Recognition of RNA by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids

The S9.6 antibody is broadly used to detect RNA:DNA hybrids but has significant affinity for double-stranded RNA. The impact of this off-target RNA binding activity has not been thoroughly investigated, especially in the context of immunofluorescence microscopy. We report that S9.6 immunofluorescence signal observed in fixed human cells arises predominantly from ribosomal RNA, not RNA:DNA hybrids. S9.6 staining was unchanged by pretreatment with the RNA:DNA hybrid–specific nuclease RNase H1, despite verification in situ that S9.6 recognized RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to RNase T1, which specifically degrades RNA. Additional imaging and biochemical data indicate that the prominent cytoplasmic and nucleolar S9.6 signal primarily derives from ribosomal RNA. Importantly, genome-wide maps obtained by DNA sequencing after S9.6-mediated DNA:RNA immunoprecipitation (DRIP) are RNase H1 sensitive and RNase T1 insensitive. Altogether, these data demonstrate that imaging using S9.6 is subject to pervasive artifacts without pretreatments and controls that mitigate its promiscuous recognition of cellular RNAs.     

Humanization of fibroblast growth factor 1 single‐chain antibody and validation for its antitumorigenic efficacy in breast cancer and glioma cells

Single‐chain variable fragment (scFv) antibodies are the smallest immunoglobulins with high antigen‐binding affinity. We have previously reported that fibroblast growth factor 1 played pivotal roles in cancer development and generated a mouse scFv (mscFv1C9) could effectively prohibit cancer cell proliferation in vitro and in vivo. Here, we further humanized this scFv (hscFv1C9) using a structure‐guided complementarity determining region grafting strategy. The purified hscFv1C9 maintained similar antigen‐binding affinity and specificity as mscFv1C9, and it was capable of inhibiting growth of different tumours in vitro and in vivo. These data strongly suggested that hscFv1C9 has antitumour potentials.     

A new human catalytic antibody Se‐scFv‐2D8 and its selenium‐containing single domains with high GPX activity

Glutathione peroxidase (GPX) is a well‐known antioxidant selenoenzyme, which can catalyze the reduction of a variety of hydroperoxides and consequently protect cells and other biological tissues against oxidative damage. Many attempts have been made to mimic its function, and a human catalytic antibody Se‐scFv‐B3 with GPX activity has been prepared in our previous study. This time, a new clone 2D8 that bound specifically to the glutathione analog GSH‐S‐DNPBu was selected again by using the technology of phage display antibody library, and then scFv‐2D8 was successfully expressed in soluble form and purified using Ni²⁺‐immobilized metal affinity chromatography. After being converted into selenium‐containing scFv by chemically modification, it showed higher GPX activity than previous abzyme Se‐scFv‐B3. The heavy chain variable fragment of scFv‐2D8 was also prepared and converted into selenium‐containing protein using the same method. This selenium‐containing single‐domain antibody showed some GPX activity and, to the best of our knowledge, is the first human single‐domain abzyme with GPX activity, which lays a foundation for preparing GPX abzyme with human origin, lower molecular weight and higher activity. Copyright © 2009 John Wiley & Sons, Ltd.     

Promoting α‐secretase cleavage of beta‐amyloid with engineered proteolytic antibody fragments

Deposition of beta‐amyloid (Aβ) is considered as an important early event in the pathogenesis of Alzheimer's Disease (AD), and reduction of Aβ levels by various therapeutic approaches is actively being pursued. A potentially non‐inflammatory approach to facilitate clearance and reduce toxicity is to hydrolyze Aβ at its α‐secretase site. We have previously identified a light chain fragment, mk18, with α‐secretase‐like catalytic activity, producing the 1–16 and 17–40 amino acid fragments of Aβ40 as primary products, although hydrolysis is also observed following other lysine and arginine residues. To improve the specific activity of the recombinant antibody by affinity maturation, we constructed a single chain variable fragment (scFv) library containing a randomized CDR3 heavy chain region. A biotinylated covalently reactive analog mimicking α‐secretase site cleavage was synthesized, immobilized on streptavidin beads, and used to select yeast surface expressed scFvs with increased specificity for Aβ. After two rounds of selection against the analog, yeast cells were individually screened for proteolytic activity towards an internally quenched fluorogenic substrate that contains the α‐secretase site of Aβ. From 750 clones screened, the two clones with the highest increase in proteolytic activity compared to the parent mk18 were selected for further study. Kinetic analyses using purified soluble scFvs showed a 3‐ and 6‐fold increase in catalytic activity (k_cat/K_M) toward the synthetic Aβ substrate compared to the original scFv primarily due to an expected decrease in K_M rather than an increase in k_cat. This affinity maturation strategy can be used to select for scFvs with increased catalytic specificity for Aβ. These proteolytic scFvs have potential therapeutic applications for AD by decreasing soluble Aβ levels in vivo. © 2009 American Institute of Chemical Engineers. Biotechnol. Prog., 2009     

A novel anti‐prion protein monoclonal antibody and its single‐chain fragment variable derivative with ability to inhibit abnormal prion protein accumulation in cultured cells

mAbs T1 and T2 were established by immunizing PrP gene ablated mice with recombinant MoPrP of residues 121–231. Both mAbs were cross‐reactive with PrP from hamster, sheep, cattle and deer. A linear epitope of mAb T1 was identified at residues 137–143 of MoPrP and buried in PrP^C expressed on the cell surface. mAb T1 showed no inhibitory effect on accumulation of PrP^Sc in cultured scrapie‐infected neuroblastoma (ScN2a) cells. In contrast, mAb T2 recognized a discontinuous epitope ranged on, or structured by, residues 132–217 and this epitope was exposed on the cell surface PrP^C. mAb T2 showed an excellent inhibitory effect on PrP^Sc accumulation in vitro at a 50% inhibitory concentration of 0.02 μg/ml (0.14 nM). The scFv form of mAb T2 (scFv T2) was secreted in neuroblastoma (N2a58) cell cultures by transfection through eukaryotic secretion vector. Coculturing of ScN2a cells with scFv T2‐producing N2a58 cells induced a clear inhibitory effect on PrP^Sc accumulation, suggesting that scFv T2 could potentially be an immunotherapeutic tool for prion diseases by inhibition of PrP^Sc accumulation.     

Phage-derived fully human antibody scFv fragment directed against human vascular endothelial growth factor receptor 2 blocked its interaction with VEGF

Vascular endothelial growth factor receptor 2 (VEGFR‐2) plays a critical role in tumor angiogenesis. None therapeutic antibodies targeting VEGFR‐2 are available in clinical use. Herein, we describe the screening of a new single‐chain antibody fragment (scFv) targeting extracellular domain 3 of human VEGFR‐2 (kinase insert domain‐containing receptor [KDR]3) from Griffin phage display scFv library. A comprehensive sequence analysis was performed to assign the framework and complementary‐determining regions. The scFv exerted particular binding sites to KDR3 on molecular docking, and the binding affinity was further convinced by binding analysis both in quantitative ELISA and real‐time kinetic determination by biosensors (K_D = 40 nM). Finally, the scFv was revealed to inhibit VEGF‐stimulated proliferation of human umbilical vein endothelial cells (HUVECs; IC₅₀ = 5 nM) and to inhibit HUVEC migration significantly at 17 nM. Taken together, our results indicate that we have successfully isolated a scFv which differentially recognizes KDR3 and has potential clinical applications in the treatment of angiogenesis related diseases. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 981–989, 2012     

RNase H eliminates R‐loops that disrupt DNA replication but is nonessential for efficient DSB repair

In Saccharomyces cerevisiae, genome stability depends on RNases H1 and H2, which remove ribonucleotides from DNA and eliminate RNA–DNA hybrids (R‐loops). In Schizosaccharomyces pombe, RNase H enzymes were reported to process RNA–DNA hybrids produced at a double‐strand break (DSB) generated by I‐PpoI meganuclease. However, it is unclear if RNase H is generally required for efficient DSB repair in fission yeast, or whether it has other genome protection roles. Here, we show that S. pombe rnh1? rnh201? cells, which lack the RNase H enzymes, accumulate R‐loops and activate DNA damage checkpoints. Their viability requires critical DSB repair proteins and Mus81, which resolves DNA junctions formed during repair of broken replication forks. “Dirty” DSBs generated by ionizing radiation, as well as a “clean” DSB at a broken replication fork, are efficiently repaired in the absence of RNase H. RNA–DNA hybrids are not detected at a reparable DSB formed by fork collapse. We conclude that unprocessed R‐loops collapse replication forks in rnh1? rnh201? cells, but RNase H is not generally required for efficient DSB repair.     

Construction and characterization of two anti‐sweetener single chain antibodies using radioligand binding,fluorescence and circular dichroism spectroscopy

Two single‐chain antibodies (scFv) that bind the superpotent sweetener ligand, NC‐174, were generated from mouse monoclonal antibodies (mAb) NC6.8 (IgG, κ) and NC10.14 (IgG, λ). These scFv were constructed by cloning the variable region sequences of the mAb, connecting them in tandem with a 25‐amino‐acid polypeptide linker, and expressing them in E. coli using the pET‐11a system. The recombinant proteins were purified using Ni²⁺–NTA–agarose by virtue of a hexahistidine sequence introduced to the C‐terminus of the heavy chain variable region during the cloning process. The secondary structure and ligand binding properties of the two scFv, the parent mAbs and proteolytically derived Fab fragments were examined using radioligand binding, circular dichroism (CD) and fluorescence spectroscopy. The far‐UV CD spectra of both scFv possessed predominantly β character, as did those of the Fab, and the near‐UV CD spectral data for scFvNC10.14, NC6.8 and NC10.14 Fab indicated that chromophore perturbation occurred upon ligand binding. The affinity constants determined for the two scFv, Fab and mAb were nearly equivalent. Copyright © 1999 John Wiley & Sons, Ltd.     

Antigen‐responsive regulation of Cell motility and migration via the signalobodies based on c‐Fms and c‐Mpl

Since cell migration plays critical roles in development and homeostasis of the body, artificial control of cell migration would be promising for the treatment of various diseases related to migration. To this end, we previously developed single‐chain Fv (scFv)/receptor chimeras, named signalobodies, which can control cell fates via a specific antigen that is different from natural cytokines. Although a conventional chemotaxis chamber assay revealed that several signalobodies based on receptor tyrosine kinases transduced antigen‐dependent migration signals, we have never performed direct observation of the cells to obtain more information on overall properties of cell motility and migration. In this study, we utilized murine pro‐B Ba/F3 cells expressing either a scFv‐Fms or scFv‐Mpl signalobody, and compared their migratory characteristics. We employed a lipid–polyethylene glycol conjugate to softly immobilize the suspension cells on a slide, which facilitated direct observation of chemokinetic activity of the cells. Consequently, both cells markedly exhibited chemokinesis in response to a specific antigen. In addition, the cells were subjected to a stable antigen‐concentration gradient to observe horizontal directional cell migration in real time. The results showed that the cells expressing scFv‐Fms underwent directional migration toward a positive antigen‐concentration gradient. Taken together, we successfully demonstrated antigen‐responsive regulation of cell motility and migration via the signalobodies. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:411–417, 2014     

Catalytically inactive,purified RNase H1: A specific and sensitive probe for RNA–DNA hybrid imaging

R-loops are three-stranded nucleic acid structures with both physiological and pathological roles in cells. R-loop imaging generally relies on detection of the RNA–DNA hybrid component of these structures using the S9.6 antibody. We show that the use of this antibody for imaging can be problematic because it readily binds to double-stranded RNA (dsRNA) in vitro and in vivo, giving rise to nonspecific signal. In contrast, purified, catalytically inactive human RNase H1 tagged with GFP (GFP-dRNH1) is a more specific reagent for imaging RNA–DNA hybrids. GFP-dRNH1 binds strongly to RNA–DNA hybrids but not to dsRNA oligonucleotides in fixed human cells and is not susceptible to binding endogenous RNA. Furthermore, we demonstrate that purified GFP-dRNH1 can be applied to fixed cells to detect hybrids after their induction, thereby bypassing the need for cell line engineering. GFP-dRNH1 therefore promises to be a versatile tool for imaging and quantifying RNA–DNA hybrids under a wide range of conditions.     

Redesigning of anti‐c‐Met single chain Fv antibody for the cytoplasmic folding and its structural analysis

Typically, single chain Fv antibodies are unable to fold properly under a reducing cytoplasm because of the reduction of disulfide bonds. The inability to fold limits both the production of the functional scFvs and their targeting against antigens, which are generally executed in a reducing cytoplasm. In this study, the target scFv CDR was grafted with stable human consensus framework sequences, which enabled the generation of a foldable scFv in a reducing cytoplasm of Escherichia coli. Additionally, the structural features affecting the folding efficiency of the engineered scFv were identified by analyzing the predicted structure. An anti‐c‐Met scFv, which was a cytoplasmic non‐foldable protein, was redesigned as the model system. This study confirmed that the engineered anti‐c‐Met scFv was folded into its native form in the cytoplasm of E. coli BL21(DE3) without a significant loss in the specific binding activity against c‐Met antigen. The structures of the wild‐type anti‐c‐Met scFv and the engineered scFv were predicted using homology modeling. A comparative analysis based on the sequence and structure showed that the hydrophobicity of 12 solvent exposed residues decreased, and two newly formed salt bridges might have improved the folding efficiency of the engineered scFv under the reducing condition. Biotechnol. Bioeng. 2010; 106: 367–375. © 2010 Wiley Periodicals, Inc.     

Generation of high-affinity fully human anti-interleukin-8 antibodies from its cDNA by two-hybrid screening and affinity maturation in yeast

We have developed a technology for rapidly generating novel and fully human antibodies by simply using the antigen DNA. A human single‐chain variable fragment (scFv) antibody library was constructed in a yeast two‐hybrid vector with high complexity. After cloning cDNA encoding the mature sequence of human interleukin‐8 (hIL8) into the yeast two‐hybrid system vector, we have screened the human scFv antibody library and obtained three distinct scFv clones that could specifically bind to hIL8. One clone was chosen for further improvement by a novel affinity maturation process using the error‐prone PCR of the scFv sequence followed by additional rounds of yeast two‐hybrid screening. The scFv antibodies of both primary and affinity‐matured scFv clones were expressed in E. coli. All purified scFvs showed specific binding to hIL8 in reciprocal coimmunoprecipitation and ELISA assays. All scFvs, as well as a fully human IgG antibody converted from one of the scFv clones and expressed in the mammalian cells, were able to effectively inhibit hIL8 in neutrophil chemotaxis assays. The technology described can generate fully human antibodies with high efficiency and low cost.     

Different combinations of atomic interactions predict protein‐small molecule and protein‐DNA/RNA affinities with similar accuracy

Interactions between proteins and other molecules play essential roles in all biological processes. Although it is widely held that a protein's ligand specificity is determined primarily by its three‐dimensional structure, the general principles by which structure determines ligand binding remain poorly understood. Here we use statistical analyses of a large number of protein?ligand complexes with associated binding‐affinity measurements to quantitatively characterize how combinations of atomic interactions contribute to ligand affinity. We find that there are significant differences in how atomic interactions determine ligand affinity for proteins that bind small chemical ligands, those that bind DNA/RNA and those that interact with other proteins. Although protein‐small molecule and protein‐DNA/RNA binding affinities can be accurately predicted from structural data, models predicting one type of interaction perform poorly on the others. Additionally, the particular combinations of atomic interactions required to predict binding affinity differed between small‐molecule and DNA/RNA data sets, consistent with the conclusion that the structural bases determining ligand affinity differ among interaction types. In contrast to what we observed for small‐molecule and DNA/RNA interactions, no statistical models were capable of predicting protein?protein affinity with >60% correlation. We demonstrate the potential usefulness of protein‐DNA/RNA binding prediction as a possible tool for high‐throughput virtual screening to guide laboratory investigations, suggesting that quantitative characterization of diverse molecular interactions may have practical applications as well as fundamentally advancing our understanding of how molecular structure translates into function. Proteins 2015; 83:2100–2114. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

GFP‐SCFV: Expression and possible applications as a tool for experimental investigations of atherosclerosis

Experimental studies on atherosclerosis are crucial for investigating its pathophysiology, defining new therapeutic targets, and developing new drugs and diagnostic tools. Thus, many imaging markers have been developed and introduced in experimental studies. The main advantage of these new tools is that they allow the noninvasive diagnosis of atherosclerotic vascular disease. Here, we describe the cloning, expression, purification, and stabilization of a chimeric protein specifically designed to probe cells and tissues for the presence of LDL(?), a relevant marker of atherosclerosis. The DNA sequence that encodes the anti‐LDL(?) scFv, previously obtained from a hybridoma secreting an anti‐LDL(?) monoclonal antibody, was inserted into the bacterial vector pET‐28a(+) in tandem with a DNA sequence encoding GFP. The recombinant protein was expressed in high yields in E. coli as inclusion bodies. The applicability of GFP‐scFv was assessed by ELISA, which determined its affinity for LDL(?) and confocal microscopy, that showed macrophage uptake of the protein along with LDL(?). In conclusion, our data suggest that the anti‐LDL(?) GFP‐scFv chimeric protein could be useful in studies on atherogenesis as well as for developing diagnostic tools for atherosclerosis. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1206–1213, 2014     

Improving GPX activity of selenium‐containing human single‐chain Fv antibody by site‐directed mutation based on the structural analysis

Glutathione peroxidase (GPX) is one of the important members of the antioxidant enzyme family. It can catalyze the reduction of hydroperoxides with glutathione to protect cells against oxidative damage. In previous studies, we have prepared the human catalytic antibody Se‐scFv‐B3 (selenium‐containing single‐chain Fv fragment of clone B3) with GPX activity by incorporating a catalytic group Sec (selenocysteine) into the binding site using chemical mutation; however, its activity was not very satisfying. In order to try to improve its GPX activity, structural analysis of the scFv‐B3 was carried out. A three‐dimensional (3D) structure of scFv‐B3 was constructed by means of homology modeling and binding site analysis was carried out. Computer‐aided docking and energy minimization (EM) calculations of the antibody‐GSH (glutathione) complex were also performed. From these simulations, Ala44 and Ala180 in the candidate binding sites were chosen to be mutated to serines respectively, which can be subsequently converted into the catalytic Sec group. The two mutated protein and wild type of the scFv were all expressed in soluble form in Escherichia coli Rosetta and purified by Ni²⁺‐immobilized metal affinity chromatography (IMAC), then transformed to selenium‐containing catalytic antibody with GPX activity by chemical modification of the reactive serine residues. The GPX activity of the mutated catalytic antibody Se‐scFv‐B3‐A180S was significantly increased compared to the original Se‐scFv‐B3. Copyright © 2009 John Wiley & Sons, Ltd.     

Impact of single-chain Fv antibody fragment affinity on nanoparticle targeting of epidermal growth factor receptor-expressing tumor cells

To determine the importance of single-chain Fv (scFv) affinity on binding, uptake, and cytotoxicity of tumor-targeting nanoparticles, the affinity of the epidermal growth factor receptor (EGFR) scFv antibody C10 was increased using molecular evolution and yeast display. A library containing scFv mutants was created by error-prone PCR, displayed on the surface of yeast, and higher affinity clones selected by fluorescence activated cell sorting. Ten mutant scFv were identified that had a 3-18-fold improvement in affinity (KD=15-88 nM) for EGFR-expressing A431 tumor cells compared to C10 scFv (KD=264 nM). By combining mutations, higher affinity scFv were generated with KD ranging from 0.9 nM to 10 nM. The highest affinity scFv had a 280-fold higher affinity compared to that of the parental C10 scFv. Immunoliposome nanoparticles (ILs) were prepared using EGFR scFv with a 280-fold range of affinities, and their binding and uptake into EGFR-expressing tumor cells was quantified. At scFv densities greater than 148 scFv/IL, there was no effect of scFv affinity on IL binding and uptake into tumor cells, or on cytotoxicity. At lower scFv densities, there was less uptake and binding for ILs constructed from the very low affinity C10 scFv. The results show the importance of antibody fragment density on nanoparticle uptake, and suggest that engineering ultrahigh affinity scFv may be unnecessary for optimal nanoparticle targeting.     

Epitope‐specificity of recombinant antibodies reveals promiscuous peptide‐binding properties

Protein–peptide interactions are a common occurrence and essential for numerous cellular processes, and frequently explored in broad applications within biology, medicine, and proteomics. Therefore, understanding the molecular mechanism(s) of protein–peptide recognition, specificity, and binding interactions will be essential. In this study, we report the first detailed analysis of antibody–peptide interaction characteristics, by combining large‐scale experimental peptide binding data with the structural analysis of eight human recombinant antibodies and numerous peptides, targeting tryptic mammalian and eukaryote proteomes. The results consistently revealed that promiscuous peptide‐binding interactions, that is, both specific and degenerate binding, were exhibited by all antibodies, and the discovery was corroborated by orthogonal data, indicating that this might be a general phenomenon for low‐affinity antibody–peptide interactions. The molecular mechanism for the degenerate peptide‐binding specificity appeared to be executed through the use of 2–3 semi‐conserved anchor residues in the C‐terminal part of the peptides, in analogue to the mechanism utilized by the major histocompatibility complex–peptide complexes. In the long‐term, this knowledge will be instrumental for advancing our fundamental understanding of protein–peptide interactions, as well as for designing, generating, and applying peptide specific antibodies, or peptide‐binding proteins in general, in various biotechnical and medical applications.     

Cytosolic RNA:DNA hybrids activate the cGAS–STING axis

Intracellular recognition of non‐self and also self‐nucleic acids can result in the initiation of potent pro‐inflammatory and antiviral cytokine responses. Most recently, cGAS was shown to be critical for the recognition of cytoplasmic dsDNA. Binding of dsDNA to cGAS results in the synthesis of cGAMP(2′–5′), which then binds to the endoplasmic reticulum resident protein STING. This initiates a signaling cascade that triggers the induction of an antiviral immune response. While most studies on intracellular nucleic acids have focused on dsRNA or dsDNA, it has remained unexplored whether cytosolic RNA:DNA hybrids are also sensed by the innate immune system. Studying synthetic RNA:DNA hybrids, we indeed observed a strong type I interferon response upon cytosolic delivery of this class of molecule. Studies in THP‐1 knockout cells revealed that the recognition of RNA:DNA hybrids is completely attributable to the cGAS–STING pathway. Moreover, in vitro studies showed that recombinant cGAS produced cGAMP upon RNA:DNA hybrid recognition. Altogether, our results introduce RNA:DNA hybrids as a novel class of intracellular PAMP molecules and describe an alternative cGAS ligand next to dsDNA.     

High‐efficiency affinity precipitation of multiple industrial mAbs and Fc‐fusion proteins from cell culture harvests using Z‐ELP‐E2 nanocages

Affinity precipitation using Z‐elastin‐like polypeptide‐functionalized E2 protein nanocages has been shown to be a promising alternative to Protein A chromatography for monoclonal antibody (mAb) purification. We have previously described a high‐yielding, affinity precipitation process capable of rapidly capturing mAbs from cell culture through spontaneous, multivalent crosslinking into large aggregates. To challenge the capabilities of this technology, nanocage affinity precipitation was investigated using four industrial mAbs (mAbs A–D) and one Fc fusion protein (Fc A) with diverse molecular properties. A molar binding ratio of 3:1 Z:mAb was sufficient to precipitate >95% mAb in solution for all molecules evaluated at ambient temperature without added salt. The effect of solution pH on aggregation kinetics was studied using a simplified two‐step model to investigate the protein interactions that occur during mAb–nanocage crosslinking and to determine the optimal solution pH for precipitation. After centrifugation, the pelleted mAb–nanocage complex remained insoluble and was capable of being washed at pH ≥ 5 and eluted with at pH < 4 with >90% mAb recovery for all molecules. The four mAbs and one Fc fusion were purified from cell culture using optimal process conditions, and >94% yield and >97% monomer content were obtained. mAb A–D purification resulted in a 99.9% reduction in host cell protein and >99.99% reduction in DNA from the cell culture fluids. Nanocage affinity precipitation was equivalent to or exceeded expected Protein A chromatography performance. This study highlights the benefits of nanoparticle crosslinking for enhanced affinity capture and presents a robust platform that can be applied to any target mAb or Fc‐containing proteins with minimal optimization of process parameters.