Direct in Vivo Screening of Intrabody Libraries Constructed on a Highly Stable Single-chain Framework

Abstract

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...

Effect of single-chain antibody targeting of the ligand-binding domain in the anaplastic lymphoma kinase receptor

“…Thus, we used a yeast two-hybrid approach to select scFvs that recognize this LBD epitope and hypothesized that such scFvs may show inhibitory effects on the ligand/receptor interaction. scFvs were initially selected from a library of randomized complementarity-determining region 3 (CDR-3) of the heavy chain (Auf der Maur et al , 2002). The library was constructed within an scFv framework (FW) (Auf der Maur et al , 2004) that served as the negative control in the experiments reported here.…”

“…We thus applied our yeast two-hybrid, antigen–antibody interaction screening technology to isolate antigen-binding scFvs by screening a human scFv library of randomized synthetic CDR-H3 sequences (Auf der Maur et al , 2002). The library was constructed on an scFv-FW that had been previously isolated by a yeast screening system (Auf der Maur et al , 2001, 2004).…”

“…The last two residues were kept constant because of their structural importance (Chothia and Lesk, 1987). The scFv library was cloned in a yeast expression vector (pLibl) as a C-terminal fusion to the transcriptional activation domain of Gal4 (Auf der Maur et al , 2002). …”

Understanding differences between synthetic and natural antibodies can help improve antibody engineering

“…In other studies, specific Abs were obtained from libraries with introduced diversity only to CDRH3. [28][29][30] However, the relative importance of CDRH3 compared to other CDRs has been recently revisited in numerous studies. Large scale analyses 39,46 of Abs have assessed the role of CDRH3.…”

“…[20][21][22][23] In addition, many synthetic libraries are based on a single, [24][25][26] or limited set of V region frameworks 17,27 and many introduce diversification only to CDRH3. [28][29][30] Similarly, some libraries limit the introduced diversity to only 2-4 amino acids per position. 31,32 A critical question, therefore, in designing synthetic libraries is to what extent the resulting Abs are similar to natural Abs in the way they recognize and bind the Ag.…”