Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection
TL;DR: The success of C1-N1N2 in discriminating activated Rho in immunofluorescence studies implies that this new tool, in collaboration with currently used RhoA and B antibodies, has the potential to analyze Rho activation in cell function and tumor development.
Abstract: The Rho GTPases A, B and C proteins, members of the Rho family whose activity is regulated by GDP/GTP cycling, function in many cellular pathways controlling proliferation and have recently been implicated in tumorigenesis. Although overexpression of Rho GTPases has been correlated with tumorigenesis, only their GTP-bound forms are able to activate the signalling pathways implicated in tumorigenesis. Thus, the focus of much recent research has been to identify biological tools capable of quantifying the level of cellular GTP-bound Rho, or determining the subcellular location of activation. However useful, these tools used to study the mechanism of Rho activation still have limitations. The aim of the present work was to employ phage display to identify a conformationally-specific single chain fragment variable (scFv) that recognizes the active, GTP-bound, form of Rho GTPases and is able to discriminate it from the inactive, GDP-bound, Rho in endogenous settings. After five rounds of phage selection using a constitutively activated mutant of RhoB (RhoBQ63L), three scFvs (A8, C1 and D11) were selected for subsequent analysis. Further biochemical characterization was pursued for the single clone, C1, exhibiting an scFv structure. C1 was selective for the GTP-bound form of RhoA, RhoB, as well as RhoC, and failed to recognize GTP-loaded Rac1 or Cdc42, two other members of the Rho family. To enhance its production, soluble C1 was expressed in fusion with the N-terminal domain of phage protein pIII (scFv C1-N1N2), it appeared specifically associated with GTP-loaded recombinant RhoA and RhoB via immunoprecipitation, and endogenous activated Rho in HeLa cells as determined by immunofluorescence. We identified an antibody, C1-N1N2, specific for the GTP-bound form of RhoB from a phage library, and confirmed its specificity towards GTP-bound RhoA and RhoC, as well as RhoB. The success of C1-N1N2 in discriminating activated Rho in immunofluorescence studies implies that this new tool, in collaboration with currently used RhoA and B antibodies, has the potential to analyze Rho activation in cell function and tumor development.
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TL;DR: This work describes the first fully synthetic phage display library of humanized llama single domain antibody (NaLi-H1: Nanobody Library Humanized 1), based on a humanized synthetic singledomain antibody (hs2dAb) scaffold optimized for intracellular stability, which provides high affinity binders without animal immunization.
Abstract: Antibodies are proteins that form part of an animal’s immune system and can identify and help eradicate infections. These proteins are also needed at many stages in biological research and represent one of the most promising tools in medical applications, from diagnostics to treatments. Traditionally, antibodies have been collected from animals that had been previously injected with a target molecule that the antibodies must recognize. An alternative strategy that uses bacteria and bacteria-infecting viruses instead of animals was developed several decades ago and allows researchers to obtain antibodies more quickly. However, the majority of the scientific community view these “in vitro selected antibodies” as inferior to those produced via the more traditional approach. Moutel, Bery et al. set out to challenge this widespread opinion, using a smaller kind of antibody known as nanobodies. The proteins were originally found in animals like llamas and camels and are now widely used in biological research. One particularly stable nanobody was chosen to form the backbone of the in vitro antibodies, and the DNA that encodes this nanobody was altered to make the protein more similar to human antibodies. Moutel, Bery et al. then changed the DNA sequence further to make billions of different versions of the nanobody, each one slightly different from the next in the region that binds to the target molecules. Transferring this DNA into bacteria resulted in a library (called the NaLi-H1 library) of bacterial clones that produce the nanobodies displayed at the surface of bacteria-infecting viruses. Moutel, Bery et al. then screened this library against various target molecules, including some from tumor cells, and showed that the fully in vitro selected antibodies worked just as well as natural antibodies in a number of assays. The in vitro antibodies could even be used to track, or inactivate, proteins within living cells. The NaLi-H1 library will help other researchers obtain new antibodies that bind strongly to their targets. The approaches developed to create the library could also see more people decide to create their own synthetic libraries, which would accelerate the identification of new antibodies in a way that is cheaper and requires fewer experiments to be done using animals. These in vitro selected antibodies could help to advance both fundamental and medical research.
219 citations
Cites methods from "Identification of a GTP-bound Rho s..."
...…libraries, immune or naı̈ve llama VHH libraries (Monegal et al., 2012; Olichon and Surrey, 2007) or from scFv libraries (Dimitrov et al., 2008; Goffinet et al., 2008; Nizak et al., 2003a) we then rationally designed CDR diversity with fixed CDR1 and CDR2 size and four CDR3 sizes (9, 12, 15…...
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TL;DR: This work grades every paper published in 2008 on a scale from A to F and outlines what features make a biosensor article fabulous, middling or abysmal and focuses on a few experimental, analysis and presentation mistakes that are alarmingly common.
Abstract: Optical biosensor technology continues to be the method of choice for label-free, real-time interaction analysis. But when it comes to improving the quality of the biosensor literature, education should be fundamental. Of the 1413 articles published in 2008, less than 30% would pass the requirements for high-school chemistry. To teach by example, we spotlight 10 papers that illustrate how to implement the technology properly. Then we grade every paper published in 2008 on a scale from A to F and outline what features make a biosensor article fabulous, middling or abysmal. To help improve the quality of published data, we focus on a few experimental, analysis and presentation mistakes that are alarmingly common. With the literature as a guide, we want to ensure that no user is left behind.
192 citations
TL;DR: The approaches used for antibody purification are critically examined with the aim of providing the reader with the principles and practical insights required to understand the intricacies of the procedures.
Abstract: The global antibody market has grown exponentially due to increasing applications in research, diagnostics and therapy. Antibodies are present in complex matrices (e.g. serum, milk, egg yolk, fermentation broth or plant-derived extracts). This has led to the need for development of novel platforms for purification of large quantities of antibody with defined clinical and performance requirements. However, the choice of method is strictly limited by the manufacturing cost and the quality of the end product required. Affinity chromatography is one of the most extensively used methods for antibody purification, due to its high selectivity and rapidity. Its effectiveness is largely based on the binding characteristics of the required antibody and the ligand used for antibody capture. The approaches used for antibody purification are critically examined with the aim of providing the reader with the principles and practical insights required to understand the intricacies of the procedures. Affinity support matrices and ligands for affinity chromatography are discussed, including their relevant underlying principles of use, their potential value and their performance in purifying different types of antibodies, along with a list of commercially available alternatives. Furthermore, the principal factors influencing purification procedures at various stages are highlighted. Practical considerations for development and/or optimizations of efficient antibody-purification protocols are suggested.
161 citations
TL;DR: It is shown that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody phage display, and the highly diverse universal antibody gene libraries HAL9/10 were constructed using an optimized scFv phagemid vector design.
Abstract: Antibody phage display is a proven key technology that allows the generation of human antibodies for diagnostics and therapy. From naive antibody gene libraries - in theory - antibodies against any target can be selected. Here we describe the design, construction and characterization of an optimized antibody phage display library. The naive antibody gene libraries HAL9 and HAL10, with a combined theoretical diversity of 1.5×1010 independent clones, were constructed from 98 healthy donors using improved phage display vectors. In detail, most common phagemids employed for antibody phage display are using a combined His/Myc tag for detection and purification. We show that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody phage display. For several published antibody libraries, the selected number of kappa scFvs were lower compared to lambda scFvs, probably due to a lower kappa scFv or Fab expression rate. Deletion of a phenylalanine at the end of the CL linker sequence in our new phagemid design increased scFv production rate and frequency of selected kappa antibodies significantly. The HAL libraries and 834 antibodies selected against 121 targets were analyzed regarding the used germline V-genes, used V-gene combinations and CDR-H3/-L3 length and composition. The amino acid diversity and distribution in the CDR-H3 of the initial library was retrieved in the CDR-H3 of selected antibodies showing that all CDR-H3 amino acids occurring in the human antibody repertoire can be functionally used and is not biased by E. coli expression or phage selection. Further, the data underline the importance of CDR length variations. The highly diverse universal antibody gene libraries HAL9/10 were constructed using an optimized scFv phagemid vector design. Analysis of selected antibodies revealed that the complete amino acid diversity in the CDR-H3 was also found in selected scFvs showing the functionality of the naive CDR-H3 diversity.
105 citations
Additional excerpts
...pCANTAB3his [50], pCES [51], pHEN2 [52], pHAL14 [31], pIT2 [53],...
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TL;DR: Therapeutic approaches targeting Rho GTPase signaling may provide a novel alternative for anti-HCC therapy.
Abstract: Rho GTPases are major regulators of signal transduction pathways and play key roles in processes including actin dynamics, cell cycle progression, cell survival and gene expression, whose deregulation may lead to tumorigenesis. A growing number of in vitro and in vivo studies using tumor-derived cell lines, primary tumors and animal cancer models strongly suggest that altered Rho GTPase signaling plays an important role in the initiation as well as in the progression of hepatocellular carcinoma (HCC), one of the deadliest human cancers in the world. These alterations can occur at the level of the GTPases themselves or of one of their regulators or effectors. The participation into the tumorigenic process can occur either through the over-expression of one of these components which presents an oncogenic activity as illustrated with RhoA and C or through the attenuation of the expression of a component presenting tumor suppressor activity as for Cdc42 or the RhoGAP, DLC-1. Consequently, these observations reflect the heterogeneity and the complexity of liver carcinogenesis. Recently, pharmacological approaches targeting Rho GTPase signaling have been used in HCC-derived models with relative success but remain to be validated in more physiologically relevant systems. Therefore, therapeutic approaches targeting Rho GTPase signaling may provide a novel alternative for anti-HCC therapy.
97 citations
References
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TL;DR: Rho GTPases are molecular switches that control a wide variety of signal transduction pathways in all eukaryotic cells and their ability to influence cell polarity, microtubule dynamics, membrane transport pathways and transcription factor activity is probably just as significant.
Abstract: Rho GTPases are molecular switches that control a wide variety of signal transduction pathways in all eukaryotic cells. They are known principally for their pivotal role in regulating the actin cytoskeleton, but their ability to influence cell polarity, microtubule dynamics, membrane transport pathways and transcription factor activity is probably just as significant. Underlying this biological complexity is a simple biochemical idea, namely that by switching on a single GTPase, several distinct signalling pathways can be coordinately activated. With spatial and temporal activation of multiple switches factored in, it is not surprising to find Rho GTPases having such a prominent role in eukaryotic cell biology.
4,616 citations
"Identification of a GTP-bound Rho s..." refers background in this paper
...Rho GTPases control a wide variety of signal transduction pathways regulating many fundamental processes of cell biology, such as organization of the actin cytoskeleton [2], gene expression, cell proliferation and survival [3]....
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TL;DR: The RAS oncogenes are members of a large family of small GTPases that bind GTP and hydrolyse it to GDP and the switching between these two states regulates a wide range of cellular processes.
Abstract: The RAS oncogenes were identified almost 20 years ago. Since then, we have learnt that they are members of a large family of small GTPases that bind GTP and hydrolyse it to GDP. This is then exchanged for GTP and the cycle is repeated. The switching between these two states regulates a wide range of cellular processes. A branch of the RAS family--the RHO proteins--is also involved in cancer, but what is the role of these proteins and would they make good therapeutic targets?
1,451 citations
"Identification of a GTP-bound Rho s..." refers background in this paper
...Rho GTPases control a wide variety of signal transduction pathways regulating many fundamental processes of cell biology, such as organization of the actin cytoskeleton [2], gene expression, cell proliferation and survival [3]....
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TL;DR: This work created highly diverse repertoires of heavy and light chains entirely in vitro from a bank of human V gene segments and generated a large synthetic repertoire of Fab fragments displayed on filamentous phage to help dissect the contributions of biological mechanisms and structural features governing V gene usage in vivo.
Abstract: Antibody fragments of moderate affinity (approximately microM) can be isolated from repertoires of approximately 10(8) immunoglobulin genes by phage display and rounds of selection with antigen, and the affinities improved by further rounds of mutation and selection. Here, as an alternative strategy, we attempted to isolate high affinity human antibodies directly from large repertoires. We first created highly diverse repertoires of heavy and light chains entirely in vitro from a bank of human V gene segments and then, by recombination of the repertoires in bacteria, generated a large (close to 6.5 x 10(10)) synthetic repertoire of Fab fragments displayed on filamentous phage. From this repertoire we isolated Fab fragments which bound to a range of different antigens and haptens, and with affinities comparable with those of antibodies from a secondary immune response in mice (up to 4 nM). Although the VH-26 (DP-47) segment was the most commonly used segment in both artificial and natural repertoires, there were also major differences in the pattern of segment usage. Such comparisons may help dissect the contributions of biological mechanisms and structural features governing V gene usage in vivo.
1,286 citations
"Identification of a GTP-bound Rho s..." refers background or methods in this paper
...1 library [24], one clone (C1) was selected for its ability to discriminate between the activated versus the unactivated form of RhoB, as well as RhoA and RhoC, but not other Rho family members....
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...1 library [24], a human synthetic VH+VL scFv phage library in a phagemid vector, and the positive control Escherichia coli TG1 (TG1 containing an anti-tyroglobulin clone) were generously provided by Fiona Sait of The Medical Research Council (Cambridge, England)....
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TL;DR: IMGT, the international ImMunoGeneTics information system, was created in 1989 by Marie-Paule Lefranc, Laboratoire d'ImmunoGénétique Moléculaire LIGM at Montpellier, France, in order to standardize and manage the complexity of immunogenetics data.
Abstract: The international ImMunoGeneTics information system® (IMGT) (http://imgt.cines.fr), created in 1989, by the Laboratoire d'ImmunoGenetique Moleculaire LIGM (Universite Montpellier II and CNRS) at Montpellier, France, is a high-quality integrated knowledge resource specializing in the immunoglobulins (IGs), T cell receptors (TRs), major histocompatibility complex (MHC) of human and other vertebrates, and related proteins of the immune systems (RPI) that belong to the immunoglobulin superfamily (IgSF) and to the MHC superfamily (MhcSF). IMGT includes several sequence databases (IMGT/LIGM-DB, IMGT/PRIMER-DB, IMGT/PROTEIN-DB and IMGT/MHC-DB), one genome database (IMGT/GENE-DB) and one three-dimensional (3D) structure database (IMGT/3Dstructure-DB), Web resources comprising 8000 HTML pages (IMGT Marie-Paule page), and interactive tools. IMGT data are expertly annotated according to the rules of the IMGT Scientific chart, based on the IMGT-ONTOLOGY concepts. IMGT tools are particularly useful for the analysis of the IG and TR repertoires in normal physiological and pathological situations. IMGT is used in medical research (autoimmune diseases, infectious diseases, AIDS, leukemias, lymphomas, myelomas), veterinary research, biotechnology related to antibody engineering (phage displays, combinatorial libraries, chimeric, humanized and human antibodies), diagnostics (clonalities, detection and follow up of residual diseases) and therapeutical approaches (graft, immunotherapy and vaccinology). IMGT is freely available at http://imgt.cines.fr.
1,137 citations
TL;DR: It is shown that Ratl fibroblasts expressing activated Val-12 Racl (Racl with valine at residue 12) display all the hallmarks of malignant transformation and that Rac is essential for transformation by Ras, indicating that oncogenic Ras drives both the Rac and MAP-kinase pathways, which cooperate to cause transformation.
Abstract: The GTPase Rac1 is a key component in the reorganization of the actin cytoskeleton that is induced by growth factors or oncogenic Ras1. Here we investigate the role of Rac1 in cell transformation and show that Rat1 fibroblasts expressing activated Val-12 Rac1 (Rac1 with valine at residue 12) display all the hallmarks of malignant transformation. In a focus-forming assay in NIH3T3 fibroblasts to measure the efficiency of transformation, we found that dominant-negative Asn-17 Rac1 inhibited focus formation by oncogenic Ras, but not by RafCAAX, a Raf kinase targeted to the plasma membrane by virtue of the addition of a carboxyterminal localization signal from K-Ras. This indicates that Rac is essential for transformation by Ras. In addition, Val-12 Rac1 synergizes strongly with RafCAAX in focus-formation assays, indicating that oncogenic Ras drives both the Rac and MAP-kinase pathways, which cooperate to cause transformation.
889 citations
"Identification of a GTP-bound Rho s..." refers background in this paper
...Activated Rho proteins cooperate strongly with oncogenes Ras and Raf in focus-formation assays, but either fail to independently induce transformation or else exhibit weak transforming activity [6-9]....
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