Characterization of an antibody scFv that recognizes fibrillar insulin and beta-amyloid using atomic force microscopy

Fibrillar amyloid is the hallmark feature of many protein aggregation diseases, such as Alzheimer's and Parkinson's diseases. A monoclonal single-chain variable fragment (scFv) targeting insulin fibrils was isolated using phage display technology and an atomic force microscopy (AFM) mica substrate. Specific targeting of the scFv to insulin fibrils but not monomers or other small oligomeric forms, under similar conditions, was demonstrated both by enzyme-linked immunosorbent assays and AFM recognition imaging. The scFv also recognizes beta-amyloid fibrils, a hallmark feature of Alzheimer's disease. The results suggest that the isolated scFv possibly targets a shared fibrillar motif-probably the cross-beta-sheet characteristic of amyloid fibrils. The techniques outlined here provide additional tools to further study the process of fibril formation. The scFvs isolated can have potential use as diagnostic or therapeutic reagents for protein aggregation diseases.     

Investigation of human keratinocyte cell adhesion using atomic force microscopy

Desmosomal junctions are specialized structures critical to cellular adhesion within epithelial tissues. Disassembly of these junctions is seen consequent to the development of autoantibodies directed at specific desmosomal proteins in blistering skin diseases such as pemphigus. However, many details regarding cell junction activity under normal physiological and disease conditions remain to be elucidated. Because of their complex structure, desmosomal junctions are not well suited to existing techniques for high-resolution three-dimensional structure-function analyses. Here, atomic force microscopy (AFM) is used for detailed characterization and visualization of the cell junctions of human epithelial cells. We demonstrate the ability to image the detailed three-dimensional structure of the cell junction at high magnification. In addition, the effect of specific antibody binding to desmosomal components of the cell junction is studied in longitudinal analyses before and after antibody treatment. We show that antibodies directed against desmoglein 3 (a major component of the desmosomal structural unit, and the major target of autoantibodies in patients with pemphigus vulgaris) are associated with changes at the cell surface of the human keratinocytes and alterations within keratinocyte intercellular adhesion structures, supporting the assertion that cell structures and junctions are modified by antibody binding. The present study indicates that the molecular structure of gap junctions can be more completely analyzed and characterized by AFM, offering a new technological approach to facilitate a better understanding of disease mechanisms and potentially monitor therapeutic strategies in blistering skin diseases.From the Clinical EditorDisassembly of desmosomal junctions is seen in blistering skin diseases such as Pemphigus. This present study demonstrates that the molecular structure of gap junctions can be more completely analyzed and characterized by atomic force microscopy.     

人源性肺癌噬菌体展示抗体库的构建

目的应用噬菌体展示技术,构建天然人源抗肺癌噬菌体抗体组合文库,筛选能与肺癌细胞特异结合的抗体。方法用RNA提取试剂盒提取淋巴细胞RNA,以Oligo DT为引物反转录合成cDNA,以半套式PCR扩增轻链和重链可变区抗体基因并重组到原核表达载体中,通过噬菌体外壳蛋白形成融合蛋白,形成噬菌体抗体组合文库。结果电泳显示扩增的mRNA有清晰的28s、18s和5.8s的条带,600~700bp片段者为重组克隆;噬菌体展示抗体库大小在108~107之间;优化电转化条件,最终得到库容为1.2×108cfu,双链重组率为41%的抗体库。结论成功构建人源抗肺癌噬菌体展示文库,为下一步筛选具有肺癌细胞特异亲和力的人源性单克隆抗体奠定了基础。     

Development of diagnostically and therapeutically useful human antibody medicines by phage display system

Phage display has been utilized for making recombinant antibody fragments (Fab or single chain Fv) of human, mouse, or other origins. After construction of an antibody combinatorial library, antigen-specific recombinant antibody fragments can be easily isolated by biopanning of the phage library displaying antibody fragment fused with viral coat protein III against antigen proteins, antigen-expressing live cells, or fixed cells. Using this technique, a variety of human recombinant antibody fragments can be retrieved from bone marrow cells, lymph node cells, or peripheral blood cells of patients with infectious diseases, autoimmune diseases, and cancer. To develop diagnostically and therapeutically useful human antibody medicines, we should first select recombinant antibody fragments not only with antigen-binding activity but also with bioactivity such as virus or toxin neutralization, or tumor-specific cytotoxicity. To achieve this goal, several steps in antibody phage display may be improved: 1) a larger library should be constructed for possible isolation of minor populations present in the repertoire; 2) the biopanning procedure should be improved for isolation of antibody fragments reactive with immunologically minor epitopes; 3) the screening procedure should be based on the measurement of the bioactivity as well as the antigen-binding activity; 4) if necessary, the affinity and specificity of selected antibody fragments should be improved. In this review, I discuss how to isolate clinically useful recombinant antibody fragments efficiently using a phage display system introducing our achievements.     

从噬菌体单链抗体库中筛选MMP7单链抗体

目的:从人天然噬菌体单链抗体库中筛选与MMP7特异结合的单链抗体(scFv),并对其特异性及活性进行分析。方法:转化MMP7质粒,分离、纯化与鉴定表达的MMP7。以MMP7为靶标对人天然噬菌体单链抗体库进行4轮的筛选富集,初步筛选到能与MMP7结合的scFv。通过ELISA和Western对所获scFv的特异性进行分析。结果:分离纯化鉴定了MMP7,筛选获得了结合MMP7的人噬菌体抗体。ELISA和Western表明筛选到的scFv特异性较好。结论:利用噬菌体抗体库技术可直接获得特异人源MMP7抗体,为抗MMP7单抗药物的研发提供了新的可能性。     

humMR1, a highly specific humanized single chain antibody for targeting EGFRvIII

Production of an efficient humanized single chain antibody is reported here to specifically target EGFRvIII, a truncated receptor expressed in a wide variety of human cancers. CDR loops of MR1, a phage display-derived murine single chain antibody developed against this mutant receptor, were grafted on human frameworks that had been selected based on similarity to MR1 in terms of two distinct parameters, variable domain protein sequence and CDR canonical structures. Moreover, two point mutations were introduced in CDR-H2 and CDR-H3 loops of the humanized antibody to destroy its cross-reactivity to wild-type EGFR. The resultant antibody, referred to as humMR1, was found by MTT assay, ELISA and western blot techniques to be highly specific for EGFRvIII. The affinity of this antibody for EGFRvIII-specific 14-amino acid synthetic peptide and HC2 cells were measured to be 1.87 × 10¹⁰ and 2.17 × 10¹⁰/M respectively. This humanized antibody leads to 78.5% inhibition in proliferation of EGFRvIII-overexpressing cells.     

A peptide accelerating the conversion of plasminogen activator inhibitor-1 to an inactive latent state

The serpin plasminogen activator inhibitor-1 (PAI-1) is a specific inhibitor of plasminogen activators and a potential therapeutic target in cancer and cardiovascular diseases. Accordingly, formation of a basis for development of specific PAI-1-inactivating agents is of great interest. One possible inactivation mode for PAI-1 is conversion to the inactive, so-called latent state. We have now screened a phage-displayed peptide library with PAI-1 as bait and isolated a 31-residue cysteine-rich peptide that will be referred to as paionin-4. A recombinant protein consisting of paionin-4 fused to domains 1 and 2 of the phage coat protein g3p caused a 2- to 3-fold increase in the rate of spontaneous inactivation of PAI-1. Paionin-4-D1D2 bound PAI-1 with a K(D) in the high nanomolar range. Using several biochemical and biophysical methods, we demonstrate that paionin-4-D1D2-stimulated inactivation consists of an acceleration of conversion to the latent state. As demonstrated by site-directed mutagenesis and competition with other PAI-1 ligands, the binding site for paionin-4 was localized in the loop between alpha-helix D and beta-strand 2A. We also demonstrate that a latency-inducing monoclonal antibody has an overlapping, but not identical binding site, and accelerates latency transition by another mechanism. Our results show that paionin-4 inactivates PAI-1 by a mechanism clearly different from other peptides, small organochemical compounds, or antibodies, whether they cause inactivation by stimulating latency transition or by other mechanisms, and that the loop between alpha-helix D and beta-strand 2A can be a target for PAI-1 inactivation by different types of compounds.     

On the mechanism of targeting of phage fusion protein-modified nanocarriers: only the binding peptide sequence matters

The integration of pharmaceutical nanocarriers with phage display techniques is emerging as a new paradigm for targeted cancer nanomedicines. We explored the direct use of landscape phage fusion proteins for the self-assembly of phage-derived binding peptides to liposomes for cancer cell targeting. The primary purpose of this study was to elucidate the targeting mechanism with a particular emphasis on the relative contributions of the two motifs that make up the landscape phage fusion protein (a binding peptide and the phage pVIII coat protein) to the targeting efficiency. Using transmission electron microscopy and dynamic light scattering, we confirmed the formation of phage-liposomes. Using FACS analysis, fluorescence microscopy, and fluorescence photospectrometry, we found that liposomes modified with MCF-7-specific phage fusion proteins (MCF-7 binding peptide, DMPGTVLP, fused to the phage PVIII coat protein) provided a strong and specific association with target MCF-7 cancer cells but not with cocultured, nontarget cells including C166-GFP and NIH3T3. The substitution for the binding peptide fused to phage pVIII coat protein abolished the targeting specificity. The addition of free binding peptide, DMPGTVLP, competitively inhibited the interaction of MCF-7-specific phage-liposomes with target MCF-7 cells but showed no reduction of MCF-7-associated plain liposomes. The proteolysis of the binding peptide reduced MCF-7 cell-associated phage-liposomes in a proteinase K (PK) concentration-dependent manner with no effect on the binding of plain liposomes to MCF-7 cells. Overall, only the binding peptide motif was involved in the targeting specificity of phage-liposomes. The presence of phage pVIII coat protein did not interfere with the targeting efficiency.     

New technologies in therapeutic antibody development

The development of therapeutic antibodies took a sharp turn with the introduction of phage display technology over a decade ago. Antibodies are used in a whole range of disease fields, such as autoimmunity, cancer, inflammation and infectious diseases. Now, the first antibody derived from phage display technology has been approved in the US by the Food and Drug Administration. The antibody industry is continuously developing new and robust discovery platforms and novel antibody formats, which points to the versatility of antibodies as therapeutic and diagnostic agents.     

Screening of phage displayed human liver cDNA library against dexamethasone

This paper described an attempt to establish a new method to screen the target biomolecule from phage displayed cDNA library against small molecule drug insoluble in water. Dexamethasone was selected as the model drug, and the screening was carried out in an Eppendorf tube packed with the drug. The whole procedure was monitored by PCR with the enriched specific phage clone as the template. After four rounds screening, the PCR products of selected phages with the lengths of 400 and 600 bp were sequenced, and revealed identical sequences with cytochrome c oxidase subunit III and albumin respectively by GenBank searching. Furthermore, frontal analysis-capillary electrophoresis (FA-CE) was performed to study the interaction between dexamethasone and albumin, and the binding constant was calculated to be 1.153 x 10(3), validating the weak specific interaction between the drug and the target protein. All these results demonstrated that with insoluble drug as the solid phase directly, the screening of target large molecule expressed in phage display cDNA library was feasible, which might pave an easy way to screen the candidate drug targets.     

Construction and characterization of a highly reactive chicken-derived single-chain variable fragment (scFv) antibody against Staphylococcus aureus developed with the T7 phage display system

The purpose of this study was to construct a single-chain variable fragment (scFv) antibody from chicken egg yolk immunoglobulin (IgY) by means of genetic engineering and subsequent panning for a specific antibody against Staphylococcus aureus.Methods and resultsWe amplified the scFv using blood and spleen obtained from 100-day-old Roman chickens immunized with inactivated S. aureus and subsequently constructed a T7 phage display antibody library using phage display technology. Four non-repeated blood scFv and 6 spleen scFv were obtained following 3 rounds of panning of the T7 phage display antibody library, enzyme-linked immunosorbent assay and sequencing. These 10 scFv were cloned into the prokaryotic expression vector pCold I with expression induced at a low temperature. Four soluble proteins were obtained. Among them, soluble protein SF_V6 derived from the spleen showed good reactivity against S. aureus using indirect ELISA and produced a particularly strong antibacterial effect in vitro.ConclusionWe were successful in isolating a highly specific scFv antibody against S. aureus from the spleen phage display library. This study provides a simple and rapid method for the quick preparation of a large number of antibodies against S. aureus and provides the foundation for the positioning of antibodies in the organism and the study of the antibacterial mechanism through which the antibody functions.     

Development of a human antibody fragment directed against the alpha folate receptor as a promising molecule for targeted application

Alpha folate receptor (FRα) is currently under investigation as a target for the treatment of patients with non-small-cell lung cancer (NSCLC), since it is highly expressed in tumor cells but is largely absent in normal tissue. In this study, a novel human variable domain of a heavy-chain (VH) antibody fragment specific to FRα was enriched and selected by phage bio-planning. The positive phage clone (3A102 VH) specifically bound to FRα and also cross-reacted with FRβ, as tested by ELISA. Clone 3A102 VH was then successfully expressed as a soluble protein in an E. coli shuffle strain. The obtained soluble 3A102 VH demonstrated a high affinity for FRα with affinity constants (K_aff) values around 7.77 ± 0.25 × 10⁷ M⁻¹, with specific binding against both FRα expressing NSCLC cells and NSCLC patient-derived primary cancer cells, as tested by cell ELISA. In addition, soluble 3A102 VH showed the potential desired property of a targeting molecule by being internalized into FRα-expressing cells, as observed by confocal microscopy. This study inspires the use of phage display to develop human VH antibody (Ab) fragments that might be well suited for drug targeted therapy of NSCLC and other FRα-positive cancer cells.     

Monoclonal antibodies as targeting and therapeutic agents: prospects for liver transplantation, hepatitis and hepatocellular carcinoma

1. Monoclonal antibodies (mAbs) of high specificity and stability have become key resources in the therapeutic, diagnostic and drug discovery fields to treat various immunological disorders and malignancies of different organs. 2. The latest genetic engineering technology applied in antibody design and production, such as phage display technology and genetically modified mouse, have revolutionized the clinical applicability and feasibility of the use of mAbs in humans. 3. Innovative antibody products in the forms of single-chain or super-humanized antibody therapeutics having a higher affinity for target antigens and minimal antigenicity in hosts have been introduced for experimental purposes and/or clinical trials. 4. Although there are successful examples of antibody therapeutics in the market, the use of mAbs in treating hepatitis-related disease and hepatocellular carcinoma is rare and remains to be exploited.     

In vitro optimization of liposomal nanocarriers prepared from breast tumor cell specific phage fusion protein

Fusion proteins created by phage display peptides with tumor cell specificity and the pVIII major coat protein of filamentous phages have been explored recently as a simple and cost-effective means for preparing tumor-targeted liposomes that improve the cytotoxicity of anticancer drugs in vitro. The next step in the development of this approach is the optimization of the liposome composition for the maximum targeting activity and subsequent testing in vivo. This study aimed to investigate the impact of preparation protocols, lipid composition and phage protein content on the targeting efficiency of phage protein-modified liposomes. Analysis of size, zeta potential and morphology was used to investigate the effect of preparation protocols on the stability and homogeneity of the phage liposomes. A previously developed coculture targeting assay and a factorial design approach were used to determine the role of lipid composition of the liposomal membrane on the target cell specificity of the phage liposomes. Western blot combined with proteinase K treatment detected the orientation of targeted phage protein in liposomal membrane. Phage protein, DPPG and PEG_2k-PE showed positive effects on target specificity of phage liposomes. The results served to identify optimal formulation that offer an improved liposomal affinity for target tumor cells over the non-optimized formulation.     

Liposomes targeted by fusion phage proteins

Targeting of nanocarriers has long been sought after to improve the therapeutic indices of anticancer drugs. Here we provide the proof of principle for a novel approach of nanocarrier targeting through their fusion with target-specific phage coat proteins. The source of the targeted phage coat proteins are landscape phage libraries--collections of recombinant filamentous phages with foreign random peptides fused to all 4000 copies of the major coat protein. We exploit in our approach the intrinsic physicochemical properties of the phage major coat protein as a typical membrane protein. Landscape phage peptides specific for specific tumors can be obtained by affinity selection, and purified fusion coat proteins can be assimilated into liposomes to obtain specific drug-loaded nanocarriers. As a paradigm for inceptive experiments, a streptavidin-specific phage peptide selected from a landscape phage library was incorporated into approximately 100-nm liposomes. Targeting of liposomes was proved by their specific binding to streptavidin-coated beads.     

Identification and characterization of peptide probes directed against PKCα conformations

Abstract: Phage display is a powerful technology that allows identification of high affinity peptides that bind specifically to a given molecular target. Using a highly complex peptide display library, we have identified separate classes of peptides that bind to protein kinase C alpha (PKCα) only under activation conditions. Furthermore, peptide binding was specific to PKCα and not to any of the other closely related PKC isoforms. The conformational and isoform specificity of the peptide binding was demonstrated using surface plasmon resonance as well as time‐resolved fluorescence assays. Kinase assays showed that these peptides were not direct substrates for PKC nor did they inhibit phosphorylation of PKC substrates. These peptides are most likely directed against protein–protein interaction sites on PKC. The data presented here offers another example of application of phage display technology to identify conformation‐dependent peptide probes against therapeutically important drug targets. These peptides are ideally suited to be used as surrogate ligands to identify compounds that bind specifically to PKCα, as well as conformational probes to detect activated forms of PKCα.     

VGFR噬菌体疫苗的构建及抗肿瘤效果

目的构建重组血管内皮生长因子受体(VGFR)／中心实验激酶功能区受体(KDR)噬菌体疫苗并且观察其抗肿瘤效果。方法将人源VGFR、KDR的膜外部分插入到T7噬菌体的基因组中,作为一种融合蛋白展示在T7噬菌体外壳上,从而构建了一种新型的抗肿瘤疫苗。ELlSA方法检测抗VGFR、KDR 抗体,C57小鼠经过四次免疫,接种Lewis肺癌后记录肿瘤生长情况,评价疫苗效果。结果成功从人 A549细胞中可克隆出VGFR、KDR基因,并且构建了相应的噬菌体疫苗。ELISA方法检测到了免疫小鼠血清中有特异性抗体的产生,滴度可达104,肿瘤生长曲线显示噬菌体疫苗有明显的抑制肿瘤生长的作用。结论提示利用噬菌体展示技术构建的噬菌体疫苗的应用是一种非常有效的抗肿瘤方法。     

Nanomedicine and protein misfolding diseases

Misfolding and self assembly of proteins in nano-aggregates of different sizes and morphologies (nano-ensembles, primarily nanofilaments and nano-rings) is a complex phenomenon that can be facilitated, impeded, or prevented, by interactions with various intracellular metabolites, intracellular nanomachines controlling protein folding and interactions with other proteins. A fundamental understanding of molecular processes leading to misfolding and self-aggregation of proteins involved in various neurodegenerative diseases will provide critical information to help identify appropriate therapeutic routes to control these processes. An elevated propensity of misfolded protein conformation in solution to aggregate with the formation of various morphologies impedes the use of traditional physical chemical approaches for studies of misfolded conformations of proteins. In our recent alternative approach, the protein molecules were tethered to surfaces to prevent aggregation and AFM force spectroscopy was used to probe the interaction between protein molecules depending on their conformations. It was shown that formation of filamentous aggregates is facilitated at pH values corresponding to the maximum of rupture forces. In this paper, a novel surface chemistry was developed for anchoring of amyloid beta (Abeta) peptides at their N-terminal moieties. The use of the site specific immobilization procedure allowed to measure the rupture of Abeta-Abeta contacts at single molecule level. The rupture of these contacts is accompanied by the extension of the peptide chain detected by a characteristic elasto-mechanical component of the force-distance curves. Potential applications of the nanomechanical studies to understanding the mechanisms of development of protein misfolding diseases are discussed.     

Monoclonal antibody therapy of inflammatory bowel disease.

Animal models of inflammatory bowel disease have provided insight in the regulation of mucosal inflammation. This has resulted in novel therapeutic approaches that specifically target a single inflammatory mediator. Monoclonal antibody therapy has been used in steroid refractory Crohn's disease patients. AntiTNF antibody administration induced complete remissions and few side effects were observed. Although these findings need to be confirmed in controlled and long term treatment studies they may guide the development of specific and effective therapies for these diseases.     

噬菌体肽库展示的应用及其最新进展

噬菌体肽库展示技术是近年来发展起来的用丝状噬菌体展示外源肽的一项新技术 ,被广泛应用于研究蛋白质与蛋白质之间的相互作用 ,新药的开发等各个领域。该文就该技术的原理、发展历史以及目前的常见应用作一简要概述