Note: Descriptions are shown in the official language in which they were submitted.
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HUMAN CUB-DOI~I~AIN-CONTAINING PROTEIN AND GENE
ENCODING THE SAME
The present invention claims the benefit of U.S.
Provisional Application Numbers 60/160,285 and 60/183,583
which were filed October 19, 1999 and February 18, 2000
respectively and are herein incorporated in their entirety.
1. INTRODUCTION
The present invention relates to the discovery,
identification, and characterization of novel human
polynucleotides encoding proteins sharing sequence similarity
with mammalian proteins having CUB domains. 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 sequences, 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, or the treatment of
physiological disorders, or diseases.
2. BACKGROUND OF THE INVENTION
The CUB domain is an extracellular domain (ECD) present
in variety of diverse proteins such as bone morphogenetic
protein 1, proteinases, spermadhesins, complement
subcomponents, and neuronal recognition molecules. Given the
importance of these functions, CUB proteins have been
associated with, inter alia, regulating development,
modulating cellular processes, and preventing infectious
disease.
3 5 3 . SUNIrIARY OF THE INVENTION
The present invention relates to the discovery,
identification, and characterization of nucleotides that
encode novel human proteins, and the corresponding amino acid
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sequences of these proteins. The novel human proteins (NHPs)
described for the first time herein share structural
similarity with animal CUB domain proteins, coagulation
factors V and XIII, milk fat globule-EGF factor 8,
transcriptional repressor AE-binding protein-1, and
neuropilins 1 and 2 (which, like the presently described
protein, contain both CUB and discoidin domains).
The novel human nucleic acid (cDNA) sequences described
herein, encode proteins/open reading frames (ORFs) of 487,
586, and 539 amino acids in length (see SEQ ID NOS: 2, 4, and
6 respectively).
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, 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 NHPs
(e. g., expression constructs that place the described gene
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.
Several knockout ES cell lines have been produced that contain
a gene trap mutation in a murine ortholog/homolog of the
disclosed NHPs.
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.
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4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES
The Sequence Listing provides the sequences of several
NHP ORFs encoding the described NHP amino acid sequences. SEQ
ID N0:7 describes a NHP ORF and flanking sequences.
5. DETAILED DESCRIPTION OF THE INVENTION
The NHPs described for the first time herein are novel
proteins that are expressed in, inter alia, human cell lines,
and human prostate, pituitary, fetal brain, brain, thymus,
spleen, lymph node, trachea, kidney, fetal liver, thyroid,
adrenal gland, salivary gland, stomach, small intestine,
colon, muscle, heart, mammary gland, adipose, skin, esophagus,
bladder, cervix, rectum, and testis cells.
The described sequences were compiled from gene trapped
cDNAs, genomic sequence, and clones isolated from human brain,
adipose, testis, and placenta cDNA libraries (Edge Biosystems,
Gaithersburg, MD, and Clontech, Palo Alto, CA). 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 genes,
including the specifically described NHPs, and 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 in
which all or a portion of the signal sequence is deleted; (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 or ligand binding domain, accessory
protein/self-association domain, etc.) fused to another
peptide or polypeptide; or (e) therapeutic or diagnostic
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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), or a contiguous exon splice junction
first described in the Sequence Listing, 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/0.1o 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, at p. 2.10.3) and
encodes a functionally equivalent gene product. Additionally
contemplated are any nucleotide sequences that hybridize to
the complement of the DNA sequence that encode and express an
amino acid sequence presented in the Sequence Listing under
moderately stringent conditions, e.g., washing in 0.2xSSC/0.1o
SDS at 42°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. Patent
No. 5,837,458). The invention also includes degenerate
nucleic acid variants of the disclosed NHP polynucleotide
sequences.
Additionally contemplated are polynucleotides encoding a
NHP ORF, or its functional equivalent, encoded by a
polynucleotide sequence that is 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
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BLAST sequence comparison analysis using, for example, the GCG
sequence analysis package using standard default settings).
The invention also includes nucleic acid molecules,
preferably DNA molecules, that hybridize to, and are therefore
the complements of, the described NHP gene 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 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. The oligonucleotides, typically
between about 16 to about 40 (or any whole number within the
stated range) nucleotides in length may partially overlap each
other and/or a NHP sequence may be represented using
oligonucleotides that do not overlap. Accordingly, the
described NHP polynucleotide sequences shall typically
comprise at least about two or three distinct oligonucleotide
sequences of at least about 18, and preferably about 25,
nucleotides in length that are each first disclosed in the
described Sequence Listing. Such oligonucleotide sequences
may 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.
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For oligonucleotide probes, highly stringent conditions
may refer, e.g., to washing in 6xSSC/0.05o 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 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.
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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. 215:327-330). 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. 16:3209), 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.
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, N.Y.; and Ausubel et al., 1989, Current
Protocols in Molecular Biology, Green Publishing Associates
and Wiley Interscience, N.Y.
Alternatively, suitably labeled NHP nucleotide probes can
be used to screen a human genomic library using appropriately
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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 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 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,
such as, for example, testis tissue). 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.
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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)
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, for 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.
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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 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.)
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 NHP are
likely to cross-react with a corresponding mutant NHP gene
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.
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
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elements known to those skilled in the art that drive and
regulate expression. Such regulatory elements include but are
not limited to the human 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 a NHP, as well as compounds or
nucleotide constructs that inhibit expression of a NHP gene
(transcription factor inhibitors, antisense and 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 NHPs or NHP
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 a NHP, but can also
identify compounds that trigger NHP-mediated activities or
pathways.
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Finally, the NHP products can be used as therapeutics.
For example, soluble derivatives such as NHP peptides/domains
corresponding to NHP, 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
a NHP could activate or effectively antagonize the endogenous
NHP receptor. 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 NHP, 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 T~iE NHP SEQUENCES
The cDNA sequences and corresponding deduced amino acid
sequences of the described NHPs are presented in the Sequence
Listing. SEQ ID N0:7 describes a NHP ORF as well as flanking
regions. The NHP nucleotides were obtained from human cDNA
libraries using probes and/or primers generated from human
gene trapped sequence tags, and genomic sequence. Expression
analysis has provided evidence that the described NHP can be
expressed a variety of human cells as well as gene trapped
human cells.
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5.2 NHPs AND NIiP 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, for 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 disease.
The Sequence Listing discloses the amino acid sequences
encoded by the described NHP polynucleotides. The NHPs
display an initiator methionines in DNA sequence contexts
consistent with a translation initiation site, and several of
the ORFs display a consensus signal sequence which can
indicate that the described NHP ORFs are secreted proteins, or
can be membrane associated.
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 NHPs encoded by a NHP nucleotide
sequence 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 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, NY, herein incorporated
by reference) are generically representative of all the
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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 can 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, a NHP peptide or NHP
polypeptide is thought to be a soluble or secreted molecule,
the peptide or polypeptide can be recovered from the culture
media. Such expression systems also encompass engineered host
cells that express 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
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the structural and functional characteristics of a 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 encoding 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 produced for the
generation of pharmaceutical compositions of and/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 & 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
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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 can 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).
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
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an entire NHP sequence 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 Bittner 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
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, WI38, 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
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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 a NHP product. Such engineered cell lines may
be particularly useful in screening and evaluation of
compounds that affect the endogenous activity of a 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-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.
Nat!. Acad. Sci. USA 78:1527); gpt, which confers resistance
to mycophenolic acid !Mulligan & Berg, 1981, Proc. Nat!. Acad.
Sci. USA 78:2072); neo, which confers 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).
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.,
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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 Ni2+~nitriloacetic
acid-agarose columns and histidine-tagged proteins are
selectively eluted with imidazole-containing buffers.
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')z 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 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 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 sequence 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 a NHP),
truncated NHP polypeptides (NHP in which one or more domains
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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, 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, diptheria 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 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
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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.
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 334:544-
546) can be adapted to produce single chain antibodies against
NHP sequence 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.
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')2 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 (Huse 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. 147(8):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" a NHP and, therefore,
bind and activate or neutralize a receptor. Such anti-
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idiotypic antibodies or Fab fragments of such anti-idiotypes
can be used in therapeutic regimens involving a NHP signaling
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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