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 -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.
Adenovirus (Ad) vectors are of utility for many therapeutic applications. Strategies have been developed to alter adenoviral tropism to achieve a cell-specific gene delivery capacity employing fiber modifications allowing genetic incorporation of targeting motifs. In this regard, single chain antibodies (scFv) represent potentially useful agents to achieve targeted gene transfer. However, the distinct biosynthetic pathways that scFv and Ad capsid proteins are normally routed through have thus far been problematic with respect to scFv incorporation into the Ad capsid. Utilization of stable scFv, which also maintain correct folding and thus functionality under intracellular reducing conditions, could overcome this restriction. We genetically incorporated a stable scFv into a de-knobbed, fibritin-foldon trimerized Ad fiber and demonstrated selective targeting to the cognate epitope expressed on the membrane surface of cells. We have shown that the scFv employed in this study retains functionality and that stabilizing the targeting molecule, per se, is critical to allow retention of antigen recognition in the adenovirus capsid-incorporated context. Gene Therapy (2006) 13, 88-94.
The tyrosine kinase receptor anaplastic lymphoma kinase (ALK) and its ligand, the growth factor pleiotrophin (PTN), are highly expressed during the development of the nervous system and have been implicated in the malignant progression of different tumor types. Here, we describe human single-chain variable fragment (scFv) antibodies that target the ligand-binding domain (LBD) in ALK and show the effect in vitro and in vivo. The ALK LBD was used as a bait in a yeast two-hybdrid system to select human scFv from a library with randomized complementarity-determining region 3 domains. Surface plasmon resonance showed high-affinity binding of the selected scFv. The anti-ALK scFv competed for binding of PTN to ALK in intact cells and inhibited PTN-dependent signal transduction through endogenous ALK. Invasion of an intact endothelial cell monolayer by U87MG human glioblastoma cells was inhibited by the anti-ALK scFv. In addition, the growth of established tumor xenografts in mice was reversed after the induction of the conditional expression of the anti-ALK scFv. In archival malignant brain tumors expression levels of ALK and PTN were found elevated and appear correlated with poor patient survival. This suggests a rate-limiting function of the PTN/ALK interaction that may be exploited therapeutically.
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.
MTF-1 is a zinc finger transcription factor that mediates the cellular response to heavy metal stress; its targeted disruption in the mouse leads to liver decay and embryonic lethality at day E14. Recently, we have sequenced the entire MTF-1 gene in the compact genome of the pufferfish Fugu rubripes. Here we have defined the promoter sequences of human and mouse MTF-1 and the genomic structure of the mouse MTF-1 locus. The transcription unit of MTF-1 spans 42 kb (compared to 8.5 kb in Fugu) and is located downstream of the gene for a phosphatase (INPP5P) in mouse, human, and fish. In all of these species, the MTF promoter region has the features of a CpG island. In both mouse and human, the 5' untranslated region harbors conserved short reading frames of unknown function. RNA mapping experiments revealed that in these two species, MTF-1 mRNA is transcribed from a cluster of multiple initiation sites from a TATA-less promoter without metal-responsive elements. Transcription from endogenous and transfected MTF-1 promoters was not affected by heavy metal load or other stressors, in support of the notion that MTF-1 activity is regulated at the posttranscriptional level. Tissue Northern blots normalized for poly A+ RNA indicate that MTF-1 is expressed at similar levels in all tissues, except in the testes, that contain more than 10-fold higher mRNA levels.
Single-chain Fv antibody fragments (scFv) represent a convenient antibody format for intracellular expression in eukaryotic or prokaryotic cells. These so-called intrabodies have great potential in functional genomics as a tool to study the function of newly identified proteins in vivo, for example by binding-induced modulation of their activity or by blocking interactions with other proteins. However, the intracellular expression and activity of many single-chain Fvs are limited by their instability and folding efficiency in the reducing intracellular environment, where the highly conserved intrachain disulfide bonds do not form. In the present work, we used an in vivo selection system to isolate novel antigen-binding intrabodies. We screened two intrabody libraries carrying a randomized third hypervariable loop onto the heavy chain of a stable framework, which had been further optimized by random mutagenesis for better behavior in the selection system, and we biophysically characterized the selected variants to interpret the outcome of the selection. Our results show that singleframework intrabody libraries can be directly screened in vivo to rapidly select antigen-specific intrabodies.Antibodies are pivotal components of the vertebrate immune system that can bind to almost any molecule with a high degree of specificity and affinity. These characteristics have been exploited to turn the natural antibodies into powerful biotechnological tools in diagnostic and therapeutic applications. Advances in recombinant DNA technology have facilitated the manipulation, cloning, and expression of the antibody genes in a wide variety of hosts (1-3). Several forms of antibodies have been constructed to obtain derivatives that carry the binding site in a smaller assembly. One of the minimal forms still retaining the full binding site is the single-chain Fv fragment (scFv) 1 (4 -6). In the scFv form, the variable regions of the heavy and the light chains, which bear the hypervariable loops (or complementarity determining regions (CDR)), are connected by a flexible linker, allowing the expression of the protein from a single cDNA sequence.Natural antibodies, which are secreted by plasma cells, have evolved to function in an extracellular environment. In contrast to full-length, double-chain antibodies, the scFv antibody form can in principle be readily expressed also in the cytoplasm of eukaryotic cells and directed to any compartment to target intracellular proteins and thus evoke specific biological effects. Hence, intracellular scFvs, also known as "intrabodies," might have a great potential in functional genomics by blocking or modulating the activity of a growing number of newly identified proteins, thereby contributing to the understanding of their functions. In the long run, intrabodies might even be used in therapeutic applications, possibly in gene therapy settings.However, there are only few examples of successful applications (7-13), and the cytoplasmic expression of scFvs is generally confronted with the difficultie...
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