Design and selection of an intrabody library produced de-novo for the non-structural protein NSP5 of rotavirus

Intracellular antibodies or intrabodies have great potential in protein knockout strategies for intracellular antigens. We applied the Intracellular Antibody Capture Technology for the direct selection in yeast of a mouse scFv library (V(L)-V(H) format) constructed from animals immunised with recombinant non-structural protein NSP5 of Rotavirus. We selected five different intracellular antibodies (ICAbs), which specifically recognize Delta2, an NSP5 deletion mutant used as bait. The anti-NSP5 ICAbs were well expressed both in yeast and mammalian cells as cytoplasmic or nuclear-tagged forms. By immunofluorescence and co-immunoprecipitation assays we characterised the intracellular interaction of the five anti-NSP5 ICAbs with the co-expressed antigens.     

Making cell-permeable antibodies (Transbody) through fusion of protein transduction domains (PTD) with single chain variable fragment (scFv) antibodies: potential advantages over antibodies expressed within the intracellular environment (Intrabody)

Over the past decade, there has been growing interest in the use of antibodies against intracellular targets. This is currently achieved through recombinant expression of the single chain variable fragment (scFv) antibody format within the cell, which is commonly referred to as an intrabody. This possesses a number of inherent advantages over RNA interference (iRNA). Firstly, the high specificity and affinity of intrabodies to target antigens is well-established, whereas iRNA has been frequently shown to exert multiple non-specific effects. Secondly, intrabodies being proteins possess a much longer active half-life compared to iRNA. Thirdly, when the active half-life of the intracellular target molecule is long, gene silencing through iRNA would be slow to yield any effect, whereas the effects of intrabody expression would be almost instantaneous. Lastly, it is possible to design intrabodies to block certain binding interactions of a particular target molecule, while sparing others. There is, however, various technical challenges faced with intrabody expression through the application of recombinant DNA technology. In particular, protein conformational folding and structural stability of the newly-synthesized intrabody within the cell is affected by reducing conditions of the intracellular environment. Also, there are overwhelming safety concerns surrounding the application of transfected recombinant DNA in human clinical therapy, which is required to achieve intrabody expression within the cell. Of particular concern are the various viral-based vectors that are commonly-used in genetic manipulation. A novel approach around these problems would be to look at the possibility of fusing protein transduction domains (PTD) to scFv antibodies, to create a 'cell-permeable' antibody or 'Transbody'. PTD are short peptide sequences that enable proteins to translocate across the cell membrane and be internalized within the cytosol, through atypical secretory and internalization pathways. There are a number of distinct advantages that a 'Transbody' would possess over conventional intrabodies expressed within the cell. For a start, 'correct' conformational folding and disulfide bond formation can take place prior to introduction into the target cell. More importantly, the use of cell-permeable antibodies or 'Transbodies' would avoid the overwhelming safety and ethical concerns surrounding the direct application of recombinant DNA technology in human clinical therapy, which is required for intrabody expression within the cell. 'Transbodies' introduced into the cell would possess only a limited active half-life, without resulting in any permanent genetic alteration. This would allay any safety concerns with regards to their application in human clinical therapy.     

Selection of Recombinant, Library-Derived Antibody Fragments against p24 for Human Immunodeficiency Virus Type 1 Diagnostics

By application of combinatorial library technology, we generated the first recombinant antibody fragments directed against the major capsid protein p24 of human immunodeficiency virus type 1 (HIV-1). A library of single-chain Fv fragments (scFvs) was constructed by using the antibody variable-region (V) genes of B cells derived from the spleen of a viral lysate-immunized mouse. Antibodies were selected by panning or by enrichment with biotinylated antigen, yielding four different families of antibody fragments. The different types of scFvs were characterized by affinity measurements, by antigen recognition on Western blots, and by pepscan analysis. The epitope of one of the scFvs is located near the residues involved in CypA binding, thereby making it an attractive candidate for therapeutic applications. Comparison of the V gene sequence of this scFV with that of a previously described monoclonal antibody reactive against this immunodominant epitope revealed the usage of the identical combination of V_H and V_κ regions. Thus, this is one of the rare examples in which the original combination in a library-derived antibody fragment was retrieved. After appropriate affinity and format improvements, the best of our recombinant scFvs may form the basis for a sensitive p24 assay as a measure of viral load. In addition, anti-p24 scFvs could be expressed as intracellular antibodies (intrabodies) to aid in the treatment of HIV infections.     

The Therapeutic Potential of Intrabodies in Neurologic Disorders

Single-chain Fv and single-domain antibodies retain the binding specificity of full-length antibodies but they can be cloned, selected, engineered, and manipulated as genes. When expressed intracellularly in mammalian cells these intracellular antibodies, or intrabodies, have the potential to alter the folding, interactions, modifications, or subcellular localization of their targets. These reagents have previously been developed as therapeutics against cancer and HIV. Since misfolded and accumulated intracellular proteins characterize several major neurodegenerative disorders, including Huntington disease (HD) and Parkinson disease, these disorders are prime candidates for intrabody therapy. In this article we review the extension of intrabody technology to the nervous system. Studies of HD have been used to develop the approach and anti-synuclein strategies are in the early stages of development. Such neurodegenerative diseases are therefore poised for engineered antibody approaches, which can provide a pipeline of novel therapeutics and new drug discovery tools.     

噬菌体展示技术在抗体亲和力成熟中的应用进展

抗体因其高度的特异性在诊断和靶向治疗上一直备受青睐,相关行业发展也是突飞猛进。噬菌体展示技术作为一种抗体库构建技术,不仅可应用于特异性抗体的筛选,也可应用于对已获得的低亲和力抗体进行亲和成熟的研究。该方法主要利用VH和VL的随机重组,能在一定程度上模拟体内抗体亲和力成熟的过程,使噬菌体展示技术在提升抗体亲和力方面拥有许多优势,以下对噬菌体展示技术在亲和力成熟方面的应用进行综述。     

A universal strategy for stable intracellular antibodies

The expression of intracellular antibodies (intrabodies) in mammalian cells has provided a powerful tool to manipulate microbial and cellular signalling pathways in a highly precise manner. However, several technical hurdles have thus far restricted their more widespread use. In particular, single-chain antibodies (scFvs) have been reported to fold poorly in the reducing environment of the cytoplasm and as such there has been a reluctance to use scFv-phage libraries as a source of intrabodies unless a preselection step was applied to identify these rare scFvs that could fold properly in the absence of disulfide bonds. Recently, we reported that scFvs can be efficiently expressed within the cytoplasm of bacteria when fused at the C-terminus of the Escherichia coli maltose-binding protein (MBP). Here, we demonstrate that such MBP-scFvs are similarly stabilized when expressed in the mammalian cell cytoplasm as well as other compartments. This was demonstrated by comparing MBP-scFv fusions to the corresponding unfused scFvs that activate a defective beta-galactosidase enzyme, others that neutralize the wild-type beta-galactosidase enzyme, and an antibody that blocks the epidermal growth factor receptor. In all cases, the MBP-scFvs significantly outperformed their unfused counterparts. Our results suggest that fusion of scFvs to MBP, and possibly to other "chaperones in the context of a fusion protein", may provide a universal approach for efficient expression of intrabodies in the mammalian cell cytoplasm. This strategy should allow investigators to bypass much of the in vitro scFv characterization that is often not predictive of in vivo intrabody function and provide a more efficient use of large native and synthetic scFv-phage libraries already in existence to identify intrabodies that will be active in vivo.     

Predicting antigenic peptides suitable for the selection of phage antibodies

Selection from phage antibody libraries can be considered to be an in vitro immune system in which the antibody response is reduced to the bare minimum of antigen recognition. Using selections of antibodies on peptides from a phage antibody library, we investigated what constitutes peptide antigenicity in the context of the antibody-protein binding site. We selected polyclonal antibodies in a high throughput format against 44% of 90 overlapping peptides derived from three different proteins. Of these, 33% of peptides (epitopic peptides) were able to select antibodies that recognized the protein from which the peptides were derived. Although no algorithm was able to predict all epitopic peptides, solvent accessibility was the best predictor in this cell-free antibody selection context. We subsequently applied solvent accessibility to successfully predict epitopic peptides from p53 and Znf217, and showed that such peptide selected single-chain antibodies were able to recognize soluble p53 in ELISA and Znf217 in a western blot. This is likely to have considerable utility in functional genomics and proteomics where it should be possible to select antibodies against gene products on the basis of deduced amino acid sequence in a high throughput fashion.     

Development of an antibody proteomics system using a phage antibody library for efficient screening of biomarker proteins

Proteomics-based analysis is currently the most promising approach for identifying biomarker proteins for use in drug development. However, many candidate biomarker proteins that are over- or under-expressed in diseased tissues are found by such a procedure. Thus, establishment of an efficient method for screening and validating the more valuable targets is urgently required. Here, we describe the development of an "antibody proteomics system" that facilitates the screening of biomarker proteins from many candidates by rapid preparation of cross-reacting antibodies using phage antibody library technology. Using two-dimensional differential in-gel electrophoresis analysis, 16 over-expressed proteins from breast cancer cells were identified. Specifically, proteins were recovered from the gel pieces and a portion of each sample was used for mass spectrometry analysis. The remainder was immobilized onto a nitrocellulose membrane for antibody-expressing phage enrichment and selection. Using this procedure, antibody-expressing phages against each protein were successfully isolated within two weeks. The expression profiles of the identified proteins were then acquired by immunostaining of breast tumor tissue microarrays with the antibody-expressing phages. Using this approach, expression of Eph receptor A10, TRAIL-R2 and Cytokeratin 8 in breast tumor tissues were successfully validated. These results demonstrate the antibody proteomics system is an efficient method for screening tumor-related biomarker proteins.     

Evidence for the stabilizing effect of antibodies on the subunit association of glyceraldehyde-3-phosphate dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenases isolated from baker's yeast and from rat skeletal muscle were shown to have no common antigenic determinants revealed with antibodies elicited in rabbits. Fab fragments of rabbit anti-yeast enzyme antibodies have no effect on the dehydrogenase activity of the antigen. This is consistent with the results previously obtained with a combination of the rat muscle enzyme with specific antibodies.

Tetrameric yeast apo-glyceraldehyde-3-phosphate dehydrogenase immobilized on the solid support by means of interaction with specific antibodies covalently bound to the matrix retains the full catalytic activity and is capable of dissociating into dimers at low temperature in the presence of ATP. The dissociation is accompanied by the inactivation of dimers which become solubilized, whereas those bound to the matrix remain active even in the cold in the presence of ATP.

The tetrameric molecule of yeast glyceraldehyde-3-phosphate dehydrogenase, covalently bound to CNBractivated Sepharose via one subunit, is completely protected from dissociation by three molecules of specific antibodies bound per tetramer.

The binding of four molecules of Fab fragments of specific antibodies per tetramer protects the rat muscle apo-glyceraldehyde-3-phosphate dehydrogenase from NaCl-induced cold inactivation and from thermoinactivation in solution.

The binding of two molecules of anti-rat muscle enzyme antibodies to a hybrid tetramer that consists of a yeast dimer covalently bound to Sepharose, and of a rat muscle dimer, prevents the dissociation of the immobilized molecule (that is, solubilization of the rat muscle dimer).

The gel-filtration pattern of the complexes of the rat muscle apoenzyme with Fab fragments of specific antibodies points to a shift of the equilibrium between the tetrameric and dimeric forms of the enzyme in solution towards the tetrameric form.

On the basis of these observations, we conclude that specific antibodies strengthen subunit interactions in the oligomer by means of stabilization of the native structure of individual subunits.     

Proteome Analysis Based on Human Recombinant Antibody Microarrays

Protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform high-throughput global proteome analysis. A chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. We aim to develop protein microarrays based on human recombinant scFv antibody fragments for global proteome analysis. The concept of comparing proteomic maps of healthy versus diseased samples will allow disease-specific proteins to be detected. In fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. However, the complexity of proteomes, containing several thousands of different proteins, is a problem. Here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. To this end, an anticytokine antibody array was developed and human dendritic cells (±activation) was used as model system. The results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. Furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. Due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. In more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. The results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable.     

噬菌体抗体库技术在肿瘤诊断及治疗中的应用

噬菌体抗体库技术是近10年发展起来的一种抗体制备的新技术。它最大的优点是实现了直接将基因型（genotype）和表型（phenotype）联系在一起，能够在体外模拟体内的抗体生成过程，使抗体工程技术进入了一个新的时代。随着噬菌体抗体库技术的不断发展和成熟，它在肿瘤的免疫诊断和免疫治疗中显示出了广阔的应用前景。     

Cytoplasmic expression and specific binding of the VH/VL single domain intrabodies in transfected NIH3T3 cells

Intracellularly expressed antibody fragments (intrabodies) have been utilized as powerful tools not only for clinical applications but also for the functional analysis of proteins inside the cell. Among several types of intrabodies developed so far, single domain types composed of only the variable regions (V_H or V_L) of antibodies are the smallest and thus the easiest to design. In this study, four types of single domain intrabodies were evaluated against a cytosolic protein, Wiskott-Aldrich syndrome protein (WASP), in gene-transfected NIH3T3 cells. These single domains were composed of the V_H and V_L region with or without their leader sequences. Although these single domains were expressed at similar levels in NIH3T3 cells, the binding activity to the cytosolic target was higher in the single domain constructs with leader sequences. These results suggest the usefulness of the single domain intrabody constructs to analyze the functional domains of cytosolic proteins in cells.     

Engineered antibody therapies to counteract mutant huntingtin and related toxic intracellular proteins

The engineered antibody approach to Huntington's disease (HD) therapeutics is based on the premise that significantly lowering the levels of the primary misfolded mutant protein will reduce abnormal protein interactions and direct toxic effects of the misfolded huntingtin (HTT). This will in turn reduce the pathologic stress on cells, and normalize intrinsic proteostasis. Intracellular antibodies (intrabodies) are single-chain (scFv) and single-domain (dAb; nanobody) variable fragments that can retain the affinity and specificity of full-length antibodies, but can be selected and engineered as genes. Functionally, they represent a protein-based approach to the problem of aberrant mutant protein folding, post-translational modifications, protein-protein interactions, and aggregation. Several intrabodies that bind on either side of the expanded polyglutamine tract of mutant HTT have been reported to improve the mutant phenotype in cell and organotypic cultures, fruit flies, and mice. Further refinements to the difficult challenges of intraneuronal delivery, cytoplasmic folding, and long-term efficacy are in progress. This review covers published studies and emerging approaches on the choice of targets, selection and engineering methods, gene and protein delivery options, and testing of candidates in cell and animal models. The resultant antibody fragments can be used as direct therapeutics and as target validation/drug discovery tools for HD, while the technology is also applicable to a wide range of neurodegenerative and other diseases that are triggered by toxic proteins.     

A Systems Approach to the Proteomic Identification of Novel Cancer Biomarkers

The proteomics field has experienced rapid growth with technologies achieving ever increasing accuracy, sensitivity, and throughput, and with availability of computational tools to address particular applications. Given that the proteome represents the most functional component encoded for in the genome, a systems approach to disease investigations and biomarker identification benefits substantially from integration of proteome level studies. Here we present proteomic approaches that have allowed systematic searches for potential cancer markers by integrating cancer cell profiling with additional sources of data, as illustrated with recent studies of ovarian cancer.     

Extended half-life and elevated steady-state level of a single-chain Fv intrabody are critical for specific intracellular retargeting of its antigen, caspase-7

8 h) and high steady-state levels of protein accumulation, while the H2 intrabodies had a half-life of 2 h and less protein at steady state. These results suggest that the choice of sFv as an intrabody depends critically on the intracellular sFv protein having an extended half-life and elevated steady-state level. Thus, extended half-life must be considered together with sFv antibody specificity and affinity when choosing an optimal sFv intrabody for functional studies of cellular proteins.     

An Assay for Mannan-Binding Lectin-Associated Serine Protease 3, MASP-3

In analogy to DNA microarrays, protein microarrays offer a new distinct possibility to perform sensitive high-throughput global proteome analysis. However, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. The analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. We have recently generated a human recombinant single-chain Fv antibody library, genetically constructed around one framework, the nCoDeR-library, containing 2 × 1010 clones. Single framework antibody fragments (sinFabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). However, the choice of framework is critical. We have shown that the selected nCoDeR framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. Furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. An in-house-designed substrate, macroporous silicon coated with nitrocellulose (MAP3-NC7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. We have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. Using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinFab-molecules to Ni2⁺-coated solid supports, thereby allowing nonpurified probes to be directly applied.