The intracellular antibody capture technology (IACT): towards a consensus sequence for intracellular antibodies

Visintin, Michela; Settanni, Giovanni; Maritan, Amos; Graziosi, Sergio; Marks, James D.; Cattaneo, Antonino

doi:10.1006/jmbi.2002.5392

Cited by 129 publications

(118 citation statements)

References 59 publications

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“…In previous reports, the two-hybrid approach for detecting antigen-antibody interactions has mainly been used to identify sufficiently stable binders from a pool of scFvs, which had been first selected by antigen-specific phage display from large libraries (39,47,48). This double-step approach was necessary because only a fraction of a typical scFv library will generally have sufficient stability for intracellular application, and yeast cannot be readily used to screen very large libraries.…”

Section: Discussionmentioning

confidence: 99%

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

Maur¹,

Zahnd²,

Fischer³

et al. 2002

Journal of Biological Chemistry

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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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Section: Discussionmentioning

confidence: 99%

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

Maur¹,

Zahnd²,

Fischer³

et al. 2002

Journal of Biological Chemistry

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“…33 Because the low efficiency with which yeast can be transformed does not allow the screening of complex libraries, a pre-selection of scFvs by panning in E. coli must be performed. The clones obtained from the panning are then subjected to a yeast two-hybrid screen and later screened in a mammalian twohybrid system.…”

Section: O N O T D I S T R I B U T Ementioning

confidence: 99%

“…The clones obtained from the panning are then subjected to a yeast two-hybrid screen and later screened in a mammalian twohybrid system. 33,35 Another strategy for selection of intrabodies makes use of the twin-arginine translocation machinery. The twin-arginine pathway (tat pathway) enables the translocation of proteins already folded in the cytoplasm through the bacterial cytoplasmic membrane.…”

Section: O N O T D I S T R I B U T Ementioning

confidence: 99%

Targeting antibodies to the cytoplasm

Marschall

Frenzel

Schirrmann

et al. 2011

mAbs

110

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“…Newly developed ways of building antibody libraries, especially in the scFv format, and new methods for selection from those libraries, including phage display, yeast twohybrid and mammalian two-hybrid, solved some of the problems by facing the development of intrabodies as therapeutic agents. The development in the selection methods led to the definition of an intrabody consensus sequence, which improved expression properties in vivo (Marasco, 1997b;Tse et al, 2002;Visintin et al, 2002;Stocks, 2005).…”

Section: Intracellular Antibodies (Intrabodies)mentioning

confidence: 99%

Novel antibodies as anticancer agents

2007

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In recent years antibodies, whether generated by traditional hybridoma technology or by recombinant DNA strategies, have evolved from Paul Ehrlich's 'magic bullets' to a modern age 'guided missile'. In the recent years of immunologic research, we are witnessing development in the fields of antigen screening and protein engineering in order to create specific anticancer remedies. The developments in the field of recombinant DNA, protein engineering and cancer biology have let us gain insight into many cancer-related mechanisms. Moreover, novel techniques have facilitated tools allowing unique distinction between malignantly transformed cells, and regular ones. This understanding has paved the way for the rational design of a new age of pharmaceuticals: monoclonal antibodies and their fragments. Antibodies can select antigens on both a specific and a high-affinity account, and further implementation of these qualities is used to target cancer cells by specifically identifying exogenous antigens of cancer cell populations. The structure of the antibody provides plasticity resonating from its functional sites. This review will screen some of the many novel antibodies and antibody-based approaches that are being currently developed for clinical applications as the new generation of anticancer agents.

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The intracellular antibody capture technology (IACT): towards a consensus sequence for intracellular antibodies

Cited by 129 publications

References 59 publications

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

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

Targeting antibodies to the cytoplasm

Novel antibodies as anticancer agents

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