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  Antibodies that bind to an epitope on the huntington's disease proteinUSPTO Application #: 20080107657Title: Antibodies that bind to an epitope on the huntington's disease protein Abstract: The present invention relates generally to the generation and characterization of anti-huntingtin antibodies binding an epitope on the Huntington's disease protein. The invention further relates to the use of such anti-huntingtin antibodies in the diagnosis and treatment of Huntington's disease. (end of abstract) Agent: Knobbe Martens Olson & Bear LLP - Irvine, CA, US Inventors: Ali Khoshnan, Jan Ko, Paul H. Patterson USPTO Applicaton #: 20080107657 - Class: 424144100 (USPTO) Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Immunoglobulin, Antiserum, Antibody, Or Antibody Fragment, Except Conjugate Or Complex Of The Same With Nonimmunoglobulin Material, Monoclonal Antibody Or Fragment Thereof (i.e., Produced By Any Cloning Technology), Binds Receptor, Receptor Integral To Or Derived From A Lymphocytic Or Lymphocytic-like Cell (e.g., Nk Cell, Etc.) The Patent Description & Claims data below is from USPTO Patent Application 20080107657. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATIONS [0001] The present application is a continuation of U.S. patent application Ser. No. 10/354,246, which in turn claims priority under 35 U.S.C. .sctn.119(e) from U.S. provisional application No. 60/353,032, filed on Jan. 28, 2002. All of the priority applications are hereby incorporated by reference in their entirety. BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present invention relates generally to antibodies to Huntington's disease protein as well as methods and means for making and using such antibodies. [0004] 2. Description of the Related Art [0005] Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disorder that is caused by the extension of a polyglutamine (polyQ) tract in exon 1 the protein huntingtin (Htt) to a length of greater than 40 units (Reddy et al. Trends Neurosci. 22:248-255 (1999)). The huntingtin gene is known and the subject of U.S. Pat. No. 5,693,757. Mutant Htt with greater than 40 CAG repeats gains a toxic function and induces death in subpopulations of neurons in the striatum and cortex (Zoghbi et al. Annu. Rev. Neurosci. 23:217-247 (2000); Tobin et al. Trends Cell Biol. 10:531-536 (2000)). Neuronal death in HD has been attributed not only to polyQ toxicity, but also to activation of caspases, interference with transcriptional machinery, and sequestration/inactivation of wild-type Htt and other important cellular factors. [0006] A hallmark of HD and other polyQ diseases is the formation of insoluble protein aggregates in affected neurons (Ross Neuron 19:1147-1150 (1997); Wanker Biol. Chem. 937-942 (2000). Immunohistochemistry and subcellular fractionation indicate that Htt is normally located in the cytoplasm while the mutant form of Htt is also found in aggregates in the nucleus (Ferrigno et al. Neuron 26:9-12 (2000)). A major component of the aggregates in HD is the N terminus exon 1 of mutant Htt. As normal huntingtin protein is localized in the cytoplasm and mutant huntingtin protein is found in aggregates, also known as and referred to as inclusions, in the nucleus (Ferrigno et al., Neuron, 26:9-12 (2000)), translocation of mutant huntingtin protein to the nucleus is believed to be important in the pathogenesis of HD. [0007] Because there is no current treatment available for this disease, there is a clear need for new treatments for Huntington's disease. Molecules that block the toxic effects of Htt itself or the lethal consequences of its binding to other proteins have good potential for therapeutic use. Thus, antibodies may serve as treatments for Huntington's disease. An antibody termed 1C2 is described in WO 97/17445. Finkbeiner (U.S. Pat. No. 6,291,652) provides antibodies specific for proteins having polyglutamine expansions. In particular, Finkbeiner provides antibodies having a higher affinity than an antibody identified as 1C2. SUMMARY OF THE INVENTION [0008] In one aspect, the invention involves antibodies, specifically monoclonal antibodies including antibody fragments, such as single-chain variant fragments, and mimetics thereof (including intrabodies), to the huntingtin protein. Preferred biological activities of the antibodies include the capability of preventing cell death or apoptosis, preventing mutant huntingtin protein aggregation and the regulating the toxic effects of mutant huntingtin protein that are associated with neurodegenerative disease. In one embodiment, the antibodies bind specifically to an epitope within a polyproline region of the huntingtin protein comprising greater than 5 consecutive proline residues and are capable of inhibiting aggregation of huntingtin protein. In another embodiment, the antibodies bind specifically to an epitope within the polyglutamine region of the huntingtin protein comprising greater than 6 consecutive glutamine residues and are capable of stimulating aggregation of huntingtin protein. In another embodiment, the antibodies specifically interact with an amino acid epitope within the carboxy terminus of the protein encoded by exon 1 of the huntingtin protein, said carboxy terminus comprising the amino acid sequence of SEQ ID NO: 2. In another embodiment, the antibodies are in association with a therapeutically acceptable carrier. The single-chain variant antibody fragments are encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5 and 6. [0009] The methods of the invention involve the treatment of an individual, preferably a patient, more preferably a mammalian patient and even more preferably a human mammalian patient, having or suspected of having Huntington's disease by administering a therapeutically effective amount of an antibody, such as a single-chain variant fragment, or antibody composition comprising a single-chain variant fragment to the individual. The antibody compositions of the methods are preferably delivered intracranially, for example, by injection directly into brain tissue or by injection into the cerebrospinal fluid. [0010] The methods of the invention may also involve the treatment of Huntington's disease by expressing anti-huntingtin antibodies, including single-chain variant fragments, in cells expressing mutant huntingtin protein. Nucleic acids encoding the subject antibodies and methods for their expression, including in therapeutic treatment protocols, are provided. Nucleic acids of the invention can be introduced into a host cell using various viral vectors and non-viral delivery techniques for expression of the nucleic acid encoding the antibody in brain tissue. BRIEF DESCRIPTION OF THE DRAWINGS [0011] FIG. 1 shows epitope mapping of anti-huntingtin antibodies MW1-MW8 by peptide array which includes both human and mouse huntingtin peptides. Two rows of peptide dot blots are shown for each of the MW1-MW8 anti-huntingtin antibodies with the upper row corresponding to the peptides shown at the top of the figure, and the lower row corresponding to the peptides shown at the bottom of the figure. The three types of epitope of the huntingtin protein are underlined in the corresponding peptide sequences ______=polyQ; ...=polyP; .sub.------=C terminus). [0012] FIG. 2 shows a diagram of the epitope mapping results from the peptide array analysis in FIG. 1. The results of the peptide array are displayed on a linear diagram of the normal human huntingtin amino acid sequence (SEQ ID NO: 1). [0013] FIG. 3 shows a Western blot of normal (WT) and transgenic 94Q knock-in (94Q) mouse cerebellum extracts using anti-huntingtin antibodies MW1-MW8. Control antibodies 1C2 and 1F8 were used to identify mutant huntingtin protein, and 2166 antibody was used to identify both mutant and normal huntingtin protein. [0014] FIG. 4 shows a Western blot of normal (HD7) and Huntington's disease (HD2) human lymphoblastoma cell extracts using anti-huntingtin antibodies MW1-MW8. Control antibodies 1C2 and 1F8 were used to identify mutant huntingtin protein, and 2166 antibody was used to identify both mutant and normal huntingtin protein. [0015] FIGS. 5A-5E show immunofluorescence staining patterns of MW1 anti-huntingtin and control 1C2 antibodies in wild-type (WT) and R6/2 transgenic cortex (R6), having mutant spinal cord neurons. FIG. 5A shows the level of background immunostaining in the absence of primary antibody. FIG. 5B show immunostaining of MW1 and 1C2 antibodies in cortical neurons. FIGS. 5D and 5E shows immunostaining of MW1 and 1C2 antibodies in fresh frozen R6/1 cortex sections, respectively. [0016] FIGS. 6A-6H show immunofluorescence staining patterns of MW2-MW5 anti-huntingtin and control 1F8 antibodies in wild-type (WT) and R6/2 transgenic cortex (R6), having mutant spinal cord neurons. MW2 (FIG. 6A), MW3 (FIG. 6B), MW4 (FIG. 6C), MW5 (FIG. 6D) and control 1F8 (FIG. 6E) antibodies exhibit similar patterns, neuronal Golgi complex staining, when used to stain spinal cord sections. MW3 staining of paraformaldehyde fixed spinal cord sections from R6/2 mice is shown in FIG. 6F. MW3 staining of wild-type and R6/2 mutant brain sections are shown in FIGS. 6G and 6H, respectively. [0017] FIGS. 7A-7I show immunofluorescence staining patterns of MW6-MW8 anti-huntingtin antibodies in wild-type (WT) and mutant transgenic R6/2 (R6) spinal cord and brain. FIGS. 7E-7H show a confocal series of MW7 staining. MW6 shows punctate staining of the neuropil in WT (FIG. 7A) and R6/2 spinal cord while MW7 shows punctate staining of the perinuclear or nuclear membrane in WT (FIG. 7C) and R6/2 (FIG. 7D) brain. MW8 shows staining of neuronal inclusions in R6/2 (8-week old) fixed cortex sections (FIG. 7J). [0018] FIG. 8 shows a diagram illustrating the binding patterns of the MW1-MW8 anti-huntingtin antibodies to the huntingtin protein as analyzed by peptide array and immunohistochemical staining in vivo. The domain structure of the diagram of the huntingtin protein is from left to right as follows: the N-terminus, the polyQ domain, the polyP domain and the C-terminus. [0019] FIG. 9 shows coimmunoprecipitation of expressed MW scFv proteins from lysates of 293 cells cotransfected with Htt exon 1-EGFP, either 25-residue polyQ (PQ25) or 103-residue polyQ (PQ103), and a Flag-scFv or Flag-I.kappa.B.alpha.. [0020] FIG. 10 shows expression of hMW9 scFv and a control scFv (C) as analyzed by in vitro transcription and translation of hMW9 scFv in the presence of .sup.35S-methionine. The scFv was incubated with 5 .mu.g of recombinant GST-HDx-1 bound to gluthathione beads and subsequent analysis of the scFv that bound to the glutathione beads by SDS-PAGE and autoradiography. Continue reading... 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