Selection of antibodies for intracellular function using a two-hybridin vivosystem

Abstract

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.intrabodies ͉ single-chain Fv ͉ therapy ͉ functional genomics

Antineoplastic effect of anti-erbB-2 intrabody is not correlated with scFv affinity for its target

“…Such methods have included genetic techniques of deriving cDNA directly from hybridomas [21][22][23][24][25]34 as well as biopanning of phage scFv 33,35 libraries and others. 38 These methods have allowed direct isolation of scFvs against virtually any subcellular target protein. Thus, unlike antisense molecules and ribozymes, whereby specific recognition and ablation of target oncogenes sequences must be empirically determined, scFvs may be reliably derived from accessible substrate systems for use as intrabodies.…”

Intracellular Single-Chain Variable Fragments Directed to the Src Homology 2 Domains of Syk Partially Inhibit FcεRI Signaling in the RBL-2H3 Cell Line

“…system (25) to select the potential Syk in vivo binders. This experiment revealed that all the scFv that bound Syk in vitro did not necessarily bind to Syk in the yeast two-hybrid system, and only two scFv named G4G11 and G4E4 showed a very good performance in binding to Syk in the cytoplasm of yeast cells (Fig.…”

“…All strains and plasmids were described elsewhere (25). Briefly, LexA fusion baits were prepared in the plasmid pBTM116.…”

“…The aim of our study was the targeting of Syk in vivo, and for that purpose we needed scFv fragments that exhibit good solubility and stability in the cytoplasm of mammalian cells. To identify the potential Syk in vivo binders, we used a yeast two-hybrid selection system (25) as an intermediate selection step after the in vitro selection and before the expression in mammalian cells. This additional selection step permitted to identify two scFv, named G4G11 and G4E4, which showed the best binding to Syk in the cytoplasm of yeast cells (Fig.…”

“…The coding sequences of the scFv G4G11, G4E4, and G6G2 were cut from the pHEN2 recombinant vectors by using the restriction sites SfiI and NotI, and they were cloned into pVP16* vector. For the two-hybrid analysis, competent cells of Saccharomyces cerevisiae strain L40 were prepared as described (25), and positive clones were selected by using auxotrophic markers for both plasmids and for lysine and histidine prototropy. Histidine-positive colonies and controls were lysed in liquid nitrogen and assayed for ␤-gal activity on filters, as described (25).…”