid, a metabolite of imidacloprid that showed 11.5%. The average recoveries of the phage ELISA for imidacloprid in water and soil samples were in the ranges of 74.6 to 86.3% and 72.5 to 93.6%, respectively. The results of the competitive phage ELISA for imidacloprid in the fortified samples agreed well with those of a high-performance liquid chromatography (HPLC) method. The simple phage-displayed peptide technology has been proven to be a convenient and efficient method for the development of an alternative format of ELISA for small molecules.     

Genome‐wide profiling of humoral immune response to Coxiella burnetii infection by protein microarray

Comprehensive evaluation of the humoral immune response to Coxiella burnetii may identify highly needed diagnostic antigens and potential subunit vaccine candidates. Here we report the construction of a protein microarray containing 1901 C. burnetii ORFs (84% of the entire proteome). This array was probed with Q‐fever patient sera and naïve controls in order to discover C. burnetii‐specific seroreactive antigens. Among the 21 seroreactive antigens identified, 13 were significantly more reactive in Q‐fever cases than naïve controls. The remaining eight antigens were cross‐reactive in both C. burnetii infected and naïve patient sera. An additional 64 antigens displayed variable seroreactivity in Q‐fever patients, and underscore the diversity of the humoral immune response to C. burnetii. Nine of the differentially reactive antigens were validated on an alternative immunostrip platform, demonstrating proof‐of‐concept development of a consistent, safe, and inexpensive diagnostic assay alternative. Furthermore, we report here the identification of several new diagnostic antigens and potential subunit vaccine candidates for the highly infectious category B alphaproteobacteria, C. burnetii.     

Suppression of humoral immune responses by synthetic C3a peptides

Synthetic oligopeptides based on the COOH-terminal sequence of native human C3a were examined for the ability to suppress antigen-specific and polyclonal antibody responses. The synthetic peptides were found to qualitatively mimic the suppressive actions of human C3a, but proved to be less active on a molar basis. Comparison of the activity of the peptides with native C3a indicates that the most potent peptide is C3a 57-77 with C3a 65-77, C3a 70-77, C3a[Ala 71,72]70-77, and C3a 73-77 being less active, respectively. These results indicate that the region of the C3a molecule responsible for immunosuppression is located in the COOH region.     

Cyclic peptides identified by phage display are competitive inhibitors of the tRNA-dependent amidotransferase of Helicobacter pylori

In Helicobacter pylori, the heterotrimeric tRNA-dependent amidotransferase (GatCAB) is essential for protein biosynthesis because it catalyzes the conversion of misacylated Glu-tRNA^Gln and Asp-tRNA^Asn into Gln-tRNA^Gln and Asn-tRNA^Asn, respectively. In this study, we used a phage library to identify peptide inhibitors of GatCAB. A library displaying loop-constrained heptapeptides was used to screen for phages binding to the purified GatCAB. To optimize the probability of obtaining competitive inhibitors of GatCAB with respect to its substrate Glu-tRNA^Gln, we used that purified substrate in the biopanning process of the phage-display technique to elute phages bound to GatCAB at the third round of the biopanning process. Among the eluted phages, we identified several that encode cyclic peptides rich in Trp and Pro that inhibit H. pylori GatCAB in vitro. Peptides P10 and P9 were shown to be competitive inhibitors of GatCAB with respect to its substrate Glu-tRNA^Gln, with K_i values of 126 and 392 μM, respectively. The docking models revealed that the Trp residues of these peptides form π-π stacking interactions with Tyr81 of the synthetase active site, as does the 3′-terminal A76 of tRNA, supporting their competitive behavior with respect to Glu-tRNA^Gln in the transamidation reaction. These peptides can be used as scaffolds in the search for novel antibiotics against the pathogenic bacteria that require GatCAB for Gln-tRNA^Gln and/or Asn-tRNA^Asn formation.     

利用噬菌体随机肽库筛选可结合志贺毒素B亚单位的短肽序列

以制备的重组志贺毒素B亚单位(StxB)为靶标,利用噬菌体展示亲和淘选技术,经4轮筛选,从随机十二肽库中筛选到与StxB结合的一批噬菌体克隆,对特异结合活性较高的27个噬菌体克隆的表面展示肽进行序列测定,其中A6序列出现16次,A9和A3序列分别出现2次和3次。为评价筛选克隆中和毒素毒性的能力,将展示肽出现频率最高的A6噬菌体克隆,体外与志贺毒素孵育进行动物试验,动物存活率达33.3%,表明毒素的毒性得到部分抑制,A6短肽可能发展成为志贺毒素的拮抗剂。