Gueorguieva, D. et al. Identification of single domain, Bax-specific intrabodies that confer resistance to mammalian cells against oxidative-stress-induced apoptosis. FASEB. J. 20, 2636-2638

Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada
The FASEB Journal (Impact Factor: 5.04). 01/2007; 20(14):2636-8. DOI: 10.1096/fj.06-6306fje
Source: PubMed

ABSTRACT Bax is a proapoptotic protein implicated in cell death involved in several neurodegenerative diseases. Intracellularly expressed antibody (Ab) fragments (intrabodies) inhibiting Bax function would have potential for developing therapeutics for the aforementioned diseases and can serve as research tools. We report identification, cloning, and functional characterization of several Bax-specific single-domain antibodies (sdAbs). These minimal size Ab fragments, which were isolated from a llama V(H)H phage display library by panning, inhibited Bax function in in vitro assays. Importantly, as intrabodies, these sdAbs, which were stably expressed in mammalian cells, were nontoxic to their host cells and rendered them highly resistant to oxidative-stress-induced apoptosis. The intrabodies prevented mitochondrial membrane potential collapse and apoptosis after oxidative stress in the host cells. These anti-Bax V(H)Hs could be used as tools for studying the role of Bax in oxidative-stress-induced apoptosis and for developing novel therapeutics for the degenerative diseases involving oxidative stress.

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Available from: Siyaram Pandey, Dec 17, 2013
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    • "The stability of Nbs is perfectly suited for the production of intrabodies that require their expression in the reducing environment of the cytoplasm. Bax-specific intrabodies have been developed to prevent the oxidative stress-induced apoptosis that is implicated in neurodegenerative diseases.44 The targeting of intracellular proteins may also proceed with the fusion of Nbs to peptides, thus mediating translocation across the cell membrane – although intrachain disulfide bonding could hamper unfolding and translocation. "
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    • "In fact, their high intrinsic stability allows them to fold properly in reducing environments that do not allow disulfide bond formation such as cell cytoplasm or nucleus. Consequently, and unlike conventional antibody fragments, most sdAbs can efficiently be used as intracellular antibodies (intrabodies) to probe or block a specific epitope of an intracellular protein (Muyldermans 2001; Gueorguieva et al. 2006; Rothbauer et al. 2006; Verheesen et al. 2006; Groot et al. 2008; Serruys et al. 2009). Last but not least, the genes of these fragments show a large degree of homology with the VH3 subset of human VH genes (Ewert et al. 2002; Su et al. 2002), which confer them a low antigenicity in humans, a very attractive feature for immunotherapy approaches. "
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    ABSTRACT: Monoclonal antibodies are now widely recognized as therapeutic molecules and more than 25 molecules have been approved in the United States and other countries. Despite these successes, the clinical activity of these molecules is still far from optimal and new solutions have to be found, especially in the field of cancer therapy. The potential of bispecific antibodies, capable of simultaneously binding two different antigens has been clearly demonstrated over the years but the difficulty to produce large amounts of homogenous preparations has hindered a wider development of this approach. A variety of new recombinant formats is currently changing the situation. Single domain antibodies derived from IgG devoid of light chain naturally occurring in camelids and sharks, or produced by antibody engineering from human sources, are endowed with outstanding physical properties that qualifies them as ideal building blocks to elaborate multivalent and/or multispecific molecules. This chapter aims at giving an overview of the published work using single domain antibodies to create bispecific antibodies.
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    • "This is a critical stage for the inhibition of apoptosis due to the fact that Bax channel formation is required for the destabilization of the mitochondria, and subsequent release of cytotoxic factors [92]. These inhibitory compounds were modeled after single-domain antibodies that were able to bind specifically to Bax [93]. They are small enough to have the potential to cross the blood brain barrier and are not susceptible to proteolysis. "
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