Antigen‐independent selection of stable intracellular single‐chain antibodies

Abstract

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.

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

“…8). In a previous report (46), we have presented a method for the direct in vivo testing and selection of intrabody frameworks independently of their antigen-binding specificity. These pre-selected frameworks can evidently Only residues V H 109 -136 were randomized.…”

Direct Phage to Intrabody Screening (DPIS): Demonstration by Isolation of Cytosolic Intrabodies Against the TES1 Site of Epstein Barr Virus Latent Membrane Protein 1 (LMP1) that Block NF-κB Transactivation

“…These have included a protein fragment complementation assay for selection of antibody -antigen interactions in the cytoplasm of E. coli 40 and a yeast two-hybrid system as a method to select antibodies for intracellular function in eukaryotic cells. 36,39 This latter assay has been used to further characterize structural variants of known intrabodies for improvements in stability, solubility and in vivo performance 39,55,56 and in one study for the de novo isolation of a single antigen-specific intrabody. 38 In a modification of this technique known as intracellular antibody capture (IAC), in vitro phage display is used as a pre-selection enrichment step to adjust for the difference between the complexity of antibody phage libraries and the transformation efficiency in yeast.…”

Camelized Rabbit-derived VH Single-domain Intrabodies Against Vif Strongly Neutralize HIV-1 Infectivity

“…4,5 Intrabodies have particular promise in the area of functional genomics by directly blocking a protein function or by interfering with protein -protein interactions, thereby contributing to the understanding of a growing number of newly identified proteins. 6,7 In the long term, intrabodies may even find an enormous broad therapeutic application as in gene therapy setting.…”

“…3 The preserved intrachain disulfide bridges of heavy and light chains do not form in scFvs expressed in the cytoplasm, thus resulting in unstable intrabodies that are non-functional inside the cell. 6,7,20 Therefore, only intrinsically very soluble and stable scFv fragments will be able to fold correctly in sufficient amounts to be active as intrabodies. 21 At this time, no rules or consistent predictions can be established about intrabodies that will tolerate the reducing cellular environments.…”