The Development of Intrabody Trap Technology (ITT) for Functional Genomics

1.1 - The Aim of the Research. Antibodies can be ectopically expressed as secreted proteins in cells that do not normally express them to interfere with extracellular antigens and/or as intracellular proteins targeted to different intracellular compartments to inhibit intracellular gene products: this has lead to an effective protein knock-out technology. Expression of antibodies inside cells has been used successfully to ablate protein function. The performance of antibodies that are intracellularly expressed is however somewhat unpredictable because the reducing environment of the cell cytoplasm in which they are forced to work prevents some antibodies, but not others, to fold properly. For this reason, a selection procedure for the isolation of antibodies able to fold correctly and to bind antigens under condition of intracellular expression would be highly desirable. The development of this novel selection procedure could exploit methods to monitor intracellular protein \u2013 protein interactions, for instance the yeast two-hybrid technology. Such a system would greatly facilitate the isolation of candidate antibodies for intracellular antibody down-stream applications in the system of interest, and would lend itself to the development of cell-based high-throughput screening procedures in functional genomics applications. In view of this, the creation of a new technology that allows the isolation of intracellular scFv fragments, based on their ability to bind antigen under conditions of intracellular expression has become an important goal both for functional genomics and for gene therapy, to fulfill the requirement of antibodies with improved thermodynamic stability and solubility properties. 1.2 - Results The following chapters will describe all the steps taken in order to achieve the final goal: the development of the intrabody trap technology (ITT), an in vivo selection and assay procedure for functional intracellular antibodies using a two-hybrid approach. In the first part of the project we find that several characterized antibodies can bind their target antigen in eukaryotic cells when expressed in the two-hybrid format and we have been able to isolate intracellular binders from panels of scFv, all of which can bind antigen in vitro. Furthermore, we showed a model selection in which a single chain scFv was isolated from a mixture of half a million clones, indicating that this is a robust procedure that should facilitate isolation of intrabody specificity from complex mixtures. The results form the basis of the \u201cintrabody trap technology\u201d, whereby many different, specific and functional antibodies can be isolated in vivo under condition of intracellular expression. The second part of the thesis reports the demonstration that ITT can be effectively applied to the de novo selection of functional intrabodies, by performing a real selection from polyclonal scFv fragments partially enriched from a large naive phage library by antigen panning. The experimental procedure described in this thesis has allowed the selection of intrabodies against a protein antigen involved in Alzheimer\u2019s disease (AD), the microtubule associated protein tau, the main components of the paired helical filaments (PHFs), found in neurofibrillary lesions in AD brains. In particular the aim of the work was to isolate scFv fragments against a tau fragment, which displays apoptotic capacity in different cellular context

Using IMPrESS to guide policy change in multiple sclerosis

The International MultiPlE Sclerosis Study (IMPrESS) studied the significant impact of multiple sclerosis (MS) on the health and well-being of both people with the disease and their caregivers, along with its broader socioeconomic impact. Results confirmed that there is an urgent need to achieve better outcomes for people with MS. This paper uses results from the IMPrESS to present new international evidence on the socioeconomic burden of MS and discuss the merits of a likely paradigm shift in the management of MS towards the use of better (and more accurate) diagnostic follow-up to monitor disease progression and the earlier use of disease-modifying treatments (DMTs) to achieve better clinical, quality-of-life and socioeconomic results for individuals

Gephyrin Selective Intrabodies as a New Strategy for Studying Inhibitory Receptor Clustering

The microtubule-binding protein gephyrin is known to play a pivotal role in targeting and clustering postsynaptic inhibitory receptors. Here, the Intracellular Antibodies Capture Technology (IATC) was used to select two single-chain antibody fragments or intrabodies, which, fused to nuclear localization signals (NLS), were able to efficiently and selectively remove gephyrin from glycine receptor (GlyR) clusters. Co-transfection of NLS-tagged individual intrabodies with gephyrin-enhanced green fluorescent protein (EGFP) in HEK 293 cells revealed a partial relocalization of gephyrin aggregates onto the nucleus or in the perinuclear area. When expressed in cultured neurons, these intrabodies caused a significant reduction in the number of immunoreactive GlyR clusters, which was associated with a decrease in the peak amplitude of glycine-evoked whole cell currents as assessed with electrophysiological experiments. Hampering protein function at a posttranslational level may represent an attractive alternative for interfering with gephyrin function in a more spatially localized manner

Value assessment of disease-modifying therapies for Relapsing-Remitting Multiple Sclerosis: HTA evidence from seven OECD countries

This study systematically compares HTA recommendations on a number of disease–modifying therapies for patients with Relapsing-Remitting Multiple Sclerosis. We analysed publicly available HTA reports for nine medicine-indication pairs across seven OECD countries using a methodological framework enabling systematic analysis of HTA recommendations. The analysis was conducted based on a number of value dimensions, including clinical and economic variables, as well as several other dimensions of value beyond cost-effectiveness. The material was qualitatively and quantitatively coded following the different stages of HTA decision-making process. Fifty-seven medicine-indication pairs were assessed across the study countries. Of those, eight medicine indication-pairs reported diverging HTA recommendations. Although HTA recommendations were based on the same evidence submitted in most cases, significant variations were identified in interpretation and acceptance of evidence resulting in different uncertainties raised and different ways of addressing them. Uncertainties arose both in terms of the clinical and the economic evidence, including the design of key trials or the data quality in economic models. Beyond costs and effects, additional dimensions of value had an impact in the direction of recommendations, however with different magnitude across countries. We show that there is heterogeneity across countries in HTA for evaluating DMTs for RRMS with a lack of standardised methods in evaluating clinical and economic evidence and the use of social value judgments to inform decision-making

Selection of antibodies for intracellular function using a two-hybrid in vivo system

Expression of antibodies inside cells has been used successfully to ablate protein function. This finding suggests that the technology should have an impact on disease treatment and in functional genomics where proteins of unknown function are predicted from genomic sequences. A major hindrance is the paucity of antibodies that function in eukaryotic cells, presumably because the antibodies fold incorrectly in the cytoplasm. To overcome this problem, we have developed an in vivo assay for functional intracellular antibodies using a two-hybrid approach. In this assay, antibody, as single-chain Fv (scFv) linked to a transcriptional transactivation domain, can interact with a target antigen, linked to a LexA-DNA binding domain, and thereby activate a reporter gene. We find that several characterized antibodies can bind their target antigen in eukaryotic cells in this two-hybrid format, and we have been able to isolate intracellular binders from among sets of scFv that can bind antigen in vitro. Furthermore, we show a model selection in which a single scFv was isolated from a mixture of half a million clones, indicating that this is a robust procedure that should facilitate capture of antibody specificities from complex mixtures. The approach can provide the basis for de novo selection of intracellular scFv from libraries, such as those made from spleen RNA after immunization with antigen, for intracellular analysis of protein function based only on genomic or cDNA sequences