ENGINEERED NANOBODIES WITH PROGRAMMABLE TARGET ANTIGEN PROTEOLYSIS (PTAP) FUSIONS REGULATE INTRACELLULAR ALPHA-SYNUCLEIN IN VITRO AND IN VIVO.

Alpha-synuclein (αSyn) aggregation and the formation of Lewy pathology (LP) is a foundational pathophysiological phenomenon in synucleinopathies. Delivering therapeutic single-chain and single-domain antibodies that bind pathogenic targets can disrupt intracellular aggregation. The fusion of antibody fragments to a negatively-charged proteasomal targeting motif (PEST) creates bifunctional constructs that enhance both solubility and turnover. With sequence-specific point mutations of PEST sequences that modulate proteasomal degradation efficiency, we report the creation of Programmable Target Antigen Proteolysis (PTAP) technology that can provide graded control over the levels of target antigens. We have previously demonstrated our lead anti-αSyn intrabody, VH14-PEST, is capable of reducing the pathological burden of synucleinopathy in vitro and in vivo. Here, we report a family of fully humanized VH14-PTAP constructs for controllable, therapeutic targeting of intracellular α-Syn. In cells, we demonstrate successful target engagement and efficacy of VH14-hPEST intrabodies, and validate proof-of-principle in human cells using 3D human organoids derived from PD-patient induced pluripotent stem cells (iPSC). In two synuclein-based rat models, PTAP intrabodies attenuated nigral αSyn pathology, preserved nigrostriatal dopaminergic tone, and slowed the propagation of αSyn pathology. These data demonstrate the potency of intracellular αSyn targeting as a method to alleviate pathology and highlight the potential clinical utility of PTAP intrabodies.

Enhanced CNS transduction from AAV.PHP.eB infusion into the cisterna magna of older adult rats compared to AAV9.

The development of high efficiency, central nervous system (CNS) targeting AAV-based gene therapies is necessary to address challenges in both pre-clinical and clinical investigations. The engineered capsids, AAV.PHP.B and AAV.PHP.eB, show vastly improved blood-brain barrier penetration compared to their parent serotype, AAV9, but with variable effect depending on animal system, strain, and delivery route. As most characterizations of AAV.PHP variants have been performed in mice, it is currently unknown whether AAV.PHP variants improve CNS targeting when delivered intrathecally in rats. We evaluated the comparative transduction efficiencies of equititer doses (6 × 1011vg) of AAV.PHP.eB-CAG-GFP and AAV9-CAG-GFP when delivered into the cisterna magna of 6-9-month old rats. Using both quantitative and qualitative assessments, we observed consistently superior biodistribution of GFP+ cells and fibers in animals treated with AAV.PHP.eB compared to those treated with AAV9. Enhanced GFP signal was uniformly observed throughout rostrocaudal brain regions in AAV.PHP.eB-treated animals with matching GFP protein expression detected in the forebrain, midbrain, and cerebellum. Collectively, these data illustrate the benefit of intracisternal infusions of AAV.PHP.eB as an optimal system to distribute CNS gene therapies in preclinical investigations of rats, and may have important translational implications for the clinical CNS targeting.

Optimizing intracellular antibodies (intrabodies/nanobodies) to treat neurodegenerative disorders.

Intrabodies (both single-chain Fv and single-domain VH, VHH, and VL nanobodies) offer unique solutions to some of the challenges of delivery and target engagement posed by immunotherapeutics for the brain and other areas of the nervous system. The specificity, which includes the recognition of post-translational modifications, and capacity for engineering that characterize these antibody fragments can be especially well-focused when the genes encoding only the binding sites of the antibody are expressed intracellularly. Multifunctional constructs use fusions with peptides that can re-target antigen-antibody complexes to enhance both pharmacodynamic activity and intracellular solubility simultaneously. Fusions with proteolytic targeting signals, such as the PEST degron, greatly enhance potency in some cases. Stem cell transplants can be protected from exogenous misfolded proteins by stable transfection with intrabodies. Tandem expression to target two or more misfolding proteins in one treatment may be especially valuable for proteostatic disruptions due to genetic, aging, or toxic triggers. Advances in bioinformatics, screening protocols, and especially gene therapy are showing great promise for intrabody/ nanobody treatments of a full range of neurological disorders, including Alzheimer's disease and related tau dementias, Parkinson's disease and Lewy body diseases, Huntington's disease, amyotrophic lateral sclerosis, and prion diseases, among others.

Computational affinity maturation of camelid single-domain intrabodies against the nonamyloid component of alpha-synuclein.

Improving the affinity of protein-protein interactions is a challenging problem that is particularly important in the development of antibodies for diagnostic and clinical use. Here, we used structure-based computational methods to optimize the binding affinity of VHNAC1, a single-domain intracellular antibody (intrabody) from the camelid family that was selected for its specific binding to the nonamyloid component (NAC) of human α-synuclein (α-syn), a natively disordered protein, implicated in the pathogenesis of Parkinson's disease (PD) and related neurological disorders. Specifically, we performed ab initio modeling that revealed several possible modes of VHNAC1 binding to the NAC region of α-syn as well as mutations that potentially enhance the affinity between these interacting proteins. While our initial design strategy did not lead to improved affinity, it ultimately guided us towards a model that aligned more closely with experimental observations, revealing a key residue on the paratope and the participation of H4 loop residues in binding, as well as confirming the importance of electrostatic interactions. The binding activity of the best intrabody mutant, which involved just a single amino acid mutation compared to parental VHNAC1, was significantly enhanced primarily through a large increase in association rate. Our results indicate that structure-based computational design can be used to successfully improve the affinity of antibodies against natively disordered and weakly immunogenic antigens such as α-syn, even in cases such as ours where crystal structures are unavailable.

Correction: Sustained AAV9-mediated expression of a non-self protein in the CNS of non-human primates after immunomodulation.

[This corrects the article DOI: 10.1371/journal.pone.0198154.].

Proteasome-targeted nanobodies alleviate pathology and functional decline in an α-synuclein-based Parkinson's disease model.

Therapeutics designed to target α-synuclein (α-syn) aggregation may be critical in halting the progression of pathology in Parkinson's disease (PD) patients. Nanobodies are single-domain antibody fragments that bind with antibody specificity, but allow readier genetic engineering and delivery. When expressed intracellularly as intrabodies, anti-α-syn nanobodies fused to a proteasome-targeting proline, aspartate or glutamate, serine, and threonine (PEST) motif can modulate monomeric concentrations of target proteins. Here we aimed to validate and compare the in vivo therapeutic potential of gene therapy delivery of two proteasome-directed nanobodies selectively targeting α-syn in a synuclein overexpression-based PD model: VH14*PEST (non-amyloid component region) and NbSyn87*PEST (C-terminal region). Stereotaxic injections of adeno-associated viral 5-α-syn (AAV5-α-syn) into the substantia nigra (SN) were performed in Sprague-Dawley rats that were sorted into three cohorts based on pre-operative behavioral testing. Rats were treated with unilateral SN injections of vectors for VH14*PEST, NbSyn87*PEST, or injected with saline 3 weeks post lesion. Post-mortem assessments of the SN showed that both nanobodies markedly reduced the level of phosphorylated Serine-129 α-syn labeling relative to saline-treated animals. VH14*PEST showed considerable maintenance of striatal dopaminergic tone in comparison to saline-treated and NbSyn87*PEST-treated animals as measured by tyrosine hydroxylase immunoreactivity (optical density), DAT immunoreactivity (optical density), and dopamine concentration (high-performance liquid chromatography). Microglial accumulation and inflammatory response, assessed by stereological counts of Iba-1-labeled cells, was modestly increased in NbSyn87*PEST-injected rats but not in VH14*PEST-treated or saline-treated animals. Modest behavioral rescue was also observed, although there was pronounced variability among individual animals. These data validate in vivo therapeutic efficacy of vector-delivered intracellular nanobodies targeting α-syn misfolding and aggregation in synucleinopathies such as PD.

Sustained AAV9-mediated expression of a non-self protein in the CNS of non-human primates after immunomodulation.

A critical issue in transgene delivery studies is immune reactivity to the transgene- encoded protein and its impact on sustained gene expression. Here, we test the hypothesis that immunomodulation by rapamycin can decrease immune reactivity after intrathecal AAV9 delivery of a transgene (GFP) in non-human primates, resulting in sustained GFP expression in the CNS. We show that rapamycin treatment clearly reduced the overall immunogenicity of the AAV9/GFP vector by lowering GFP- and AAV9-specific antibody responses, and decreasing T cell responses including cytokine and cytolytic effector responses. Spinal cord GFP protein expression was sustained for twelve weeks, with no toxicity. Immune correlates of robust transgene expression include negligible GFP-specific CD4 and CD8 T cell responses, absence of GFP-specific IFN-γ producing T cells, and absence of GFP-specific cytotoxic T cells, which support the hypothesis that decreased T cell reactivity results in sustained transgene expression. These data strongly support the use of modest doses of rapamycin to modulate immune responses for intrathecal gene therapies, and potentially a much wider range of viral vector-based therapeutics.

Bifunctional Anti-Non-Amyloid Component α-Synuclein Nanobodies Are Protective In Situ.

Misfolding, abnormal accumulation, and secretion of α-Synuclein (α-Syn) are closely associated with synucleinopathies, including Parkinson's disease (PD). VH14 is a human single domain intrabody selected against the non-amyloid component (NAC) hydrophobic interaction region of α-Syn, which is critical for initial aggregation. Using neuronal cell lines, we show that as a bifunctional nanobody fused to a proteasome targeting signal, VH14PEST can counteract heterologous proteostatic effects of mutant α-Syn on mutant huntingtin Exon1 and protect against α-Syn toxicity using propidium iodide or Annexin V readouts. We compared this anti-NAC candidate to NbSyn87, which binds to the C-terminus of α-Syn. NbSyn87PEST degrades α-Syn as well or better than VH14PEST. However, while both candidates reduced toxicity, VH14PEST appears more effective in both proteostatic stress and toxicity assays. These results show that the approach of reducing intracellular monomeric targets with novel antibody engineering technology should allow in vivo modulation of proteostatic pathologies.

Antibody Engineering & Therapeutics 2016: The Antibody Society's annual meeting, December 11-15, 2016, San Diego, CA.

Antibody Engineering & Therapeutics, the largest meeting devoted to antibody science and technology and the annual meeting of The Antibody Society, will be held in San Diego, CA on December 11-15, 2016. Each of 14 sessions will include six presentations by leading industry and academic experts. In this meeting preview, the session chairs discuss the relevance of their topics to current and future antibody therapeutics development. Session topics include bispecifics and designer polyclonal antibodies; antibodies for neurodegenerative diseases; the interface between passive and active immunotherapy; antibodies for non-cancer indications; novel antibody display, selection and screening technologies; novel checkpoint modulators / immuno-oncology; engineering antibodies for T-cell therapy; novel engineering strategies to enhance antibody functions; and the biological Impact of Fc receptor engagement. The meeting will open with keynote speakers Dennis R. Burton (The Scripps Research Institute), who will review progress toward a neutralizing antibody-based HIV vaccine; Olivera J. Finn, (University of Pittsburgh School of Medicine), who will discuss prophylactic cancer vaccines as a source of therapeutic antibodies; and Paul Richardson (Dana-Farber Cancer Institute), who will provide a clinical update on daratumumab for multiple myeloma. In a featured presentation, a representative of the World Health Organization's INN expert group will provide a perspective on antibody naming. "Antibodies to watch in 2017" and progress on The Antibody Society's 2016 initiatives will be presented during the Society's special session. In addition, two pre-conference workshops covering ways to accelerate antibody drugs to the clinic and the applications of next-generation sequencing in antibody discovery and engineering will be held on Sunday December 11, 2016.

Transcriptional dysregulation of inflammatory/immune pathways after active vaccination against Huntington's disease.

Immunotherapy, both active and passive, is increasingly recognized as a powerful approach to a wide range of diseases, including Alzheimer's and Parkinson's. Huntington's disease (HD), an autosomal dominant disorder triggered by misfolding of huntingtin (HTT) protein with an expanded polyglutamine tract, could also benefit from this approach. Individuals can be identified genetically at the earliest stages of disease, and there may be particular benefits to a therapy that can target peripheral tissues in addition to brain. In this active vaccination study, we first examined safety and immunogenicity for a broad series of peptide, protein and DNA plasmid immunization protocols, using fragment (R6/1), and knock-in (zQ175) models. No safety issues were found. The strongest and most uniform immune response was to a combination of three non-overlapping HTT Exon1 coded peptides, conjugated to KLH, delivered with alum adjuvant. An N586-82Q plasmid, delivered via gene gun, also showed ELISA responses, mainly in the zQ175 strain, but with more variability, and less robust responses in HD compared with wild-type controls. Transcriptome profiling of spleens from the triple peptide-immunized cohort showed substantial HD-specific differences including differential activation of genes associated with innate immune responses, absence of negative feedback control of gene expression by regulators, a temporal dysregulation of innate immune responses and transcriptional repression of genes associated with memory T cell responses. These studies highlight critical issues for immunotherapy and HD disease management in general.

Structure of a single-chain Fv bound to the 17 N-terminal residues of huntingtin provides insights into pathogenic amyloid formation and suppression.

Huntington's disease is triggered by misfolding of fragments of mutant forms of the huntingtin protein (mHTT) with aberrant polyglutamine expansions. The C4 single-chain Fv antibody (scFv) binds to the first 17 residues of huntingtin [HTT(1-17)] and generates substantial protection against multiple phenotypic pathologies in situ and in vivo. We show in this paper that C4 scFv inhibits amyloid formation by exon1 fragments of huntingtin in vitro and elucidate the structural basis for this inhibition and protection by determining the crystal structure of the complex of C4 scFv and HTT(1-17). The peptide binds with residues 3-11 forming an amphipathic helix that makes contact with the antibody fragment in such a way that the hydrophobic face of this helix is shielded from the solvent. Residues 12-17 of the peptide are in an extended conformation and interact with the same region of another C4 scFv:HTT(1-17) complex in the asymmetric unit, resulting in a ß-sheet interface within a dimeric C4 scFv:HTT(1-17) complex. The nature of this scFv-peptide complex was further explored in solution by high-resolution NMR and physicochemical analysis of species in solution. The results provide insights into the manner in which C4 scFv inhibits the aggregation of HTT, and hence into its therapeutic potential, and suggests a structural basis for the initial interactions that underlie the formation of disease-associated amyloid fibrils by HTT.

Antibodies and protein misfolding: From structural research tools to therapeutic strategies.

Protein misfolding disorders, including the neurodegenerative conditions Alzheimer's disease (AD) and Parkinson's disease (PD) represent one of the major medical challenges or our time. The underlying molecular mechanisms that govern protein misfolding and its links with disease are very complex processes, involving the formation of transiently populated but highly toxic molecular species within the crowded environment of the cell and tissue. Nevertheless, much progress has been made in understanding these events in recent years through innovative experiments and therapeutic strategies, and in this review we present an overview of the key roles of antibodies and antibody fragments in these endeavors. We discuss in particular how these species are being used in combination with a variety of powerful biochemical and biophysical methodologies, including a range of spectroscopic and microscopic techniques applied not just in vitro but also in situ and in vivo, both to gain a better understanding of the mechanistic nature of protein misfolding and aggregation and also to design novel therapeutic strategies to combat the family of diseases with which they are associated. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.

Can intrabodies serve as neuroprotective therapies for Parkinson's disease? Beginning thoughts.

Misfolded proteins and subsequent protein aggregation appears to underlie a significant fraction of neurodegenerative diseases including Parkinson's disease. One of the neuropathological hallmarks of Parkinson's disease is the presence of α-syn containing intracellular inclusions known as Lewy bodies and Lewy neurites. Intrabodies are antibody fragments that have been engineered to be expressed intracellularly. They can be directed towards specific target antigens present in various subcellular locations, and have shown promise in cancer, HIV, autoimmune diseases, and Huntington's disease. More recently they have been shown to modulate abnormalities caused by aggregated α-syn in cell culture. This mini-review mainly focuses on summarizing structural and cellular effects of intrabodies shown to have affinity for different forms of α-synuclein (monomeric, oligomeric and fibrillar), as well as those exhibiting affinity for particular residues of α-synuclein (e.g., the NAC region, C terminal region).

Intrabodies as neuroprotective therapeutics.

The process of misfolding of proteins that can trigger a pathogenic cascade leading to neurodegenerative diseases largely originates intracellularly. It is possible to harness the specificity and affinity of antibodies to counteract either protein misfolding itself, or the aberrant interactions and excess stressors immediately downstream of the primary insult. This review covers the emerging field of engineering intracellular antibody fragments, intrabodies and nanobodies, in neurodegeneration. Huntington's disease has provided the clearest proof of concept for this approach. The model systems and readouts for this disorder power the studies, and the potential to intervene therapeutically at early stages in known carriers with projected ages of onset increases the chances of meaningful clinical trials. Both single-chain Fv and single-domain nanobodies have been identified against specific targets; data have allowed feedback for rational design of bifunctional constructs, as well as target validation. Intrabodies that can modulate the primary accumulating protein in Parkinson's disease, alpha-synuclein, are also reviewed, covering a range of domains and conformers. Recombinant antibody technology has become a major player in the therapeutic pipeline for cancer, infectious diseases, and autoimmunity. There is also tremendous potential for applying this powerful biotechnology to neurological diseases.

MSH3 polymorphisms and protein levels affect CAG repeat instability in Huntington's disease mice.

Expansions of trinucleotide CAG/CTG repeats in somatic tissues are thought to contribute to ongoing disease progression through an affected individual's life with Huntington's disease or myotonic dystrophy. Broad ranges of repeat instability arise between individuals with expanded repeats, suggesting the existence of modifiers of repeat instability. Mice with expanded CAG/CTG repeats show variable levels of instability depending upon mouse strain. However, to date the genetic modifiers underlying these differences have not been identified. We show that in liver and striatum the R6/1 Huntington's disease (HD) (CAG)∼100 transgene, when present in a congenic C57BL/6J (B6) background, incurred expansion-biased repeat mutations, whereas the repeat was stable in a congenic BALB/cByJ (CBy) background. Reciprocal congenic mice revealed the Msh3 gene as the determinant for the differences in repeat instability. Expansion bias was observed in congenic mice homozygous for the B6 Msh3 gene on a CBy background, while the CAG tract was stabilized in congenics homozygous for the CBy Msh3 gene on a B6 background. The CAG stabilization was as dramatic as genetic deficiency of Msh2. The B6 and CBy Msh3 genes had identical promoters but differed in coding regions and showed strikingly different protein levels. B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability. The DHFR protein, which is divergently transcribed from a promoter shared by the Msh3 gene, did not show varied levels between mouse strains. Thus, naturally occurring MSH3 protein polymorphisms are modifiers of CAG repeat instability, likely through variable MSH3 protein stability. Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of DNA repair genes may have prognostic implications for various repeat-associated diseases.

Fusion to a highly charged proteasomal retargeting sequence increases soluble cytoplasmic expression and efficacy of diverse anti-synuclein intrabodies.

Intrabodies can be powerful reagents to effect modulation of aberrant intracellular proteins that underlie a range of diseases. However, their cytoplasmic solubility can be limiting. We previously reported that overall charge and hydrophilicity can be combined to provide initial estimates of intracellular solubility, and that charge engineering via fusion can alter solubility properties experimentally. Additional studies showed that fusion of a proteasome-targeting PEST motif to the anti-huntingtin intrabody scFv-C4 can degrade mutant huntingtin proteins by directing them to the proteasome, while also increasing the negative charge. We now validate the generality of this approach with intrabodies against α-synuclein (α-syn), an important target in Parkinson disease. In this study, fusion of the PEST sequence to a set of four diverse, poorly soluble anti-α-syn intrabodies (D5E, 10H, D10 scFv, VH14 nanobody) significantly increased steady-state soluble intrabody protein levels in all cases, despite fusion with the PEST proteasomal-targeting signal. Furthermore, adding this PEST motif to the least soluble construct, VH14, significantly enhanced degradation of the target protein, α-syn~GFP. The intrabody-PEST fusion approach thus has dual advantages of potentially solubilizing intrabodies and enhancing their functionality in parallel. Empirical testing of intrabody-PEST fusions is recommended for enhancement of intrabody solubility from diverse sources.

Engineered antibody therapies to counteract mutant huntingtin and related toxic intracellular proteins.

The engineered antibody approach to Huntington's disease (HD) therapeutics is based on the premise that significantly lowering the levels of the primary misfolded mutant protein will reduce abnormal protein interactions and direct toxic effects of the misfolded huntingtin (HTT). This will in turn reduce the pathologic stress on cells, and normalize intrinsic proteostasis. Intracellular antibodies (intrabodies) are single-chain (scFv) and single-domain (dAb; nanobody) variable fragments that can retain the affinity and specificity of full-length antibodies, but can be selected and engineered as genes. Functionally, they represent a protein-based approach to the problem of aberrant mutant protein folding, post-translational modifications, protein-protein interactions, and aggregation. Several intrabodies that bind on either side of the expanded polyglutamine tract of mutant HTT have been reported to improve the mutant phenotype in cell and organotypic cultures, fruit flies, and mice. Further refinements to the difficult challenges of intraneuronal delivery, cytoplasmic folding, and long-term efficacy are in progress. This review covers published studies and emerging approaches on the choice of targets, selection and engineering methods, gene and protein delivery options, and testing of candidates in cell and animal models. The resultant antibody fragments can be used as direct therapeutics and as target validation/drug discovery tools for HD, while the technology is also applicable to a wide range of neurodegenerative and other diseases that are triggered by toxic proteins.

Nicotinamide improves motor deficits and upregulates PGC-1α and BDNF gene expression in a mouse model of Huntington's disease.

Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by an expansion of the polyglutamine (polyQ) repeat in exon-1 in the Huntingtin gene (HTT). This results in misfolding and accumulation of the huntingtin (htt) protein, forming nuclear and cytoplasmic inclusions. HD is associated with dysregulation of gene expression as well as mitochondrial dysfunction. We hypothesized that by improving transcriptional regulation of genes necessary for energy metabolism, the HD motor phenotype would also improve. We therefore examined the protective effects of nicotinamide (NAM), a well-characterized water-soluble B vitamin that is an inhibitor of sirtuin1/class III NAD(+)-dependent histone deacetylase (HDAC). In this study, both mini-osmotic pumps and drinking water deliveries were tested at 250 mg NAM/kg/day, using the B6.HDR6/1 transgenic mouse model. Results were similar for both modes of delivery, and there was no evidence of toxicity. We found that NAM treatment increased mRNA levels of brain-derived neurotrophic factor (BDNF), and Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), the master regulator of mitochondrial biogenesis. Protein levels of BDNF were also significantly increased. In addition, NAM treatment increased PGC-1α activation in HD mice, pointing to a possible mode of action as a therapeutic. Critically, NAM treatment was able to improve motor deficits associated with the HD phenotype, tested as time courses of open field, rotarod, and balance beam activities. These improvements were substantial, despite the fact that NAM did not appear to reduce htt aggregation, or to prevent late-stage weight loss. Our study therefore concludes that NAM or similar drugs may be beneficial in clinical treatment of the motor dysfunctions of HD, while additional therapeutic approaches must be added to combat the aggregation phenotype and overall physiological decline.

Bifunctional anti-huntingtin proteasome-directed intrabodies mediate efficient degradation of mutant huntingtin exon 1 protein fragments.

Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by a trinucleotide (CAG)(n) repeat expansion in the coding sequence of the huntingtin gene, and an expanded polyglutamine (>37Q) tract in the protein. This results in misfolding and accumulation of huntingtin protein (htt), formation of neuronal intranuclear and cytoplasmic inclusions, and neuronal dysfunction/degeneration. Single-chain Fv antibodies (scFvs), expressed as intrabodies that bind htt and prevent aggregation, show promise as immunotherapeutics for HD. Intrastriatal delivery of anti-N-terminal htt scFv-C4 using an adeno-associated virus vector (AAV2/1) significantly reduces the size and number of aggregates in HDR6/1 transgenic mice; however, this protective effect diminishes with age and time after injection. We therefore explored enhancing intrabody efficacy via fusions to heterologous functional domains. Proteins containing a PEST motif are often targeted for proteasomal degradation and generally have a short half life. In ST14A cells, fusion of the C-terminal PEST region of mouse ornithine decarboxylase (mODC) to scFv-C4 reduces htt exon 1 protein fragments with 72 glutamine repeats (httex1-72Q) by ~80-90% when compared to scFv-C4 alone. Proteasomal targeting was verified by either scrambling the mODC-PEST motif, or via proteasomal inhibition with epoxomicin. For these constructs, the proteasomal degradation of the scFv intrabody proteins themselves was reduced<25% by the addition of the mODC-PEST motif, with or without antigens. The remaining intrabody levels were amply sufficient to target N-terminal httex1-72Q protein fragment turnover. Critically, scFv-C4-PEST prevents aggregation and toxicity of httex1-72Q fragments at significantly lower doses than scFv-C4. Fusion of the mODC-PEST motif to intrabodies is a valuable general approach to specifically target toxic antigens to the proteasome for degradation.