U.S. patent application number 10/798773 was filed with the patent office on 2005-01-27 for novel human kinases and polynucleotides encoding the same.
Invention is credited to Abuin, Alejandro, Walke, D. Wade, Xie, Oiongshu, Yu, Xuanchuan.
Application Number | 20050019885 10/798773 |
Document ID | / |
Family ID | 23112809 |
Filed Date | 2005-01-27 |
United States Patent
Application |
20050019885 |
Kind Code |
A1 |
Yu, Xuanchuan ; et
al. |
January 27, 2005 |
Novel human kinases and polynucleotides encoding the same
Abstract
Novel human polynucleotide and polypeptide sequences are
disclosed that can be used in therapeutic, diagnostic, and
pharmacogenomic applications.
Inventors: |
Yu, Xuanchuan; (Conroe,
TX) ; Xie, Oiongshu; (Chestnut Hill, MA) ;
Abuin, Alejandro; (The Woodlands, TX) ; Walke, D.
Wade; (Spring, TX) |
Correspondence
Address: |
Lance K. Ishimoto
LEXICON GENETICS INCORPORATED
8800 Technology Forest Place
The Woodlands
TX
77381
US
|
Family ID: |
23112809 |
Appl. No.: |
10/798773 |
Filed: |
March 11, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10798773 |
Mar 11, 2004 |
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10141634 |
May 8, 2002 |
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6734010 |
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60289727 |
May 9, 2001 |
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Current U.S.
Class: |
435/183 ;
536/23.2 |
Current CPC
Class: |
C12N 9/12 20130101 |
Class at
Publication: |
435/183 ;
536/023.2 |
International
Class: |
C07H 021/04; C12N
009/00 |
Claims
1. (Cancelled)
2. (Cancelled)
3. (Cancelled)
4. (Cancelled)
5. A substantially isolated protein having the kinase activity of
the protein shown in SEQ. ID NO:2 or SEQ ID NO:4, which is encoded
by a nucleotide sequence that hybridizes to SEQ ID NO:1 or SEQ ID
NO:3 under highly stringent conditions.
6. An isolated protein comprising the amino acid sequence of SEQ ID
NO:2 or SEQ ID NO:4.
7. An antibody that specifically recognizes a protein comprising
the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.
Description
[0001] The present application claims the benefit of U.S.
Provisional Application No. 60/289,727, which was filed on May 9,
2001, and is herein incorporated by reference in its entirety.
1. INTRODUCTION
[0002] The present invention relates to the discovery,
identification, and characterization of novel human polynucleotides
encoding proteins sharing sequence similarity with animal kinases.
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
overexpress 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, which can be used for diagnosis, drug screening,
clinical trial monitoring, the treatment of diseases and disorders,
and cosmetic or nutriceutical applications.
2. BACKGROUND OF THE INVENTION
[0003] Kinases mediate the phosphorylation of a wide variety of
proteins and compounds in the cell. Along with phosphatases,
kinases are involved in a range of regulatory pathways. Given the
physiological importance of kinases, they have been subject to
intense scrutiny and are proven drug targets.
3. SUMMARY OF THE INVENTION
[0004] The present invention relates to the discovery,
identification, and characterization of nucleotides that 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 animal kinases,
including, but not limited to, adenylate kinases and
phosphotransferases. Accordingly, the described NHPs encode novel
kinases having homologues and orthologs across a range of phyla and
species.
[0005] The novel human polynucleotides described herein encode open
reading frames (ORFs) encoding proteins of 479 and 94 amino acids
in length (see, respectively, SEQ ID NOS:2 and 4).
[0006] 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 NHPs,
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 open reading frame or
regulatory sequence replacement constructs) or to enhance the
expression of the described NHPS (e.g., expression constructs that
place the described polynucleotide under the control of a strong
promoter system), and transgenic animals that express a NHP
sequence, 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 cell
("ES cell") 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:1-4 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. In addition, animals in which the unique
NHP sequences described in SEQ ID NOS:1-4 are "knocked-out" provide
a unique source in which to elicit antibodies to homologous and
orthologous proteins, which would have been previously viewed by
the immune system as "self" and therefore would have failed to
elicit significant antibody responses. To these ends, gene trapped
knockout ES cells have been generated in murine homologs of the
described NHPs.
[0007] Additionally, the unique NHP sequences described in SEQ ID
NOS:1-4 are useful for the identification of protein coding
sequences, and mapping a unique gene to a particular chromosome.
These sequences identify biologically verified exon splice
junctions, as opposed to splice junctions that may have been
bioinformatically predicted from genomic sequence alone. The
sequences of the present invention are also useful as additional
DNA markers for restriction fragment length polymorphism (RFLP)
analysis, and in forensic biology.
[0008] 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 and/or NHP products, 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
[0009] The Sequence Listing provides the sequence of the novel
human ORFs encoding the described novel human kinase proteins.
5. DETAILED DESCRIPTION OF THE INVENTION
[0010] The NHPs described for the first time herein are novel
proteins that are expressed in, inter alia, human cell lines and
human fetal brain, brain, pituitary, cerebellum, spinal cord,
thymus, spleen, lymph node, trachea, lung, kidney, fetal liver,
prostate, testis, thyroid, adrenal gland, pancreas, salivary gland,
stomach, colon, skeletal muscle, heart, uterus, placenta, mammary
gland, skin, esophagus, bladder, cervix, rectum, pericardium,
hypothalamus, ovary, fetal kidney, fetal lung, gall bladder,
tongue, aorta, 6-, 9-, and 12-week old embryos, adenocarcinoma,
osteosarcoma, embryonic carcinoma, umbilical vein, and
microvascular endothelial cells. Additional expression studies
suggest that the NHPs are also expressed in human bone marrow and
adipose cells. The described sequences were compiled from sequences
available in GENBANK, and cDNAs generated from human mammary gland
mRNAs (Edge Biosystems, Gaithersburg, Md.) that were identified
using primers generated from human genomic DNA.
[0011] The present invention encompasses the nucleotides presented
in the Sequence Listing, host cells expressing such nucleotides,
the expression products of such nucleotides, and: (a) nucleotides
that encode mammalian homologs of the described nucleotides,
including the specifically described NHPs, and the NHP products;
(b) nucleotides that encode one or more portions of a NHP 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; (d) nucleotides that
encode chimeric fusion proteins containing all or a portion of a
coding region of a NHP, or one of its domains (e.g., a
receptor/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.
[0012] As discussed above, the present invention includes 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 NaHPO.sub.4, 7% sodium
dodecyl sulfate (SDS), 1 mM EDTA at 65.degree. C., and washing in
0.1.times.SSC/0.1% SDS at 68.degree. C. (Ausubel et al., eds.,
1989, Current Protocols in Molecular Biology, Vol. I, Green
Publishing Associates, Inc., and John Wiley & Sons, Inc., N.Y.,
at p. 2.10.3) and encodes a functionally equivalent expression
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 conditions, e.g., washing in
0.2.times.SSC/0.1% SDS at 42.degree. C. (Ausubel et al., 1989,
supra), yet still encode 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.
Pat. Nos. 5,837,458 or 5,723,323 both of which are herein
incorporated by reference). The invention also includes degenerate
nucleic acid variants of the disclosed NHP polynucleotide
sequences.
[0013] 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 to
corresponding regions of SEQ ID NO:1 (as measured by BLAST sequence
comparison analysis using, for example, the GCG sequence analysis
package, as described herein, using default parameters).
[0014] The invention also includes nucleic acid molecules,
preferably DNA molecules, that hybridize to, and are therefore the
complements of, the described NHP-encoding polynucleotides. Such
hybridization conditions can be highly stringent or less highly
stringent, as described herein. 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 bases long, 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 reaction (PCR) to screen libraries,
isolate clones, and prepare cloning and sequencing templates,
etc.
[0015] Alternatively, such NHP oligonucleotides can be used as
hybridization probes for screening libraries, and assessing gene
expression patterns (particularly using a microarray or
high-throughput "chip" format). Additionally, a series of 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:1-4
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:1-4, 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. Pat.
Nos. 5,700,637, 5,556,752, 5,744,305, 4,631,211, 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.
[0016] Addressable arrays comprising sequences first disclosed in
SEQ ID NOS:1-4 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 the production technology. The length of these
probes is usually within a range of between about 8 to about 2000
nucleotides. The probes typically comprise about 60 nucleotides or
can comprise about 25 nucleotides from the sequences first
disclosed in SEQ ID NOS:1-4.
[0017] For example, a series of NHP 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 olignucleotide 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.
[0018] 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:1-4 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.
[0019] Probes consisting of sequences first disclosed in SEQ ID
NOS:1-4 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 recognition of drug dependent changes in gene
expression that are modulated through pathways distinct from the
intended target of the drug. These unique sequences therefore also
have utility in defining and monitoring both drug action and
toxicity.
[0020] As an example of utility, the sequences first disclosed in
SEQ ID NOS:1-4 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:1-4 in silico, and by comparing previously collected genetic
databases and the disclosed sequences using computer software known
to those in the art.
[0021] Thus the sequences first disclosed in SEQ ID NOS:1-4 can be
used to identify mutations associated with a particular disease,
and also in diagnostic or prognostic assays.
[0022] 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 sequence(s) first disclosed in
SEQ ID NOS:1-4. 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,
Mich., etc.), can optionally be used in conjunction with one or
more discrete nucleotide sequence(s) present in the sequence that
can be described by the relative position of the sequence relative
to one or more additional sequence(s) or one or more restriction
sites present in the disclosed sequence.
[0023] For oligonucleotide probes, highly stringent conditions may
refer, e.g., to washing in 6.times.SSC/0.05% sodium pyrophosphate
at 37.degree. C. (for 14-base oligos), 48.degree. C. (for 17-base
oligos), 55.degree. C. (for 20-base oligos), and 60.degree. C. (for
23-base oligos). These nucleic acid molecules may encode or act as
NHP antisense molecules, useful, for example, in NHP gene
regulation and/or as antisense primers in amplification reactions
of NHP nucleic acid sequences. With respect to NHP gene regulation,
such techniques can be used to regulate biological functions.
Further, such sequences can be used as part of ribozyme and/or
triple helix sequences that are also useful for NHP gene
regulation.
[0024] Inhibitory antisense or double stranded oligonucleotides can
additionally comprise at least one modified base moiety that is
selected from the group including, but not limited to,
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluraci- l, 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-isopenten- yladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
[0025] 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.
[0026] In yet another embodiment, the antisense oligonucleotide
will comprise at least one modified phosphate backbone selected
from the group including, but not limited to, a phosphorothioate, a
phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a
phosphordiamidate, a methylphosphonate, an alkyl phosphotriester,
and a formacetal or analog thereof.
[0027] In yet another embodiment, the antisense oligonucleotide is
an .alpha.-anomeric oligonucleotide. An .alpha.-anomeric
oligonucleotide forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual .beta.-units, the
strands run parallel to each other (Gautier et al., 1987, Nucl.
Acids Res. 15:6625-6641). The oligonucleotide is a
2'-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.
15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987,
FEBS Lett. 215:327-330). Alternatively, double stranded RNA can be
used to disrupt the expression and function of a targeted NHP.
[0028] 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. 16:3209), and methylphosphonate
oligonucleotides can be prepared by use of controlled pore glass
polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. USA
85:7448-7451), etc.
[0029] 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, Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (and periodic updates
thereof), and Ausubel et al., 1989, supra.
[0030] 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 genomid 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.
[0031] For example, the present sequences can be used in
restriction fragment length polymorphism (RFLP) analysis to
identify specific individuals. In this technique, an individual's
genomic DNA is digested with one or more restriction enzymes, and
probed on a Southern blot to yield unique bands for identification
(as generally described in U.S. Pat. No. 5,272,057, incorporated
herein by reference). In addition, the sequences of the present
invention can be used to provide polynucleotide reagents, e.g., PCR
primers, targeted to specific loci in the human genome, which can
enhance the reliability of DNA-based forensic identifications by,
for example, providing another "identification marker" (i.e.,
another DNA sequence that is unique to a particular individual).
Actual base sequence information can be used for identification as
an accurate alternative to patterns formed by restriction enzyme
generated fragments.
[0032] 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,
genomic DNA and/or cDNA obtained by reverse transcription of mRNA
prepared from, for example, human or non-human cell lines or tissue
known to express, or suspected of expressing, an allele of a NHP
gene.
[0033] The PCR product can be subcloned and sequenced to ensure
that the amplified sequences represent the sequence 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.
[0034] 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 to express, or suspected of expressing, 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.
[0035] A cDNA encoding a mutant NHP sequence 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 to express, or suspected of expressing,
a NHP, 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 sequence. 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
mutation(s) responsible for the loss or alteration of function of
the mutant NHP gene product can be ascertained.
[0036] Alternatively, a genomic library can be constructed using
DNA obtained from an individual'suspected of carrying, or known to
carry, a mutant NHP allele (e.g., a person manifesting a
NHP-associated phenotype such as, for example, behavioral
disorders, immune disorders, obesity, high blood pressure, etc.),
or a cDNA library can be constructed using RNA from a tissue known
to express, or suspected of expressing, 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
sequences can then be purified and subjected to sequence analysis
according to methods well-known to those skilled in the art.
[0037] Additionally, an expression library can be constructed
utilizing cDNA synthesized from, for example, RNA isolated from a
tissue known to express, or suspected of expressing, a mutant NHP
allele in an individual suspected of carrying, or known to carry,
such a mutant allele. In this manner, gene products made by the
putatively mutant tissue may 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 and Lane, eds.,
1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Press,
Cold Spring Harbor, N.Y.).
[0038] 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 expression 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 expression product. Library clones
detected via their reaction with such labeled antibodies can be
purified and subjected to sequence analysis according to methods
well-known in the art.
[0039] An additional application of the described novel human
polynucleotide sequences is their use in the 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. Pat. Nos. 5,830,721, 5,837,458, 6,117,679,
and 5,723,323, which are herein incorporated by reference in their
entirety.
[0040] 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, baculovirus as described in U.S. Pat. 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 sequence 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 or 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 .alpha.-mating factors.
[0041] Where, as in the present instance, some of the described NHP
peptides or polypeptides are thought to be cytoplasmic or nuclear
proteins (although processed forms or fragments can be secreted or
membrane associated), expression systems can be engineered that
produce soluble derivatives of a NHP (corresponding to a NHP
extracellular and/or intracellular domains, or truncated
polypeptides lacking one or more hydrophobic domains) and/or NHP
fusion protein products (especially NHP-Ig fusion proteins, i.e.,
fusions of a NHP domain to an IgFc), NHP antibodies, and
anti-idiotypic antibodies (including Fab fragments), that can be
used in therapeutic applications. Preferably, the above expression
systems are engineered to allow the desired peptide or polypeptide
to be recovered from the culture media.
[0042] The present invention also encompasses antibodies and
anti-idiotypic antibodies (including Fab fragments), antagonists
and agonists of a NHP, as well as compounds or nucleotide
constructs that inhibit expression of a NHP sequence (transcription
factor inhibitors, antisense and ribozyme molecules, or open
reading frame sequence 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.).
[0043] 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 a NHP in the
body. The use of engineered host cells and/or animals can offer an
advantage in that such systems allow not only for the
identification of compounds that bind to the endogenous
receptor/ligand of a NHP, but can also identify compounds that
trigger NHP-mediated activities or pathways.
[0044] 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 a soluble NHP, a NHP-IgFc fusion protein, or an anti-idiotypic
antibody (or its Fab) that mimics the NHP, could activate or
effectively antagonize an endogenous NHP or a protein interactive
therewith. Nucleotide constructs 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.
[0045] Various aspects of the invention are described in greater
detail in the subsections below.
5.1 THE NHP SEQUENCES
[0046] The cDNA sequences and corresponding deduced amino acid
sequences of the described NHPs are presented in the Sequence
Listing.
[0047] Expression analysis has provided evidence that the described
NHPs can be expressed in a relatively wide range of human tissues.
In addition to adenylate kinases, the described NHPs also share
significant similarity to a range of additional kinase families,
including kinases associated with signal transduction, from a
variety of phyla and species.
[0048] A number of polymorphisms were identified during the
sequencing of the NHPs, including: a G/C polymorphism at the region
represented by nucleotide position number 427 of, for example, SEQ
ID NO:1, which can result in a glu or gln being present at
corresponding amino acid (aa) position 143 of, for example, SEQ ID
NO:2;and a C/G polymorphism at the region represented by nucleotide
position number 483 of, for example, SEQ ID NO:1, both of which
result in a val being present at corresponding aa position 161 of,
for example, SEQ ID NO:2. The present invention contemplates NHPs
comprising any or all of the above polymorphisms, as well as all
combinations and permutations of the described polymorphisms.
[0049] The gene encoding the described NHPs is apparently encoded
on human chromosome 9 (see GENBANK accession no. AL161726).
Accordingly, the described sequences are also useful for mapping
and identifying the coding regions of the human genome, and for
defining exon splice junctions.
[0050] Given the physiological importance of protein kinases, they
have been subject to intense scrutiny, as exemplified and discussed
in U.S. Pat. Nos. 5,756,289 and 5,817,479, and 6,340,583, herein
incorporated by reference in their entirety, which additionally
describe a variety of uses and applications for the described
NHPs.
[0051] 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.
[0052] 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 and Wagner, 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
82:6148-6152); 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, Transgenic Animals, Intl. Rev.
Cytol. 115:171-229, which is incorporated by reference herein in
its entirety.
[0053] The present invention provides for transgenic animals that
carry a NHP transgene in all their cells, as well as animals that
carry a transgene in some, but not all their cells, i.e., mosaic
animals or somatic cell transgenic animals. A transgene may be
integrated as a single transgene, or in concatamers, e.g.,
head-to-head tandems or head-to-tail tandems. A 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 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.
[0054] 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).
[0055] 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 265:103-106. 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.
[0056] Once transgenic animals have been generated, the expression
of the recombinant NHP gene may be assayed utilizing standard
techniques. Initial screening may be 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 that 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.
[0057] The present invention also provides for "knock-in" animals.
Knock-in animals are those in which a polynucleotide sequence
(i.e., a gene or a cDNA) that the animal does not naturally have in
its genome is inserted in such a way that it is expressed. Examples
include, but are not limited to, a human gene or cDNA used to
replace its murine ortholog in a mouse, a murine cDNA used to
replace the murine gene in a mouse, and a human gene or cDNA or
murine cDNA that is tagged with a reporter construct used to
replace the murine ortholog or gene in a mouse. Such replacements
can occur at the locus of the murine ortholog or gene, or at
another specific site. Such knock-in animals are useful for the in
vivo study, testing and validation of, intra alia, human drug
targets, as well as for compounds that are directed at the same,
and therapeutic proteins.
5.2 NHPS AND NHP POLYPEPTIDES
[0058] NHPs, NHP polypeptides, NHP 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, for the identification of other cellular gene
products related to a NHP, and as reagents in assays for screening
for compounds that can be used 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 therapeutic agents.
[0059] The Sequence Listing discloses the amino acid sequences
encoded by the described NHP-encoding polynucleotides. The NHPs
display initiator methionines that are present in DNA sequence
contexts consistent with eucaryotic translation initiation
sites.
[0060] The NHP amino acid sequences of the invention include the
amino acid sequences 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 herein 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
the Sequence Listing, when taken together with the genetic code
(see, for example, Table 4-1 at page 109 of "Molecular Cell
Biology", 1986, J. Darnell et al., eds., Scientific American Books,
New York, N.Y., 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.
[0061] 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
modify a NHP substrate, the ability to effect an identical or
complementary downstream pathway, or a change in cellular
metabolism (e.g., proteolytic activity, ion flux, 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 herein, but that result in a silent change,
thus producing a functionally equivalent expression 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.
[0062] A variety of host-expression vector systems can be used to
express the NHP nucleotide sequences of the invention. Where the
NHP peptide or polypeptide can exist, or has been engineered to
exist, as a soluble or secreted molecule, the soluble NHP
peptide-or polypeptide can be recovered from the culture media.
Such expression ompas 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 a NHP, but to
assess biological activity, e.g., in certain drug screening
assays.
[0063] 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 nucleotide 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 NHP nucleotide sequences and promoters
derived from the genome of mammalian cells (e.g., metallothione in
promoter) or from mammalian viruses (e.g., the adenovirus late
promoter; the vaccinia virus 7.5K promoter).
[0064] 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 produced for the generation of pharmaceutical
compositions of or containing a 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 lacZ coding region so that a fusion protein is
produced; pIN vectors (Inouye and Inouye, 1985, Nucleic Acids Res.
13:3101-3109; Van Heeke and Schuster, 1989, J. Biol. Chem.
264:5503-5509); and the like. pGEX vectors may 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
expression product can be released from the GST moiety.
[0065] In an exemplary insect system, Autographa californica
nuclear polyhedrosis virus (AcNPV) is used as a vector to express
foreign polynucleotide sequences. The virus grows in Spodoptera
frugiperda cells. A NHP coding sequence can be cloned individually
into a non-essential region (for example the polyhedrin gene) of
the virus and placed under control of an AcNPV promoter (for
example the polyhedrin promoter). Successful insertion of a 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. Pat. No.
4,215,051).
[0066] 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 sequence 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 and 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, may be provided. Furthermore, the initiation
codon should 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).
[0067] In addition, a host cell strain may be chosen that modulates
the expression of the inserted sequences, or modifies and processes
the expression product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be 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 expression products. Appropriate cell lines or host
systems can be chosen to ensure the desired modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells that possess the cellular machinery for the
desired processing of the primary transcript, glycosylation, and
phosphorylation of the expression product may be used. Such
mammalian host cells include, but are not limited to, CHO, VERO,
BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, human cell
lines.
[0068] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
that stably express the NHP sequences described herein can be
engineered. Rather than using expression vectors that 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
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 that 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.
[0069] 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-guanine
phosphoribosyltransferase (Szybalska and Szybalski, 1962, Proc.
Natl. Acad. Sci. USA 48:2026), and adenine
phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes,
which can be employed in tk.sup.-, hgprt.sup.- or aprt.sup.- 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, Proc. Natl. Acad.
Sci. USA 77:3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA
78:1527); gpt, which confers resistance to mycophenolic acid
(Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo,
which confess resistance to the aminoglycoside G-418
(Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1); and hygro,
which confers resistance to hygromycin (Santerre et al., 1984, Gene
30:147).
[0070] Alternatively, any fusion protein can be readily purified by
utilizing an antibody specific for the fusion protein being
expressed. Another exemplary system 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 sequence of interest is
subcloned into a vaccinia recombination plasmid such that the
sequence'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
Ni.sup.2+ nitriloacetic acid-agarose columns, and histidine-tagged
proteins are selectively eluted with imidazole-containing
buffers.
[0071] Also encompassed by the present invention are fusion
proteins that direct a 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 an appropriate
signal sequence to a NHP would also transport a NHP to a desired
location within the cell. Alternatively targeting of a NHP or its
nucleic acid sequence might be achieved using lippsome or lipid
complex based delivery systems. Such technologies are described in
"Liposomes: A Practical Approach", New, R.R.C., ed., Oxford
University Press, N.Y., and in U.S. Pat. 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 NHPs to a target site or desired
organ, where they cross the cell membrane and/or the nucleus where
the NHPs can exert their functional activity. This goal may be
achieved by coupling of a NHP to a cytokine or other ligand that
provides targeting specificity, and/or to a protein transducing
domain (see generally U.S. Provisional Patent Application Ser. Nos.
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, and can optionally be
engineered to include nuclear localization signals.
[0072] Additionally contemplated are oligopeptides that are modeled
on an amino acid sequence first described in the Sequence Listing.
Such NHP oligopeptides are generally between about 10 to about 100
amino acids long, or between about 16 to about 80 amino acids long,
or between about 20 to about 35 amino acids long, or any variation
or combination of sizes represented therein that incorporate a
contiguous region of sequence first disclosed in the Sequence
Listing. Such NHP oligopeptides can be of any length disclosed
within the above ranges and can initiate at any amino acid position
represented in the Sequence Listing.
[0073] The invention also contemplates "substantially isolated" or
"substantially pure" proteins or polypeptides. By a "substantially
isolated" or "substantially pure" protein or polypeptide is meant a
protein or polypeptide that has been separated from at least some
of those components that naturally accompany it. Typically, the
protein or polypeptide is substantially isolated or pure when it is
at least 60%, by weight, free from the proteins and other
naturally-occurring organic molecules with which it is naturally
associated in vivo. Preferably, the purity of the preparation is at
least about 75%, or at least about 90%, or at least about 99%, by
weight. A substantially isolated or pure protein or polypeptide may
be obtained, for example by extraction from a natural source, by
expression of a recombinant nucleic acid encoding the protein or
polypeptide, or by chemically synthesizing the protein or
polypeptide.
[0074] Purity can be measured by any appropriate method, e.g.,
column chromatography such as immunoaffinity chromatography using
an antibody specific for the protein or polypeptide, polyacrylamide
gel electrophoresis, or HPLC analysis. A protein or polypeptide is
substantially free of naturally associated components when it is
separated from at least some of those contaminants that accompany
it in its natural state. Thus, a polypeptide that is chemically
synthesized or produced in a cellular system different from the
cell from which it naturally originates will be, by definition
substantially free from its naturally associated components.
Accordingly, substantially isolated or pure proteins or
polypeptides include eukaryotic proteins synthesized in E. coli,
other prokaryotes, or any other organism in which they do not
naturally occur.
5.3 ANTIBODIES TO NHP PRODUCTS
[0075] Antibodies that specifically recognize one or more epitopes
of a NHP, 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').sub.2 fragments,
fragments produced by a Fab expression library, anti-idiotypic
(anti-Id) antibodies, and epitope-binding fragments of any of the
above.
[0076] The antibodies of the invention can be used, for example, in
the detection of a 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 a NHP. Such
antibodies may also be utilized in conjunction with, for example,
compound screening schemes for the evaluation of the effect of test
compounds on expression and/or activity of a NHP expression
product. Additionally, such antibodies can be used in conjunction
with gene therapy to, for example, evaluate normal and/or
engineered NHP-expressing cells prior to their introduction into a
patient. Such antibodies may additionally be used in methods for
the inhibition of abnormal NHP activity. Thus, such antibodies may
be utilized as a part of treatment methods.
[0077] For the production of antibodies, various host animals may
be immunized by injection with a NHP, a NHP peptide (e.g., one
corresponding to a functional domain of a NHP), a truncated NHP
polypeptide (NHP in which one or more domains have been deleted),
functional equivalents of a NHP or mutated variants of a 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.
[0078] Monoclonal antibodies, which are homogeneous populations of
antibodies to a particular antigen, can be obtained by any
technique that 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 256:495-497; and U.S. Pat. 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, and IgD, and any subclass thereof. The hybridomas
producing the mAbs 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.
[0079] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad.
Sci. USA 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. Pat. Nos. 6,114,598, 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 U.S. Pat. No. 6,150,584 and respective
disclosures, which are herein incorporated by reference in their
entirety.
[0080] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 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 expression
products. Single chain antibodies are formed by linking the heavy
and light chain fragments of the Fv region via an amino acid
bridge, resulting in a single chain polypeptide.
[0081] Antibody fragments that recognize specific epitopes may be
generated by known techniques. For example, such fragments include,
but are not limited to: F(ab').sub.2 fragments, which can be
produced by pepsin digestion of an antibody molecule; and Fab
fragments, which can be generated by reducing the disulfide bridges
of F(ab').sub.2 fragments. Alternatively, Fab expression libraries
may be constructed (Huse et al., 1989, Science, 246:1275-1281) to
allow rapid and easy identification of monoclonal Fab fragments
with the desired specificity.
[0082] 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 and
Bona, 1993, FASEB J. 7:437-444; and Nissinoff, 1991, J. Immunol.
147:2429-2438). For example, antibodies that bind to a NHP domain
and competitively inhibit the binding of NHP to its cognate
receptor/ligand can be used to generate anti-idiotypes that "mimic"
the NHP and, therefore, bind, activate, or neutralize a NHP, NHP
receptor, or NHP ligand. Such anti-idiotypic antibodies, or Fab
fragments of such anti-idiotypes, can be used in therapeutic
regimens involving a NHP-mediated pathway.
[0083] Additionally given the high degree of relatedness of
mammalian NHPs, the presently described knock-out mice (having
never seen a NHP, and thus never been tolerized to a NHP) have a
unique utility, as they can be advantageously applied to the
generation of antibodies against the disclosed mammalian NHPs
(i.e., a NHP will be immunogenic in NHP knock-out animals).
[0084] 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.
Sequence CWU 1
1
4 1 1440 DNA homo sapiens 1 atggacgcca ctatcgcccc gcaccgtatc
ccccccgaga tgccccagta cggggaggag 60 aaccacatct tcgagttgat
gcagaacatg ctggagcaac tcctgatcca ccagcccgaa 120 gatcccatcc
ccttcatgat ccagcacttg catagagaca acgacaatgt gcccaggatt 180
gtaatattag gtccacccgc ctcagggaaa acaacaatag caatgtggct ctgcaaacat
240 ctgaacagca gtctcctcac cctggagaac ctgatcttaa atgagttttc
ctatacggcc 300 accgaagcca gaaggcttta tctgcaaagg aagacagttc
ccagcgcgct gctcgtccag 360 ctgattcagg aacgcctggc tgaagaggat
tgcatcaagc agggctggat tctggatggc 420 atccctgaga cgcgtgagca
ggctctgagg atccagaccc tggggatcac acccagacac 480 gtcattgtgc
tgagtgctcc agacacggtc ctgatcgaga gaaacttggg gaagagaatc 540
gaccctcaaa ctggagagat ttatcacacc acctttgact ggccacccga atctgaaatc
600 cagaaccgtc tcatggtgcc agaggacatc tcagagctgg agacggctca
gaaactgctg 660 gagtatcata ggaacatcgt cagggtcatt ccctcctacc
ccaaaatcct caaagtcatc 720 agtgctgacc agccatgtgt ggacgtcttc
taccaggctc tgacctatgt ccaaagcaac 780 catcgtacta atgccccgtt
caccccgagg gtgctgctgc tcgggcctgt gggcagtggg 840 aaaagtctgc
aggccgccct cctggcccag aaatacaggc ttgtcaatgt ctgctgtggg 900
caactgctga aagaggctgt ggcagatagg accacgtttg gcgagctcat ccagcccttc
960 tttgaaaagg agatggcagt tcctgacagc ctcctcatga aggtgctgag
ccagcgcctg 1020 gaccagcagg actgcatcca gaaaggctgg gtgctacacg
gcgtcccgcg ggacctcgac 1080 caggcacacc tgctgaaccg cctgggctac
aatcccaaca gggtgttttt cctgaatgtg 1140 ccatttgatt ccatcatgga
gcggctgact ctgagaagaa ttgatccagt cactggggaa 1200 aggtaccacc
tcatgtacaa gccacctccc accatggaga tccaggctcg cctcctgcag 1260
aacccaaagg atgctgaaga gcaggtcaag ctgaaaatgg acctgttcta caggaactca
1320 gctgacttgg agcagttgta tgggtcggcc atcaccctca atggggacca
ggacccatac 1380 acagtcttcg aatacatcga gagtgggatc attaatcccc
tgcccaagaa aatcccctga 1440 2 479 PRT homo sapiens 2 Met Asp Ala Thr
Ile Ala Pro His Arg Ile Pro Pro Glu Met Pro Gln 1 5 10 15 Tyr Gly
Glu Glu Asn His Ile Phe Glu Leu Met Gln Asn Met Leu Glu 20 25 30
Gln Leu Leu Ile His Gln Pro Glu Asp Pro Ile Pro Phe Met Ile Gln 35
40 45 His Leu His Arg Asp Asn Asp Asn Val Pro Arg Ile Val Ile Leu
Gly 50 55 60 Pro Pro Ala Ser Gly Lys Thr Thr Ile Ala Met Trp Leu
Cys Lys His 65 70 75 80 Leu Asn Ser Ser Leu Leu Thr Leu Glu Asn Leu
Ile Leu Asn Glu Phe 85 90 95 Ser Tyr Thr Ala Thr Glu Ala Arg Arg
Leu Tyr Leu Gln Arg Lys Thr 100 105 110 Val Pro Ser Ala Leu Leu Val
Gln Leu Ile Gln Glu Arg Leu Ala Glu 115 120 125 Glu Asp Cys Ile Lys
Gln Gly Trp Ile Leu Asp Gly Ile Pro Glu Thr 130 135 140 Arg Glu Gln
Ala Leu Arg Ile Gln Thr Leu Gly Ile Thr Pro Arg His 145 150 155 160
Val Ile Val Leu Ser Ala Pro Asp Thr Val Leu Ile Glu Arg Asn Leu 165
170 175 Gly Lys Arg Ile Asp Pro Gln Thr Gly Glu Ile Tyr His Thr Thr
Phe 180 185 190 Asp Trp Pro Pro Glu Ser Glu Ile Gln Asn Arg Leu Met
Val Pro Glu 195 200 205 Asp Ile Ser Glu Leu Glu Thr Ala Gln Lys Leu
Leu Glu Tyr His Arg 210 215 220 Asn Ile Val Arg Val Ile Pro Ser Tyr
Pro Lys Ile Leu Lys Val Ile 225 230 235 240 Ser Ala Asp Gln Pro Cys
Val Asp Val Phe Tyr Gln Ala Leu Thr Tyr 245 250 255 Val Gln Ser Asn
His Arg Thr Asn Ala Pro Phe Thr Pro Arg Val Leu 260 265 270 Leu Leu
Gly Pro Val Gly Ser Gly Lys Ser Leu Gln Ala Ala Leu Leu 275 280 285
Ala Gln Lys Tyr Arg Leu Val Asn Val Cys Cys Gly Gln Leu Leu Lys 290
295 300 Glu Ala Val Ala Asp Arg Thr Thr Phe Gly Glu Leu Ile Gln Pro
Phe 305 310 315 320 Phe Glu Lys Glu Met Ala Val Pro Asp Ser Leu Leu
Met Lys Val Leu 325 330 335 Ser Gln Arg Leu Asp Gln Gln Asp Cys Ile
Gln Lys Gly Trp Val Leu 340 345 350 His Gly Val Pro Arg Asp Leu Asp
Gln Ala His Leu Leu Asn Arg Leu 355 360 365 Gly Tyr Asn Pro Asn Arg
Val Phe Phe Leu Asn Val Pro Phe Asp Ser 370 375 380 Ile Met Glu Arg
Leu Thr Leu Arg Arg Ile Asp Pro Val Thr Gly Glu 385 390 395 400 Arg
Tyr His Leu Met Tyr Lys Pro Pro Pro Thr Met Glu Ile Gln Ala 405 410
415 Arg Leu Leu Gln Asn Pro Lys Asp Ala Glu Glu Gln Val Lys Leu Lys
420 425 430 Met Asp Leu Phe Tyr Arg Asn Ser Ala Asp Leu Glu Gln Leu
Tyr Gly 435 440 445 Ser Ala Ile Thr Leu Asn Gly Asp Gln Asp Pro Tyr
Thr Val Phe Glu 450 455 460 Tyr Ile Glu Ser Gly Ile Ile Asn Pro Leu
Pro Lys Lys Ile Pro 465 470 475 3 285 DNA homo sapiens 3 atggagcggc
tgactctgag aagaattgat ccagtcactg gggaaaggta ccacctcatg 60
tacaagccac ctcccaccat ggagatccag gctcgcctcc tgcagaaccc aaaggatgct
120 gaagagcagg tcaagctgaa aatggacctg ttctacagga actcagctga
cttggagcag 180 ttgtatgggt cggccatcac cctcaatggg gaccaggacc
catacacagt cttcgaatac 240 atcgagagtg ggatcattaa tcccctgccc
aagaaaatcc cctga 285 4 94 PRT homo sapiens 4 Met Glu Arg Leu Thr
Leu Arg Arg Ile Asp Pro Val Thr Gly Glu Arg 1 5 10 15 Tyr His Leu
Met Tyr Lys Pro Pro Pro Thr Met Glu Ile Gln Ala Arg 20 25 30 Leu
Leu Gln Asn Pro Lys Asp Ala Glu Glu Gln Val Lys Leu Lys Met 35 40
45 Asp Leu Phe Tyr Arg Asn Ser Ala Asp Leu Glu Gln Leu Tyr Gly Ser
50 55 60 Ala Ile Thr Leu Asn Gly Asp Gln Asp Pro Tyr Thr Val Phe
Glu Tyr 65 70 75 80 Ile Glu Ser Gly Ile Ile Asn Pro Leu Pro Lys Lys
Ile Pro 85 90
* * * * *