Article Published in the Author Account of

Martin R Stocks

Intrabodies: Turning the Immune System Inside Out for New Discovery Tools and Therapeutics

Abstract: It is estimated that 63% of drug targets are intracellular and cannot be reached by antibody drugs and many other therapeutic agents. Intrabody (single-chain antibody or its fragments) produced intracellularly is a promising technology that could bring forth intracellular therapeutics in addition to being an important research tool.



Introduction

The human immune system has evolved over millennia to effectively deal with a huge diversity of threats to the integrity of the body. Antibodies, which are the principal agents of the humoral immune system, are masterpieces of evolutionary engineering, combining precise discrimination of target molecules or structures with potent binding capacities. The function of an alien molecule can either be blocked directly by an antibody binding it, or the antibody can recruit effector functions from the immune system to bring about the neutralization, destruction, or removal of the molecule. The potential value of antibodies is nowhere more evident than in the production of new medicines. Technological developments have allowed for the isolation and large scale production of single antibody molecules that can target disease-relevant molecules or structures either in the body fluids or on the surface of cells, resulting in highly precise and potent therapeutic outcomes and, as a result, antibodies currently form the fastest growing group of new drugs being developed and brought to market.

Naturally occurring antibodies are secreted from B cells and function exclusively in the extracellular fluids. However, a substantial proportion of diseases result from perturbations within cells, out of the reach of normal antibodies. A crude analysis of a human proteomic database (Figure 1) suggests that the current armamentarium of the pharmaceutical industry can only readily access approximately 37% of all potential targets. The remaining 63% are intracellular and tend to act in multi-protein complexes or in non-catalytic roles. These types of target may require intervention either at the genetic level or by agents capable of disrupting protein-protein interactions, a role eminently suited to antibodies and their functional fragments. It has long been a dream of biomedical scientists to bring the power of antibody engineering to bear on the treatment of such diseases by re-engineering antibody molecules to be expressed within cells, but only recently has the technological know-how been developed to make such intracellular antibodies, or intrabodies, a reality.

How to Make an Intrabody

Naturally occurring antibodies are complex, highly structured, multi-chain proteins that are generated through equally complex biosynthetic pathways. The structure of an antibody is critical to its function and, in most cases, the polypeptide chains of an antibody are held together by both intra-chain and inter-chain covalent disulphide bonds (Figure 2). Although the individual polypeptides of an antibody can be forced to express within the cytoplasm of a cell, these disulfide bridges cannot form due to the reducing intracellular environment, so not only do the polypeptides fail to assemble into an intact antibody, but also the individual chains generally fail to form the correct intrinsic structure to be able to function as an antibody component. Fortunately, the discovery of rare hyper-stable structures within the population of antibody genes and advances in the structural formulation of recombinant antibodies, in particular the development of single-polypeptide forms of antibody sub-fragments (single-chain Fv and single domain; see Figure 1 for explanation) have allowed the engineering of antibody structures that are more amenable to intracellular expression. If an antibody fragment has the capacity to fold correctly in the absence of disulphide bridges, and can be expressed as a single polypeptide, then there is a good chance that it will function inside a cell.

Until recently, predicting which antibody genes would behave appropriately was impossible, so, in order to isolate such fragments, it was necessary to develop intracellular screening systems that were capable of isolating such molecules empirically. The yeast 2-hybrid system, which was originally designed to find interacting pairs in protein networks, has been invaluable in this regard. Essentially, a target protein (the bait) is expressed as a fusion protein with the DNA binding domain of a yeast transcription factor, and libraries of antibody fragments (the prey) are co-expressed as fusions with a transcriptional activation domain. Binding of antibody to antigen in the yeast nucleus reconstitutes the 2 parts of the transcription factor and drives the expression of reporter genes - usually auxotrophic markers and/or enzymes which can be assayed for levels of activity. A key strength of this approach is that the target antigen does not have to be expressed and purified prior to selection, as it is expressed from a cDNA sequence as part of the screening process.

A variation of this approach, utilizing mammalian transcription factor fragments and assayed in mammalian cells is used as a secondary screen and gives an extra level of stringency, ensuring that the intrabodies isolated by the combined methods will function as desired in the intracellular environment. This system has been used extensively to generate intrabodies from naive libraries of antibody fragments, and analysis of the isolated antibody genes has led in turn to the definition of a subset of antibody gene families that have the capacity to function intracellularly. These sequences are now being utilized to generate bespoke libraries that will contain a high proportion of intrabodies and therefore make the whole screening process more tractable (Tse et al., 2002).

Using Intrabodies as Research Tools

The capacity of intrabodies to block intermolecular interactions gives them the potential to be extremely potent research tools for the pharmaceutical industry. Figure 3 shows how intrabodies might be used throughout the drug discovery process, from discovery and validation of the target molecule to guiding the drug discovery process, as well as producing potential therapeutic entities in themselves. In the area of target validation, intrabodies offer the intriguing possibility of selectively blocking the functions of a complex target molecule on an epitope by epitope basis, which would provide a much greater depth of information than gene-based knockdown technologies. Gene-based knockdown technologies, such as RNAi, antisense, and genetic knockouts are currently the most widely used target validation tools, but can be somewhat crude in that they provide only a comparison of gene-on versus gene-off phenotypes (or more accurately, gene-high versus gene-low) and give no information on the functions of individual domains or on the intermolecular interactions that the target is engaged in during its normal and pathological functions. Intrabodies are therefore eminently suited as a second layer of validation, providing a much more detailed profile of a target’s functions (Visintin et al., 2004). In addition to the elucidation of target function, intrabodies can also be applied to the process of discovering lead compounds that bind and disrupt the function of the target molecule. For instance, an intrabody that produces a desired phenotype when bound to the target intracellularly, can then be used in vitro to define critical structures and surface elements of the target by fragment selection and/or co-crystalization, thereby providing data to guide the rational design of small-molecule drugs. Such an intrabody could also be used in drug screens by searching for entities that were capable of displacing the intrabody from its target epitope, and of course the intrabody itself would be a therapeutic lead molecule (Stocks, 2005).

Using Intrabodies as Therapeutics

The ability of intrabodies to block protein-protein interactions has directed much research to their application in areas of therapeutic need which are considered difficult for standard drugs, in particular oncology, neurodegeneration, and antivirals (Lobato and Rabbitts, 2004).

In oncology, intrabodies specific for H-RAS, cyclin E and the NS3 protein of Hepatitis C virus have all been shown to block transformation in cell culture. There is a variation on standard intrabodies in which the antibody fragment is expressed into the lumen of the endoplasmic reticulum (ER) and then retained within the ER/golgi complex by peptide tags that engage the golgi’s retention and recycling system. It has been used to prevent the surface expression of the folate receptor on ovarian cancer cells, resulting in growth inhibition. A similar approach has been used to knock-down expression of the tie2 receptor in a mouse xenograft tumor model, inhibiting angiogenesis within the tumor mass and thereby preventing the tumor from growing.

In the field of neurobiology (Miller and Messer, 2005), intracellular antibodies that can inhibit or prevent the polymerization or aggregation of candidate pathological proteins in degenerative diseases are showing some promise. Intrabodies to huntingtin have been shown to inhibit aggregation of the protein in cell-based models of Huntington disease, as have an anti-a-synuclein intrabodies in Parkinson’s disease models. ER-expressed antibodies to the b-amyloid precursor protein, which is involved in the pathogenesis of Alzheimer’s disease, have been shown to inhibit the production of b-amyloid either by blocking the main cleavage site or by preventing the protein from leaving the ER. Other areas where intrabodies are being actively investigated include HIV and Hepatitis C infection, transplantation (downregulating the expression of MHC molecules) and, outside the medical world, in preventing viral infections in crop plants.

Challenges for Intrabody Research

Although the application of intrabodies to drug development is potentially of huge value, there remain many issues that need to be resolved before the technology becomes fully integrated into the mainstream of drug discovery and development. The critical element in making intrabodies a technology of choice for target discovery and validation is the capacity to rapidly and reliably generate panels of intrabodies to any given target. Until recently an unachievable goal, the development, standardization, and automation of new screening protocols, along with the capacity to build bespoke libraries of intrabodies, are now making such a technology platform possible. The ability to readily generate specific intrabodies also impacts upon therapeutic applications of the technology. Until recently, the majority of such research has been reagent-led, with the work following on from the serendipitous discovery of an antibody with intracellular capabilities. New intrabody libraries, and screening protocols, are ushering in an era where clinically oriented work can start with a target-led approach.

However, the one overarching issue for the clinical application of intrabodies is that of delivery. At present, intrabodies are fundamentally a gene-based system with expression being the optimum method of getting the intrabodies into cells and to their site of action. Although great progress is being made in the development of both viral (Lundstrom, 2004) and non-viral (Conwell and Huang, 2005) cellular transduction systems for therapeutic application, there remains great suspicion within the general public, and a degree of skepticism within the scientific and medical communities about the whole premise of gene-based therapy, from its capacity to efficiently deliver a payload at the required site of action to its general safety. Recent safety scares during gene-therapy trials have led to the apparent abandonment of rational risk-benefit analysis and have severely harmed the capacity of commercial entities in particular to develop gene-based technologies such as intrabodies. It is unclear when, or even if, this situation will improve.

One possible way around the issues of gene-based delivery is the development of technologies that allow the direct delivery of protein therapeutics across the cell membrane. Advances in liposome and other encapsulation technologies offer the potential to deliver either gene or protein to cells and the recent development of protein transduction domains (PTDs — see text box) offer the intriguing possibility of intrabodies becoming an injectable drug.

Conclusions

Intrabodies constitute a powerful technology that can bring great benefit to those who are willing and able to embrace the complexity of their generation and manipulation. In the short term, they can be applied within basic research and in the drug discovery arena to define and probe the critical elements of a disease related target. Direct therapeutic application is still some way off, but is nonetheless a real possibility, especially in areas where traditional drugs and approaches struggle to intervene. The technology is still developing and the continued advancement can only bring the day of their routine use closer. For further discussion and sources of information, see Stocks (2006).

References and Further Readings

Conwell CC, Huang L. Recent advances in non-viral gene delivery. Advances in Genetics 53PA:1-18, 2005.

Lobato MN, Rabbitts TH. Intracellular antibodies as specific reagents for functional ablation: future therapeutic molecules. Current Molecular Medicine 4(5):519-528, 2004.

Lundstrom K. Gene therapy applications of viral vectors. Technology in Cancer Research and Treatment 3:467-477, 2004.

Matsushita M, Matsui H: Protein transduction technology. Journal of Molecular Medicine 83(5):324-328, 2005.

Miller TW, Messer A. Intrabody applications in neurological disorders: progress and future prospects. Molecular Therapy 12(3):394-401, 2005.

Stocks MR. Intracellular antibodies: a revolution waiting to happen. Current Opinion in Molecular Therapeutics 8(1):17-23, 2006.

Stocks M. Intrabodies as drug discovery tools and therapeutics. Current Opinion in Chemical Biology 9(4):359-365, 2005.

Tse E, Chung G, Rabbitts TH. Isolation of antigen-specific intracellular antibody fragments as single chain Fv for use in mammalian cells. Methods in Molecular Biology 185:433-446, 2002.

Visintin M, Quondam M, Cattaneo A. The intracellular antibody capture technology: towards the high-throughput selection of functional intracellular antibodies for target validation. Methods 34(2):200-214, 2004.

[Discovery Medicine, 5(30):538-543, 2005]

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