U.S. patent application number 11/849851 was filed with the patent office on 2008-05-08 for antibodies that bind to an epitope on the huntington's disease protein.
This patent application is currently assigned to California Institute of Technology. Invention is credited to Ali Khoshnan, Jan Ko, Paul H. Patterson.
Application Number | 20080107657 11/849851 |
Document ID | / |
Family ID | 29739412 |
Filed Date | 2008-05-08 |
United States Patent
Application |
20080107657 |
Kind Code |
A1 |
Khoshnan; Ali ; et
al. |
May 8, 2008 |
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.
Inventors: |
Khoshnan; Ali; (South
Pasedena, CA) ; Ko; Jan; (Arcadia, CA) ;
Patterson; Paul H.; (Altandena, CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Assignee: |
California Institute of
Technology
Pasadena
CA
91125
|
Family ID: |
29739412 |
Appl. No.: |
11/849851 |
Filed: |
September 4, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10354246 |
Jan 28, 2003 |
|
|
|
11849851 |
Sep 4, 2007 |
|
|
|
60353032 |
Jan 28, 2002 |
|
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Current U.S.
Class: |
424/144.1 ;
530/388.22 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 2317/74 20130101; C07K 16/18 20130101; C07K 2317/34 20130101;
C07K 2317/622 20130101; A61P 25/14 20180101; A61K 2039/54 20130101;
C07K 2317/21 20130101 |
Class at
Publication: |
424/144.1 ;
530/388.22 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/28 20060101 C07K016/28 |
Claims
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 and wherein
the antibody is capable of inhibiting aggregation of the huntingtin
protein.
2. The monoclonal antibody of claim 1, in association with a
therapeutically acceptable carrier.
3. A method for treatment of Huntington's disease, comprising
administering to a patient an effective amount of a monoclonal
antibody of claim 1.
4. The method of claim 3 wherein said monoclonal antibody is a
single-chain variant fragment encoded by the nucleotide sequence of
SEQ ID NO: 5.
5. The method of claim 3 wherein the patient is a mammalian
patient.
6. The method of claim 5 wherein the mammalian patient is
human.
7. The method of claim 6 wherein the antibody is delivered
intracranially.
8. The method of claim 7 wherein the antibody is injected directly
into brain tissue.
9. The method of claim 7 wherein the antibody is injected into the
cerebrospinal fluid.
10. A method for treatment of Huntington's disease, comprising
expression of a monoclonal antibody of claim 1 in cells expressing
mutant huntingtin protein.
11. The method of claim 10 wherein said monoclonal antibody is a
single-chain variant fragment encoded by the nucleotide sequence of
SEQ ID NO: 5.
Description
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.
[0021] FIG. 11 shows immunofluorescence staining of 293 cells
transfected with MW1, MW2 and MW7 scFvs or a control empty scFv
vector (C) with anti-Flag antibodies two days after transfection
and subsequent fixation.
[0022] FIG. 12 shows the effects of the expression of hMW9 scFv,
empty plasmid (C) or control plasmid (cscFv) and HDx-1, containing
103 polyQ and fused to GFP in human 293 cells as analyzed by
fluorescence microscopy.
[0023] FIG. 13 shows colocalization of MW1, MW2 or MW7 scFv with
mutant Htt in 293 cells cotransfected with mutant Htt fused to EGFP
tag and scFv tagged with a Flag tag.
[0024] FIG. 14 shows colocalization of MW8 scFv with mutant Htt in
293 cells cotransfected with mutant Htt fused to EGFP tag and scFv
tagged with a Flag tag.
[0025] FIG. 15 shows inhibition of Htt-induced cell death in 293
cells with MW7 scFv and enhancement of Htt-induced cell death in
293 cells with MW1 or MW2 scFvs. 293 cells were transfected with
Htt exon 1-EGFP and an empty vector (control) or one of the
anti-huntingtin scFvs, MW1, MW2 or MW7 tagged with a Flag tag. The
transfected cells were visualized by GFP fluorescence, and dying
cells by TUNEL staining. The presence of MW7 scFv decreases the
number of TUNEL+ cells.
[0026] FIG. 16 shows inhibition of Htt-induced cell death in 293
cells with MW8 scFv. 293 cells were transfected with Htt exon
1-EGFP and an empty vector (control) or MW8 anti-huntingtin scFv,
tagged with a Flag tag. The transfected cells were visualized by
GFP fluorescence, and dying cells by TUNEL staining. The presence
of MW8 scFv decreases the number of TUNEL+ cells.
[0027] FIG. 17 shows a chart representing quantitation of the
effects of the expression of anti-huntingtin antibodies, MW1, MW2
and MW7 on mutant Htt toxicity. MW1 and MW2 exacerbated Htt-induced
cell death while MW7 inhibited Htt toxicity.
[0028] FIGS. 18A-18B show reduction of aggregation of mutant
huntingtin protein in 293 cells as analyzed by Western blotting.
Lysates of 293 cells transfected with mutant Htt and an scFv, MW1,
MW2 or MW7, were subjected to high-speed centrifugation and were
analyzed by Western blotting with anti-HD.sub.1-17 antibodies. The
Htt in the pellet that can be solubilized by SDS treatment is about
80 kDa, whereas the Htt that cannot be solubilized does not enter
the gel and is visualized as a band at the top of the gel (FIG.
18A). FIG. 18B shows that the level of soluble Htt in the cleared
lysates does not appear to be affected by expression of MW1, MW2 or
MW7 scFv expression.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0029] 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).
[0030] 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.
[0031] Nucleic acid sequences encoding the subject antibodies, as
well as methods for their expression, including in therapeutic
treatment protocols, are also provided.
[0032] 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.
[0033] 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.
[0034] 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.
DEFINITIONS
[0035] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. See,
e.g. Singleton et al., Dictionary of Microbiology and Molecular
Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994);
Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold
Springs Harbor Press (Cold Springs Harbor, N.Y. 1989). For purposes
of the present invention, the following terms are defined
below.
[0036] "Huntingtin" and "Htt" refer broadly to the huntingtin gene
and the protein encoded by the huntingtin gene, including mutant
and variant forms as well as native forms. "Variants" are
biologically active polypeptides having an amino acid sequence
which differs from the sequence of a native sequence polypeptide.
Native sequence human huntingtin protein is described, for example,
by The Huntington's Disease Collaborative Research Group in Cell
72:971-983 (1993) as well as in Li et al. Nature 378:398-402 (1995)
and WO 02/29408. The number of polyglutamine repeats in native
huntingtin protein is known to vary, from about 13 to about 36
glutamine residues in the polyQ region of native human protein.
Native sequence murine Htt is described, for example, in Lin et al.
Hum. Mol. Genet. 3 (1), 85-92 (1994) and typically comprises about
7 glutamine residues in the polyQ region. Particular variants of
the huntingtin gene will comprise different numbers of CAG repeats,
resulting in variation in the polyglutamine region of the
huntingtin protein.
[0037] "Mutant huntingtin protein" refers to huntingtin protein
which differs in some respect from the native sequence huntingtin
protein. Typically, mutant huntingtin will comprise an expanded
polyglutamine or polyproline region compared to the native form. A
preferred mutant huntingtin protein has an expanded polyglutamine
region of 40 or more glutamine residues.
[0038] As used herein, "nucleic acid" is defined as RNA or DNA that
encodes a protein or peptide of the invention, particularly an
antibody to the huntingtin protein, is complementary to a nucleic
acid sequence encoding such peptides, hybridizes to such a nucleic
acid and remains stably bound to it under appropriate stringency
conditions, exhibits at least about 50%, 60%, 70%, 75%, 85%, 90% or
95% nucleotide sequence identity across the open reading frame, or
encodes a polypeptide sharing at least about 50%, 60%, 70% or 75%
sequence identity, preferably at least about 80%, and more
preferably at least about 85%, and even more preferably at least
about 90 or 95% or more identity with the peptide sequences.
Specifically contemplated are genomic DNA, cDNA, mRNA and antisense
molecules, as well as nucleic acids based on alternative backbones
or including alternative bases whether derived from natural sources
or synthesized.
[0039] As used herein, the terms nucleic acid, polynucleotide and
nucleotide are interchangeable and refer to any nucleic acid,
whether composed of phosphodiester linkages or modified linkages
such as phosphotriester, phosphoramidate, siloxane, carbonate,
carboxymethylester, acetamidate, carbamate, thioether, bridged
phosphoramidate, bridged methylene phosphonate, bridged
phosphoramidate, bridged phosphoramidate, bridged methylene
phosphonate, phosphorothioate, methylphosphonate,
phosphorodithioate, bridged phosphorothioate or sultone linkages,
and combinations of such linkages. The terms nucleic acid,
polynucleotide and nucleotide also specifically include nucleic
acids composed of bases other than the five biologically occurring
bases (adenine, guanine, thymine, cytosine and uracil).
[0040] The terms "replicable expression vector" and "expression
vector" refer to a piece of DNA, usually double-stranded, which may
have inserted into it a piece of foreign DNA. Foreign DNA is
defined as heterologous DNA, which is DNA not naturally found in
the host cell. The vector is used to transport the foreign or
heterologous DNA into a suitable host cell. Once in the host cell,
the vector can replicate independently of the host chromosomal DNA,
and several copies of the vector and its inserted (foreign) DNA may
be generated. In addition, the vector contains the necessary
elements that permit translating the foreign DNA into a
polypeptide. Many molecules of the polypeptide encoded by the
foreign DNA can thus be rapidly synthesized.
[0041] The term "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, a ribosome binding site, and
possibly, other as yet poorly understood sequences. Eukaryotic
cells are known to utilize promoters, polyadenylation signals, and
enhancer.
[0042] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or a secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, then synthetic oligonucleotide adaptors or linkers are used
in accord with conventional practice.
[0043] "Biological property" or "biological activity" is a
biological function caused by an antibody or other compound of the
invention. With regard to the anti-huntingtin protein antibodies,
biological activity refers, in part, to the ability to specifically
bind to the huntingtin protein. Other preferred biological
activities include prevention of cell death or apoptosis,
prevention of mutant huntingtin aggregation and the ability to
regulate the toxic effects of mutant huntingtin protein that are
associated with neurodegenerative disease.
[0044] "Antibodies" (Abs) and "immunoglobulins" (Igs) are
glycoproteins having the same structural characteristics. While
antibodies exhibit binding specificity to a specific antigen,
immunoglobulins include both antibodies and other antibody-like
molecules that lack antigen specificity. Polypeptides of the latter
kind are, for example, produced at low levels by the lymph system
and at increased levels by myelomas.
[0045] "Antigen" when used herein refers to a substance, such as a
particular peptide or protein, that can bind to a specific
antibody. Preferred antigens include huntingtin protein, mutant
huntingtin protein, and fragments thereof.
[0046] "Native antibodies" and "native immunoglobulins" are usually
heterotetrameric glycoproteins, composed of two identical light (L)
chains and two identical heavy (H) chains. Each light chain is
linked to a heavy chain by one covalent disulfide bond. while The
number of disulfide linkages varies among the heavy chains of
different immunoglobulin isotypes. Each heavy and light chain also
has regularly spaced intra-chain disulfide bridges. Each heavy
chain has at one end a variable domain (V.sub.H) followed by a
number of constant domains. Each light chain has a variable domain
at one end (V.sub.L) and a constant domain at its other end. The
constant domain of the light chain is aligned with the first
constant domain of the heavy chain, and the light-chain variable
domain is aligned with the variable domain of the heavy chain.
Particular amino acid residues are believed to form an interface
between the light- and heavy-chain variable domains.
[0047] The term "antibody" herein is used in the broadest sense and
specifically covers human, non-human (e.g. murine) and humanized
monoclonal antibodies, including full length monoclonal antibodies,
polyclonal antibodies, multi-specific antibodies (e.g., bispecific
antibodies), and antibody fragments, including intrabodies, so long
as they exhibit the desired biological activity.
[0048] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the antibody will be purified (1) to greater than 95%
by weight of antibody as determined by the Lowry method, and
terminal or internal amino acid sequence by use of a spinning cup
sequenator, or (3) to homogeneity by SDS-PAGE under reducing or
nonreducing conditions using Coomassie blue or, preferably, silver
stain. Isolated antibody includes the antibody in situ within
recombinant cells since at least one component of the antibody's
natural environment will not be present. Ordinarily, however,
isolated antibody will be prepared by at least one purification
step.
[0049] "Antibody fragments" comprise a portion of a full-length
antibody, generally the antigen binding or variable domain thereof.
Examples of antibody fragments include Fab, Fab', F(ab').sub.2, and
Fv fragments; diabodies; intrabodies; linear antibodies;
single-chain antibody molecules; and multi-specific antibodies
formed from antibody fragments.
[0050] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of antibodies wherein the
individual antibodies comprising the population are identical
except for possible naturally occurring mutations that may be
present in minor amounts. Monoclonal antibodies are highly specific
and are directed against a single antigenic site. In addition,
monoclonal antibodies may be made by any method known in the art.
For example, the monoclonal antibodies to be used in accordance
with the present invention may be made by the hybridoma method
first described by Kohler et al., Nature 256:495 (1975), or may be
made by recombinant DNA methods (see, e.g., U.S. Pat. No.
4,816,567). The "monoclonal antibodies" may also be isolated from
phage antibody libraries using the techniques described in Clackson
et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol.
222:581-597 (1991), for example. The monoclonal antibodies herein
specifically include antibody fragments, such as single-chain Fv or
scFv antibody fragments.
[0051] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins) in which a portion of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass.
Fragments of chimeric antibodies are also included provided they
exhibit the desired biological activity (U.S. Pat. No. 4,816,567;
and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855
(1984)).
[0052] "Humanized" forms of non-human (e.g., murine) antibodies are
antibodies that contain minimal sequence derived from non-human
immunoglobulin. Humanized antibodies are generally human
immunoglobulins in which hypervariable region residues are replaced
by hypervariable region residues from a non-human species such as
mouse, rat, rabbit or non-human primate having the desired
specificity, affinity, and capacity. Framework region (FR) residues
of the human immunoglobulin may be replaced by corresponding
non-human residues. In addition, humanized antibodies may comprise
residues that are not found in either the recipient antibody or in
the donor antibody. In general, the humanized antibody will
comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the
hypervariable regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FRs are those of
a human immunoglobulin sequence. The humanized antibody optionally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin. For further
details, see Jones et al., Nature 321:522-525 (1986); Reichmann et
al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.
2:593-596 (1992).
[0053] "Single-chain Fv" or "scFv" antibody fragments typically
comprise the V.sup.H and V.sub.L domains of a monoclonal antibody,
wherein these domains are present in a single polypeptide chain.
Generally, the Fv polypeptide further comprises a polypeptide
linker between the V.sub.H and V.sub.L domains which enables the
scFv to form the desired structure for antigen binding. For a
review of scFv see Pluckthun in The Pharmacology of Monoclonal
Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New
York, pp. 269-315 (1994).
[0054] The term "epitope" is used to refer to binding sites for
(monoclonal or polyclonal) antibodies on protein antigens. There
are many methods known in the art for mapping and characterizing
the location of epitopes on proteins, including solving the crystal
structure of an antibody-antigen complex, competition assays, gene
fragment expression assays, and synthetic peptide-based assays, as
described, for example, in Chapter 11 of Harlow and Lane, Using
Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1999. A competition ELISA assay is
specifically described in Example 1. According to the gene fragment
expression assays, the open reading frame encoding the protein is
fragmented either randomly or by specific genetic constructions and
the reactivity of the expressed fragments of the protein with the
antibody to be tested is determined. The gene fragments may, for
example, be produced by PCR and then transcribed and translated
into protein in vitro, in the presence of radioactive amino acids.
The binding of the antibody to the radioactively labeled protein
fragments is then determined by immunoprecipitation and gel
electrophoresis. Certain epitopes can also be identified by using
large libraries of random peptide sequences displayed on the
surface of phage particles (phage libraries). Alternatively, a
defined library of overlapping peptide fragments can be tested for
binding to the test antibody in simple binding assays. The latter
approach is suitable to define linear epitopes of about 5 to 15
amino acids.
[0055] An antibody binds "essentially the same epitope" as a
reference antibody, when the two antibodies recognize identical or
sterically overlapping epitopes. The most widely used and rapid
methods for determining whether two epitopes bind to identical or
sterically overlapping epitopes are competition assays, which can
be configured in all number of different formats, using either
labeled antigen or labeled antibody. Usually, the antigen is
immobilized on a 96-well plate, and the ability of unlabeled
antibodies to block the binding of labeled antibodies is measured
using radioactive or enzyme labels. A competition ELISA assay is
disclosed in Example 1.
[0056] The term amino acid or amino acid residue, as used herein,
refers to naturally occurring L amino acids or to D amino acids as
described further below with respect to variants. The commonly used
one- and three-letter abbreviations for amino acids are used herein
(Bruce Alberts et al., Molecular Biology of the Cell, Garland
Publishing, Inc., New York (3d ed. 1994)).
[0057] Hybridization is preferably performed under "stringent
conditions" which means (1) employing low ionic strength and high
temperature for washing, for example, 0.015 sodium chloride/0.0015
M sodium citrate/0.1% sodium dodecyl sulfate at 50.degree. C., or
(2) employing during hybridization a denaturing agent, such as
formamide, for example, 50% (vol/vol) formamide with 0.1% bovine
serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium
phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM
sodium citrate at 42.degree. C. Another example is use of 50%
formamide, 5.times.SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM
sodium phosphate (pH 6/8), 0.1% sodium pyrophosphate,
5.times.Denhardt's solution, sonicated salmon sperm DNA (50
.mu.g/ml), 0.1% SDS, and 10% dextran sulfate at 42.degree. C., with
washes at 42.degree. C. in 0.2.times.SSC and 0.1% SDS.
[0058] As used herein, "treatment" is a clinical intervention made
in response to a disease, disorder or physiological condition
manifested by a patient, particularly Huntington's disease. The aim
of treatment includes the alleviation or prevention of symptoms,
slowing or stopping the progression or worsening of a disease,
disorder, or condition and/or the remission of the disease,
disorder or condition. "Treatment" refers to both therapeutic
treatment and prophylactic or preventative measures. Those in need
of treatment include those already affected by a disease or
disorder or undesired physiological condition as well as those in
which the disease or disorder or undesired physiological condition
is to be prevented.
[0059] In the methods of the present invention, the term "control"
and grammatical variants thereof, are used to refer to the
prevention, partial or complete inhibition, reduction, delay or
slowing down of an unwanted event, such as the presence or onset of
Huntington's disease.
[0060] The term "effective amount" refers to an amount sufficient
to effect beneficial or desirable clinical results.
[0061] The term "flag-tagged" when used herein refers to a chimeric
polypeptide comprising a single-chain variable region fragment Ab
(scFv) fused to a "flag epitope." The flag epitope has enough
residues to provide an epitope against which an antibody may bind
for detection purposes (Chiang et al., Pept. Res., 6:62-64 (1993)),
but is also short enough such that it does not interfere with the
activity of the scFv to which it is fused.
Antibodies to Huntingtin
[0062] 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.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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).
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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).
[0077] 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.
[0078] 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)).
[0079] 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.
[0080] 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.
[0081] 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.
[0082] 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.
[0083] 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).
[0084] 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).
[0085] 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.
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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.
[0090] 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.
[0091] 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.
[0092] 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.
Screening for Antibodies with Desired Properties
[0093] Once antibodies to the immunogen have been produced, they
may be screened for desirable biological properties, such as high
affinity binding to the desired antigen, specific binding to
particular mutant forms of the huntingtin protein, the ability to
prevent cell death or apoptosis associated with mutant huntingtin
protein, and/or the ability to prevent aggregation of mutant
huntingtin protein.
Prevention of Cell Death or Apoptosis
[0094] In a preferred embodiment, antibodies are identified that
reduce the level of cell death associated with expression of mutant
huntingtin protein. This activity may be observed in a model system
for Huntington's disease, for example using terminal
deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)
staining. Such an assay is described in Example 2, below and in
Khoshnan et al., PNAS, 99:1002-1007 (2002), the entire contents of
which are incorporated herein by reference in its entirety.
[0095] Specifically, extensive cellular DNA degradation is a
characteristic event which often occurs in the early stages of
apoptosis and is mediated by a Ca2+-dependent endonuclease. As
cleavage of the DNA in apoptotic cells results in double-stranded
DNA fragments and single strand breaks, the degraded DNA may be
detected by labeling methods. For example, enzymatic labeling of
the free 3'-OH termini of the cellular DNA with modified
nucleotides using exogenous enzymes, such as terminal
deoxynucleotidyl transferase, is used to detect DNA strand breaks.
The labeled DNA may be subsequently analyzed by immunocytochemistry
(ICC), such as flow cytometry, fluorescence microscopy or light
microscopy. Accordingly, preferred antibodies that reduce the level
of apoptosis in a model system for Huntington's disease may be
selected using the TUNEL staining.
Prevention of Mutant Huntingtin Aggregation
[0096] In a preferred embodiment of the invention, anti-huntingtin
antibodies, particularly those that are directed to the polyproline
region, are identified that have the ability to inhibit the
aggregation of huntingtin in vivo. Aggregation of the huntingtin
protein is associated with Huntington's disease and is present in
affected neurons. Aggregation may be evaluated by examining the
amount of huntingtin protein that is precipitated from cell lysates
by centrifugation. The amount of aggregation is analyzed by
subjecting the lysates that were subjected to centrifugation to
SDS-PAGE. An exemplary assay is described in Example 2 and in
Khoshnan et al., PNAS, 99:1002-1007 (2002).
Diagnostic Applications
[0097] The subject antibodies, binding fragments and mimetics
thereof find use in immunoassays that are capable of providing for
the detection of huntingtin or mutant huntingtin protein in a
sample. In such assays, the sample suspected of comprising the
huntingtin or mutant huntingtin protein of interest will typically
be obtained from a subject, such as a human subject, suspected of
suffering from the disease of interest or at risk for developing
the disease of interest. The sample is generally a physiological
sample from the patient such as blood or tissue. Depending on the
nature of the sample, it may or may not be pretreated prior to
assay, as will be apparent to one of skill in the art.
[0098] A number of different immunoassay formats are known in the
art and may be employed in detecting the presence of protein of
interest in a sample. Immunoassays of interest include Western
blots on protein gels or protein spots on filters, where the
antibody is labeled, as is known in the art. A variety of protein
labeling schemes are known in the art and may be employed, the
particular scheme and label chosen being the one most convenient
for the intended use of the antibody, e.g. immunoassay. Examples of
labels include labels that permit both the direct and indirect
measurement of the presence of the antibody. Examples of labels
that permit direct measurement of the antibody include radiolabels,
such as .sup.3H or .sup.125I, fluorescent dyes, beads,
chemiluminescers and colloidal particles. Examples of labels which
permit indirect measurement of the presence of the antibody include
enzymes where a substrate may provided for a colored or fluorescent
product. For example, the antibodies may be labeled with a
covalently bound enzyme capable of providing a detectable product
signal after addition of suitable substrate. Instead of covalently
binding the enzyme to the antibody, the antibody may be modified to
comprise a first member of specific binding pair which specifically
binds with a second member of the specific binding pair that in
conjugated to the enzyme, e.g. the antibody may be covalently bound
to biotin and the enzyme conjugate to streptavidin. Examples of
suitable enzymes for use in conjugates include horseradish
peroxidase, alkaline phosphatase, malate dehydrogenase and the
like. Where not commercially available, such antibody-enzyme
conjugates are readily produced by techniques known to those
skilled in the art.
[0099] Other immunoassays include those based on a competitive
formats, as are known in the art. One such format would be where a
solid support is coated with the polyproline region containing
protein, including for example the mutant huntingtin protein.
Labeled antibody is then combined with a sample suspected of
comprising protein of interest to produce a reaction mixture which,
following sufficient incubation time for binding complexes to form,
is contacted with the solid phase bound protein. The amount of
labeled antibody which binds to the solid phase will be
proportional to the amount of protein in the sample, and the
presence of protein may therefore be detected. Other competitive
formats that may be employed include those where the sample
suspected of comprising protein is combined with a known amount of
labeled protein and then contacted with a solid support coated with
antibody specific for the protein. Such assay formats are known in
the art and further described in both Guide to Protein
Purification, supra, and Antibodies, A Laboratory Manual (Cold
Springs Harbor Press (Cold Springs Harbor, N.Y. 1989)).
[0100] In immunoassays involving solid supports, the solid support
may be any compositions to which antibodies or fragments thereof
can be bound, which is readily separated from soluble material, and
which is otherwise compatible with the overall immunoassay method.
The surface of such supports may be solid or porous and of any
convenient shape. Examples of suitable insoluble supports to which
the receptor is bound include beads, e.g. magnetic beads, membranes
and microtiter plates. These are typically made of glass, plastic
(e.g. polystyrene), polysaccharides, nylon or nitrocellulose.
Microtiter plates are especially convenient because a large number
of assays can be carried out simultaneously, using small amounts of
reagents and samples.
[0101] Before adding patient samples or fractions thereof, the
non-specific binding sites on the insoluble support i.e. those not
occupied by the first antibody, are generally blocked. Preferred
blocking agents include non-interfering proteins such as bovine
serum albumin, casein, gelatin, and the like. Alternatively,
detergents, such as Tween, NP40 or TX100 may be used at
non-interfering concentrations.
[0102] It is particularly convenient in a clinical setting to
perform the immunoassay in a self-contained apparatus, and such
devices are provided by the subject invention. A number of such
devices and methods for their use are known in the art. The
apparatus will generally employ a continuous flow-path over a
suitable filter or membrane, and will have at least three regions,
a fluid transport region, a sample region, and a measuring region.
The sample region is prevented from fluid transfer contact with the
other portions of the flow path prior to receiving the sample.
After the sample region receives the sample, it is brought into
fluid transfer relationship with the other regions, and the fluid
transfer region contacted with fluid to permit a reagent solution
to pass through the sample region and into the measuring region.
The measuring region may have bound to it a first antibody. The
second, labeled antibody combined with the assayed sample is
introduced and the sandwich assay performed as above.
Screening to Identify Compounds with a Desired Biological
Activity
[0103] The subject antibodies, binding fragments and mimetics
thereof also find use in screening applications designed to
identify agents or compounds that are capable of modulating, e.g.
inhibiting, the binding interaction between the protein to which
the antibody binds and a cellular target. For example, the subject
antibodies find use in screening assays that identify compounds
capable of modulating the interaction between mutant huntingtin
protein and its cellular targets. In such assays, the subject
antibody is contacted with mutant huntingtin protein in the
presence of a candidate modulation agent and any resultant binding
complexes between the antibody and the mutant huntingtin protein
are detected. The results of the assay are then compared with a
control. Those agents which change the amount of binding complexes
that are produced upon contact are identified as agents that
modulate the binding activity of mutant huntingtin protein and
therefore are potential therapeutic agents. Of interest in many
embodiments is the identification of agents that inhibit, at least
to some extent, the binding of mutant huntingtin protein with its
target. In many assays, at least one of the protein or antibody is
attached to a solid support and at least one of these members is
labeled, where supports and labels are described supra.
[0104] In other assays, the ability of a candidate compound to
disrupt or enhance the biological activity of an anti-huntingtin
antibody is measured. For example, the ability of a candidate
compound to prevent or enhance the inhibition of cell death,
apoptosis or aggregation normally produced by an anti-huntingtin
antibody may be measured.
[0105] A variety of different candidate agents may be screened by
the above screening methods. Candidate agents encompass numerous
chemical classes, though typically they are organic molecules,
preferably small organic compounds having a molecular weight of
more than 50 and less than about 2,500 daltons. Candidate agents
comprise functional groups necessary for structural interaction
with proteins, particularly hydrogen bonding, and typically include
at least an amine, carbonyl, hydroxyl or carboxyl group, preferably
at least two of the functional chemical groups. The candidate
agents often comprise cyclical carbon or heterocyclic structures
and/or aromatic or polyaromatic structures substituted with one or
more of the above functional groups. Candidate agents are also
found among biomolecules including peptides, saccharides, fatty
acids, steroids, purines, pyrimidines, derivatives, structural
analogs or combinations thereof.
[0106] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides and oligopeptides.
Alternatively, libraries of natural compounds in the form of
bacterial, fungal, plant and animal extracts are available or
readily produced. Additionally, natural or synthetically produced
libraries and compounds are readily modified through conventional
chemical, physical and biochemical means, and may be used to
produce combinatorial libraries. Known pharmacological agents may
be subjected to directed or random chemical modifications, such as
acylation, alkylation, esterification, amidification, etc. to
produce structural analogs.
Methods of Treatment
[0107] An individual suffering from Huntington's disease may be
treated using antibodies of the present invention or compounds
identified in screens using the antibodies. By treatment is meant
at least an amelioration of the symptoms associated with the
pathological condition afflicting the host, where amelioration is
used in a broad sense to refer to at least a reduction in the
magnitude of a parameter, e.g. symptom, associated with the
pathological condition being treated, such as neuronal cell death.
As such, treatment includes situations where the pathological
condition, or at least symptoms associated therewith, are
completely inhibited, e.g. prevented from happening, or stopped,
e.g. terminated, such that the host no longer suffers from the
pathological condition, or at least the symptoms that characterize
the pathological condition.
[0108] A variety of individuals are treatable according to the
subject methods. Generally such individuals are "mammals" or
"mammalian," where these terms are used broadly to describe
organisms which are within the class mammalia, including the orders
carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs,
and rats), and primates (e.g., humans, chimpanzees, and monkeys).
In many embodiments, the individuals will be humans.
[0109] In certain embodiments, the methods of treatment involve
administration of an effective amount of a compound that modulates,
e.g. inhibits, the interaction of a mutant huntingtin protein, with
its cellular targets. The compound is preferably an antibody of the
invention that targets the polyproline region of the huntingtin
protein, the polyglutamine region of the huntingtin protein or an
epitope within the c-terminal sequence of exon 1 of the huntingtin
protein. In a preferred embodiment the antibodies are human or
humanized, such that any undesirable immune response in the patient
is minimized.
[0110] The anti-huntingtin antibodies may be administered using any
convenient protocol capable of resulting in the desired therapeutic
activity. Thus, the agent can be incorporated into a variety of
formulations for therapeutic administration. More particularly, the
agents of the present invention can be formulated into
pharmaceutical compositions by combination with appropriate,
pharmaceutically acceptable carriers or diluents (Remington: The
Science and Practice of Pharmacy, 19.sup.th Edition, Alfonso, R.,
ed., Mack Publishing Co. (Easton, Pa.: 1995)), and may be
formulated into preparations in solid, semi-solid, liquid or
gaseous forms, such as tablets, capsules, powders, granules,
ointments, solutions, suppositories, injections, inhalants and
aerosols.
[0111] Anti-huntingtin protein antibodies can also be administered
by inhalation. Commercially available nebulizers for liquid
formulations, including jet nebulizers and ultrasonic nebulizers
are useful for such administration. Anti-huntingtin protein
antibodies can also be aerosolized using a fluorocarbon formulation
and a metered dose inhaler, or inhaled as a lyophilized and milled
powder.
[0112] The anti-huntingtin antibodies to be used for in vivo
administration must be sterile. The sterility may be accomplished
by filtration using sterile filtration membranes, prior to or
following lyophilization and reconstitution. The anti-huntingtin
antibodies may be stored in lyophilized form or in solution.
[0113] The anti-huntingtin antibody compositions may be placed into
a container with a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[0114] In pharmaceutical dosage forms, the antibodies or other
compounds may be used alone or in appropriate association, as well
as in combination with other pharmaceutically active or inactive
compounds. The following methods and excipients are merely
exemplary and are in no way limiting.
[0115] For oral preparations, the agents can be used alone or in
combination with appropriate additives to make tablets, powders,
granules or capsules, for example, with conventional additives,
such as lactose, mannitol, corn starch or potato starch; with
binders, such as crystalline cellulose, cellulose derivatives,
acacia, corn starch or gelatins; with disintegrators, such as corn
starch, potato starch or sodium carboxymethylcellulose; with
lubricants, such as talc or magnesium stearate; and if desired,
with diluents, buffering agents, moistening agents, preservatives
and flavoring agents.
[0116] The agents can be formulated into preparations for injection
by dissolving, suspending or emulsifying them in an aqueous or
nonaqueous solvent, such as vegetable or other similar oils,
synthetic aliphatic acid glycerides, esters of higher aliphatic
acids or propylene glycol; and if desired, with conventional
additives such as solubilizers, isotonic agents, suspending agents,
emulsifying agents, stabilizers and preservatives.
[0117] The agents can be utilized in aerosol formulation to be
administered via inhalation. The compounds of the present invention
can be formulated into pressurized acceptable propellants such as
dichlorodifluoromethane, propane, nitrogen and the like.
[0118] Furthermore, the agents can be made into suppositories by
mixing with a variety of bases such as emulsifying bases or
water-soluble bases. The compounds of the present invention can be
administered rectally via a suppository. The suppository can
include vehicles such as cocoa butter, carbowaxes and polyethylene
glycols, which melt at body temperature, yet are solidified at room
temperature.
[0119] Each dosage for human and animal subjects will preferably
contain a predetermined quantity of compounds of the present
invention calculated in an amount sufficient to produce the desired
effect, in association with a pharmaceutically acceptable diluent,
carrier or vehicle. The specifications for the novel unit dosage
forms of the present invention depend on the particular compound
employed and the effect to be achieved, and the pharmacodynamics
associated with each compound in the host.
[0120] The pharmaceutically acceptable excipients, such as
vehicles, adjuvants, carriers or diluents, are readily available to
the public. Moreover, pharmaceutically acceptable auxiliary
substances, such as pH adjusting and buffering agents,
antioxidants, low molecular weight (less than about 10 residues)
polypeptides, tonicity adjusting agents, stabilizers, wetting
agents and the like, are readily available to the public.
"Carriers" when used herein refers to pharmaceutically acceptable
carriers, excipients or stabilizers which are nontoxic to the cell
or mammal being exposed to the carrier at the dosages and
concentrations used.
[0121] Administration of the agents can be achieved in various
ways, including intracranial, either injected directly into the
brain tissue or injected into the cerebrospinal fluid, oral,
buccal, rectal, parenteral, intraperitoneal, intradermal,
transdermal, intracheal, intracerebral, etc., administration. The
antibodies may be administered in combination with one or more
additional therapeutic agents. Administration may be chronic or
intermittent, as deemed appropriate by the supervising
practitioner, particularly in view of any change in the disease
state or any undesirable side effects. Administration "in
combination with" one or more further therapeutic agents includes
both simultaneous (at the same time) and consecutive administration
in any order. "Chronic" administration refers to administration of
the agent in a continuous manner while "intermittent"
administration refers to treatment that is not done without
interruption.
[0122] In a particular embodiment, antibodies of the invention are
administered by intracranial injection. The injection will
typically be directly into affected brain regions or into the
cerebrospinal fluid.
[0123] An effective amount of an antibody or compound of the
present invention to be employed therapeutically will depend, for
example, upon the therapeutic objectives, the route of
administration, and the condition of the patient. Accordingly, it
will be necessary for the therapist to titer the dosage and modify
the route of administration as required to obtain the optimal
therapeutic effect. A typical daily dosage might range from about 1
.mu.g/kg to up to 100 mg/kg or more, depending on the factors
mentioned above. Typically, the clinician will administer a
molecule of the present invention until a dosage is reached that
provides the required biological effect. The progress of this
therapy is easily monitored by conventional assays.
[0124] Also provided by the subject invention are methods of
treating Huntington's disease conditions by expressing antibodies,
particularly intrabodies, i.e. non-secreted forms of the subject
antibodies, e.g. scFv analogs of the subject antibodies, as
described supra, in cells expressing mutant huntingtin protein.
Intrabodies and methods for their use in the treatment of disease
conditions are described in U.S. Pat. Nos. 5,851,829 and 5,965,371,
the disclosures of which are herein incorporated by reference. In
such methods, a nucleic acid encoding the antibody or intrabody,
generally in the form of an expression cassette that includes a
sequence encoding the antibody domains of interest, such as the
V.sub.H and V.sub.L domains, as well as other components, e.g.
promoters, linkers, intracellular localization domains or
sequences, etc., is introduced into the target cells in which
antibody or intrabody production is desired. The nucleic acid is
introduced into the target cells using any convenient methodology,
e.g. through use of a vector, such as a viral vector, liposome
vector, by biolistic transfection and the like, where suitable
vectors are well known in the art. Viral and/or non-viral methods
of delivering the nucleic acid encoding the intrabody to the cell
may be used.
[0125] A wide variety of non-viral vehicles for delivery of a
polynucleotide encoding an antibody of the present invention are
known in the art and are encompassed in the present invention. A
nucleic acid encoding an anti-huntingtin antibody or intrabody can
be delivered to a cell as naked DNA (U.S. Pat. No. 5,692,622; WO
97/40163). Alternatively, a the nucleic acid can be delivered to a
cell by association with one or more of a variety of substances
including, but not limited to cationic lipids; biocompatible
polymers, including natural polymers and synthetic polymers;
lipoproteins; polypeptides; polysaccharides; lipopolysaccharides;
artificial viral envelopes; metal particles; and bacteria. The
nucleic acid could also be delivered as a microparticle. Mixtures
or conjugates of these various substances can also be used as
delivery vehicles. The nucleic acid can be associated
non-covalently or covalently with these delivery agents. It is
possible to target liposomes to a particular cell type.
[0126] Viral vectors include, but are not limited to, DNA viral
vectors such as those based on adenoviruses, herpes simplex virus,
poxviruses such as vaccinia virus, and parvoviruses, including
adeno-associated virus; and RNA viral vectors, including, but not
limited to, retroviral vectors. Retroviral vectors include, for
example, murine leukemia virus, and lentiviruses such as human
immunodeficiency virus. Naldini et al., Science 272:263-267
(1996).
[0127] In a particular embodiment, the nucleic acid encoding the
intrabody that is to be expressed is inserted into a viral vector.
Preferred viral constructs are based on a retroviral genome, more
preferably a lentiviral genome as these viruses are able to infect
both dividing and non-dividing cells. The vector is transfected
into packaging cells and recombinant retrovirus is collected. The
recombinant retrovirus is then contacted with the cells in which
expression of the intrabody is desired. For example, the virus may
be injected intracranially or into the cerebrospinal fluid. In a
particular embodiment, the virus is injected directly into brain
regions that are known to be affected by Huntington's disease.
[0128] Following introduction of the nucleic acid into the target
cells, the nucleic acid is allowed to be expressed in the target
cell, whereby intrabodies that specifically bind to the protein of
interest, e.g. mutant huntingtin protein, are produced in the cell.
Production of the intrabodies interferes with the activity of the
protein, e.g. mutant huntingtin protein, and thereby treats the
host suffering from the disease condition.
EXAMPLES
[0129] Further details of the invention can be found in the
following example, which further defines the scope of the
invention. The following examples, including the experiments
conducted and achieved are provided for illustrative purposes only
and are not to be construed as limiting upon the present invention.
All references cited throughout the specification, are hereby
expressly incorporated by reference in their entirety.
Example 1
Anti-Huntingtin Antibodies
[0130] A. Production of Anti-Huntingtin Antibodies
[0131] The antigens used and the isotypes of the MW monoclonal
anti-huntingtin antibodies are summarized in Table 1.
[0132] 1. Immunization
[0133] For generation of anti-huntingtin antibodies, six-week-old
Balb/c female mice were primed and boosted at 2 week intervals by
intraperitoneal injection of antigen emulsified in adjuvant (RIBI
Immunochem, Hamilton, Mont., USA). Three different methods were
used for the generation of the anti-huntingtin antibodies.
[0134] a. DRPLA-19Q or DRPLA-35Q
[0135] For generation of mAbs, herein referred to as MW (for Milton
Wexler) mAbs, MW1, MW2 and MW5, mice were injected with antigen
proteins that were expressed from two constructs comprising the
polyQ domain (19 or 35 repeats) of huntingtin and 34 amino acids of
the dentatorubralpalliodoluysian atrophy (DRPLA) gene fused to
glutathione-S-transferase (GST) (Onodera et al., FEBS Lett,
399:135-139 (1996)). Test bleeds were obtained 7 days after very
other injection. A final series of boosts were performed without
adjuvant. Spleen cells were isolated from the mouse 3 days after
the final boost and fused with HL-1 murine myeloma cells (Ventrex,
Portland, Me. USA) using polyethylene glycol (PEG 1500,
Boehringer-Mannheim, Mannheim, Germany) (Lebron et al., J.
Immunol., 222:59-63 (1999)). Using enzyme linked substrate assay
(ELISA) to screen against these antigens versus GST alone, three
hybridomas were selected for cloning.
[0136] b. Expanded PolyQ Domain of Exon 1 of Huntingtin Protein in
Soluble Form
[0137] For generation of mAbs, MW3, MW4 and MW6, mice were
immunized as described above with protein that was soluble in
aqueous solution and was expressed from a construct comprising the
expanded polyQ domain (67 glutamine repeats) of Htt exon 1 (67Q)
fused to GST (GST-HDx67Q). Spleen cells were isolated from the mice
3 days after the final boost and fused with HL-1 murine myeloma
cells.
[0138] c. Expanded PolyQ Domain of Exon 1 of Huntingtin Protein in
Soluble and Aggregated Form
[0139] For generation of mAbs, MW7 and MW8, mice were immunized
with the same Htt exon 1 (67Q) protein fused to GST (GST-HDx67Q).
However, boosting of the mice was performed with an aggregated form
of exon 1 having 67 Q repeats of the huntingtin protein (67Q),
prepared by removing the GST. Spleen cells were isolated from the
mice 3 days after the final boost and fused with HL-1 murine
myeloma cells.
[0140] 2. Selection of Hybridomas
[0141] Three hybridomas generated from mice immunized with proteins
expressed from the two constructs containing the polyQ domain (19
or 35 repeats) and 34 amino acids of the
dentatorubralpalliodoluysian atrophy (DRPLA) gene fused to
glutathione-S-transferase (GST) were selected for cloning. mAbs
from these hybridomas were termed MW1, MW2, and MW5.
[0142] The hybridomas generated from mice immunized with
GST-HDx67Q, and from mice immunized with the same GST-HDx67Q
antigen and boosted with an aggregated form that lacked the GST
were both screened by ELISA using the antigen, GST-HDx67Q and GST
alone, and by Western blotting of extracts from the Huntington's
disease (HD) lymphoblastoma cell line HD2. mAbs, MW3, MW4 and MW6
were generated from hybridomas generated from mice immunized with
GST-HDx67Q while mAbs, MW7 and MW8 were generated from hybridomas
generated from mice immunized with GST-HDx67Q and boosted with an
aggregated form of GST-HDx67Q that lacked GST.
[0143] a. ELISA
[0144] The hybridomas were analyzed by ELISA using the antigen,
GST-HDx67Q and GST alone. MW3, MW4 and MW6 bound the injected
protein, GST-HDx67Q, but did not bind to GST alone. MW7 and MW 8
were selected for having a positive ELISA signal with
GST-HDx67Q.
[0145] b. Western Blots
[0146] For the Western blots, lymphoblasts from control (HD7) and
HD patients (HD2) were cultured in Isscove's modified Dulbecco's
medium (Irvine Scientific, Irvine, Calif. USA) supplemented with
15% fetal calf serum and 2 mM glutamine. Lymphoblasts or cerebella
from mice were homogenized in 300 mM NaCl, 1 mM EDTA, 0.5% Triton
X-100, 50 mM Tris, pH 7.0, with complete protease inhibitor
cocktail (Boehringer Mannheim). The homogenates were centrifuged
(14,300 rpm for 10 min). The protein concentrations of the
supernatants were determined by BCA assay (Pierce, Rockford, Ill.,
USA). The protein in the supernatants were concentrated by
precipitation at 70.degree. C. for 3 min. The precipitates were
resuspended in 6 M urea at one half of the original supernatant
volume and concentrated sodium dodecyl sulfate (SDS) dissociation
buffer added to achieve a final concentration of 5%
2-mercaptoethanol, 1.5% SDS, and 5% glycerol. Samples were heated
at 95.degree. C. for 10 minutes and subjected to SDS polyacrylamide
gel electrophoresis (PAGE) on 5% gels (Laemmlie, E. K., Nature,
227:105-132 (1970)). Gels were electrotransferred to nitrocellulose
membrane (Schleicher & Schuell, Keene, N.H., USA) overnight
with cooling. These membranes were then preblocked with 1% blocking
reagent (Boehringer Mannheim) and incubated with the MW mAbs
(undiluted hybridoma supernatants), MAB2166 (Chemicon, Temecula,
Calif., USA; 1/1000 dilution), 1F8 ascites fluid (M. MacDonald;
1/1000 dilution), overnight at room temperature. Blots were washed
with 0.5% Tween-20 in phosphate-buffered saline (PBS) for 10
minutes 3 times before incubation with biotinylated goat anti-mouse
immunoglobulin (Ig) G+IgM (Chemicon), diluted 1/1000 in blocking
buffer, for 1 hour at room temperature. After washing, the blots
were incubated with horseradish peroxidase-strepavidin (Chemicon)
in blocking buffer for 1 hour at room temperature. Blots were
developed using 4-chloro-1-naphthol. TABLE-US-00001 TABLE 1
GENERATION AND CHARACTERIZATION OF ANTI-HUNTINGTIN (Htt) MONOCLONAL
ANTIBODIES (mAbs) Antigen MAb Isotype Epitope Immunoblot ICC
DRPLA-19Q MW1 IgG2b polyQ Mutant Htt Cytoplasm DRPLA-35Q and MW2
IgM polyQ Mutant Htt Golgi TRX-35Q MW5 IgM polyQ Mutant Htt + other
Golgi bands HDx-67Q (soluble) MW3 IgM polyQ Mutant Htt + other
Golgi bands MW4 IgM polyQ Mutant Htt + other Golgi bands MW6 IgM
polyQ Band below 350 kD + Cytoplasm variable size band HDx-67Q
(soluble 1.sup.st MW7 IgM polyP 350 kD + 130 kD Perinuclear in
wild-type and boost with and lower mouse brain; inclusions
aggregate) in R6/2 brain MW8 IgG2a AEEPLHRPK ? No staining in
wild-type mouse brain; inclusions in R6/2 brain
[0147] B. Characterization of Anti-Htt Antibodies
[0148] 1. Epitope Mapping
[0149] To determine the epitopes recognized by these mAbs, we
utilized arrays of dot blots that contain overlapping 14mer
peptides synthesized from the first 91 amino acids of normal human
Htt (containing a 23 polyQ domain). The first dot contained the
peptide corresponding to amino acids 1-14, the second dot contained
the peptide corresponding to 4-17, the third dot contained the
peptide corresponding to 7-20, etc.
[0150] Each of the MW1-MW6 mAbs specifically bound one of three
single, contiguous epitopes in the Htt sequence (FIG. 1). MW1-MW6
bound peptides that contain >6 glutamines and were specific for
the polyQ region. As the antigens used to generate the MW1-MW6 mAbs
contained other amino acids in numbers equal or greater than the
polyQ domain, the polyQ domain may be highly antigenic or may be
prominently displayed in soluble protein fragments.
[0151] As summarized in FIG. 2, MW7 specifically binds peptides
that contain the polyP domain in Htt. There are two of these
domains in exon 1, and MW7 binds all peptides with >5
consecutive prolines. MW8, in contrast, binds specifically to an
eight amino acid stretch, AEEPLHRP (SEQ ID NO: 2), near the
Cterminus of exon 1. MW7 and MW8 mAbs did not bind the polyQ domain
in Htt.
[0152] 2. Western Blots
[0153] To determine if MW1-MW8 mAbs were able to distinguish
between normal and mutant Htt containing the expanded polyQ, the
MW1-MW8 mAbs were tested in parallel for binding, as analyzed by
immunoblotting, to brain extracts of a wild-type mouse and a
knock-in transgenic mouse that expresses a mouse human chimeric Htt
exon 1 construct that contains 94Q repeats (94Q knock-in mouse)
(Menalled et al., Exp. Neurol., 162:328-342 (2000)) (FIG. 3) and
extracts of a lymphoblastoma cell line from a human HD patient
(HD2) that expressed both normal and mutant Htt and a
lymphoblastoma cell line from human non-HD patient (HD7) that
expressed only normal Htt (FIG. 4). Control antibodies, 1C2
(Chemicon MAb1574; Trottier et al., Nat. Genet., 10:1040110 (1995))
and 1F8 (Wheeler et al., Hum. Mol. Genet., 9:503-513 (2000)) were
used to identify mutant Htt, and Ab2166 (Chemicon) were used to
identify both mutant and normal Htt.
[0154] The extracts used and the procedure for immunoblotting was
performed as described above.
[0155] MW1-MW6 specifically bound the polyQ epitope in Htt with
MW1-MW5 preferentially binding to the expanded repeat mutant form
of Htt rather than normal Htt on Western blots. More specifically,
the mAbs MW1 and MW2 displayed a very specific binding pattern
similar to the pattern for 1C2, strongly staining the expanded
mutant polyQ form of Htt that is approximately 350 kD in size in
mouse brain extracts from the 94Q mice and did not bind the normal
polyQ form in mouse brain extracts from WT mice (FIG. 3). MW1 and
MW2 mAbs also specifically bound the 350 kDa form of mutant Htt in
extracts from HD2 cells (FIG. 4).
[0156] MW3-MW5 displayed a very specific binding pattern similar to
the pattern for 1F8 in mouse brain extracts. MW3-MW5 specifically
bound the expanded mutant repeat form of Htt rather than normal Htt
as well as other bands of lower molecular weights which may be
breakdown products of Htt with different conformations (FIG. 3).
MW3-MW5 mAbs also specifically bound the 350 kDa form of mutant Htt
in extracts from HD2 cells (FIG. 4).
[0157] MW6 specifically bound in normal and HD human lymphoblastoma
cell extracts an antigen that has a size varying from about 250-300
kDa which may be a breakdown product of Htt.
[0158] MW7 bound the expanded repeat mutant form of Htt in mouse
brain extracts (FIG. 3). In human lymphoblastoma cell extracts, MW7
bound the high molecular weight Htt very weakly, but binds strongly
one or two smaller molecular weight proteins which are present in
normal (HD7) and Huntington's disease (HD2) human lymphoblastoma
cell extracts (FIG. 4) at roughly equivalent levels.
[0159] MW8 did not detectably bind any proteins in mouse brain
extracts nor in human lymphoblastoma cell extracts examined by
immunoblotting.
[0160] 3. Immunostaining
[0161] Light microscopic immunohistochemistry was done with
10-.mu.m sections of 4% paraformaldehyde fixed R6/2 tissue or fresh
frozen R6/1 tissue. Briefly, 8-10-week-old R6/1, R6/2 or control
littermates (Jackson Laboratory; Mangiarini et al., Cell,
87:493-506 (1996)) were anesthetized with Phenobarbital, perfused
with PBS followed by 4% paraformaldehyde or PBS only. Brains were
removed and frozen on dry ice with O.C.T. compound (Sakura Finetek,
Torrance, Calif., USA). R6/2 contains human Htt exon 1 with 144
polyQ repeats while R6/1 contains 116 repeats and displays symptoms
at a later age than R6/2. Fixed sections were incubated with mAbs
MW3-8 or 1F8 ( 1/1000). PBS washed sections were incubated with
Hi-Fluorescence goat anti-mouse IgG (Antibodies, Inc., Davis,
Calif., USA) and DTAF goat anti mouse IgG+IgM (Chemicon) in
blocking buffer (2% bovine serum albumin, 5% normal goat serum).
Fresh frozen sections were incubated with ascites of MW1 or MW2 at
1/1000, or 1C2 (Chemicon MAB1574) at 1/1000, in blocking buffer.
Biotinylated goat anti-mouse IgG+IgM and fluorescein
isothiocyanatestreptavidin were used. Light microscopic images were
captured using a digital camera (SPOT, Diagnostic Instruments,
Sterling Heights, Mich., USA) attached to an epi-fluorescent
microscope (Leica DMLB, Deerfield, Ill., USA). Thirty-five
micrometer 4% paraformaldehyde fixed floating sections were
processed using the same secondary Abs as above and subjected to
confocal microscopy (Leica DM IRB/E, Leica confocal software).
[0162] MW1 and control 1C2 antibodies displayed primarily punctate
cytoplasmic staining of neurons (FIGS. 5B and 5D) in wild-type and
R6/2 transgenic brain sections. Neuropil staining with MW1 which
was also apparent was specific because controls omitting the
primary antibody were largely negative under the same staining and
photographic conditions (FIG. 5a).
[0163] MW2-MW5 and control 1F8 antibodies displayed little or no
staining of the neuropil, but stained neuronal Golgi complex in
wild-type spinal cord section as shown in FIGS. 6A-6E and in R6/2
transgenic spinal cord sections (MW3 staining is shown in FIG. 6F)
with no difference in staining between wild-type and mutant
transgenic spinal cord with MW3-MW5 antibodies. However, MW3, MW4
and MW5 staining in R6/2 brain sections (MW3 staining is shown in
FIG. 6H) was less than staining in wild-type brain sections (MW3
staining is shown in FIG. 6G).
[0164] MW6 displayed very strong punctate staining of neuropil and
strong homogeneous staining of neuronal cytoplasm in wild-type
(FIG. 7A) and mutant spinal cord (FIG. 7B), with no obvious
difference in staining between wild-type and mutant spinal cord.
MW6 antibodies did not strongly stain neuronal nucleus.
[0165] MW7 displayed punctate perinuclear or nuclear membrane
staining in wild-type (FIG. 7C) and mutant brain sections (FIG. 7D)
with weaker punctate perinuclear or nuclear membrane staining, but
more prominent nuclear inclusion staining in mutant R6/2 brain
sections. The perinuclear staining is shown in a confocal
microscope series (FIGS. 7E-7H).
[0166] MW8 mAbs displayed nuclear inclusion staining in R6/2 brain
sections (FIG. 7J), but did not stain nuclear inclusions in
wild-type brain sections (FIG. 71). MW8 mAbs also stained small
inclusions in the neuropil.
[0167] In summary, MW1-MW6 mAbs did not stain nuclear inclusions
well in brain sections while MW7 and MW8 mAbs stained nuclear
inclusions in brain sections of mice expressing a human chimeric
Htt exon 1 construct with 94Q repeats.
[0168] 4. Summary
[0169] Both the epitope mapping and histochemical results are
summarized in FIG. 8. The availability of four regions of exon 1 of
Htt, the N-terminal 17 amino acids, the polyQ domain, the polyP
domain and the C-terminal domain, for Ab binding was different in
the Golgi, perinuclear and nuclear subcellular compartments. The
N-terminal 17 amino acids of exon 1 of Htt was available for Ab
binding in the Golgi, perinuclear and nuclear subcompartments. In
the spinal cord neuronal Golgi complex of both WT and R6/2 mice,
the polyQ domain was available, but the adjacent polyP and
C-terminal domains were occluded. In the perinuclear region of
neurons of R6/2 mice, the polyP domain was available for Ab
binding, but the C-terminus was occluded. Within the nucleus of
neurons in the R6/2 (but not wild-type) mice, the polyQ domain was
occluded, but the adjacent N-terminal, C-terminal and polyP domains
were open for Ab binding.
Example 2
Anti-Htt Antibody Fragments
[0170] To examine the effects of the anti-huntingtin (Htt)
antibodies on the biological activities of Htt exon 1, we generated
single-chain variable region fragment Abs (scFvs) for MW1 and MW2
anti-Htt antibodies, which recognizes the polyQ Http epitope, MW7
anti-Htt antibody, which recognizes the polyP domains of Htt exon 1
and MW8 anti-Htt antibody, which recognizes an 8 amino acid epitope
near the C-terminus of the huntingtin protein. Human
anti-huntingtin hMW9 antibody was isolated from a human scFvs phage
library using recombinant mutant huntingtin protein. The scFvs for
MW1, MW2, MW7 and MW8, expressed in E. coli were tested for binding
to Htt on immunoblots, and the scFv for hMW9 were tested for
binding to His-HDx in vitro. Positive clones were selected for
further characterization in mammalian cells.
[0171] A. Generation of scFvs
[0172] 1. MW1, MW2, MW7 and MW8 scFvs
[0173] For generation of MW1, MW2, MW7 and MW8 scFvs, total RNA was
extracted from hybridoma cell lines secreting each of the anti-Htt
MW mAbs, and mRNA was purified by using oligo-dT columns (Qiagen,
Valencia, Calif.). Complementary cDNA was produced for each mRNA
pool by using random hexanucleotide primers. The cDNAs served as
sources of DNA to amplify both variable region heavy (VH) and
variable region light (VL) chains for each mAb by using primers
complementary to the consensus sequences flanking each domain
(Amersham Pharmacia) and PCR technology. To generate recombinant
single-chain fragment Abs, the amplified VH and VL of each mAb were
linked by a 45-mer nucleotide encoding Gly-Ser. These scFv genes
were cloned into the M13 phagemid, pCANTBE5 (Amersham Pharmacia),
and used to transform Escherichia coli, strain TG15, which supports
production of recombinant phage. The amplified recombinant phage
population was selected for binding on immunoblots to Htt
exon-1-glutathione S-transferase (GST)-containing a 67-polyQ
repeat. Phage that specifically bound Htt were eluted and used to
reinfect TG15 E. coli. Individual clones were tested again for Htt
binding, and the nucleotide sequence of positive clones was
determined by dideoxynucleotide chain-termination method. The
nucleotide sequence of MW1, MW2, MW7 and MW8 scFVs are represented
by SEQ ID NOs: 3, 4, 5 and 6, respectively.
[0174] 2. Human MW9 scFv Antibody
[0175] For generation of hMW9 scFv, the cDNA for mutant huntingtin
exon 1 (HDx) fused to a His tag was expressed in E. coli and
purified on nickel columns. The purified mutant protein was
subjected to SDS-PAGE and transferred to nitrocellulose membranes.
The nitrocellulose membranes were incubated with a human
single-chain fragment variables (scFv) phage library encoding
.about.9.times.10.sup.10 clones. Phage bound to HDx were selected
and amplified by infection of susceptible E. coli for 5 rounds.
Finally, amplified clones were selected with a recombinant GST-HDX
in a solution-based assay. Individual clones were isolated,
expressed and recombinant scFvs were tested for binding to His-HDx
in vitro. Positive clones that bound in vitro were co-expressed
with mutant HDx-1 in a tissue culture model of Huntington disease
and results were evaluated for inhibition of cell death. hMW9 scFv
expression inhibits aggregation and cell death induced by mutant
HDx in this model.
[0176] B. Characterization of scFvs
[0177] 1. Expression Analysis
[0178] a. MW1, MW2, MW7 and MW8 scFvs
[0179] To test for scFv expression of the MW1, MW2, MW7 and MW8
anti-Htt antibodies, 293 cells were transfected with the
Flag-tagged scFvs and cell lysates were analyzed by Western
blotting and intracellular staining.
[0180] The reading frame for the scFvs were each subcloned into the
mammalian plasmid pcDNA3.1 in frame with the Flag epitope for
detection purposes (Chiang et al., Pept. Res., 6:62-64 (1993)).
Selected clones were amplified and used to transfect 293 cells that
were grown in DMEM supplemented with 10% heat inactivated bovine
serum, 2 mM glutamine, 1 mM streptomycin and 100 international
units of penicillin. Cells were grown in 6-well plates to about 70%
confluence and transfected with a total of 2 .mu.g of DNA by using
lipofectamine, following the manufacturer's recommendations
(Invitrogen). Expression of the scFvs were examined by Western blot
analysis of the transfected cell extracts by using an anti-Flag Ab
(Sigma).
[0181] Full-length proteins for MW1, MW2 and MW7 (FIG. 9A) scFvs
were detected using anti-Flag Ab.
[0182] b. hMW9 scFv
[0183] To test for scFv expression of the hMW9 anti-Htt antibodies,
control and hMW9 scFvs were expressed by in vitro transcription and
translation in the presence of .sup.35S-methionine. Equal amounts
of each were incubated with 5 .mu.g of recombinant GST-HDx-1 bound
to glutathione beads in a buffer containing mild detergent and
glycerol. Following incubations for 3 hours at room temperature,
the beads were washed 5 times in the buffer with mild detergent and
glycerol. The scFvs that were bound to the beads were extracted and
subjected to SDS-PAGE and autoradiography (FIG. 10).
[0184] 2. Histological Analysis
[0185] For histological examination of 293 cells transfected with
Flag-tagged scFvs, transfected cells were fixed in 4%
paraformaldehyde for 30 minutes at 4.degree. C., permeabilized in
70% methanol at -20.degree. C. for 1 hour, and incubated with
anti-Flag Ab (1:1000) for 2 hours. Cells expressing scFvs were
detected by a goat anti-mouse Ab conjugated to Alexa 594 (Molecular
Probes), and examined with a confocal microscope.
[0186] Histological examination revealed that the MW1, MW2 and MW7
(FIG. 11) scFvs have a predominantly cytoplasmic distribution.
[0187] 3. Cell Viability
[0188] Because other proteins besides Htt contain polyQ and polyP
domains, it was of interest to test whether expression of the scFvs
had an effect on cell viability.
[0189] To determine the effects of scFvs on cell viability, human
293 cells were cotransfected with each scFv and a plasmid encoding
enhanced green fluorescent protein (EGFP; CLONTECH) as a
transfection marker to readily detect which cells were transfected
(Nucifora et al., Science, 291:2423-2428 (2001)). Viable cells that
expressed GFP were counted 4 days after transfection by using a
fluorescence microscope.
[0190] After 4 days of growth, the mean cell counts from at least
30 microscope fields in six wells each revealed no significant
differences between the control (112.+-.6), and the scFvs for MW1
(97.+-.3), MW2 (117.+-.4), and MW7 (113.+-.5). Accordingly, scFv
expression did not affect cell growth or viability.
[0191] 4. Interaction of Anti-Htt Antibody Fragments with Htt in
Living Cells
[0192] a. Coimmunoprecipitation
[0193] To determine whether the scFvs interact with Htt in living
cells, flag-tagged scFvs or flag-tagged I.kappa.B.alpha., a
control, were coexpressed in 293 cells with Htt exon 1 containing
either 25 polyQ repeats (PQ25) or 103 polyQ repeats (PQ103), fused
to EGFP and subjected to coimmunoprecipitation analysis. The scFvs
and I.kappa.B.alpha. in Triton X-100 cell extracts were
precipitated with anti-Flag Ab, and the precipitates were subjected
to SDS-PAGE. The SDS-PAGE gels were analyzed for the presence of
Htt exon 1 by Western blotting using anti-Flag Ab (FIG. 9A) and
antibodies specific for the N-terminal 17 amino acids of exon 1 of
the huntingtin protein (FIG. 9B).
[0194] Coimmunoprecipitation experiments were performed with 293
cell lysates cotransfected as described above. Briefly, cells were
harvested 24 hours after transfection and lysed by sonication in
buffer A (25 mM Hepes, pH 7.4/2.5 mM MgCl.sub.2/50 mM NaCl/1 mM
EDTA/1% Triton X-100). After clearing the lysates by centrifugation
at 14,300 rpm (Eppendorf microcentrifuge) for 10 minutes at
4.degree. C., 200 .mu.g of each lysate was incubated at 4.degree.
C. with rocking for 2 hours with a 40-.mu.l slurry of anti-Flag Ab
coupled to protein A beads. The beads were then washed five times
in buffer A by using centrifugation at 5,000 rpm, and the complexes
were resolved on SDS-PAGE. For Western blotting, rabbit anti-HD1-17
(Mende-Mueller et al., J. Neurosci., 21:1830-1837 (2001)) and
anti-Flag (1:1000; Sigma) were used as the primary Abs. Secondary
antibodies conjugated to horseradish peroxidase (HRP) were used to
detect the reactive protein bands by enhanced chemiluminescence
(Santa Cruz Biotechnology). The SDS-PAGE gels were first probed
with anti-flag antibodies for the presence of the scFvs and then
stripped and reprobed with an antibody specific for the N-terminal
17 amino acids of exon 1 of the huntingtin protein for the presence
of mutant PQ 103 huntingtin about 80 kDa and mutant PQ25 huntingtin
about 50 kDa.
[0195] As shown in FIG. 9A, similar amounts of each of the MW1, MW2
and MW7 scFvs, which migrate with a molecular mass of about 35 kDa
and I.kappa.B.alpha. which migrates with a molecular mass of about
43 kDa were precipitated with the anti-Flag Ab (FIG. 9A), and
mutant Htt exon 1 (PQ103) coimmunoprecipitates with each scFv (FIG.
9B; 86-kDa bands). Similar results were obtained when Htt exon 1
with a 25-Q stretch (PQ25) was used for transfection (FIG. 9B; 50
kDa bands). As a negative control, in cells expressing Flag-tagged
I.kappa.B.alpha., I.kappa.B.alpha. was precipitated from the
extract by the anti-Flag Ab (FIG. 9A; 44-kDa band), but Htt was not
coprecipitated (FIG. 9B). The bands below 30 kDa in FIG. 9B likely
represented nonspecific staining of the precipitating Ab.
[0196] 5. Colocalization
[0197] To confirm binding of the scFvs to mutant huntingtin protein
and to localize the sites of interaction within cells, we used
confocal microscopy to examine 293 cells cotransfected with mutant
exon 1 of the huntingtin protein having 103 Q repeats and fused to
EGFP (103-Q Htt-EGFP) and each anti-Htt scFv.
[0198] For colocalization experiments, the scFvs and 103-Q
Htt-EGFP, obtained from the Cure Huntington Disease Initiative
Resource Bank (Univ. of California, Los Angeles; Steffan et al.,
Proc. Natl. Acad. Sci. USA, 97:6763-6768 (2000)), were
cotransfected in 293 cells grown on coverslips. 24 hours after
transfection, cells were fixed, stained, and examined as described
above. Depending on the experiment, 50-70% of the cells expressed
EGFP.
[0199] Although the MW1, MW2 and MW7 (FIG. 11) scFvs and MW8 scFvs
were distributed throughout the cytoplasm in the absence of Htt,
when cotransfected with 103-Q Htt-EGFP, the MW1, MW2 and MW7 (FIG.
13) and MW8 (FIG. 14) scFvs were concentrated in the perinuclear
region and colocalized with 103-Q Htt-EGFP.
[0200] 6. Effects of Anti-Htt Antibody Fragments on Htt-Induced
Cell Death
[0201] To evaluate the effect of anti-Htt scFvs on the toxic
effects of mutant Htt, we examined by terminal
deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)
staining of 293 cells cotransfected with 103-Q Htt-EGFP and each
scFv.
[0202] Two days after cotransfection, 293 cells were fixed as
described above and washed three times in PBS. The TUNEL reaction
consisted of 25 units of terminal deoxynucleotidyltransferase and 1
mM dUTP conjugated to tetramethylrhodamine (Roche Molecular
Biochemicals) in 1.times. buffer/2.5 mM CoCl.sub.2 in a final
volume of 50 .mu.l (according to manufacturer's instructions).
Coverslips with fixed cells were laid over the reaction mixture and
incubated at 37.degree. C. in a humidified incubator. Samples were
washed four times with PBS, mounted on microscope slides, and
examined by confocal microscopy. TUNEL-positive cells that were
expressing mutant Htt exon 1 were counted from at least 16
independent microscope fields with a .times.20 objective lens in
four separate experiments. The data were analyzed by using EXCEL
software to determine the standard deviation and the P value (t
test).
[0203] Cells expressing 103-Q Htt-EGFP along with an empty scFv
vector displayed significant TUNEL staining, and apoptotic bodies
were observed starting about 12 hours after transfection (FIG. 13,
control column). TUNEL staining was even more dramatic in the
presence of MW1 or MW2 scFv and mutant Htt (FIG. 15). MW1 or MW2
scFv binding to the polyQ domain accentuated the toxicity of mutant
Htt.
[0204] Expression of MW7 scFv (FIG. 15) or MW8 scFv (FIG. 16)
inhibited the toxicity of mutant Htt. These experiments were done
under the same conditions as in FIG. 9A, which demonstrated
equivalent expression of MW1, MW2 and MW7 scFvs in the cells.
[0205] To quantify the effects of scFv expression on mutant Htt
toxicity, we counted TUNEL+ cells. The increase in mutant
Htt-induced TUNEL staining in the presence of MW1 and -2 scFvs is
38% and 67%, respectively (P<0.05) (FIG. 17). In contrast, the
number of TUNEL+ cells in the presence of MW7 scFv is reduced to
33% of the control (P<0.05) (FIG. 15), and the number of TUNEL+
cells in the presence of MW8 scFv is reduced from 72 apoptotic
bodies to 26 apoptotic bodies (FIG. 16). Thus, although the
anti-polyQ mAbs MW1 and MW2 scFvs accentuate the toxicity of mutant
Htt, expression of the anti-polyP mAb MW7 and MW8 scFvs inhibits
the toxicity of mutant Htt.
[0206] To determine the effects of hMW9 scFv on mutant Htt cell
toxicity, human 293 cells were cotransfected with the 103-Q
Htt-EGFP and hMW9 or as controls, an empty plasmid or a control
scFv that does not bind to exon 1 of the huntingtin protein (HDx-1)
by lipofectamine. 103-Q Htt-EGFP, was cotransfected with an empty
plasmid (FIG. 12; EGFP-103Q-HDx-1+C), hMW9 (FIG. 12;
EGFP-103Q-HDx-1+MW9) or cscFv, a control that does not bind to
HDx-1 (FIG. 12; EGFP-103Q-HDx-1+cscFv). Two days post-transfection
cells were examined by a fluorescent microscope. Cells remained
intact in the presence of hMW9 (middle panel) when compared to the
presence of a control that does not bind to HDx-1 (bottom panel).
Without hMW9, mutant Htt results in cell toxicity and the presence
of apoptotic bodies (FIG. 12, top panel). In the presence of hMW9,
cells transfected with mutant Htt are healthy and have less
apoptotic bodies (FIG. 12, middle panel).
[0207] 7. Effects of Anti-Htt Antibody Fragments on Aggregation of
Mutant Htt
[0208] To evaluate the effects of scFv expression on mutant Htt
aggregation in 293 cells, Htt aggregation was evaluated
biochemically by examining the amount of Htt that precipitated from
cell lysates by centrifugation at 150,000.times.g for 30 min.
[0209] For aggregation studies, 293 cells were cotransfected with
mutant Htt exon 1 and an scFv were harvested 48 hours after
transfection. Cells were lysed by sonication in buffer A. Lysates
were centrifuged at 150,000.times.g in an SW55 rotor (Beckman
Instruments, Fullerton, Calif.) for 30 minutes (Nucifora et al.,
Science, 291:2423-2428 (2001)). Pellets were dissolved in sample
buffer containing 2% SDS, boiled and subjected to SDS-PAGE, and
transferred to nitrocellulose membranes for immunoblotting
analysis. Aggregates were detected with anti-HD1-17 polyclonal
antibody (Mende-Mueller et al., J. Neurosci., 21:1830-1837
(2001)).
[0210] The pellets contained aggregated Htt (or Htt that is bound
to large structures) that can be solubilized by SDS treatment (FIG.
18A, 80-kDa bands), as well as Htt that remained insoluble after
boiling in SDS and cannot enter the gel (FIG. 18A, top of gel).
Both such species of pelleted Htt were detected in extracts of
cells transfected with mutant Htt exon 1 alone (FIG. 18A).
Aggregation increased when mutant Htt exon 1 was coexpressed with
MW1 scFv or MW2 scFv. Very little aggregated Htt was found in the
presence of MW7, MW8 or hMW9 scFv. Scanning the bands at the top of
the gel in FIG. 18 yielded values in arbitrary units of 68.8 for
MW1, 54.3 for MW2, 0.2 for MW7, and 48.8 for no scFv.
[0211] Accordingly, coexpression of MW7 scFv interfered with
aggregation of mutant Htt exon 1, and there was a qualitative
correlation between the effects of the scFvs on Htt aggregation and
toxicity. The expression of MW7 did not cause a depletion in the
level of soluble Htt (FIG. 18B).
[0212] Coexpression of hMW9 also interfered with aggregation of
mutant Htt exon 1.
Sequence CWU 1
1
6 1 91 PRT Homo sapiens 1 Met Ala Thr Leu Glu Lys Leu Met Lys Ala
Phe Glu Ser Leu Lys Ser 1 5 10 15 Phe Gln Gln Gln Gln Gln Gln Gln
Gln Gln Gln Gln Gln Gln Gln Gln 20 25 30 Gln Gln Gln Gln Gln Gln
Gln Gln Pro Pro Pro Pro Pro Pro Pro Pro 35 40 45 Pro Pro Pro Gln
Leu Pro Gln Pro Pro Pro Gln Ala Gln Pro Leu Leu 50 55 60 Pro Gln
Pro Gln Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Gly Pro 65 70 75 80
Ala Val Ala Glu Glu Pro Leu His Arg Pro Lys 85 90 2 8 PRT Homo
sapiens 2 Ala Glu Glu Pro Leu His Arg Pro 1 5 3 746 DNA Homo
sapiens 3 atggcccagg tcaaactgca ggagtctggg ggaggcttag tgcagcctgg
agggtccctg 60 aaactctcct gtgcagcctc tggattcact ttcagagact
attatatgta ttgggttcgc 120 cagactccag agaagaggct ggagtgggtc
gcattcatta gtaatggtgg tggtagcacc 180 tattatccag acactgtaaa
gggccgattc accatctcca gagacaatgc caagaacacc 240 ctgtacctgc
aaatgagccg tctgaagtct gaggacacag ccatgtatta ctgtgcaaga 300
gggaggggct acgtatggtt tgcttactgg ggccaaggga ccacggtcac cgtcttctca
360 ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg gatcggacat
tgtgctaacc 420 cagtctccag cttccttagc tgtatctctg gggcagaggg
ccaccatctc atacagggcc 480 agcaaaagtg tcagtacatc tggctatagt
tatatgcact ggaaccaaca gaaaccagga 540 cagccaccca gactcctcat
ctatcttgta tccaacctag aatctggggt ccctgccagg 600 ttcagtggca
gtgggtctgg gacagacttc accctcaaca tccatcctgt ggaggaggag 660
gatgctgcaa cctattactg tcagcacatt agggagctta cacgttcgga ggaggcacca
720 agctggaaat caaacgggcg gccgca 746 4 761 DNA Homo sapiens 4
atggcccagg tgaaactgca ggagtcagga cctgagctga agaagcctgg agagacagtc
60 aagatctcct gcaaggcttc tgggtatacc ttcacaaact atggaatgaa
ctgggtgaag 120 caggctccag gaaagggttt aaagtggatg ggctggataa
acacctacac tggagagcca 180 acatatgctg atgactccaa gggacggttt
gccttctctt tggaaacctc tgccagcact 240 gcctatttgc agatcaacaa
cctcaaaaat gaggacatgg ctacatattt ctgtgcaaga 300 aggggattac
tgtttgctta ctggggccaa gggaccacgg tcaccgtctc ctcaggtgga 360
ggcggttcag gcggaggtgg ctctggcggt ggcggaggtg gctctggcgg tggcggatcg
420 gacatcgagc tcactcagtc tccaacttcc ttagctgtat ctctggggca
gagggccacc 480 atctcataca gggccagcaa aagtgtcagt acatctggct
atagttatat gcactggaac 540 caacagaaac caggacagcc acccagactc
ctcatctatc ttgtatccaa cctagaatct 600 ggggtccctg ccaggttcag
tggcagtggg tctgggacag acttcaccct caacatccat 660 cctgtggagg
aggaggatgc tgcaacctat tactgtcagc acattaggga gcttacacgt 720
tcggaggggg gacaaagttg gaaataaaac gggcggccgc a 761 5 826 DNA Homo
sapiens 5 atggactaca aggacgacga tgacaaggtg gcccaggtca agctgcagga
gtctggagga 60 ggcttggtgc aacctggagg atccatgaaa ctctcttgtg
ctgcctctgg attcactttt 120 agtgacgcct ggatggactg ggtccgccag
tctccagaga aggggctgag tggggttgct 180 gaaattagaa gcaaagctaa
taatcatgca acatactatg ctgagtctgt gaaagggagg 240 ttcaccatct
caagagatga ttccaaaagt agtgtctacc tgcaaatgaa cagcttaaga 300
gctgaagaca ctggcattta ttactgtatc tatgcggggt ttgcttactg gggccaaggg
360 accacggtca ccgtctcctc aggtggaggc ggttcaggcg gaggtggctc
tggcggtggc 420 ggatcggaca tcgagctcac tcagtctcca tcctccctgg
ctatgtcagt aggacagaag 480 gtcactatga gctgcaagtc cagtcagagc
cttttaaata gtagcaatca aaagaactat 540 ttggcctggt accagcagaa
accaggacag tctcctaaac ttctggtata ctttgcatcc 600 actagggaat
ctggagtccc tgatcgcttc ataggcagtg gatctgggac agatttcact 660
cttaccatca gcagtgtgca ggctgaagac ctggcagatt acttctgtca gcaacattat
720 agcactccgt ggacgttcgg tggaggcacc aagctggaaa tcaaacgggg
acaaagttgg 780 aaataaaacg gtgggggacc aagctggaaa taaaacgggc ggccgc
826 6 729 DNA Homo sapiens 6 atggcccagg tgcagctgca ggagtcaggg
ggaggcttag tgaagcctgg agggtccctg 60 aaactctcct gtgcagcctc
tggattcact ttcagtgact attacatgta ttgggttcgc 120 cagactccgg
aaaagaggct ggagtgggtc gcaaccatta gtgatggtgg tagttacacc 180
tactatccag acaatatgaa ggggcgattc accatctcca gagacaatgc caagaacaac
240 ctgtacctgc aaatgagcag tctgaagtct gaggatacag ccatgtattt
ttgtgcaaga 300 gatctgggga aatggggcca aggcaccacg gtcaccgtct
cctcaggtgg aggcggttca 360 ggcggaggtg gctctggcgg tggcggatcg
gacatcgagc tcactcagtc tccaacttcc 420 ttagctgtat ctctggggca
gagggccacc atctcataca gggccagcaa aagtgtcagt 480 acatctggct
atagttatat gcactggaac caacagaaac caggacagcc acccagactc 540
ctcatctatc ttgtatccaa cctagaatct ggggtccctg ccaggttcag tggcagtggg
600 tctgggacag acttcaccct caacatccat cctgtggagg aggaggatgc
tgcaacctat 660 tactgtcagc acattaggga gcttacacgt tcggaggggg
accaagctgg aaataaaacg 720 ggcggccgc 729
* * * * *