Adrian Auf Der Maur

Correlation between in Vitro Stability and in Vivo Performance of Anti-GCN4 Intrabodies as Cytoplasmic Inhibitors

A cellular assay system for measuring the activity of cytoplasmically expressed anti-GCN4 scFv fragments directed against the Gcn4p dimerization domain was established in the budding yeast Saccharomyces cerevisiae. The inhibitory potential of different constitutively expressed anti-GCN4 scFv intrabodies was monitored by measuring the activity of β-galactosidase expressed from a GCN4-dependent reporter gene. The in vivo performance of these scFv intrabodies in specifically decreasing reporter gene activity was related to their in vitro stability, measured by denaturant-induced equilibrium unfolding. A framework-engineered stabilized version showed significantly improved activity, while a destabilized point mutant of the anti-GCN4 wild-type showed decreased effects in vivo. These results indicate that stability engineering can result in improved performance of scFv fragments as intrabodies. Increasing the thermodynamic stability appears to be an essential factor for improving the yield of functional scFv in the reducing environment of the cytoplasm, where the conserved intradomain disulfides of antibody fragments cannot form.

Antigen-independent selection of stable intracellular single-chain antibodies.

The intracellular expression of single‐chain Fv antibody fragments (scFv) in eukaryotic cells has an enormous potential in functional genomics and therapeutics [Marasco (1997) Gene Ther. 4, 11–15; Richardson and Marasco (1995) Trends Biotechnol. 13, 306–310]. However, the application of these so‐called intrabodies is currently limited by their unpredictable behavior under the reducing conditions encountered inside eukaryotic cells, which can affect their stability and solubility properties [Wörn et al. (2000) J. Biol. Chem. 275, 2795–2803; Biocca et al. (1995) Bio/Technology 13, 1110–1115]. We present a novel system that enables selection of stable and soluble intrabody frameworks in vivo without the requirement or knowledge of antigens. This system is based on the expression of single‐chain antibodies fused to a selectable marker that can control gene expression and cell growth. Our results show that the activity of a selectable marker fused to well characterized scFvs [Wörn et al. (2000) J. Biol. Chem. 275, 2795–2803] correlates with the solubility and stability of the scFv moieties. This method provides a unique tool to identify stable and soluble scFv frameworks, which subsequently serve as acceptor backbones to construct intrabody complementarity determining region libraries by randomization of hypervariable loops.

Quenching accumulation of toxic galactose-1-phosphate as a system to select disruption of protein-protein interactions in vivo

The reverse two-hybrid system has been developed to readily identify molecules or mutations that can disrupt protein-protein interactions in vivo. This system is generally based on the interaction-dependent activation of a reporter gene, whose product inhibits the growth of the engineered yeast cell. Thus, disruption of the interaction between the hybrid proteins can be positively selected because, by reducing the expression of the negative marker gene, it allows cell growth. Although several counter-selectable marker genes are currently available, their application in the reverse two-hybrid system is generally confronted with technical and practical problems such as low selectivity and relatively complex experimental procedures. Thus, the characterization of more reliable and simple counter-selection assays for the reverse two-hybrid system continues to be of interest. We have developed a novel counter-selection assay based on the toxicity of intracellular galactose-1-phosphate, which accumulates upon expression of a galactokinase-encoding GAL1 reporter gene in the absence of transferase activity. Decreased GAL1 gene expression upon dissociation of interacting proteins causes reduction of intracellular galactose-1-phosphate concentrations, thus allowing cell growth under selective conditions.

Target validation through protein- domain knockout - applications of intracellularly stable single-chain antibodies

The human genome project has delivered a large number of genes and respective proteins that await to be validated as potential drug targets.Such a complexity has made target validation the main bottleneck in today’s drug development process. In addition, the majority of these new potential targets are proteins that function intracellularly.Approaches such as gene knockout, antisense RNA or RNA interference (RNAi) are currently used to validate candidate drug targets by analysing the effects of their deletion. Intrabodies (single-chain antibodies expressed within the cell) present an attractive alternative for directly modulating protein function in vivo. In particular, intrabodies can be used to target specific domains of a protein and perform so-called ‘protein-domain knockouts’, thus allowing the dissection of the varied functions of multi-domain proteins.

Intrabodies: Development and Application in Functional Genomics and Therapy

The human genome project has led to the identification of a large number of genes and respective proteins, thus providing the pharmaceutical industry with thousands of new potential drug targets, most of which function in intracellular compartments (Lander et al., 2001; Venter et al., 2001). This fact opens new perspectives for therapy of human diseases; however, it also demands reliable approaches for understanding the role and the function of these new genes and proteins (functional genomics) and for identifying those that can be validated as drug targets (target validation). Different experimental tools are currently used for investigating the function of these new intracellular proteins, including their potential role in disease, and for evaluating them as potential drug targets. The classical way to investigate the function of genes, and thereby determine the physiological and pathological relevance of gene products, is to interfere with their expression (Ihle, 2000). Approaches such as gene knockout, antisense oligonucleotide or RNA interference (RNAi) are currently used to study gene and protein function and to validate candidate drug targets by analysing the effects of their deletion. One limitation of all these techniques is that they eliminate all functions of a target gene product at once, thus making it difficult to dissect potentially distinct roles of different domains and to mimic the effects of a small molecule that presumably will act at a specific domain of the protein (Kamb and Caponigro, 2001).