Intrabody applications in neurological disorders: progress and future prospects

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Intrabody Applications in Neurological Disorders: Progress and Future Prospects Todd W. Miller and Anne Messer* Wadsworth Center, New York State Department of Health, and Department of Biomedical Sciences, University at Albany, Albany, NY 12201, USA *To whom correspondence and reprint requests should be addressed at the Wadsworth Center/David Axelrod Institute, P.O. Box 22002, 120 New Scotland Avenue, Albany, NY 12201-2002, USA. Fax: +1 (518) 402 4773. E-mail: [email protected].

Available online 16 June 2005

Single-chain Fv and single-domain antibodies retain the binding specificity of full-length antibodies, but they can be expressed as single genes in phage or yeast surface-display libraries, thus allowing efficient in vitro selection from a naive human repertoire. Selected genes can then be expressed intracellularly in mammalian cells as intrabodies, with the potential for alteration of the folding, interactions, modifications, or subcellular localization of their targets. These reagents have been developed as therapeutics against cancer and HIV. Since misfolded and accumulated intracellular proteins characterize a wide range of neurodegenerative disorders, they are also potentially useful intrabody targets. Here, we review the extension of intrabody technology to the nervous system, in which studies of Huntington’s disease have been used to develop the approach, and anti-synuclein and -B-amyloid strategies are in the early stages of development. Research on several other neurodegenerations, including Parkinson’s, Alzheimer’s, and prion diseases, provides support for the development of intrabodies directed against specific targets, or possibly against more common downstream targets, as novel therapeutics and as drug discovery tools. Key Words: intrabody, antibody, therapy, neurological, scFv, DAB, Alzheimer’s, Parkinson’s, polyglutamine, Huntington’s, prion, amyloid

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recent Published Studies Using Intrabodies against Neurodegenerative Disease Targets . Huntington’s Disease (HD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Parkinson’s Disease (PD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alzheimer’s Disease (AD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Choice of Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Why Use Intrabodies? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods of Selecting and Improving Functional Intrabodies . . . . . . . . . . . . . . . . Potential Intrabody Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

INTRODUCTION Several recent publications have highlighted the potential to use engineered intracellular antibody (Ab) fragments (intrabodies) to develop novel therapies for neurodegenerative diseases. As many neurological disor-

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ders are mediated by abnormal intracellular proteins, intrabodies may serve as therapeutics to target selectively neurological disease proteins and as tools to understand disease mechanisms for drug discovery. The concept of genetic engineering of Ab fragments, in which nucleic

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acid sequences encoding the antigen-binding sites of Abs can allow intracellular synthesis of small proteins that recognize critical epitopes, has been thoroughly reviewed in several recent papers [1–3]. Intrabodies have already been investigated as treatments for a variety of conditions, including HIV infection [4,5], tumor growth [2,6], and tissue transplantation [7]; they are also being tested in clinical trials for cancer [8]. This review discusses the recent extension of this powerful engineered Ab technology to neurological diseases.

RECENT PUBLISHED STUDIES USING INTRABODIES NEURODEGENERATIVE DISEASE TARGETS

AGAINST

Intrabody studies are showing promise and potential for several neurological diseases. Published literature using intrabodies against neurological disease proteins is summarized in Table 1. Here, we review the findings of intrabody studies for Huntington’s disease, synucleinopathies, and Alzheimer’s and prion diseases. Huntington’s Disease (HD) Based on the hypothesis that the intrabody technology already in clinical trials for cancer and HIV therapies can be applied to neurodegenerative diseases, a collaboration between the Messer and Huston labs used a large naive human spleen single-chain Fv (scFv) phage-display library [9] to examine the behavior of intrabodies against both the expanded polyglutamine and the amino-terminal flanking regions of huntingtin, the disease protein causing the autosomal dominant trinucleotide repeat disease HD. There is no effective treatment for HD, and current therapies treat only the symptoms of the disease. Intrabodies selected against mutant polyglutamine showed both a lack of specificity for huntingtin and a degree of generalized toxicity. However, one intrabody, anti-HD-C4, selected against amino-terminal residues 1– 17 adjacent to the polyglutamine of huntingtin, successfully counteracted in situ length-dependent huntingtin aggregation in three different cell lines [10], as well as in TABLE 1: Current literature on intrabodies and antibody fragments targeting disease proteins of central nervous system disorders Neurological disorder

Protein target

Publications

Synucleinopathies (Parkinson’s disease) Huntington’s disease Alzheimer’s disease Tauopathiesa Prion diseasesa Amyloidogenic diseasesb

a-Synuclein

[17–19]

Huntingtin h-Amyloid Tau Prion Amyloid conformation

[9–14,58] [21–23,59] [25] [27,33,34] [60,61]

a Disorders for which scFvs or DABs have been selected, but for which efficacy in disease model systems has not been reported. b Amyloidogenic diseases include Alzheimer’s, Huntington’s, Parkinson’s, prion and other diseases.

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organotypic slice culture models [11]. The latter study also demonstrated functional protection against mutant huntingtin-specific malonate toxicity. Recently, complete rescue of the eclosion deficit in a Drosophila model of HD, with partial rescue of life span and delay of neuronal degeneration, was achieved by transgenic expression of the anti-HD-C4 intrabody in the intact nervous system (Wolfgang et al., submitted for publication). Further studies of functional protection continue to show promise, with no apparent toxicity. The power of this therapeutic approach has been confirmed and extended in an independent study showing that mouse intrabodies to the polyproline region, flanking the polyglutamine on the carboxyl-terminal side (generated from monoclonal Abs), also reduced aggregation and apoptosis, while intrabodies to the expanded polyglutamine itself were toxic [12]. More recently, additional intrabodies were isolated using a human scFv yeast surface-display library. A single variable light chain domain (VL-DAB), derived from an scFv selected against the amino-terminal 20 amino acid residues of huntingtin, inhibited aggregation and increased cell survival similar to that described in the assays above, with the potential for simplified further engineering [13,14]. Since the tools for structural analysis of the precise effects of different intrabodies on the folding and interactions of these intrinsically highly aggregation-prone proteins are still in the early stages of development, all of the existing studies have approached the generation of anti-HD intrabodies from an empirical perspective. Analyses such as those by Dahlgren and colleagues, which combine atomic force microscopy with surface interactions to examine nanofibril formation of mutant huntingtin, should allow more structure-driven engineering in the near future [15]. Parkinson’s Disease (PD) A similar strategy was utilized in the development of anti-a-synuclein intrabodies for use in understanding and possibly treating synucleinopathies, a group of neurodegenerative disorders characterized by the intracellular accumulation of a-synuclein-positive fibrillar aggregates (Lewy bodies) that includes PD and several dementias [16]. While there are some surgical and pharmacological treatments for PD, none address the underlying process of progressive neurodegeneration, which will eventually render them ineffective. Emadi and co-workers reported that an scFv selected against monomeric a-synuclein prevents the formation of highmolecular-weight oligomers, protofibrils, and aggregates in vitro [17]. Zhou and colleagues similarly demonstrated that an anti-monomeric-a-synuclein scFv preferentially binds to and stabilizes a-synuclein in a monomeric form, rather than in multimeric (pathogenic) states [18]. The latter intrabody also rescued a cell-adhesion defect linked to a-synuclein overexpression in cultured cells,

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indicating functional correction. Maguire-Zeiss and coworkers have begun testing conformation-specific anti-asynuclein scFvs, which could help to clarify the toxic asynuclein species [19], an important step in drug discovery. Alzheimer’s Disease (AD) AD is the most prominent neurodegenerative disorder associated with aging, with rare early onset disease occurring in younger individuals. This disorder is characterized by the accumulation of extracellular and intracellular fibrils, respectively containing h-amyloid and tau [20]; such fibrils are arguably associated with disease. Liu and colleagues have demonstrated that an anti-h-amyloid scFv can prevent aggregation in a cell-free system, inhibiting subsequent toxicity in neuronal cells [21,22]. Recent work by Paganetti and co-workers showed that intracellularly expressed scFv intrabodies targeting a cleavage site in the h-amyloid precursor protein prevented the generation of h-amyloid [23]. These observations indicate that h-amyloid is produced intracellularly, and the pathway by which it is produced may be effectively targeted using intrabodies. Binding of scFv or DAB protein to extracellular h-amyloid, so as to prevent deposition, plaque formation, and pathogenesis, may also be beneficial, although these would not strictly be considered intrabodies. Tau fibril formation has also been linked to several other forms of dementia [24]. Anti-tau intrabodies have been selected by yeast intracellular Ab capture technology [25], but their efficacy in disease systems remains unknown. Prions There are several forms of prion diseases that occur in humans, including Creutzfeld–Jakob disease, Gerstmann–Straussler disease, familial fatal insomnia, and the transmissible forms, kuru and new variant Creutzfeld– Jakob disease. The disease mechanism put forth by S. B. Prusiner identifies an abnormal conformation of the prion protein (PrPsc) as the toxic species, by which this infectious protein can induce normal prion protein (PrP) to change conformation and to adopt the same abnormal features, eliciting a cascade effect and accumulation of PrPsc [26]. In a model of passive immunization, Heppner and colleagues demonstrated that transgenic expression of secreted anti-PrP Abs protected mice against PrPsc infection [27]. This mode of rescue likely utilized extracellular Ab–PrP interaction to prevent disease. Since prion amyloid has also been detected in the cytosol [28,29], and the disease mechanism may involve intracellular pathways [30–32], intracellular Ab expression may also be effective. An scFv version of the therapeutic Ab has been generated [27], although the efficacy of this scFv has not been reported. Other antiPrP scFvs that may bind PrP and block the pathogenic PrPsc cascade have been selected from phage-display

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libraries [33,34], but their utility in preventing disease is also unknown.

CHOICE

OF

TARGETS

The binding of an intrabody to its target protein may elicit any of several possible effects. The intrabody may sterically prevent interactions of the target with other protein partners, or may stabilize or destabilize the target, thus preventing or facilitating turnover. Intrabody binding may alter folding of the target, possibly leading to altered stability or interaction capacity. The intrabody can act as a signal to facilitate degradation of the intrabody–target complex as a misfolded protein. Intrabodies may also be labeled with cellular localization sequences to retarget antigens to select subcellular compartments [10,35]. The genetic manipulability of intrabodies and the rich diversity of intrabody libraries provide a convenient platform from which to investigate the role of protein isoforms and conformations in pathogenesis and to generate candidate therapeutics. Table 2 summarizes how intrabodies could be applied in several important neurodegenerative diseases to (1) alter protein folding and/or interactions directly, (2) alter post-translational modifications, (3) prevent pathogenic proteolysis, or (4) utilize epitopes common to abnormal amyloid protein conformations. Importantly, intrabody targeting of disease proteins, rather than of enzymes involved in the pathological modification of such proteins, leaves said enzymes free to perform other cellular functions.

WHY USE INTRABODIES? As drug discovery tools, the conformational specificity of Ab-based reagents may allow the identification and validation of novel drug targets. The assessment and development of more effective small-molecule drugs will also be facilitated via complementation and competition assays of these candidates in the presence of defined intrabodies. As potential direct therapeutics, intrabodies are distinguished from small-molecule and peptide drugs by their enhanced target binding specificity and stability. Intrabodies may prove to be less immunogenic than other protein/peptide therapeutics, particularly when selected from a human library. RNA interference can also provide reductions of target protein levels, but the conformational selectivity of the Ab fragments allows a broader, proteomic approach that may be particularly advantageous in neurodegenerative diseases, in which misfolded and abnormally modified versions of otherwise normal proteins are the toxic species. Intrabodies may also be well-suited to complement other classes of therapies in multi-drug treatments. Acting at a specific, known stage of a pathogenic cascade, they could counteract pathogenesis when transfected

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TABLE 2: Intrabodies may prevent pathogenesis by a variety of mechanisms through interaction with a disease-causing protein, including the prevention of protein misfolding, alteration of post-translational modifications, and prevention of pathogenic proteolysis of the disease protein Mechanism of intrabody action Preventing disease protein misfolding

Altering post-translational modifications

Prevent pathogenic proteolysis

Identification and removal of toxic amyloid species

Applicable neurological diseases Polyglutamine diseases [62–69] Alzheimer’s disease, tauopathies [70–76] Parkinson’s disease, synucleinopathies [77–79] Prion diseases [26,28,29,80–85] Alzheimer’s disease, tauopathies [16,86–88] SCA-1 [89,90] Parkinson’s disease, synucleinopathies [91–97] Alzheimer’s disease [98–100] Polyglutamine diseases [52] Parkinson’s disease, synucleinopathies [101,102] Polyglutamine diseases Alzheimer’s disease [60,103,104]a Parkinson’s disease Prion diseases

Listed are several of the more prominent disorders for which intrabodies have been, or could theoretically be, investigated using such therapeutic mechanisms, as well as publications supporting the potential use of intrabodies for such disorders. a Amyloid protein conformations recognizable by the same set of antibodies are common to several disorders.

into specific types or regions of neurons. Small molecules (possibly used at lower, less toxic doses than as single therapies) could complement intrabody effects in the most vulnerable cells, while providing sufficient, broader protection to less vulnerable cell types that would not receive intrabody genes or protein.

METHODS OF SELECTING AND IMPROVING FUNCTIONAL INTRABODIES An scFv is generated by cloning the variable domains of an Ab and then joining the single-domain cDNA sequences with DNA encoding a flexible linker [36,37], thereby allowing an scFv (approximately 250 amino acids, 29 kDa) to be expressed from a single gene. Single variable domains of an Ab (DAB) can similarly be utilized [38]. Intrabody generation from a monoclonal Ab, using the RNA from a hybridoma cell line to clone the relevant VH and VL variable domains, allows the creation of an intrabody of known specificity [39]. This approach is advantageous because monoclonal Abs to specific forms of proteins important in neuropathology have been characterized [40,41]. For broader coverage, and to uncover novel epitopes, phage or yeast surface-display libraries created from populations of Ab-expressing cells (e.g., naRve spleens, peripheral blood lymphocytes) can be selected in vitro at high throughput [9,42]. Small proteins and peptides can be used for scFv/DAB screening in vitro, using various solvents to elicit specific structures. Targets can also be screened on surfaces that mimic intracellular locations and chemistries, such as membranes. This can even accommodate some membrane epitopes themselves, although soluble proteins are most readily screened against in vitro.

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Unfortunately, scFv or DAB clones isolated using these techniques are not guaranteed to yield functional intrabodies, primarily due to misfolding of the fragment intracellularly, with an additional contribution from unformed intrachain disulfide bonds in the reducing cytoplasmic environment [43,44]. Additional engineering may therefore be required. First-round library screening can be followed by bintracellular Ab capture,Q which uses a two-hybrid screening for antigen–Ab interaction in the cytoplasm of yeast [45]. This has the advantage that intrabodies must be competent for yeast cytoplasmic expression—further diversification of the pool may be necessary if there are no suitable candidates in the original. An alternative approach to improvement is to take an intrabody that appears to have important intraneuronal properties and engineer it for enhanced performance. The three hypervariable complementarity-determining regions of each variable domain can be grafted into scFv consensus scaffoldings that have been selected for improved intracellular folding and stability, mostly for cancer studies [3,46]. This approach would need to be confirmed in neurons, since their protein handling mechanisms might differ from the types of cells that were used for the development of these scaffolds. These bdesignerQ scaffoldings are also an appealing option because they are undergoing toxicity testing in humans as part of the cancer work. A strategy of site-directed elimination of the cysteine residues in a DAB selected against the amino-terminal 20 residues of huntingtin, followed by random mutagenesis to revitalize high-affinity binding, generated an intrabody whose efficacy has been increased by a factor of 5 to 10 in an intracellular aggregation assay, blocking mutant huntingtin toxicity in two functional assays [13]. These

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data suggest that the use of such an iterative mutagenesis and testing strategy, with future intrabodies that show promising preliminary properties in cellular assays, could be worthwhile, since it would facilitate the more efficient utilization of intrabodies in vivo. Antibody fragments may be further engineered to produce derivatives with higher affinity, lower dissociation rate, or increased half-life through targeted mutagenesis of complementarity-determining regions, light-chain shuffling [47], DNA shuffling for molecular evolution [48], BIAcore-driven selection [49], or errorprone PCR [50]. In vitro affinity measurements are not necessarily reflective of intracellular affinity, although in most cases, selection for higher affinity would impart greater efficacy. Long intrabody half-life has been reported to be even more critical [51]. Selection against an antigen of interest is not currently being performed intracellularly in mammalian cells. Hence, the selection of desirable candidates does not guarantee the same antigen-binding properties when they are expressed as intrabodies in mammalian cells due to the complexity, dynamism, and irreproducibility of the intracellular environment of various mammalian cell types. Validation in cultured mammalian cell types, especially neurons, is an important first step in the use of intrabodies, either as therapeutics or for drug discovery. Expression as transgenes in the intact nervous systems of lower organisms, such as Caenorhabditis elegans or Drosophila, provides an additional level of testing that can include survival. Genetic enhancer-suppressor screening for mechanisms can also be superimposed on such studies, greatly enhancing their value.

POTENTIAL INTRABODY TOXICITY Many of the intrabodies already developed or proposed for the above studies target sequences that are shared by normal and mutant proteins; this is particularly true for

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those that have been designed to prevent misfolding or abnormal interactions, rather than to remove altered proteins. The full-length forms of many such proteins may be essential. Therefore, an intrabody targeting a proteolytic fragment may be more therapeutically effective than one specific for a much larger full-length species in a setting in which both species exist. For HD, this situation is especially advantageous: proteolytic cleavage of polyglutamine disease proteins is thought to result in the release of toxic, expanded polyglutamine-containing fragments with no known essential functions [52]. Longterm expression of virally delivered anti-HD-C4 intrabody in mouse brain did not elicit any obvious toxicity (Fig. 1). Careful toxicity testing will be required prior to clinical use, and levels of intrabody that reduce—rather than eliminate—the abnormal protein load may strike a balance between preservation of adequate normal function and removal of the offending species.

DELIVERY A major obstacle to intrabody applications in neurological disorders resides in the delivery of the protein at a sufficient concentration, and to sufficient numbers of target cells, in the nervous system. Engineering can improve the efficiency of intrabody function, which would reduce the required concentration, as was successfully demonstrated for an anti-HD VL-DAB [53]. Proteins can also be modified with protein transduction domains (PTDs) to facilitate intracellular uptake [54]. There is still significant controversy as to whether such PTD-tagged proteins can accumulate at useful levels free in the cytoplasm. They would also need to be present for long periods of time at sufficient concentrations to serve as therapeutics for most neurological diseases, although PTDs could be useful for short-term mechanistic studies. Gene therapy using recombinant viral vectors, based primarily on adeno-associated viruses, has recently been

FIG. 1. In vivo expression of an anti-huntingtin scFv intrabody in mouse brain. Four-week-old mice were stereotactically injected intrastriatally with 106 IU (2 Al of 109 IU/ml) of rabies virus glycoprotein-pseudotyped equine infectious anemia virus [57] encoding cytomegalovirus promoter-driven anti-huntingtin scFv (antiHD-C4 [10], left hemisphere). Mice were perfused at 3 months post-injection, and 30-Am microtome sections were immunostained for the HA-tagged C4 scFv. Expression was robust in striatum and cortex in the left hemisphere, suggesting that both striatal and corticostriatal neurons were transduced. The right hemisphere, injected with control virus, did not show HA immunoreactivity. Ctx, cortex; Str, striatum; V, ventricle.

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extended to the brain in several human clinical trials [55]. We have recently achieved widespread neuronal expression of an intrabody in mouse cortex and striatum mediated by intrastriatal administration of a rabies virus glycoprotein-pseudotyped recombinant equine infectious anemia (lenti)viral vector [56,57]; Fig. 1 shows anti-HDC4 scFv intrabody [10] gene expression driven by a cytomegalovirus promoter. This is the first demonstration of expression of an intrabody in a mammalian nervous system. Clinical trials have also tested adenoviral delivery of an intrabody gene as a treatment for ovarian cancer [8]; adenoviral vectors may be considered for gene delivery to glial cells for the treatment of neurological disorders, but their lack of neurotropism may render them undesirable as vectors for the disorders discussed herein.

PERSPECTIVE These new classes of intrabody reagents that can be highly engineered, both as direct therapeutics and as tools for further drug discovery, should be a valuable addition to the existing approaches to a wide range of neurological diseases, most of which currently have very few viable long-term treatment options. Intrabodies are being tested in clinical trials for peripheral diseases [8], and neurotropic viral vectors are being evaluated in the clinic for disorders of the central nervous system [55]. The rapidly advancing fields of virus-mediated gene therapy and neurological diseases hold great promise for intrabody therapy for the nervous system in the near future. ACKNOWLEDGMENTS We thank Nicholas D. Mazarakis and Oxford Biomedica, plc., for the use of the EIAV delivery vector and William J. Wolfgang for helpful discussions of the manuscript. Work in the authorsT lab was supported in part by grants from NIH/ NINDS, the Hereditary Disease Foundation/Cure HD Initiative, HuntingtonTs Disease Society of America, and the High Q Foundation. RECEIVED FOR PUBLICATION JANUARY 7, 2005; ACCEPTED APRIL 8, 2005.

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