A human scFv antibody generation pipeline for proteome research

The functional decryption of the human proteome is the challenge which follows the sequencing of the human genome. Specific binders to every human protein are key reagents for this purpose. In vitro antibody selection using phage display offers one possible solution that can meet the demand for 25,000 or more antibodies, but needs substantial standardisation and minimalisation. To evaluate this potential, three human, naive antibody gene libraries (HAL4/7/8) were constructed and a standardised antibody selection pipeline was set up. The quality of the libraries and the selection pipeline was validated with 110 antigens, including human, other mammalian, fungal or bacterial proteins, viruses or haptens. Furthermore, the abundance of VH, kappa and lambda subfamilies during library cloning and the E. coli based phage display system on library packaging and the selection of scFvs was evaluated from the analysis of 435 individual antibodies, resulting in the first comprehensive comparison of V gene subfamily use for all steps of an antibody phage display pipeline. Further, a compatible cassette vector set for E. coli and mammalian expression of antibody fragments is described, allowing in vivo biotinylation, enzyme fusion and Fc fusion.     

Development of recombinant antibody-based enzyme-linked immunosorbent assay (ELISA) for the detection of skatole

The occurrence of boar taint and the European Commission recommendation to discontinue the surgical castration of pigs by the year 2018 creates an urgent need for new analytical methods that are simple, affordable, and suitable for field testing. We describe the generation and engineering of a skatole-specific antibody derived from a synthetic antibody library and the development of ELISA for its detection. The immunoassay is capable of detecting skatole with IC50 of 222 μg L⁻¹, which is within the analytical threshold level suggested for skatole, and with low cross-reactivity interference from other indolic compounds.     

Use of phage display and high-density screening for the isolation of an antibody against the 51-kDa subunit of complex I

Monoclonal antibodies play an increasingly important role in structural biology. In this report, we develop the use of phage display technology for the isolation of an antibody that binds to a specific subunit of a macromolecular assembly. Antibodies that bind to the intact complex are selected from a phage display library and screened with a high-density Western blot to identify a subunit-specific binder. Conventional Western blotting and competition ELISA are then used to confirm the identity of the target subunit and that the antibody binds to the native protein complex and not to an epitope that is only revealed when the antibody is immobilized for phage selection. Using this technique, monoclonal scFv and Fab fragments have been produced that bind to the 51-kDa subunit of bovine complex I, a large integral membrane protein complex from mitochondria.     

“Cleavable” hapten-biotin conjugates: Preparation and use for the generation of anti-steroid single-domain antibody fragments

Antibody engineering technology has the potential to provide artificial antibodies with higher performance than conventional antibodies. Filamentous phage particles are often used to express a vast diversity of mutated antibody fragments from which clones displaying improved fragments can be isolated. We recently showed that hapten-biotin conjugates, combined via a linker involving a reductively cleavable disulfide bond, are useful for isolating phage clones displaying high-affinity anti-hapten antibody fragments. Here we prepare cleavable hapten-biotin conjugates and use them to isolate anti-hapten antibody fragments with relatively low affinities. Three diagnostically important steroids (estradiol-17β [E₂], cortisol, and 17α-hydroxyprogesterone) were each coupled with a biotin derivative containing a disulfide bond. These conjugates could be bound simultaneously by their relevant anti-steroid antibody and NeutrAvidin, and their linkers were easily cleaved by dithiothreitol (DTT) treatment. The E₂-biotin conjugate was used to generate anti-E₂ single-domain antibody fragments (sdAbs). Random point mutations were introduced by error-prone PCR into the gene fragment encoding the V_H domain of a mouse anti-E₂ antibody, and these products were expressed as phagemid particles that were reacted with the E₂-biotin conjugates that had already been immobilized on a solid-phase via NeutrAvidin. Thorough washing off of nonspecific phages and subsequent DTT treatment provided a phagemid clone that displayed a mutated sdAb with improved binding properties.     

Domain libraries: Synthetic diversity for de novo design of antibody V-regions

A completely synthetic gene library encoding the variable light (VL) immunoglobulin domains has been constructed in vitro. The library was constructed by assembling a set of six oligodeoxyribonucleotides (oligos) using the polymerase chain reaction (PCR). Three out of the six overlapping oligonucleotides were synthesized with randomized complementarity determining regions (CDR) with the codon pattern, (NNS)_n, where N is any of the four nucleotides (nt) and n is the number of codons with variation in the CDR. The framework regions, taken from the D1.3 anti-lysozyme antibody (Ab), were kept intact. Overlapping regions of approx. 20 nt, together with two additional flanking primers carrying the desired restriction sites, allowed the construction of a library in one single PCR reaction. The VL library was cloned into the phage display vector pEXmide3, and ten randomly picked clones were sequenced. These sequences exhibited complete diversity in all the three CDR and the codons for five canonical amino acid (aa) residues were kept intact and identified. Seven clones contained the full-length gene for the VL domain while deletions were observed in three clones. The restricted use of nt at the third position successfully avoided the stop codons TGA and TAA, whereas the stop codon TAG is read as Gln in an amber suppressor strain. We call this synthetic Ab diversity Domain Library, and it represents an example of syntheticlibraries with extensive, multiple randomized sequences. The use of Domain Libraries opens up the possibility for design in Ab engineering, e.g., additional CDR regions can be added or their length varied. Furthermore, the use of synthetic gene libraries, constructed with the Domain Library strategy, is not limited to the construction of synthetic Ab fragments, but can be used in the design of other types of proteins.     

Frontier of therapeutic antibody discovery:The challenges and how to face them

Therapeutic monoclonal antibodies have become an important class of modern medicines.The established technologies for therapeutic antibody discovery such as humanization of mouse antibodies,phage display of human antibody libraries and transgenic animals harboring human IgG genes have been practiced successfully so far,and many incremental improvements are being made constantly.These methodologies are responsible for currently marketed therapeutic antibodies and for the biopharma industry pipeline which are concentrated on only a few dozen targets.A key challenge for wider application of biotherapeutic approaches is the paucity of truly validated targets for biotherapeutic intervention.The efforts to expand the target space include taking the pathway approach to study the disease correlation.Since many new targets are multi-spanning and multimeric membrane proteins there is a need to develop more effective methods to generate antibodies against these difficult targets.The pharmaceutical properties of therapeutic antibodies are an active area for study concentrating on biophysical characteristics such as thermal stability and aggregation propensity.The immunogenicity of biotherapeutics in humans is a very complex issue and there are no truly predictive animal models to rely on.The in silico and T-cell response approaches identify the potential for immunogenicity;however,one needs contingency plans for emergence of antiproduct antibody response for clinical trials.     

Analysis conditions for proteomic profiling of mammalian tissue and cell extracts with antibody microarrays

Antibody microarrays are a developing tool for global proteomic profiling. A protocol was established that permits robust analyses of protein extracts from mammalian tissues and cells rather than body fluids. The factors optimized were buffer composition for surface blocking, blocking duration, protein handling and processing, labeling parameters like type of dye, molar ratio of label versus protein, and dye removal, as well as incubation parameters such as duration, temperature, buffer, and sample agitation.     

An optimized procedure for efficient phage display of antibody fragments with a low folding efficiency

We recently developed an efficient bacterial expression system for phagemid-coded antigen-binding fragments of antibody (Fabs) without the use of a helper bacteriophage. This system is characterized by an unusually long cultivation at a low temperature and gentle induction of Fab expression without the addition of the inducer isopropyl-β-D-thiogalactopyranoside (IPTG). This method allows for a high yield production of Fabs fused with phage gene III coat protein, even when the protein is defective in its folding ability. With this cultivation procedure, we aimed here at improving the production and selection efficiency of filamentous bacteriophages displaying functional Fabs on their surface (Fab-phages) that have high affinity but low folding ability. The Fab components of the Fab-phages used were clonally related but differed in their affinity and folding ability. The production of the functional Fab-phages was quantitatively evaluated under various culture conditions. With conventional phage particle preparation, the production of functional Fab-phages was significantly biased according to the folding ability of the displayed Fabs, and affinity-based biopanning was therefore unsuccessful. In contrast, with the present procedure employing cultivation at 25 °C for 16 h without IPTG induction, functional Fab-phages were produced without any such dependence on folding ability. With this optimized library, affinity-based biopanning was successful. Especially noteworthy, bead-based biopanning accurately discriminated between high affinity Fab-phages and Fab-phages with low or middling affinity. In obtaining Fab-phages with high affinity but low folding ability, these optimized procedures for both cultivation and selection were essential.     

Improved affinity coupling for antibody microarrays: engineering of double-(His)6-tagged single framework recombinant antibody fragments

Antibody-based microarray is a novel technology with great promise in biomedicine that will provide unique means to perform global proteome analysis. In the process of designing the high-density antibody microarrays required, several critical key issues have been identified that remain to be resolved. In particular, there is a great need for specific and selective approaches enabling non-purified probes to be directly purified, orientated and coupled in a generic one-step procedure directly on the chip. In this study, we report on the successful design of affinity-tagged human recombinant single-chain fragment variable antibody fragments for improved affinity coupling in array applications. By replacing the standard single-histidine (His)(6)-tag with a consecutive double-(His)(6)-tag, the binding to Ni(2+)-nitrilotriacetic acid-coated substrates was significantly improved. Surface plasmon resonance analysis showed a significantly tighter binding with at least a threefold slower dissociation. The improved binding characteristics thus enabled non-purified probes even in the format of crude expression supernatants to be directly applied thereby eliminating the need for any time-consuming pre-purification step(s) prior to the immobilization. While the double-(His)(6)-tag probes were found to be expressed equally well as compared to the single-(His)(6)-tag probes, they displayed better long-term functional on-chip stability. Taken together, the results demonstrate the generic potential of double-(His)(6)-tag recombinant antibodies for the facile fabrication of high-density antibody microarrays.     

Use of high-capacity surface with oriented recombinant antibody fragments in a 5-min immunoassay for thyroid-stimulating hormone

High-capacity surfaces can enhance analyte-binding kinetics and be beneficial for rapid immunoassays. Site-specifically immobilized, oriented recombinant single-chain Fv (scFv) and Fab antibody fragments were compared with a conventional, nonoriented monoclonal antibody (Mab) to capture antigen from serum to solid surface in a one-step, two-site thyroid-stimulating hormone (TSH) immunoassay with a 5-min incubation time. The assay used a ready-to-use dry reagent-based concept and time-resolved fluorescent measurement. TSH binding capacities were 3.0-fold (Fab) and at least 4.1-fold (scFv) higher when recombinant antibodies were used instead of Mab. Recombinant antibody fragments also produced faster kinetics (5 vs. 45-min saturation level) than Mab: 21-25% (Mab) versus 72-83% (scFv and Fab). Analytical sensitivities of the 5-min assay were 0.09 mIU/L TSH (Fab), 0.16 mIU/L TSH (scFv), and 0.26 mIU/L TSH (Mab). Between-run variabilities were 4.2-7.9% (Fab), 4.6-17.7% (scFv), and 5.5-7.2% (Mab). The assays correlated well with the AutoDELFIA hTSH (human TSH) Ultra assay (r = 0.99, n = 109). Fab was good in all aspects of immunoassay—capacity, kinetics, sensitivity, and analytical performance. As a homogeneous, stable, and small-sized binding molecule with optimized surface-coating properties as well as reduced risk for interference by heterophilic antibodies, Fab fragment is a promising and realistic immunoreagent for the future.     

Design of recombinant antibody microarrays for complex proteome analysis: choice of sample labeling-tag and solid support

Antibody-based microarray is a novel technology with great potential within high-throughput proteomics. The process of designing high-performing antibody (protein) microarrays has, however, turned out to be a challenging process. In this study, we have developed further our human recombinant single-chain variable-fragment (scFv) antibody microarray methodology by addressing two crucial technological issues, choice of sample labeling-tag and solid support. We examined the performance of a range of dyes in a one- or two-color approach on a selection of solid supports providing different surface and coupling chemistries, and surface structures. The set-ups were evaluated in terms of sensitivity, specificity, and selectivity. The results showed that a one-color approach, based on NHS-biotin (or ULS-biotin) labeling, on black polymer Maxisorb slides (or Nexterion slide H) was the superior approach for targeting low-abundant (pg/mL) analytes in nonfractionated, complex proteomes, such as human serum or crude cell supernatants. Notably, microarrays displaying adequate spot morphologies, high S/Ns, minimized nonspecific binding, and most importantly a high selectivity, specificity, and sensitivity (>or=fM range) were obtained. Taken together, we have designed the first generation of a high-performing recombinant scFv antibody microarray technology platform on black polymer Maxisorb slides for sensitive profiling of low-abundant analytes in nonfractionated biotinylated complex proteomes.     

Monoclonal antibodies: technology around the clock for new therapeutic hopes

Engineering monoclonal antibodies, now widely used in the clinic, has made it possible to develop a new generation of antibodies with optimized functional properties. These antibodies should allow a significant improvement of the treatment of diseases where only few drugs are available, if any. However, the cost of treatments with monoclonal antibodies requires further improvements in production and purification technologies, and raises the question of generic antibodies. The present review summarizes some of the technological past and present challenges in the field.     

Yeast cell surface display system for determination of humoral response to active immunization with a monoclonal antibody against EpCAM

Even though an immunogenic formulation of the murine monoclonal anti-EpCAM (epithelian cell adhesion molecule) antibody Mab 17-1A, has been shown to evoke a strong humoral immune response in both, monkey studies and early clinical trials, conventional anti-EpCAM ELISA could not identify anti-EpCAM immune response in relation to treatment with Mab17-1A. In contrast, usage of cellulose membranes prepared by SPOT technology presenting overlapping EpCAM peptides allowed the unequivocal determination of EpCAM related antibodies present in monkeys sera after immunization with IGN101. Based on such contradictory results, it was of high interest to compare obtained data to a different method for better assessment of their possible interpretation. Therefore, in the present studies, some EpCAM peptides, determined as reactive by binding of IgG isolated from sera of treated monkeys on membranes prepared by SPOT technology, were represented on yeast surface using the pYD1 yeast display vector system. Binding of biotinylated IgG from sera was detected with streptavidin–FITC and quantity of binding was determined by FACS measurement. Though using this completely different method, experiments with pre-immune and immune sera of four monkeys exemplarily are comparable to the results obtained by analysis with synthetic peptide arrays.     

Design and evaluation of a 6-mer amyloid-beta protein derived phage display library for molecular targeting of amyloid plaques in Alzheimer's disease: Comparison with two cyclic heptapeptides derived from a randomized phage display library

Amyloid plaques are the main molecular hallmark of Alzheimer's disease. Specific carriers are needed for molecular imaging and for specific drug delivery. In order to identify new low molecular weight amyloid plaque-specific ligands, the phage display technology was used to design short peptides that bind specifically to amyloid-beta protein, which is the principal component of amyloid plaques. For this purpose, a phage display library was designed from the amino acid sequence of amyloid-beta 1-42. Then, the diversity was increased by soft oligonucleotide-directed mutagenesis. This library was screened against amyloid-beta 1-42 and several phage clones were isolated. Their genomes were sequenced to identify the displayed peptides and their dissociation constants for amyloid-beta 1-42 binding were evaluated by ELISA. The two best peptides, which are derived from the C-terminus hydrophobic domain of amyloid-beta 1-42 that forms a beta-strand in amyloid fibers, were synthesized and biotinylated. After confirming their binding affinity for amyloid-beta 1-42 by ELISA, the specific interaction with amyloid plaques was validated by immunohistochemistry on brain sections harvested from a mouse model of Alzheimer's disease. The thioflavin T aggregation assay has furthermore shown that our peptides are able to inhibit the amyloid fiber formation. They are not toxic for neurons, and some of them are able to cross the blood-brain barrier after grafting to a magnetic resonance imaging contrast agent. To conclude, these peptides have high potential for molecular targeting of amyloid plaques, either as carriers of molecular imaging and therapeutic compounds or as amyloid fiber disrupting agents.     

Retracted: Kinesin family member 2C aggravates the progression of hepatocellular carcinoma and interacts with competing endogenous RNA

Kinesin family member 2C (KIF2C), a substantial mitotic regulator, has been verified to exert a malignant function in several cancers. However, its function in hepatocellular carcinoma (HCC) remains unclear. In this study, the expression profile of KIF2C in HCC was characterized through the dataset from the TCGA and clinical tissue microarrays containing 220 pairs of resected HCC tissues and adjacent nontumor tissues in our hospital. The results indicated that KIF2C was substantially higher expression in tumor tissues than adjacent nontumor tissues. High expression of KIF2C significantly correlated with large tumor (>5.0 cm) (P = .001) and implied a dismal postoperative overall survival (OS) (hazard ratio [HR] = 1.729; P = .002) in our cohort of patients. Gain and loss of function assays displayed that KIF2C promoted HCC cell proliferation, accelerated cell cycle progression, and impeded apoptosis. By bioinformatic tools and mechanistic investigation, we found that KIF2C interacted with various cell-cycle-related proteins and was significantly involved in growth-promoting pathways. KIF2C upregulated PCNA and CDC20 expression. Subsequently, we investigated the regulation of KIF2C by competing endogenous RNA and elucidated that has-miR-6715a-3p was directly bond to the 3′-untranslated region of KIF2C through dual luciferase assays, thereby inhibiting KIF2C expression. Furthermore, the long noncoding RNA GS1-358P8.4 was found to be a candidate of KIF2C for has-miR-6715a-3p binding. HCC patients with high lncRNA-GS1-358P8.4 expression had shorter OS and relapse-free survival compared to those with low expression, which was accordance with the KIF2C. Taken together, KIC2C aggravated HCC progression, it could serve as a prognostic indicator and confer a novel target for clinical treatment.     

The gene for Klebsiella bacteriophage K11 RNA polymerase: sequence and comparison with the homologous genes of phages T7, T3, and SP6

Summary We determined the nucleotide sequence of gene 1 of Klebsiella phage K11, which is a member of the T7 group of phages. The largest open reading frame corresponds to a polypeptide with 906 amino acids and a molecular weight of 100383 daltons. The deduced amino acid sequence of this polypeptide shows 71% homology to the T7 RNA polymerase (the product of T7 gene 1), 72% homology to the T3 RNA polymerase and 27% homology to the SP6 RNA polymerase. Divergent evolution was clearly most pronounced in the amino-terminal portion.     

C-terminal antibodies (CTAbs): a simple and broadly applicable approach for the rapid generation of protein-specific antibodies with predefined specificity

Recent advances in proteomic techniques have resulted in an ever-increasing need to produce antibodies. Here, to address this problem, a technically simple approach of targeting the extreme C-termini of proteins with antibodies (CTAbs) was investigated in proteins secreted by the human pathogen Streptococcus pyogenes. Target proteins were identified by a conventional proteomic approach and CTAbs produced against synthetic five amino acid peptides representing the C-terminus of each target protein. In every case where protein secretion was demonstrated (n = 20), CTAbs were successfully produced and bound specifically to the target protein (100% success rate). The apparent specificity was consistent with the structural heterogeneity of the C-termini of S. pyogenes proteins. The global specificity of CTAb binding was defined using a combinatorial library of synthetic peptides representing structural variants of the original synthetic immunogen. This is a systematic and comprehensive approach for the development of antibodies with defined specificity that can be used in a range of applications.     

Sorting protein lists with nwCompare: A simple and fast algorithm for n‐way comparison of proteomic data files

MS, the reference technology for proteomics, routinely produces large numbers of protein lists whose fast comparison would prove very useful. Unfortunately, most softwares only allow comparisons of two to three lists at once. We introduce here nwCompare, a simple tool for n‐way comparison of several protein lists without any query language, and exemplify its use with differential and shared cancer cell proteomes. As the software compares character strings, it can be applied to any type of data mining, such as genomic or metabolomic datalists.