Intrabody

Abstract: Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expansion in the number of polyglutamine-encoding CAG repeats in the gene that encodes the huntingtin (htt) protein. A property of the mutant protein that is intimately involved in the development of the disease is the propensity of the glutamine-expanded protein to misfold and generate an N-terminal proteolytic htt fragment that is toxic and prone to aggregation. Intracellular antibodies (intrabodies) against htt have been shown to reduce htt aggregation by binding to the toxic fragment and inactivating it or preventing its misfolding. Intrabodies may therefore be a useful gene-therapy approach to treatment of the disease. However, high levels of intrabody expression have been required to obtain even limited reductions in aggregation. We have engineered a single-domain intracellular antibody against htt for robust aggregation inhibition at low expression levels by increasing its affinity in the absence of a disulfide bond. Furthermore, the engineered intrabody variable light-chain (VL)12.3, rescued toxicity in a neuronal model of HD. We also found that VL12.3 inhibited aggregation and toxicity in a Saccharomyces cerevisiae model of HD. VL12.3 is significantly more potent than earlier anti-htt intrabodies and is a potential candidate for gene therapy treatment for HD. To our knowledge, this is the first attempt to improve affinity in the absence of a disulfide bond to improve intrabody function. The demonstrated importance of disulfide bond-independent binding for intrabody potency suggests a generally applicable approach to the development of effective intrabodies against other intracellular targets. Huntington's disease neurodegeneration yeast-surface display protein engineering directed evolution

Abstract: Antibodies have long been used in biomedical science as in vitro tools for the identification, purification and functional manipulation of target antigens; they have been exploited in vivo for diagnostic and therapeutic applications as well. Recent advances in antibody engineering have now allowed the genes encoding antibodies to be manipulated so that the antigen binding domain can be expressed intracellularly. The specific and high-affinity binding properties of antibodies, combined with their ability to be stably expressed in precise intracellular locations inside mammalian cells, has provided a powerful new family of molecules for gene therapy applications. These intracellular antibodies are termed ‘intrabodies’. Two clinical protocols have been approved by the RAC for the use of intrabodies in the treatment of an oncologic and an infectious disease. Their clinical use will in all likelihood become widespread if these initial studies show ‘proof in principle’. In this article, the studies from laboratories that have used intrabodies as molecular reagents for cancer therapy and for the control of infectious diseases will be reviewed and future directions of this technology will be discussed.