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.

Description of the Invention

SUMMARY OF THE INVENTION

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.

The methods of the invention involve the treatment of an individual, preferably a patient, more preferably a mammalian patient and even more prefereably 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.

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.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The huntingtin protein comprises a number of distinct regions that are believed to play a role in the toxicity of mutant Htt, as well as interaction of the Htt protein with other molecules. The present invention is based, in part, on the identification of antibodies directed to one or more distinct regions of the Htt protein that have desirable biological activities (Khoshnan et al. Proc. Natl. Acad. Sci. USA 99:1002-1007 (2002); Ko et al. Brain Res. Bull. 56:319-329 (2001), both of which are expressly incorporated herein by reference).

Antibodies, as well as other binding agents, including binding fragments and mimetics thereof (including intrabodies), that specifically bind to the Htt protein are provided. Preferred antibodies specifically bind to the polyglutamine ("polyQ") domain, polyproline ("polyP") domain or carboxy terminus of the huntingtin (Htt) protein.

Nucleic acid sequences encoding the subject antibodies, as well as methods for their expression, including in therapeutic treatment protocols, are also provided.

The preferred binding agents, e.g. antibodies, fragments and mimetics thereof, etc., bind to the huntingtin protein in a manner that differs in at least one aspect from the 1C2 antibody (Trottier et al., Nature, 10:104-110 (1995); Trottier et al., Nature, 378:403-406 (1995)). For example, and without limitation, the preferred antibodies may differ from the 1C2 antibody in terms of the epitope that they recognize or one or more of specificity, affinity and avidity.

Also provided are methods of screening compounds for the ability to modulate the activity of proteins comprising a polyglutamine repeat, particularly the huntingtin protein, as well as pharmaceutical compositions comprising such agents.

In addition, methods and devices are provided for screening samples for the presence of proteins comprising a polyglutamine repeat. In a particularly preferred embodiment, methods for identifying the presence of mutant huntingtin protein are provided. The methods may be used, for example, to diagnose a patient as someone who is, or is likely to suffer from Huntington's disease or a related disorder.

Antibodies to Huntingtin

Preferred antibodies are specific for particular epitopes on the huntingtin protein. The huntingtin protein comprises a polyglutamine-rich region close to the N-terminus of the protein, an adjacent polyproline-rich region and a carboxy-terminus region that is characterized by the sequence of SEQ ID NO: 2. DNA encoding the glutamine- and proline-rich regions of the human huntingtin protein are characterized by a polymorphic trinucleotide repeats. In particular, the polyglutamine region comprises a number of CAG repeats, encoding for glutamine residues. The CAG repeats are expanded on disease chromosomes. The adjacent polyproline region comprises polymorphic trinucleotide CCG repeats, encoding for prolines.

In the human huntingtin gene, the polymorphic CAG repeat region varies from 13 to 36 repeats and is encoded almost entirely by CAG. The mouse huntingtin gene encodes 7 consecutive glutamine residues in an imperfect repeat. In both species, the glutamine-rich region is followed by a segment with runs of prolines with interspersion of an occasional glutamine or other amino acid residue (Rubinsztein et al., Nat. Genet., 5(3):214-5 (1993), incorporated herein by reference). The polyproline regions of the huntingtin protein are well defined and found, for example, in SEQ ID NO: 5 in U.S. Pat. No. 5,693,757. These polyproline regions have sequences of at least 10 consecutive proline residues in the wild-type sequence.

More specifically, the preferred antibodies recognize an epitope within the polyglutamine-rich, polyproline-rich or carboxy-terminus domains of the huntingtin protein. By "recognize" it is meant that the antibodies bind to the huntingtin protein at the particular epitope. In many embodiments, the subject antibodies do not bind to any appreciable extent to proteins that do not share a significant degree of homology with the huntingtin protein.

The epitope specificity of the antibodies can be determined by epitope mapping as described, for example, in Ko et al., Brain Research Bulletin, 56:319-329 (2001) and in the Examples below.

Antibodies are preferably prepared by standard methods well-known in the art. The subject antibody compositions may be polyclonal, such that a heterogeneous population of antibodies differing by specificity is present, or monoclonal, in which a homogeneous population of identical antibodies that have the same specificity for the polyproline region of the huntingtin protein are present. As such, both monoclonal and polyclonal antibodies are provided by the subject invention. In many preferred embodiments, the subject antibodies are monoclonal antibodies. Specific monoclonal antibodies of interest include: MW1, MW2, MW7, MW8 and hMW9, where MW stands for "Milton Wexler," and are encoded by the nucleotide sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

Generally, an antigen or immunogen that can elicit an immune response characterized by the presence of antibodies of the subject invention is employed. The immunogen preferably comprises at least includes a portion of a protein having a polyglutamine repeat region.

In one embodiment, the immunogen is at least a portion of a wild-type or mutant huntingtin protein, comprising a polyglutamine region having at least 19 glutamine repeats. The portion of the wild-type or mutant huntingtin protein may comprise exon 1 of the huntingtin protein, referred to herein as "HDx-1." A preferred HD-1x immunogen has the sequence of SEQ ID NO: 1, and comprises a polyglutamine region, a polyproline region and a carboxy-terminus region characterized by an eight amino acid stretch having the sequence AEEPLHRP (SEQ ID NO: 2).

In another embodiment, the immunogen is at least a portion of the wild-type or mutant dentatorubral palliodoluysian atrophy (DRPLA) protein (Onodera et al., FEBS Lett., 399:135-139 (1996)). The DRPLA protein preferably comprises a polyQ domain having from 19 to 35 glutamine repeats.

In the preferred embodiments, the immunogen is present in its aggregated state. In certain embodiments, other domains are also present in the immunogens. For example, a glutathione-S-transferase domain may be present in the immunogen (Onodera et al., FEBS Lett., 399:135-139 (1996); Harris, Methods Mol Biol, 88:87-99 (1998)). Other domains may be included. For example, domains may be included that serve to facilitate purification and identification of the antigen of interest. The immunogen is typically employed in the preparation of the subject antibodies as follows.

Although methods of making monoclonal and polyclonal antibodies are well known in the art, preferred methods are briefly described herein. Variations of the following methods will be apparent to one of skill in the art.

For preparation of polyclonal antibodies, the first step is immunization of the host animal with the immunogen. To increase the immune response of the host animal, the immunogen may be combined with an adjuvant. Suitable adjuvants include alum, dextran, sulfate, large polymeric anions, oil & water emulsions, e.g. Freund's adjuvant, Freund's complete adjuvant, and the like. The immunogen may also be conjugated to synthetic carrier proteins or synthetic antigens. A variety of hosts may be immunized to produce the polyclonal antibodies. Such hosts include without limitation, rabbits, guinea pigs, other rodents such as mice or rats, sheep, goats, primates and the like. The immunogen is administered to the host, usually intradermally, with an initial dosage followed by one or more, usually at least two, additional booster dosages. Following immunization, the blood from the host is collected, followed by separation of the serum from the blood cells. The Ig present in the resultant antiserum may be further fractionated using known methods, such as ammonium salt fractionation, DEAE chromatography, and the like.

As with the preparation of polyclonal antibodies, the first step in preparing monoclonal antibodies specific for an epitope within the huntingtin protein, is to immunize a suitable host. Suitable hosts include rats, hamsters, mice, monkeys and the like, and are preferably mice. Monoclonal antibodies may be generated using the hybridoma method described by Kohler et al., Nature, 256:495 (1975) or by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567.

The immunogen is administered to the host in any convenient manner known in the art. For example, and without limitation, administration may be by subcutaneous injection with adjuvants, nitrocellulose implants comprising the immunogen or intrasplenic injections. Alternatively, lymphocytes may be immunized in vitro. The immunization protocol may be modulated to obtain a desired type of antibody, e.g. IgG or IgM, where such methods are known in the art (Kohler and Milstein, Nature, 256:495 (1975)). Booster immunizations may be made, for example one month after the initial immunization. Animals are bled and analyzed for antibody titer. Boosting may be continued until antibody production plateaus. Following immunization, plasma cells are harvested from the immunized host. Sources of plasma cells include the spleen and lymph nodes, with the spleen being preferred.

The plasma cells are then immortalized by fusion with myeloma cells to produce hybridoma cells. Fusion may be carried out by an electrocell fusion process or by using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-109, [Academic Press, 1996]). The plasma and myeloma cells are typically fused by combining the cells in a fusion medium usually in a ratio of about 10 plasma cells to 1 myeloma cell, where suitable fusion mediums include a fusion agent, e.g. PEG 1000, and the like. Following fusion, the fused cells will be selected, e.g. by growing on HAT medium.

A variety of myeloma cell lines are available. Preferably, the myeloma cell is HGPRT negative, incapable of producing or secreting its own antibodies, and growth stable. Preferred myeloma cells also fuse efficiently and support stable high-level production of antibody by the selected antibody-producing cells. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOP-21 and MC.-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol. 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York, [1987]). Specific cell lines of interest include, for example, p3U1, SP 2/0 Ag14, P3. times.63Ag8.653 (Dr. Greenberg, V.A. Hospital).

Representative hybridomas according to the subject invention include those hybridomas that secrete one of the following monoclonal antibodies: MW1, MW2, MW7, MW8 and hMW9. Each of these antibodies is described in detail below.

Following hybridoma cell production, culture supernatant from individual hybridomas is screened for reactivity with huntingtin protein, particularly mutant huntingtin protein, using standard techniques. Such screening techniques are well known in the art and include radioimmunoassay (RIA), enzyme-linked immunosorent assay (ELISA), dot blot immunoassays, Western blots and the like. The binding affinity of the monoclonal antibody may, for example, be determined by the Scatchard analysis (Munson et al., Anal. Biochem., 107:220 (1980)).

After hybridoma cells secreting antibodies with the desired specificity, affinity and/or activity are selected, the cells may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996). Culture media may be for example DMEM or RPMI-1640 medium. Alternatively, hybridomas may be grown in vitro as ascites tumors in an animal.

The desired antibody may be purified from the supernatants or ascites fluid by conventional techniques, e.g. affinity chromatography using mutant huntingtin protein bound to an insoluble support, protein A sepharose and the like.

DNA encoding the monoclonal antibody may be isolated and sequenced using conventional procedures, with the hybridoma cells serving as a source of the DNA. The isolated DNA may be introduced into host cells in culture to synthesize the monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences, Morrison, et al., Proc. Nat. Acad. Sci. 81, 6851 (1984), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. In that manner, "chimeric" or "hybrid" antibodies are prepared that have the binding specificity of an anti-Huntingtin protein described herein.

Chimeric or hybrid antibodies also may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

Human monoclonal antibodies can be made by the hybridoma method. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described, for example, by Kozbor, J. Immunol. 133, 3001 (1984), and Brodeur, et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987).

It is now possible to produce transgenic animals (e.g. mice) that are capable, upon immunization, of producing a repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy chain joining region (J.sub.H) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g. Jakobovits et al., Proc. Natl. Acad. Sci. USA 90, 2551-255 (1993); Jakobovits et al., Nature 362, 255-258 (1993).

Mendez et al. (Nature Genetics 15: 146-156 [1997]) have further improved the technology and have generated a line of transgenic mice designated as "Xenomouse II" that, when challenged with an antigen, generates high affinity fully human antibodies. This was achieved by germ-line integration of megabase human heavy chain and light chain loci into mice with deletion into endogenous J.sub.H segment as described above. The Xenomouse II harbors 1,020 kb of human heavy chain locus containing approximately 66 V.sub.H genes, complete D.sub.H and J.sub.H regions and three different constant regions (.mu., .delta. and .chi.), and also harbors 800 kb of human .kappa. locus containing 32 V.kappa. genes, J.kappa. segments and C.kappa. genes. The antibodies produced in these mice closely resemble that seen in humans in all respects, including gene rearrangement, assembly, and repertoire. The human antibodies are preferentially expressed over endogenous antibodies due to deletion in endogenous J.sub.H segment that prevents gene rearrangement in the murine locus.

Alternatively, phage display technology (McCafferty et al., Nature 348, 552-553 [1990]) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.

Binding fragments or binding mimetics of the subject antibodies may also be prepared. These fragments and mimetics preferably share the binding characteristics of the subject antibodies. "Binding characteristics" when used herein include specificity, affinity, avidity, etc. for the huntingtin protein, particularly the polyglutamine, polyproline or c-terminal region of exon 1. The subject antibodies are modified to optimize their utility, for example for use in a particular immunoassay or their therapeutic use. In one embodiment antibody fragments, such as Fv and Fab may be prepared by cleavage of the intact protein, e.g. by protease or chemical cleavage. Nucleic acid encoding the antibody fragments or binding mimetics may be identified.

Antibody fragments, such as single chain antibodies or scFvs, may also be produced by recombinant DNA technology where such recombinant antibody fragments retain the binding characteristics of the above antibodies. "Antibody fragments" when used herein refer to a portion of an intact antibody, such as the antigen binding or variable region and may include single-chain antibodies, Fab, Fab', F(ab')2 and Fv fragments, diabodies, linear antibodies, and multispecific antibodies generated from portions of intact antibodies.

Recombinantly produced antibody fragments generally include at least the V.sub.H and V.sub.L domains of the subject antibodies, so as to retain the desired binding characteristics. These recombinantly produced antibody fragments or mimetics may be readily prepared from the antibodies of the present invention using any convenient methodology, such as the methodology disclosed in U.S. Pat. Nos. 5,851,829 and 5,965,371; the disclosures of which are herein incorporated by reference. The antibody fragments or mimetics may also be readily isolated from a human scFvs phage library (Pini et al., Curr. Protein Pept. Sci., 1(2):155-69 (2000)) using huntingtin protein, particularly mutant huntingtin protein.

The invention also provides isolated nucleic acid encoding the anti-huntingtin antibodies, vectors and host cells comprising the nucleic acid, and recombinant techniques for the production of the antibodies.

For recombinant production of an antibody, the nucleic acid encoding it may be isolated and inserted into a replicable vector for further cloning and expression. DNA encoding the antibody is readily isolated and sequenced using conventional procedures. Many cloning and expression vectors are available and are well known in the art. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, e.g., as described in U.S. Pat. No. 5,534,615.

Host cells, preferably eukaryotic cells such as CHO cell or COS cells, are transformed with the above-described expression or cloning vectors for anti-huntingtin antibody production and cultured according to well-established procedures.
 

Claim 1 of 1 Claim

1. An isolated monoclonal antibody that specifically binds an epitope within a polyproline region of the huntingtin protein comprising greater than 5 consecutive proline residues; wherein said antibody is capable of inhibiting aggregation of the huntingtin protein; wherein said monoclonal antibody is a single-chain variant fragment encoded by a nucleotide sequence comprising SEQ ID NO: 5.

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