Note: Descriptions are shown in the official language in which they were submitted.
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NOVEL HUMAN ION CHANNEL PROTEINS AND
POLYNUCLEOTIDES ENCODING THE SAME
The present application claims the benefit of U.S.
Provisional Application Numbers 60/221,643 and 60/222,503
which were filed on July 28, 2000 and August 2, 2000,
respectively. These U.S. Provisional Applications are
herein incorporated by reference in their entirety.
1. INTRODUCTION
The present invention relates to the discovery,
identification, and characterization of novel human
polynucleotides encoding proteins that share sequence
similarity with mammalian ion channel proteins. The
invention encompasses the described polynucleotides, host
cell expression systems, the encoded proteins, fusion
proteins, polypeptides and peptides, antibodies to the
encoded proteins and peptides, and genetically engineered
animals that either lack or over express the disclosed
polynucleotides, antagonists and agonists of the proteins,
and other compounds that modulate the expression or
activity of the proteins encoded by the disclosed
polynucleotides that can be used for diagnosis, drug
screening, clinical trial monitoring, the treatment of
physiological disorders or diseases, and cosmetic or
nutriceutical applications.
2. BACKGROUND OF THE INVENTION
Ion channel proteins are integral membrane proteins
that mediate or facilitate the passage of materials across
the lipid bilayer. Given that ion transport has been
identified as an important regulator of mammalian
physiology, ion channel proteins are proven drug targets.
3. SUMMARY OF THE INVENTION
The present invention relates to the discovery,
identification, and characterization of nucleotides that
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encode novel human proteins, and the corresponding amino
acid sequences of these proteins. The novel human
proteins (NHPs) described for the first time herein share
structural similarity with mammalian ion channel proteins,
and particularly voltage-gated potassium channel proteins.
The novel human nucleic acid sequences described
herein, encode proteins/open reading frames (ORFs) of 545,
59 and 485 amino acids in length (see respectively, SEQ ID
NOS: 2, 4 and 7). As such, the novel polynucleotides
encode new mammalian ion channel proteins having
homologues and orthologs across a range of phyla and
species.
The invention also encompasses agonists and
antagonists of the described NHPs, including small
molecules, large molecules, mutant NHPs, or portions
thereof, that compete with native NHP, peptides, and
antibodies, as well' as nucleotide sequences that can be
used to inhibit the expression of the described NHPs
(e.g., antisense and ribozyme molecules, and gene or
regulatory sequence replacement constructs) or to enhance
the expression of the described NHP polynucleotides (e. g.,
expression constructs that place the described
polynucleotide under the control of a strong promoter
system), and transgenic animals that express a NHP
transgene, or "knock-outs" (which can be conditional) that
do not express a functional NHP. Knock-out mice can be
produced in several ways, one of which involves the use of
mouse embryonic stem cells ("ES cells") lines that contain
gene trap mutations in a murine homolog of at least one of
the described NHPs. When the unique NHP sequences
described in SEQ ID NOS:1-8 are "knocked-out" they provide
a method of identifying phenotypic expression of the
particular gene as well as a method of assigning function
to previously unknown genes. Additionally, the unique NHP
sequences described in SEQ ID NOS:1-8 are useful for the
identification of coding sequence, differentiating exons
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from introns and splice junctions as well as mapping a
unique gene to a particular chromosome.
Further, the present invention also relates to
processes for identifying compounds that modulate, i.e.,
act as agonists or antagonists, of NHP expression and/or
NHP activity that utilize purified preparations of the
described NHPs andlor NHP product, or cells expressing the
same. Such compounds can be used as therapeutic agents
for the treatment of any of a wide variety of symptoms
associated with biological disorders or imbalances.
4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES
The Sequence Listing provides the sequences of the
NHP ORFs encoding the described NHP amino acid sequences.
SEQ ID NOS:5 and 8 describe NHP ORFs as well as flanking
5' and 3' sequences.
5. DETAILED DESCRIPTION OF THE INVENTION
The NHPs described for the first time herein are
novel proteins that are expressed in various human
tissues. More particularly, NHP SEQ ID NOS:1-5 encode
novel proteins that can be found expressed in, inter alia,
human cell lines, human adipose, esophagus, cervix,
pericardium, and gene trapped human Cells.
The NHP described for the first time in SEQ ID NOS:6-
8 is a novel protein that is expressed in, inter alia,
human cell lines, human fetal brain, brain, pituitary,
cerebellum, spinal cord, thymus, spleen, lymph node, bone
marrow, trachea, lung, kidney, fetal liver, liver,
prostate, testis, thyroid, adrenal gland, pancreas,
salivary gland, stomach, small intestine, colon, skeletal
muscle, heart, uterus, placenta, mammary gland, adipose,
skin, esophagus, cervix, rectum, pericardium,
hypothalalmus, ovary, fetal kidney, and fetal lung cells.
The present invention encompasses the nucleotides
presented in the Sequence Listing, host cells expressing
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such nucleotides, the expression products of such
nucleotides, and: (a) nucleotides that encode mammalian
homologs of the described polynucleotides, including the
specifically described NHPs, and the NHP products;
(b) nucleotides that encode one or more portions of the
NHPs that correspond to functional domains, and the
polypeptide products specified by such nucleotide
sequences, including but not limited to the novel regions
of any active domain(s); (c) isolated nucleotides that
encode mutant versions, engineered or naturally occurring,
of the described NHPs in which all or a part of at least
one domain is deleted or altered, and the polypeptide
products specified by such nucleotide sequences, including
but not limited to soluble proteins and peptides in which
all or a portion of the signal (or hydrophobic
transmembrane) sequence is deleted; (d) nucleotides that
encode chimeric fusion proteins containing all or a
portion of a coding region of an NHP, or one of its
domains (e. g., a receptor or ligand binding domain,
accessory protein/self-association domain, etc.) fused to
another peptide or polypeptide; or (e) therapeutic or
diagnostic derivatives of the described polynucleotides
such as oligonucleotides,-antisense polynucleotides,
ribozymes, dsRNA, or gene therapy constructs comprising a
sequence first disclosed in the Sequence Listing. As
discussed above, the present invention includes: (a) the
human DNA sequences presented in the Sequence Listing (and
vectors comprising the same) and additionally contemplates
any nucleotide sequence encoding a contiguous NHP open
reading frame (ORF) that hybridizes to a complement of a
DNA sequence presented in the Sequence Listing under
highly stringent conditions, e.g., hybridization to
filter-bound DNA in 0.5 M NaHP04, 7o sodium dodecyl sulfate
(SDS), 1 mM EDTA at 65°C, and washing in 0.lxSSC/0.1% SDS
at 68°C (Ausubel F.M. et al., eds., 1989, Current
Protocols in Molecular Biology, Vol. I, Green Publishing
Associates, Inc., and John Wiley & sons, Inc., New York,
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at p. 2.10.3) and encodes a functionally equivalent gene
product. Additionally contemplated are any nucleotide
sequences that hybridize to the complement of a DNA
sequence that encodes and expresses an amino acid sequence
presented in the Sequence Listing under moderately
stringent condit~.ons, e.g., washing in 0.2xSSC/0.1o SDS at
42°C (Ausubel et al., 1989, supra), yet still encodes a
functionally equivalent NHP product. Functional
equivalents of a NHP include naturally occurring NHPs
present in other species and mutant NHPs whether naturally
occurring or engineered (by site directed mutagenesis,
gene shuffling, directed evolution as described in, for
example, U.S. Patent No. 5,837,458). The invention also
includes degenerate nucleic acid variants of the disclosed
NHP polynucleotide sequences.
Additionally contemplated are polynucleotides
encoding NHP ORFs, or their functional equivalents,
encoded by polynucleotide sequences that are about 99,
95, 90, or about 85 percent similar or identical to
corresponding regions of 'the nucleotide sequences of the
Sequence Listing (as measured by BLAST sequence comparison
analysis using, for example, the GCG sequence analysis
package using standard default settings).
The invention also includes nucleic acid molecules,
r
preferably DNA molecules, that hybridize to, and are
therefore the complements of, the described NHP nucleotide
sequences. Such hybridization conditions may be highly
stringent or less highly stringent, as described above.
In instances where the nucleic acid molecules are
deoxyoligonucleotides ("DNA oligos"), such molecules are
generally about 16 to about 100 bases long, or about 20 to
about 80, or about 34 to about 45 bases long, or any
variation or combination of sizes represented therein that
incorporate a contiguous region of sequence first
disclosed in the Sequence Listing. Such oligonucleotides
can be used in conjunction with the polymerase chain
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reaction (PCR) to screen libraries, isolate clones, and
prepare cloning and sequencing templates, etc.
Alternatively, such NHP oligonucleotides can be used
as hybridization probes for screening libraries, and
assessing gene expression patterns (particularly using a
micro array or high-throughput "chip" format).
Additionally, a series of the described NHP
oligonucleotide sequences, or the complements thereof, can
be used to represent all or a portion of the described NHP
sequences. An oligonucleotide or polynucleotide sequence
first disclosed in at least a portion of one or more of
the sequences of SEQ ID NOS: 1-8 can be used as a
hybridization probe in conjunction with a solid support
matrix/substrate (resins, beads, membranes, plastics,
polymers, metal or metallized substrates, crystalline or
polycrystalline substrates, etc.). Of particular note are
spatially addressable arrays (i.e., gene chips, microtiter
plates, etc.) of oligonucleotides and polynucleotides, or
corresponding oligopeptides and polypeptides, wherein at
least one of the biopolymers present on the spatially
addressable array comprises an oligonucleotide or
polynucleotide sequence first disclosed in at least one of
the sequences of SEQ ID NOS: 1-8, or an amino acid
sequence encoded thereby. Methods for attaching
biopolymers to, or synthesizing biopolymers on, solid
support matrices, and conducting binding studies thereon
are disclosed in, inter alia, U.S. Patent Nos. 5,700,637,
5,556,752, 5,744,305, 4,632,221, 5,445,934, 5,252,743,
4,713,326, 5,424,186, and 4,689,405 the disclosures of
which are herein incorporated by reference in their
entirety.
Addressable arrays comprising sequences first
disclosed in SEQ ID NOS:1-8 can be used to identify and
characterize the temporal and tissue specific expression.
of a gene. These addressable arrays incorporate
oligonucleotide sequences of sufficient length to confer
the required specificity, yet be within the limitations of
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the production technology. The length of these probes' is
within a range of between about 8 to about 2000
nucleotides. Preferably the probes consist of 60
nucleotides and more preferably 25 nucleotides from the
sequences first disclosed in SEQ ID NOS:1-8.
For example, a series of the described
oligonucleotide sequences, or the complements thereof, can
be used in chip format to represent all or a portion of
the described sequences. The oligonucleotides, typically
between about 16 to about 40 (or any whole number within
the stated range) nucleotides in length can partially
overlap each other and/or the sequence may be represented
using oligonucleotides that do not overlap. Accordingly,
the described polynucleotide sequences shall typically
comprise at least about two or three distinct
oligonucleotide sequences of at least about 8 nucleotides
in length that are each first disclosed in the described
Sequence Listing. Such oligonucleotide sequences can
begin at any nucleotide present within a sequence in the
Sequence Listing and proceed in either a sense (5'-to-3')
orientation vis-a-vis the described sequence or in an
antisense orientation.
Microarray-based analysis allows the discovery of
broad patterns of genetic activity, providing new
understanding of gene functions and generating novel and
unexpected insight into transcriptional processes and
biological mechanisms. The use of addressable arrays
comprising sequences first disclosed in SEQ ID NOS:1-8
provides detailed information about transcriptional
changes involved in a specific pathway, potentially
leading to the identification of novel components or gene
functions that manifest themselves as novel phenotypes.
Probes consisting of sequences first disclosed in SEQ
ID NOS:1-8 can also be used in the identification,
selection and validation of novel molecular targets for
drug discovery. The use of these unique sequences
permits the direct confirmation of drug targets and
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recognition of drug dependent changes in gene expression
that are modulated through pathways distinct from the
drugs intended target. These unique sequences therefore
also have utility in defining and monitoring both drug
action and toxicity.
As an example of utility, the sequences first
disclosed in SEQ ID NOS:1-8 can be utilized in microarrays
or other assay formats, to screen collections of genetic
material from patients who have a particular medical
condition. These investigations can also be carried out
using the sequences first disclosed in SEQ ID NOS:1-8 in
silico and by comparing previously collected genetic
databases and the disclosed sequences using computer
software known to those in the art.
Thus the sequences first disclosed in SEQ ID NOS:1-8
can be used to identify mutations associated with a
particular disease and also as a diagnostic or prognostic
assay.
Although the presently described sequences have been
specifically described using nucleotide sequence, it
should be appreciated that each of the sequences can
uniquely be described using any of a wide variety of
additional structural attributes, or combinations thereof.
For example, a given sequence can be described by the net
composition of the nucleotides present within a given
region of the sequence in conjunction with the presence of
one or more specific oligonucleotide sequences) first
disclosed in the SEQ ID NOS: 1-8. Alternatively, a
restriction map specifying the relative positions of
restriction endonuclease digestion sites, or various
palindromic or other specific oligonucleotide sequences
can be used to structurally describe a given sequence.
Such restriction maps, which are typically generated by
widely available computer programs (e. g., the University
of Wisconsin GCG sequence analysis package, SEQUENCHER
3.0, Gene Codes Corp., Ann Arbor, MI, etc.), can
optionally be used in conjunction with one or more
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discrete nucleotide sequences) present in the sequence
that can be described by the relative position of the
sequence relative to one or more additional sequences) or
one or more restriction sites present in the disclosed
sequence.
For oligonucleotide probes, highly stringent
conditions may refer, e.g., to washing in 6xSSC/0.05%
sodium pyrophosphate at 37°C (for 14-base oligos), 48°C
(for 17-base oligos), 55°C (for 20-base oligos), and 60°C
(for 23-base oligos). These nucleic acid molecules may
encode or act as NHP gene antisense molecules, useful, for
example, in NHP gene regulation (for and/or as antisense
primers in amplification reactions of NHP gene nucleic
acid sequences). With respect to NHP gene regulation,
such techniques can be used to regulate biological
functions. Further, such sequences may be used as part of
ribozyme and/or triple helix sequences that are also
useful for NHP gene regulation.
Inhibitory antisense or double stranded
oligonucleotides can additionally comprise at least one
modified base moiety which is selected from the group
including but not limited to 5-fluorouracil,
5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,
xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-
galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-
2-thiouracil,.beta-D-mannosylqueosine,
5'-methoxycarboxymethyluracil, 5-methoxyuracil,
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic
acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil,
4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid
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methylester, uracil-5-oxyacetic acid (v), 5-methyl-
2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil,
(acp3)w, and 2,6-diaminopurine.
The antisense oligonucleotide can also comprise at
least one modified sugar moiety selected from the group
including but not limited to arabinose, 2-fluoroarabinose,
xylulose, and hexose.
In yet another embodiment, the antisense
oligonucleotide will comprise at least one modified
phosphate backbone selected from the group consisting of a
phosphorothioate, a phosphorodithioate, a
phosphoramidothioate, a phosphoramidate, a
phosphordiamidate, a methylphosphonate, an alkyl
phosphotriester, and a formacetal or analog thereof.
In yet another embodiment, the antisense
oligonucleotide is an a-anomeric oligonucleotide. An a-
anomeric oligonucleotide forms specific double-stranded
hybrids with complementary RNA in which, contrary to the
usual (3-units, the strands run parallel to each other
(Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641).
The oligonucleotide is a 2'-0-methylribonucleotide (moue
et al., 1987, Nucl. Acids Res. 15:6131-6148), or a
chimeric RNA-DNA analogue (moue et al., 1987, FEBS Lett.
2.25:327-330). Alternatively, double stranded RNA can be
used to disrupt the expression and function of a targeted
NHP.
Oligonucleotides of the invention can be synthesized
by standard methods known in the art, e.g. by use of an
automated DNA synthesizer (such as are commercially
available from Biosearch, Applied Biosystems, etc.). As
examples, phosphorothioate oligonucleotides can be
synthesized by the method of Stein et al. (1988, Nucl.
Acids Res. 1 6:3209), and methylphosphonate
oligonucleotides can be prepared by use of controlled pore
glass polymer supports (Sarin et al., 1988, Proc. Natl.
Acad. Sci. U.S.A. 85:7448-7451), etc.
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Low stringency conditions are well known to those of
skill in the art, and will vary predictably depending on
the specific organisms from which the library and the
labeled sequences are derived. For guidance regarding
such conditions see, for example, Sambrook et al,, 1989,
Molecular Cloning, A Laboratory Manual (and periodic
updates thereof), Cold Springs Harbor Press, NY; and
Ausubel et al., 1989, Current Protocols in Molecular
Biology, Green Publishing Associates and Wiley
Interscience, NY.
Alternatively, suitably labeled NHP nucleotide probes
can be used to screen a human genomic library using
appropriately stringent conditions or by PCR. The
identification and characterization of human genomic
clones is helpful for identifying polymorphisms
(including, but not limited to, nucleotide repeats,
microsatellite alleles, single nucleotide polymorphisms,
or coding single nucleotide polymorphisms), determining
the genomic structure of a given locus/allele, and
designing diagnostic tests. For example, sequences
derived from regions adjacent to the intron/exon
boundaries of the human gene can be used to design primers
for use in amplification assays to detect mutations within
the exons, introns, splice sites (e. g., splice acceptor
and/or donor sites), etc., that can be used in diagnostics
and pharmacogenomics.
Further, a NHP gene homolog can be isolated from
nucleic acid from an organism of interest by performing
PCR using two degenerate or "wobble" oligonucleotide
primer pools designed on the basis, of amino acid sequences
within the NHP products disclosed herein. The template
for the reaction may be total RNA, mRNA, and/or cDNA
obtained by reverse transcription of mRNA prepared from
human or non-human cell lines or tissue known or suspected
to express an allele of a NHP gene.
The PCR product can be subcloned and sequenced to
ensure that the amplified sequences represent the sequence
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of the desired NHP gene. The PCR fragment can then be
used to isolate a full length cDNA clone by a variety of
methods. For example, the amplified fragment can be
labeled and used to screen a cDNA library, such as a
bacteriophage cDNA library. Alternatively, the labeled
fragment can be used to isolate genomic clones via the
screening of a genomic library.
PCR technology can also be used to isolate full
length cDNA sequences. For example, RNA can be isolated,
following standard procedures, from an appropriate
cellular or tissue source (i.e., one known, or suspected,
to express a NHP gene). A reverse transcription (RT)
reaction can be performed on the RNA using an
oligonucleotide primer specific for the most 5' end of the
amplified fragment for the priming of first strand
synthesis. The resulting RNA/DNA hybrid may then be
"tailed" using a standard terminal transferase reaction,
the hybrid may be digested with RNase H, and second strand
synthesis may then be primed with a complementary primer.
Thus, cDNA sequences upstream of the amplified fragment
can be isolated. For a review of cloning strategies that
can be used, see e.g., Sambrook et al., 1989, supra.
A cDNA encoding a mutant NHP gene can be isolated,
for example, by using PCR. In this case, the first cDNA
strand may be synthesized by hybridizing an oligo-dT
oligonucleotide to mRNA isolated from tissue known or
suspected to be expressed in an individual putatively
carrying a mutant NHP allele, and by extending the new
strand with reverse transcriptase. The second strand of
the cDNA is then synthesized using an oligonucleotide that
hybridizes specifically to the 5' end of the normal gene.
Using these two primers, the product is then amplified via
PCR, optionally cloned into a suitable vector, and
subjected to DNA sequence analysis through methods well
known to those of skill in the art. By comparing the DNA
sequence of the mutant NHP allele to that of a
corresponding normal NHP allele, the mutations)
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responsible for the loss or alteration of function of the
mutant NHP gene product can be ascertained.
Alternatively, a genomic library can be constructed
using DNA obtained from an individual suspected of or
known to carry a mutant NHP allele (e. g., a person
manifesting a NHP-associated phenotype such as, fox
example, obesity, high blood pressure, connective tissue
disorders, infertility, etc.), or a cDNA library can be
constructed using RNA from a tissue known, or suspected,
to express a mutant NHP allele. A normal NHP gene, or any
suitable fragment thereof, can then be labeled and used as
a probe to identify the corresponding mutant NHP allele in
such libraries. Clones containing mutant NHP gene
sequences can then be purified and subjected to sequence
analysis according to methods well known to those skilled
in the art.
Additionally, an expression library can be
constructed utilizing cDNA synthesized from, for example,
RNA isolated from a tissue known, or suspected, to express
a mutant NHP allele in an individual suspected of or known
to carry such a mutant allele. In this manner, gene
products made by the putatively mutant tissue can be
expressed and screened using standard antibody screening
techniques in conjunction with antibodies raised against a
normal NHP product, as described below. (For screening
techniques, see, for example, Harlow, E. and Lane, eds.,
1988, "Antibodies: A Laboratory Manual", Cold Spring
Harbor Press, Cold Spring Harbor, NY).
Additionally, screening can be accomplished by
screening with labeled NHP fusion proteins, such as, for
example, alkaline phosphatase-NHP or NHP-alkaline
phosphatase fusion proteins. In cases where a NHP
mutation results in an expressed gene product with altered
function (e. g., as a result of a missense or a frameshift
mutation), polyclonal antibodies to a NHP are likely to
cross-react with a corresponding mutant NHP gene product.
Library clones detected via their reaction with such
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labeled antibodies can be purified and subjected to
sequence analysis according to methods well known in the
art.
The invention also encompasses (a) DNA vectors that
contain any of the foregoing NHP coding sequences and/or
their complements (i.e., antisense); (b) DNA expression
vectors that contain any of the foregoing NHP coding
sequences operatively associated with a regulatory element
that directs the expression of the coding sequences (for
example, baculo virus as described in U.S. Patent No.
5,869,336 herein incorporated by reference);
(c) genetically engineered host cells that contain any of
the foregoing NHP coding sequences operatively associated
with a regulatory element that directs the expression of
the coding sequences in the host cell; and (d) genetically
engineered host cells that express an endogenous NHP gene
under the control of an exogenously introduced regulatory
element (i.e., gene activation). As used herein,
regulatory elements include, but are not limited to,
inducible and non-inducible promoters, enhancers,
operators and other elements known to those skilled in the
art that drive and regulate expression. Such regulatory
elements include but are not limited to the
cytomegalovirus (hCMV) immediate early gene, regulatable,
viral elements (particularly retroviral LTR promoters),
the early or late promoters of SV40 adenovirus, the lac
system, the trp system, the TAC system, the TRC system,
the major operator and promoter regions of phage lambda,
the control regions of fd coat protein, the promoter for
3-phosphoglycerate kinase (PGK), the promoters of acid
phosphatase, and the promoters of the yeast a-mating
factors.
The present invention also encompasses antibodies and
anti-idiotypic antibodies (including Fab fragments),
antagonists and agonists of the NHP, as well as compounds
or nucleotide constructs that inhibit expression of a NHP
gene (transcription factor inhibitors, antisense and
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ribozyme molecules, or gene or regulatory sequence
replacement constructs), or promote the expression of a
NHP (e. g., expression constructs in which NHP coding
sequences are operatively associated with expression
control elements such as promoters, promoter/enhancers,
etc . ) .
The NHPs or NHP peptides, NHP fusion proteins, NHP
nucleotide sequences, antibodies, antagonists and agonists
can be useful for the detection of mutant NHPs or
inappropriately expressed NHPs for the diagnosis of
disease. The NHP proteins or peptides, NHP fusion
proteins, NHP nucleotide sequences, host cell expression
systems, antibodies, antagonists, agonists and genetically
engineered cells and animals can be used for screening for
drugs (or high throughput screening of combinatorial
libraries) effective in the treatment of the symptomatic
or phenotypic manifestations of perturbing the normal
function of NHP in the body. The use of engineered host
cells and/or animals may offer an advantage in that such
systems allow not only for the identification of compounds
that bind to the endogenous receptor for an NHP, but can
also identify compounds that trigger NHP-mediated
activities or pathways.
Finally, the NHP products can be used as
therapeutics. For example, soluble derivatives such as
NHP peptides/domains corresponding to NHPs, NHP fusion
protein products (especially NHP-Ig fusion proteins, i.e.,
fusions of a NHP, or a domain of a NHP, to an IgFc), NHP
antibodies and anti-idiotypic antibodies (including Fab
fragments), antagonists or agonists (including compounds
that modulate or act on downstream targets in a NHP-
mediated pathway) can be used to directly treat diseases
or disorders. For instance, the administration of an
effective amount of soluble NHP, or a NHP-IgFc fusion
protein or an anti-idiotypic antibody (or its Fab) that
mimics the NHP could activate or effectively antagonize
the endogenous NHP receptor. Nucleotide constructs
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encoding such NHP products can be used to genetically
engineer host cells to express such products in vivo;
these genetically engineered cells function as
"bioreactors" in the body delivering a continuous supply
of a NHP, a NHP peptide, or a NHP fusion protein to the
body. Nucleotide constructs encoding functional NHPs,
mutant NHPs, as well as antisense and ribozyme molecules
can also be used in "gene therapy" approaches for the
modulation of NHP expression. Thus, the invention also
encompasses pharmaceutical formulations and methods for
treating biological disorders.
Various aspects of the invention are described in
greater detail in the subsections below.
5.1 THE NHP SEQUENCES '
The cDNA sequences and the corresponding deduced
amino acid sequences of the described NHPs are presented
in the Sequence Listing. The NHP nucleotide sequences
described in SEQ ID NOS:1-5 were obtained from clustered
human gene trapped sequences, ESTs, and cDNAs generated
from human testis, prostate, fetal brain, adipose mRNA
(Clontech, Palo Alto, CA, Edge Biosystems, Gaithersburg,
MD). A polymorphism was also identified including an A-
or-G transition in the sequence region corresponding to,
for example, nucleotide number 1,541 of SEQ ID N0:1 which
can result in either a glu or gly being present in the
corresponding amino acid sequence region represented by,
for example, amino acid position number 514 of SEQ ID
N0:2. The NHP sequences described in SEQ ID NOS: 1-5 are
apparently encoded on human chromosome 9.
The NHP nucleotide sequences described in SEQ ID
NOS:6-8 were obtained from clustered sequence from cDNA
clones from a human brain cDNA library and products from
human cerebellum mRNA (Clontech, Palo Alto, CA, Edge
Biosystems, Gaithersburg, MD). Several polymorphisms were
identified including an A-or-G transition in the sequence
region corresponding to, for example, nucleotide position
16
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number 271 of SEQ ID N0:6 (resulting in a asn or glu being
present at corresponding amino acid position 91 of, for
example, SEQ ID N0:7); a C-or-G transversion in the
sequence region corresponding to, for example, nucleotide
number 364 of SEQ ID N0:6 resulting in an arg or gly being
present at corresponding amino acid position 122 of, for
example, SEQ ID N0:7); a G-or-A transition in the sequence
region corresponding to, for example, nucleotide position
number 367 of SEQ ID N0:6 (resulting in a gly or ser being
present at corresponding amino acid position 123 of, for
example, SEQ ID N0:7); a T-or-A transversion in the
sequence region corresponding to, for example, nucleotide
number 699 of SEQ ID N0:6 resulting in a ser or asn being
present at corresponding amino acid position 233 of, for
example, SEQ ID N0:7); a T-or-C transition in the sequence
region corresponding to, for example, nucleotide position
number 1013 of SEQ ID N0:6 (resulting in a ile or thr
being present at corresponding amino acid position 338 of,
for example, SEQ ID N0:7); a G-or-A transition in the
sequence region corresponding to, for example, nucleotide
number 1015 of SEQ ID N0:6 resulting in an val or met
being present at corresponding amino acid position 339 of,
for example, SEQ ID N0:7); a C-or-A transversion in the
sequence region corresponding to, for example, nucleotide
position number 1397 of SEQ ID N0:6 (resulting in a pro or
his being present at corresponding amino acid position 466
of, for example, SEQ ID N0:7); a G-or-C transversion in
the sequence region corresponding to, for example,
nucleotide number 1405 of SEQ ID N0:6 resulting in a asp
or his being present at corresponding amino acid position
469 of, for example, SEQ ID N0:7); anal a G-or-T transition
in the sequence region corresponding to, for example,
nucleotide number 1419 of SEQ ID N0:6 resulting in a glu
or asp being present at corresponding amino acid position
473 of, for example, SEQ ID N0:7).
An additional application of the described novel
human polynucleotide sequences is their use in the
17
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molecular mutagenesis/evolution of proteins that are at
least partially encoded by the described novel sequences
using, for example, polynucleotide shuffling or related
methodologies. Such approaches are described in U.S.
Patents Nos. 5,830,721 and 5,837,458 which are herein
incorporated by reference in their entirety.
NHP gene products can also be expressed in transgenic
animals. Animals of any species, including, but not
limited to, worms, mice, rats, rabbits, guinea pigs, pigs,
micro-pigs, birds, goats, and non-human primates, e.g.,
baboons, monkeys, and chimpanzees may be used to generate
NHP transgenic animals.
Any technique known in the art may be used to
introduce a NHP transgene into animals to produce the
founder lines of transgenic animals. Such techniques
include, but are not limited to pronuclear microinjection
(Hoppe, P.C. and Wagner, T.E., 1989, U.S. Pat. No.
4,873,191); retrovirus mediated gene transfer into germ
lines (Van der Putten et al., 1985, Proc. Natl. Acad.
Sci., USA X2:6148-6152); gene targeting in embryonic stem
cells (Thompson et al., 1989, Cell 56:313-321);
electroporation of embryos (Lo, 1983, Mol Cell. Biol.
3:1803-1814); and sperm-mediated gene transfer (Lavitrano
et al., 1989, Cell 57:717-723); etc. For a review of such
techniques, see Gordon, 1989, firansgenic Animals, Tntl.
Rev. Cytol. 115:171-229, which is incorporated by
reference herein in its entirety.
The present invention provides for transgenic animals
that carry the NHP transgene in all their cells, as well
as animals which carry the transgene in some, but not all
their cells, i.e., mosaic animals or somatic cell
transgenic animals. The transgene may be integrated as a
single transgene or in concatamers, e.g., head-to-head
tandems or head-to-tail tandems. The transgene may also
be selectively introduced into and activated in a
particular cell type by following, for example, the
teaching of Lasko et al., 1992, Proc. Natl. Acad. Sci. USA
18 °
CA 02417642 2003-O1-28
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89:6232-6236. The regulatory sequences required for such
a cell-type specific activation will depend upon the
particular cell type of interest, and will be apparent to
those of skill in the art.
When it is desired that a NHP transgene be integrated
into the chromosomal site of the endogenous NHP gene, gene
targeting is preferred. Briefly, when such a technique is
to be utilized, vectors containing some nucleotide
sequences homologous to the endogenous NHP gene are
designed for the purpose of integrating, via homologous
recombination with chromosomal sequences, into and
disrupting the function of the nucleotide sequence of the
endogenous NHP gene (i.e., "knockout" animals).
The transgene can also be selectively introduced into
a particular cell type, thus inactivating the endogenous
NHP gene in only that cell type, by following, for
example, the teaching of Gu et al., 1994, Science,
2 65:103-106. The regulatory sequences required for such a
cell-type specific inactivation will depend upon the
particular cell type of interest, and will be apparent to~
those of skill in the art.
Once transgenic animals have been generated, the
expression of the recombinant NHP gene may be assayed
utilizing standard techniques. Initial screening may be
25~ accomplished by Southern blot analysis or PCR techniques
to analyze animal tissues to assay whether integration of
the transgene has taken place. The level of mRNA
expression of the transgene in the tissues of the
transgenic animals may also be assessed using techniques
which include but are not limited to Northern blot
analysis of tissue samples obtained from the animal, in
situ hybridization analysis, and RT-PCR. Samples of NHP
gene-expressing tissue, may also be evaluated
immunocytochemically using antibodies specific for the NHP
transgene product.
19
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5.2 NHPS AND NHP POLYPEPTIDES
NHPs, polypeptides, peptide fragments, mutated,
truncated, or deleted forms of the NHPs, and/or NHP fusion
proteins can be prepared for a variety of uses. These
uses include but are not limited to the generation of
antibodies, as reagents in diagnostic assays, the
identification of other cellular gene products related to
a NHP, as reagents in assays for screening for compounds
that can be as pharmaceutical reagents useful in the
therapeutic treatment of mental, biological, or medical
disorders and diseases. Given the similarity information
and expression data, the described NHPs can be targeted
(by drugs, oligos, antibodies, etc,) in order to treat
disease, or to therapeutically augment the efficacy of,
for example, chemotherapeutic agents used in the treatment
of breast or prostate cancer.
The Sequence Listing discloses the amino acid
sequences encoded by the described NHP polynucleotides.
The NHPs typically display have initiator methionines in
DNA sequence contexts consistent with a translation
initiation site.
The NHP amino acid sequences of the invention include
the amino acid sequence presented in the Sequence Listing
as well as analogues and derivatives thereof. Further,
corresponding NHP homologues from other species are
encompassed by the invention. In fact, any NHP protein
encoded by the NHP nucleotide sequences described above
are within the scope of the invention, as are any novel
polynucleotide sequences encoding all or any novel portion
of an amino acid sequence presented in the Sequence
Listing. The degenerate nature of the genetic code is
well known, and, accordingly, each amino acid presented in
the Sequence Listing, is generically representative of the
well known nucleic acid "triplet" codon, or in many cases
codons, that can encode the amino acid. As such, as
contemplated herein, the amino acid sequences presented in
CA 02417642 2003-O1-28
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the Sequence Listing, when taken together with the genetic
code (see, for example, Table 4-l at page 109 of
"Molecular Cell Biology", 1986, J. Darnell et al, eds.,
Scientific American Books, New York, NY, herein
incorporated by reference) are generically representative
of all the various permutations and combinations of
nucleic acid sequences that can encode such amino acid
sequences.
The invention also encompasses proteins that are
functionally equivalent to the NHPs encoded by the
presently described nucleotide sequences as judged by any
of a number of criteria, including, but not limited to,
the ability to bind and cleave a substrate of a NHP, or
the ability to effect an identical or complementary
downstream pathway, or a change in cellular metabolism
(e. g., proteolytic activity, ion flux, tyrosine
phosphorylation, etc.). Such functionally equivalent NHP
proteins include, but are not limited to, additions or
substitutions of amino acid residues within the amino acid
sequence encoded by the NHP nucleotide sequences described
above, but which result in a silent change, thus producing
a functionally equivalent gene product. Amino acid
substitutions may be made on the basis of similarity in
polarity, charge, solubility, hydrophobicity,
hydrophilicity, and/or the amphipathic nature of the
residues involved. For example, nonpolar (hydrophobic)
amino acids include alanine, leucine, isoleucine, valine,
proline, phenylalanine, tryptophan, and methionine; polar
neutral amino acids include glycine, serine, threonine,
cysteine, tyrosine, asparagine, and glutamine; positively
charged (basic) amino acids include arginine, lysine, and
histidine; and negatively charged (acidic) amino acids
include aspartic acid and glutamic acid.
A variety of host-expression vector systems can be
used to express the NHP nucleotide sequences of the
invention. Where, as in the present instance, the NHP
peptide or polypeptide is thought to be membrane protein,
21
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the hydrophobic regions of the protein can be excised and
the resulting soluble peptide or polypeptide can be
recovered from the culture media. Such expression systems
also encompass engineered host cells that express a NHP,
or functional equivalent, in situ. Purification or
enrichment of a NHP from such expression systems can be
accomplished using appropriate detergents and lipid
micelles and methods well known to those skilled in the
art. However, such engineered host cells themselves may
be used in situations where it is important not only to
retain the structural and functional characteristics of
the NHP, but to assess biological activity, e.g., in drug
screening assays.
The expression systems that may be used for purposes
of the invention include but are not limited to
microorganisms such as bacteria (e.g., E, coli, B.
subtilis) transformed with recombinant bacteriophage DNA,
plasmid DNA or cosmid DNA expression vectors containing
NHP nucleotide sequences; yeast (e. g., Saccharomyces,
Pichia) transformed with recombinant yeast expression
vectors containing NHP nucleotide sequences; insect cell
systems infected with recombinant virus expression vectors
(e. g., baculovirus) containing NHP sequences; plant cell
systems infected with recombinant virus expression vectors
(e. g., cauliflower mosaic virus, CaMV; tobacco mosaic
virus, TMV) or transformed with. recombinant plasmid
expression vectors (e.g., Ti plasmid) containing NHP
nucleotide sequences; or mammalian cell systems (e.g.,
COS, CHO, BHK, 293, 3T3) harboring recombinant expression
constructs containing promoters derived from the genome of
mammalian cells (e. g., metallothionein promoter) or from
mammalian viruses (e.g., the adenovirus late promoter; the
vaccinia virus 7.5K promoter).
In bacterial systems, a number of expression vectors
may be advantageously selected depending upon the use
intended for the NHP product being expressed. For
example, when a large quantity of such a protein is to be
22
CA 02417642 2003-O1-28
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produced for the generation of pharmaceutical compositions
of or containing NHP, or for raising antibodies to a NHP,
vectors that direct the expression of high levels of
fusion protein products that are readily purified may be
desirable. Such vectors include, but are not limited, to
the E. coli expression vector pUR278 (Ruther et al., 1983,
EMBO J. 2:1791), in which a NHP coding sequence may be
ligated individually into the vector in frame with the
lack coding region so that a fusion protein is produced;
pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res.
13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem.
264:5503-5509); and the like. pGEX vectors (Pharmacia or
American Type Culture Collection) can also be used to
express foreign polypeptides as fusion proteins with
glutathione S-transferase (GST). In general, such fusion
proteins are soluble and can easily be purified from lysed
cells by adsorption to glutathione-agarose beads followed
by elution in the presence of free glutathione. The PGEX
vectors are designed to include thrombin or factor Xa
protease cleavage sites so that the cloned target gene
product can be released from the GST moiety.
In an insect system, Autographa californica nuclear
polyhidrosis virus (AcNPV) is used as a vector to express
foreign genes. The virus grows in Spodoptera frugiperda
cells. A NHP coding sequence may be cloned individually
into non-essential regions (for example the polyhedrin
gene) of the virus and placed under control of an AcNPV
promoter (for example the polyhedrin promoter).
Successful insertion of NHP coding sequence will result in
inactivation of the polyhedrin gene and production of non
occluded recombinant virus (i.e.. virus lacking the
' proteinaceous coat coded for by the polyhedrin gene).
These recombinant viruses are then used to infect
Spodoptera frugiperda cells in which the inserted sequence
is expressed (e. g., see Smith et al., 1983, J. Virol.
46:584; Smith, U.S. Patent No. 4,215,051).
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In mammalian host cells, a number of viral-based
expression systems may be utilized. In cases where an
adenovirus is used as an expression vector, the NHP
nucleotide sequence of interest may be ligated to an
adenovirus transcription/translation control complex,
e.g.,. the late promoter and tripartite leader sequence.
This chimeric gene may then be inserted in the adenovirus
genome by in vitro or in vivo recombination. Insertion in
a non-essential region of the viral genome (e. g., region
E1 or~E3) will result in a recombinant virus that is
viable and capable of expressing a NHP product in infected
hosts (e. g., See Logan & Shenk, 1984, Proc. Natl. Acad.
Sci. USA 81:3655-3659). Specific initiation signals may
also be required for efficient translation of inserted NHP
nucleotide sequences. These signals include the ATG
initiation codon and adjacent sequences. In cases where
an entire NHP gene or cDNA, including its own initiation
codon and adjacent sequences, is inserted into the
appropriate expression vector, no additional translational
control signals may be needed. However, in cases where
only a portion of a NHP coding sequence is inserted,
exogenous translational control signals, including,
perhaps, the ATG initiation codon, must be provided.
Furthermore, the initiation codon must be in phase with
the reading frame of the desired coding sequence to ensure
translation of the entire insert. These exogenous
translational control signals and initiation codons can be
of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion
of appropriate transcription enhancer elements,
transcription terminators, etc. (See Bitter et al., 1987,
Methods in Enzymol. 153:516-544).
In addition, a host cell strain may be chosen that
modulates the expression of the inserted sequences, or
modifies and processes the gene product in the specific
fashion desired. Such modifications (e. g., glycosylation)
and processing (e.g., cleavage) of protein products may be
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CA 02417642 2003-O1-28
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important for the function of the protein. Different host
cells have characteristic and specific mechanisms for the
post-translational processing and modification of proteins
and gene products. Appropriate cell lines or host systems
can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery
for proper processing of the primary transcript,
glycosylation, and phosphorylation of the gene product may
be used. Such mammalian host cells include, but are not
limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3,
WT38, and in particular, human cell lines.
For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example,
cell lines which stably express the NHP sequences
described above can be engineered. Rather than using
expression vectors which contain viral origins of
replication, host cells can be transformed with DNA
controlled by appropriate expression control elements
(e. g., promoter, enhancer sequences, transcription
terminators, polyadenylation sites, etc.), and a
selectable marker. Following the introduction of the
foreign DNA, engineered cells may be allowed to grow for
1-2 days in an enriched media, and then are switched to a
selective media. The selectable marker in the recombinant
plasmid confers resistance to the selection and allows
cells to stably integrate the plasmid into their
chromosomes and grow to form foci which in turn can be
cloned and expanded into cell lines. This method may
advantageously be used to engineer cell lines which
express the NHP product. Such engineered cell lines may
be particularly useful in screening and evaluation of
compounds that affect the endogenous activity of the NHP
product.
A number of selection systems may be used, including
but not limited to the herpes simplex virus thymidine
kinase (Wigler, et al., 1977, Cell 11:223), hypoxanthine-
CA 02417642 2003-O1-28
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guanine phosphoribosyltransferase (Szybalska & Szybalski,
1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine
phosphoribosyltransferase (Lowy, et al., 1980, Cell
22:817) genes can be employed in tk-, hgprt- or aprt- cells,
respectively. Also, antimetabolite resistance can be used
as the basis of selection for the following genes: dhfr,
which confers resistance to methotrexate (Wigler, et al.,
1980, Natl. Acad. Sci. USA 77:3567; 0'Hare, et al., 1981,
Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers
resistance to mycophenolic acid (Mulligan & Berg, 1981,
Proc. Natl. Acad. Sci. USA 7:2072); neo, which confers
resistance to the aminoglycoside G-418 (Colberre-Garapin,
et al., 1981, J. Mol. Biol. 250:1); and hygro, which
confers resistance to hygromycin ~(Santerre, et al., 1984,
Gene 30:147).
Alternatively, any fusion protein can be readily
purified by utilizing an antibody specific for the fusion
protein being expressed. For example, a system described
by Janknecht et al. allows for the ready purification of
non-denatured fusion proteins expressed in human cell
lines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA
88:8972-8976). In this system, the gene of interest is
subcloned into a vaccinia recombination plasmid such that
the gene's open reading frame is translationally fused to
an amino-terminal tag consisting of six histidine
residues. Extracts from cells infected with recombinant
vaccinia virus are loaded onto Nip+~nitriloacetic acid-
agarose columns and histidine-tagged proteins are
selectively eluted with imidazole-containing buffers.
Also encompassed by the present invention are fusion
proteins that direct the NHP to a target organ and/or
facilitate transport across the membrane into the cytosol.
Conjugation of NHPs to antibody molecules or their Fab
fragments could be used to target cells bearing a
particular epitope. Attaching the appropriate signal
sequence to the NHP would also transport the NHP to the
desired location within the cell. Alternatively targeting
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of NHP or its nucleic acid sequence might be achieved
using liposome or lipid complex based delivery systems.
Such technologies are described in "Liposomes:A Practical
Approach", New,RRC ed., Oxford University Press, New York
and in U.S. Patents Nos. 4,594,595, 5,459,127, 5,948,767
and 6,110,490 and their respective disclosures which are
herein incorporated by reference in their entirety.
Additionally embodied are novel protein constructs
engineered in such a way that they facilitate transport of
the NHP to the target site or desired organ. This goal may
be achieved by coupling of the NHP to a cytokine or other
ligand that provides targeting specificity, and/or to a
protein transducing domain (see generally U.S.
applications Ser. No. 60/111,701 and 60/056,713, both of
which are herein incorporated by reference, for examples
of such transducing sequences) to facilitate passage
across cellular membranes if needed and can optionally be
engineered to include nuclear localization sequences when
desired.
5.3 ANTIBODIES TO NHP PRODUCTS
Antibodies that specifically recognize one or more
epitopes of a NHP, or epitopes of conserved variants of a
NHP, or peptide fragments of a NHP are also encompassed by
the invention. Such antibodies include but are not
limited to polyclonal antibodies, monoclonal antibodies
(mAbs), humanized or chimeric antibodies, single chain
antibodies, Fab fragments, F(ab')~ fragments, fragments
produced by a Fab expression library, anti-idiotypic
(anti-Id) antibodies, and epitope-binding fragments of any
of the above.
The antibodies of the invention may be used, for
example, in the detection of NHP in a biological sample
and may, therefore, be utilized as part of a diagnostic or
prognostic technique whereby patients may be tested for
abnormal amounts of NHP. Such antibodies may also be
utilized in conjunction with, for example, compound
27
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screening schemes for the evaluation of the effect of test
compounds on expression and/or activity of a NHP gene
product. Additionally, such antibodies can be used in
conjunction gene therapy to, for example, evaluate the
normal and/or engineered NHP-expressing cells prior to
their introduction into the patient. Such antibodies may
additionally be used as a method for the inhibition of
abnormal NHP activity. Thus, such antibodies may,
therefore, be utilized as part of treatment methods.
For the production of antibodies, various host
animals may be immunized by injection with a NHP, an NHP
peptide (e.g., one corresponding to a functional domain of
an NHP), truncated NHP polypeptides (NHP in which one or
more domains have been deleted), functional equivalents of
the NHP or mutated variant of the NHP. Such host animals
may include but are not limited to pigs, rabbits, mice,
goats, and rats, to name but a few. Various adjuvants may
be used to increase the immunological response, depending
on the host species, including but not limited to Freund's
adjuvant (complete and incomplete), mineral salts such as
aluminum hydroxide or aluminum phosphate, chitosan,
surface active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, and
potentially useful human adjuvants such as BCG (bacille
Calmette-Guerin) and Corynebacterium parvum.
Alternatively, the immune response could be enhanced by
combination and or coupling with molecules such as keyhole
limpet hemocyanin, tetanus toxoid, diphtheria toxoid,
ovalbumin, cholera toxin or fragments thereof. Polyclonal
antibodies are heterogeneous populations of antibody
molecules derived from the sera of the immunized animals.
Monoclonal antibodies, which are homogeneous
populations of antibodies to a particular antigen, can be
obtained by any technique which provides for the
production of antibody molecules by continuous cell lines
in culture. These include, but are not limited to, the
hybridoma technique of Kohler and Milstein, (1975, Nature
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256:495-497; and U.S. Patent No. 4,376,110), the human B-
cell hybridoma technique (Kosbor et al., 1983, Immunology
Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA
80:2026-2030), and the EBV-hybridoma technique (Cole et
al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan
R. Liss, Inc., pp. 77-96). Such antibodies may be of any
immunoglobulin class including IgG, IgM, IgE, IgA, IgD and
any subclass thereof. The hybridoma producing the mAb of
this invention may be cultivated in vitro or in vivo.
Production of high titers of mAbs in vivo makes this the
presently preferred method of production.
In addition, techniques developed for the production
of "chimeric antibodies" (Morrison et al., 1984, Proc.
Natl. Acad. Sci., 81:6851-6855; Neuberger et al., 1984,
Nature, 312:604-608; Takeda et al., 1985, Nature, 314:452-
454) by splicing the genes from a mouse antibody molecule
of appropriate antigen specificity together with genes
from a human antibody molecule of appropriate biological
activity can. be used. A chimeric antibody is a molecule
in which different portions are derived from different
animal species, such as those having a variable region
derived from a murine mAb and a human immunoglobulin
constant region. Such technologies are described in U.S.
Patents Nos. 6,075,181 and 5,877,397 and their respective
disclosures which are herein incorporated by reference in
their entirety. Also encompassed by the present invention.
is the use of fully humanized monoclonal antibodies as
described in US Patent No. 6,150,584 and respective
disclosures which are herein incorporated by reference in
their entirety.
Alternatively, techniques described for the
production of single chain antibodies (U. S. Patent
4,946,778; Bird, 1988, Science 242:423-426; Huston et al.,
1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et
al., 1989, Nature 341:544-546) can be adapted to produce
single chain antibodies against NHP gene products. Single
chain antibodies are formed by linking the heavy and light
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chain fragments of the Fv region via an amino acid bridge,
resulting in a single chain polypeptide.
Antibody fragments which recognize specific epitopes
may be generated by known techniques. For example, such
fragments include, but are not limited to: the F(ab')z
fragments which can be produced by pepsin digestion of the
antibody molecule and the Fab fragments which can be
generated by reducing the disulfide bridges of the F(ab')2
fragments. Alternatively, Fab expression libraries may be
constructed (Ruse et al., 1989, Science, 246:1275-1281) to
allow rapid and easy identification of monoclonal Fab
fragments with the desired specificity.
Antibodies to a NHP can, in turn, be utilized to
generate anti-idiotype antibodies that "mimic" a given
NHP, using techniques well known to those skilled in the
art. (See, e.g., Greenspan & Bona, 1993, FASEB J
7(5):437-444; and Nissinoff, 1991, J. Immunol.
247 0):2429-2438). For example antibodies which bind to a
NHP domain and competitively inhibit the binding of NHP to
its cognate receptor can be used to generate anti
idiotypes that "mimic" the NHP and, therefore, bind and
activate or neutralize a receptor. Such anti-idiotypic
antibodies or Fab fragments of such anti-idiotypes can be
used in therapeutic regimens involving a NHP mediated
pathway.
The present invention is not to be limited in scope
by the specific embodiments described herein, which are
intended as single illustrations of individual aspects of
the invention, and functionally equivalent methods and
components are within the scope of the invention. Indeed,
various modifications of the invention, in addition to
those shown and described herein will become apparent to
those skilled in the art from the foregoing description.
Such modifications are intended to fall within the scope
of the appended claims. All cited publications, patents,
and patent applications are herein incorporated by
reference in their entirety.
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SEQUENCE LISTING
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gccagcatccacggctggacagagggcaactataactactacatcgaggaagacgaagac180
ggsgaggaggaggaccagtggaaggacgacctggcagaagaggaccagcaggcaggggag240
gtcaccaccgccaagcccgagggccccagcgaccctccggccctgctgtccacgctgaat300
gtgaacgtgggtggccacagctaccagctggactactgcgagctggccggcttccccaag360
acgcgcctaggtcgcctggccacctccaccagccgcagccgccagctaagcctgtgcgac420
gactacgaggagcagacagacgaatacttcttcgaccgcgacccggccgtcttccagctg480
gtctacaatttctacctgtccggggtgctgctggtgctcgacgggctgtgtccgcgccgc540
ttcctggaggagctgggctactggggcgtgcggctcaagtacacgccacgctgctgccgc600
atctgcttcgaggagcggcgcgacgagctgagcgaacggctcaagatccagcacgagctg660
cgcgcgcaggcgcaggtcgaggaggcggaggaactcttccgcgacatgcgcttctacggc720
CCgCagCggCgccgcctctggaacctcatggagaagccrttctcctcggtggccgccaag780
gccatcggggtggcctccagcaccttcgtgctcgtctccgtggtggcgctggcgctcaac840
accgtggaggagatgcagcagcactcggggcagggcgagggcggcccagacctgcggccc900
atcctggagcacgtggagatgctgtgcatgggcttcttcacgctcgagtacctgctgcgc960
ctagcctccacgcccgacctgaggcgcttcgcgcgcagcgccctcaacctggtggacctg1020
gtggccatcctgccgctctaccttcagctgctgctcgagtgcttcacgggcgagggccac1080
caacgcggccagacggtgggcagcgtgggtaaggtgggtcaggtgttgcgcgtcatgcgc1140
ctcatgcgcatcttccgcatcctcaagctggcgcgccactccaccggactgcgtgccttc1200
ggcttcacgctgcgccagtgctaccagcaggtgggctgcctgctgctcttcatcgccatg1260
ggcatcttcactttctctgcggctgtctactctgtggagcacgatgtgcccagcaccaac1320
ttcactaccatcccccactcctggtggtgggccgcggtgagcatctccaccgtgggctac1380
ggagayatgtacccagagacccacctgggcaggttttttgccttcctctgcattgctttt1440
gggatcattctcaacgggatgcccatttccatcctctacaacaagttttctgattactac1500
agcaagctgaaggcttatgagtataccaccatacgcagggrgaggggagaggtgaacttc1560
atgcagagagccagaaagaagatagctgagtgtttgcttggaagcaacccacagctcacc1620
ccaagacaagagaattag 1638
<210>
2
<211>
545
<212>
PRT
<213> Sapiens
homo
<400>
2
Met Leu Ser Tyr Pro Trp
Lys Arg
Gln
Ser
Glu
Arg
Arg
Arg
Ser
Trp
1 5 10 15
1l6
CA 02417642 2003-O1-28
WO 02/10389 PCT/USO1/23827
Asn Thr Thr Glu Asn Glu Gly Ser Gln His Arg Arg Ser Ile Cys Ser
20 25 30
Leu Gly Ala Arg Ser Gly Ser Gln Ala Ser Ile His Gly Trp Thr Glu
35 . 40 45
Gly Asn Tyr Asn Tyr Tyr Tle Glu Glu Asp Glu Asp Gly Glu Glu Glu
50 55 60
Asp Gln Trp Lys Asp Asp Leu Ala Glu Glu Asp Gln Gln Ala Gly Glu
65 70 75 80
Val Thr Thr Ala Lys Pro G1u Gly Pro Ser Asp Pro Pro Ala Leu Leu
85 90 95
Ser Thr Leu Asn Val Asn Va1 Gly Gly His Ser Tyr Gln Leu Asp Tyr
100 105 110
Cys Glu Leu Ala Gly Phe Pro Lys Thr Arg Leu Gly Arg Leu Ala Thr
115 120 125
Ser Thr Ser Arg Ser Arg Gln Leu Ser Leu Cys Asp Asp Tyr Glu Glu
130 135 140
Gln Thr Asp Glu Tyr Phe Phe Asp Arg Asp Pro Ala Val Phe Gln Leu
145 150 155 160
Val Tyr Asn Phe Tyr Leu Ser Gly Val Leu Leu Val Leu Asp Gly Leu
165 170 175
Cys Pro Arg Arg Phe Leu Glu Glu Leu Gly Tyr Trp Gly Val Arg Leu
180 185 190
Lys Tyr Thr Pro Arg Cys Cys Arg Ile Cys Phe Glu Glu Arg Arg Asp
195 200 205
Glu Leu Ser Glu Arg Leu Lys Ile Gln His Glu Leu Arg Ala Gln Ala
210 215 220
Gln Val Glu Glu Ala Glu Glu Leu Phe Arg Asp Met Arg Phe Tyr Gly
225 230 235 240
Pro Gln Arg Arg Arg Leu Trp Asn Leu Met Glu Lys Pro Phe Ser Ser
245 250 255
Val Ala Ala Lys Ala Ile Gly Val Ala Ser Ser Thr Phe Val Leu Val
260 265 270
Ser Val Val Ala Leu Ala Leu Asn Thr Val Glu Glu Met Gln Gln His
275 280 285
Ser Gly Gln Gly Glu Gly Gly Pro Asp Leu Arg Pro Ile Leu Glu His
290 295 300
Val Glu Met Leu Cys Met Gly Phe Phe Thr Leu Glu Tyr Leu Leu Arg
305 310. 315 320
Leu Ala Ser Thr Pro Asp Leu Arg Arg Phe Ala Arg Ser Ala Leu Asn
325 330 335
Leu Val Asp Leu Val Ala Ile Leu Pro Leu Tyr Leu Gln Leu Leu Leu
340 345 350
Glu Cys Phe Thr Gly Glu Gly His Gln Arg Gly Gln Thr Val Gly Ser
355 360 365
Val Gly Lys Val Gly Gln Val Leu Arg Val Met Arg Leu Met Arg Ile
370 375 380
Phe Arg Ile Leu Lys Leu Ala Arg His Ser Thr Gly Leu Arg Ala Phe
385 390 395 400
Gly Phe Thr Leu Arg Gln Cys Tyr Gln Gln Val Gly Cys Leu Leu Leu
405 410 415
Phe Ile Ala Met Gly Ile Phe Thr Phe Ser Ala Ala Val Tyr Ser Val
420 425 430
Glu His Asp Val Pro Ser Thr Asn Phe Thr Thr Ile Pro His Ser Trp
435 440 445
Trp Trp Ala Ala Val Ser Ile Ser Thr Val Gly Tyr Gly Asp Met Tyr
450 455 460
Pro Glu Thr His Leu Gly Arg Phe Phe Ala Phe Leu Cys Ile Ala Phe
465 470 475 480
Gly Ile Ile Leu Asn Gly Met Pro Ile Ser Ile Leu Tyr Asn Lys Phe
485 490 495
Ser Asp Tyr Tyr Ser Lys Leu Lys Ala Tyr Glu Tyr Thr Thr Ile Arg
500 505 510
Arg Glu Arg Gly Glu Val Asn Phe Met Gln Arg Ala Arg Lys Lys Ile
2l6
CA 02417642 2003-O1-28
WO 02/10389 PCT/USO1/23827
515 520 525
Ala GIu Cys Leu Leu Gly Ser Asn Pro Gln Leu Thr Pro Arg Gln Glu
530 535 540
Asn
545
<210> 3
<211> 180
<212> DNA
<213> homo Sapiens
<400> 3
atgctcaaac agagtgagag gagacggtcc tggagctaca ggccctgtcc ggggtgctgc 60
tggtgctcga cgggctgtgt ccgcgccgct tcctggagga gctgggctac tggggcgtgc 120
ggctcaagta cacgccacgc tgctgccgca tctgcttcga ggagcggcgc gacgagctga 180
<210> 4
<211> 59
<212> PRT
<213> homo Sapiens
<400> 4
Met Leu Lys Gln Ser Glu Arg Arg Arg Ser Trp Ser Tyr Arg Pro Cys
1 5 10 15
Pro Gly Cys Cys Trp Cys Ser Thr Gly Cys Val Arg Ala Ala Ser Trp
20 25 30
Arg Ser Trp Ala Thr~Gly Ala Cys Gly Ser Ser Thr Arg His Ala Ala
35 40 45
Ala Ala Ser Ala Ser Arg Ser Gly Ala Thr Ser
50 55
<210>
<211>
2310
<212>
DNA
<213> Sapiens
homo
<400>
5
tcttcctctacctcacagggtcaagggagtgggggaggaaatgggctaagaggttctaaa60
tccctcctaacacttgcttcttccaaatcagcaagattagagcagtcaacagctgactgc120
gttcagaccctgcaggctgggctggcctgcccaggacctgagaaggggcagctccggtgg180
caatgtctgagcccctagctgtgctggtccgggctggcctctctaagacagtgcaggcca240
cgtgatccatcctcctagaggcagtgagcaggtgagggacccctacgacagccaggagga300
aaaagctaggcgtccactttccgcagccatgctcaaacagagtgagaggagacggtcctg360
gagctacaggccctggaacacgacggagaatgagggcagccaacaccgcaggagcatttg420
ctccctgggtgcccgttccggctcccaggccagcatccacggctggacagagggcaacta480
taactactacatcgaggaagacgaagacggsgaggaggaggaccagtggaaggacgacct540
ggcagaagaggaccagcaggcaggggaggtcaccaccgccaagcccgagggccccagcga600
ccctccggccctgctgtccacgctgaatgtgaacgtgggtggccacagctaccagctgga660
ctactgcgagctggccggcttccccaagacgcgcctaggtcgcctggccacctccaccag720
ccgcagccgccagctaagcctgtgcgacgactacgaggagcagacagacgaatacttctt780
cgaccgcgacccggcegtcttccagctggtctacaatttctacctgtccggggtgctgct840
ggtgctcgacgggctgtgtccgcgccgcttcctggaggagctgggctactggggcgtgcg900
gctcaagtacacgccacgctgctgccgcatctgcttcgaggagcggcgcgacgagctgag960
cgaacggctcaagatccagcacgagctgcgcgcgcaggcgcaggtcgaggaggcggagga1020
actcttccgcgacatgcgcttctacggcccgcagcggcgccgcctctggaacctcatgga1080
gaagccrttctcctcggtggccgccaaggccatcggggtggcctccagcaccttcgtgct1140
cgtctccgtggtggcgctggcgctcaacaccgtggaggagatgcagcagcactcggggca1200
gggcgagggcggcccagacctgcggcccatcctggagcacgtggagatgctgtgcatggg1260
cttcttcacgctcgagtacctgctgcgcctagcctccacgcccgacctgaggcgcttcgc1320
gcgcagcgccctcaacctggtggacctggtggccatcctgccgctctaccttcagctgct1380
gctcgagtgcttcacgggcgagggccaccaacgcggccagacggtgggcagcgtgggtaa1440
ggtgggtcaggtgttgcgcgtcatgcgcctcatgcgcatcttccgcatcctcaagctggc1500
gcgccactccaccggactgcgtgccttcggcttcacgctgcgccagtgctaccagcaggt1560
3/6
CA 02417642 2003-O1-28
WO 02/10389 PCT/USO1/23827
gggctgcctgctgctcttcatcgccatgggcatcttcactttctctgcggctgtctactc1620
tgtggagcacgatgtgcccagcaccaacttcactaccatcccccactcctggtggtgggc1680
cgcggtgagcatctccaccgtgggctacggagayatgtacccagagacccacctgggcag1740
gttttttgccttcctctgcattgcttttgggatcattctcaacgggatgcccatttccat1800
cctctacaacaagttttctgattactacagcaagctgaaggcttatgagtataccaccat1860
acgcagggrgaggggagaggtgaacttcatgcagagagccagaaagaagatagctgagtg1920
tttgcttggaagcaacccacagctcaccccaagacaagagaattagtattttataggaca1980
tgtggctggtagattccatgaacttcaaggcttcattgctctttttttaatcattatgat2040
tggcagcaaaaggaaatgtgaagcagacatacacaaaggccatttcgttcacaaagtact2100
gcctctagaaatactcattttggcccaaactcagaatgtctcatagttgctctgtgttgt2160
gtgaaacatctgaccttctcaatgacgttgatattgaaaacctgaggggagcaacagctt2220
agatttttcttgtagcttctcgtggcatctagctcaataaatatttttggacttgaaaaa2280
aaaaaaaaaaaaaaaaaaaaaaaaaaaaaa. 2310
<210>
6
<211>
1458
<212>
DNA
<213> Sapiens
Homo
<400>
6
atgagctcagcctgctgggaggccacagggagatgcaggctgggcggcgggtggatggtt60
ccaaccggttgggtccggggcctggagctcagcctgtggggtggggacccagtggtgccc120
tggagctgccgcttctgctctcagcaggatgatgggcaggacagggagaggctgacctac180
ttccagaacctgcctgagtctctgacttccctcctggtgctgctgaccacggccaacaac240
cccgatgtgatgattcctgcgtattccaagaaccgggcctatgccatcttcttcatagtc300
ttcactgtgataggaagcctgtttctgatgaacctgctgacagccatcatctacagtcag360
ttccggggctacctgatgaaatctctccagacctcgctgtttcggaggcggctgggaacc420
cgggctgcctttgaagtcctatcctccatggtgggggagggaggagccttccctcaggcc480
acccgccgaggcccgagtaccagtctccgtttctgcagagcgcccagttcctcttcggcc540
actactactttgactacctggggaacctcatcgccctggcaaacctggtgtccatttgcg600
tgttcctggtgctggatgcagatgtgctgcctgctgagcgtgatgacttcatcctgggga660
ttctcaactgcgtcttcattgtgtactacctgttggagttgctgctcaaggtctttgccc720
tgggcctgcgagggtacctgtcctaccccagcaacgtgtttgacgggctcctcaccgttg780
tcctgctggaggccggagatggtgggcctgctgtcgctgtgggacatgacccgcatgctg840
aacatgctcatcgtgttccgcttcctgcgtatcatccccagcatgaagccgatggccgtg900
gtggccagtaccgtcctgggcctggtgcagaacatgcgtgcgtttggcgggatcctggtg960
gtggtctactacgtatttgccatcattgggatcaacttgtttagaggcgtcattgtggct1020
cttcctggaaacagcagcctggcccctgccaatggctcggcgccctgtgggagcttcgag1080
cagctggagtactgggccaacaacttcgatgactttgcggctgccctggtcactctgtgg1140
aacttgatggtggtgaacaactggcaggtgtttctggatgcatatcggcgctactcaggc1200
ccgtggtccaagatctattttgtattgtggtggctggtgtcgtctgtcatctgggtcaac1260
ctgtttctggccctgattctggagaacttccttcacaagtgggacccccgcagccacctg1320
cagccccttgctgggaccccagaggccacctaccagatgactgtggagctcctgttcagg1380
gatattctggaggagcccggggagg'atgagctcacagagaggctgagccagcacccgcac1440
ctgtggctgtgcaggtga 1458
<210>
7
<211>
485
<212>
PRT
<213> Sapiens
Homo
<400>
7
Met Ser Cys Arg
Ser Ala Leu Gly
Cys Trp Gly
Glu Ala
Thr Gly
Arg
1 5 10 15
Gly Trp Leu Glu
Met Val Leu Ser
Pro Thr Leu
Gly Trp
Val Arg
Gly
20 25 30
Trp Gly Arg Phe
Gly Asp Cys Ser
Pro Val Gln
Val Pro
Trp Ser
Cys
35 40 45
Gln Asp p Arg Tyr Phe
Asp Gly Glu Arg Gln Asn
Gln As Leu Thr Leu
50 55 60
Pro Glu Thr Thr
Ser Leu Ala Asn
Thr Ser Asn
Leu Leu
Val Leu
Leu
65 70 75 80
4/6
CA 02417642 2003-O1-28
WO 02/10389 PCT/USO1/23827
Pro Asp Val Met Ile Pro Ala Tyr Ser Lys Asn Arg Ala Tyr Ala Ile
85 90 95
Phe Phe Tle Val Phe Thr Val Ile Gly Ser Leu Phe Leu Met Asn Leu
100 105 110
Leu Thr Ala Ile Ile Tyr Ser Gln Phe Arg Gly Tyr Leu Met Lys Ser
115 120 125
Leu Gln Thr Ser Leu Phe Arg Arg Arg Leu Gly Thr Arg Ala Ala Phe
130 135 140
Glu Val Leu Ser Ser Met Val Gly Glu Gly Gly A1a Phe Pro Gln Ala
145 150 155 160
Thr Arg Arg Gly Pro Ser Thr Ser Leu Arg Phe Cys Arg Ala Pro Ser
165 170 175
Ser Ser Ser Ala Thr Thr Thr Leu Thr Thr Trp Gly Thr Ser Ser Pro
180 185 190
Trp Gln Thr Trp Cys Pro Phe Ala Cys Ser Trp Cys Trp Met Gln Met
195 200 205
Cys Cys Leu Leu Ser Val Met Thr Ser Ser Trp Gly Phe Ser Thr Ala
210 215 220
Ser Ser Leu Cys Thr Thr Cys Trp Ser Cys Cys Ser Arg Ser Leu Pro
225 230 235 240
Trp Ala Cys Glu Gly Thr Cys Pro Thr Pro Ala Thr Cys Leu Thr Gly
245 250 255
Ser Ser Pro Leu Ser Cys Trp Arg Pro Glu Met Val Gly Leu Leu Ser
260 265 270
Leu Trp Asp Met Thr Arg Met Leu Asn Met Leu Ile Val Phe Arg Phe
275 280 285
Leu Arg Ile Ile Pro Ser Met Lys Pro Met Ala Val Val Ala Ser Thr
290 295 300
Val Leu Gly Leu Val Gln Asn Met Arg Ala Phe Gly Gly Ile Leu Val
305 310 315 320
Val Val Tyr Tyr Val Phe Ala Ile Ile Gly Ile Asn Leu Phe Arg Gly
325 330 335
Val Ile Val Ala Leu Pro Gly Asn Ser Ser Leu Ala Pro Ala Asn Gly
340 345 350
Ser Ala Pro Cys Gly Ser Phe Glu Gln Leu Glu Tyr Trp Ala Asn Asn
355 360 365
Phe Asp Asp Phe Ala Ala Ala Leu Val Thr Leu Trp Asn Leu Met Val
370 375 380
Val Asn Asn Trp Gln Val Phe Leu Asp Ala Tyr Arg Arg Tyr Ser Gly
385 390 395 400
Pro Trp Ser Lys Ile Tyr Phe Val Leu Trp Trp Leu Val Ser Ser Val
405 410 415
Ile Trp Val Asn Leu Phe Leu Ala Leu Ile Leu Glu Asn Phe Leu His
420 425 430
Lys Trp Asp Pro Arg Ser His Leu Gln Pro Leu Ala Gly Thr Pro Glu
435 440 445
Ala Thr Tyr Gln Met Thr Val Glu Leu Leu Phe Arg Asp Ile Leu Glu
450 455 460
Glu Pro Gly Glu Asp Glu Leu Thr Glu Arg Leu Ser Gln His Pro His
465 470 475 480
Leu Trp Leu Cys Arg
485
<210> 8
<211> 2905
<212> DNA
<213> Homo Sapiens
<400> 8
tgctcctcct gcccctcctg cctttgcccc gtagcctcac tgcttgcaca gtgcatgcaa 60
gagtcggctg cgagcaggcg aggtggcctg agggaggtca ctaggctggc tgagggcttt l20
ttgctgtggt tctgagccgg cctgcttcca ggcaccgtgt ccatgcgggt gagcggtctc 180
cctgggtgcc cactcttgcg cccggagatc ctgagtttgg tcctgtctgg ccatgagctc 240
5/6
CA 02417642 2003-O1-28
WO 02/10389 PCT/USO1/23827
agcctgctgggaggccacagggagatgcaggctgggcggcgggtggatggttccaaccgg300
ttgggtccggggcctggagctcagcctgtggggtggggacccagtggtgccctggagctg360
ccgcttctgctctcagcaggatgatgggcaggacagggagaggctgacctacttccagaa420
cctgcctgagtctctgacttccctcctggtgctgctgaccacggccaacaaccccgatgt480
gatgattcctgcgtattccaagaaccgggcctatgccatcttcttcatagtcttcactgt540
gataggaagcctgtttctgatgaacctgctgacagccatcatctacagtcagttccgggg600
ctacctgatgaaatctctccagacctcgctgtttcggaggcggctgggaacccgggctgc660
ctttgaagtcctatcctccatggtgggggagggaggagccttccctcaggccacccgccg720
aggcccgagtaccagtctccgtttctgcagagcgcccagttcctcttcggccactactac780
tttgactacctggggaacctcatcgccctggcaaacctggtgtccatttgcgtgttcctg840
gtgctggatgcagatgtgctgcctgctgagcgtgatgacttcatcctggggattctcaac900
tgcgtcttcattgtgtactacctgttggagttgctgctcaaggtctttgccctgggcctg960
cgagggtacctgtcctaccccagcaacgtgtttgacgggctcctcaccgttgtcctgctg1020
gaggccggagatggtgggcctgctgtcgctgtgggacatgacccgcatgctgaacatgct1080
catcgtgttccgcttcctgcgtatcatccccagcatgaagccgatggccgtggtggccag1140
taccgtcctgggcctggtgcagaacatgcgtgcgtttggcgggatcctggtggtggtcta1200
ctacgtatttgccatcattgggatcaacttgtttagaggcgtcattgtggctcttcctgg1260
aaacagcagcctggcccctgccaatggctcggcgccctgtgggagcttcgagcagctgga1320
gtactgggccaacaacttcgatgactttgcggctgccctggtcactctgtggaacttgat1380
ggtggtgaacaactggcaggtgtttctggatgcatatcggcgctactcaggcccgtggtc1440
caagatctattttgtattgtggtggctggtgtcgtctgtcatctgggtcaacctgtttct1500
ggccctgattctggagaacttccttcacaagtgggacccccgcagccacctgcagcccct1560
tgctgggaccccagaggccacctaccagatgactgtggagctcctgttcagggatattct1620
ggaggagcccggggaggatgagctcacagagaggctgagccagcacccgcacctgtggct1680
gtgcaggtgacgtccgggctgccrtcccagcaggggcggcaggagagagaggctggccta1740
cacaggtgcccatcatggaagaggcggccatgctgtggccagccaggcaggaagagacct1800
ttcctctgacggaccactaagctggggacaggaaccaagtcctttgcgtgtggcccaaca1860
accatctacagaacagctgctggtgcttcagggaggcgccgtgccctccgctttctttta1920
tagctgcttcagtgagaattccctcgtcgactccacagggacctttcagacaaaaatgca1980
agaagcagcggcctcccctgtcccctgcagcttccgtggtgcctttgctgccggcagccc2040
ttggggaccacaggcctgaccagggcctgcacaggttaaccgtsagacttccggggcatt2100
caggtggggatgctggtggtttgacatggatctgtctcatctattcacagctgggaatga2160
tactaatacctccgattttagcccagcaccacagggtacgttccagtttttctctctttc2220
catagctgtaaggccctttctgggaatggttctcattctccttaatctattattgggtca2280
gttttcctgcatgtccccagcctcccatcactgccacccactccccacagagatgccctg2340
ctcatccgactggggctttgactcccacactgtgtacccctcttgtgtggacgccctgct2400
gccaaaaccttcagcaaacagctttccaaatggaagttgtcactgtcaggcctttacaat2460
cagcaacagcaaaatctacatgctgctgagggtcctgcctcattaagatgcaataaatat2520
gtaagtacataaaaacagcaatagaagaaacgtaatgctttattctcaaatatgatgtct2580
acatagaaaagccaaaattattaagaatagtaagaattcacccagcactttgggaggccg2640
aggcgggtggatcatgaggtcaggagatcgagaccatcctggctaacagggtgaaacccc2700
gtctctactaaaaatacaaaaaattggccgggcgcagtggcgggcgcctgtggtcccagc2760
tactggggaggctgaggcaggagaatggcgtgaacccgggaagcggagcttgcagtgagc2820
cgagattgcgccactgcagtccgcagtccagcctgggcgacagagcgagactccgtctca2880
aaaaaaaaaaaaaaaaaaaaaaaaa 2905
6/6
