Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND MATERIALS RELATING TO
NEUROTRIMIN-LIKE POLYPEPTIDES AND POLYNUCLEOTIDES
1 TECHNICAL FIELD
The present invention provides novel polynucleotides and proteins encoded by
such
polynucleotides, along with uses for these polynucleotides and proteins, for
example in
therapeutic, diagnostic and research methods. In particular, the invention
relates to a novel
neurotrimin-like polypeptide:
2 BACKGROUND ART
Identified polynucleotide and polypeptide sequences have numerous applications
in, for
example, diagnostics, forensics, gene mapping; identification of mutations
responsible for
genetic disorders or other traits, to assess biodiversity, and to produce many
other types of data
and products dependent on DNA and amino acid sequences. Proteins are known to
have
biological activity, for example, by virtue of their secreted nature in the
case of leader sequence
cloning, by virtue of their cell or tissue source in the case of PCR-based
techniques, or by virtue
of structural similarity to other genes of known biological activity. It is to
these polypeptides
and the polynucleotides encoding them that the present invention is directed.
In particular, this
invention is directed to a novel soluble neurotrimin-like polypeptides and
polynucleotides.
During neural development, both membrane-bound and soluble proteins regulate
axonal
growth towards their targets. Integrins, cadherins and cell adhesion molecules
(CAMs) generally
promote neurite outgrowth while ephrins, semaphorins, and netrins usually
inhibit neurite
outgrowth during axonal pathfinding. Immunoglobulin superfamily members like L
1 and
NCAM are widely expressed and promote outgrowth of most neurons (Gil et al,
(1998) J.
Neurosci. 18, 9312-9325, incorporated herein by reference).
Neurotrimin is a member of the IgLON subfamily of immunoglobulin (Ig)
superfamily of
proteins that include amongst others limbic system-associated membrane protein
(LAMP), the
opioid-binding cell adhesion molecule (OBCAM), and Neurotrimin. All the member
proteins
have three Ig-like domains, exhibit significant sequence homology and have
defined and distinct
tissue distribution (Gil et al, (1998) J. Neurosci. 18, 9312-9325). Recently,
another member of
IgLON subfamily, a soluble form of CEPU-l, named CEPUS has been reported (Kim
et al,
(1999) Mol Cells 30, 270-276). LAMP is expressed by cortical and subcortical
neurons of the
limbic system, while neurotrimin is expressed mainly in sensorimotor cortex
layers IV, V and
VI, subplate, and the rostral lateral thalamus and also in pontine nucleus and
cerebellum. Tissue
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distribution of neurotrimin suggests a potential role for it in the
development of thalamocortical
and pontocerebellar projections (Struyk et al, (1995) J. Neurosci. 15, 2141-
2156).
Neurotrimin has been shown to form homomeric complexes and mediate homophilic
adhesion (Gil et al, (1998) J. Neurosci. 18, 9312-9325). Neurotrimin may form
heteromers with
other IgLON family members. Neurotrimin is highly expressed on dorsal root
ganglia and
hippocampal neurons but not on the superior cervical ganglia. Consequently,
neurotrimin
promotes the outgrowth of sensory neurons such as dorsal root ganglia that
express neurotrimin
while it inhibits growth of sympathetic neurons such as superior cervical
ganglia that express
none or low levels of neurotrimin (Gil et al, (1998) J. Neurosci. 18, 9312-
9325). Thus,
neurotrimin may play a central role in the development of nervous system.
Diagnosis of disease and determination of treatment efficacy are important
tools in
medicine. The polypeptides and polynucleotides coding for neurotrimin or parts
thereof may
provide potential treatments for disorders involving, but not limited to
cognition, memory and
learning, mood, dementia (including without limitation Alzheimer's disease,
dementia associated
with Parkinson's disease, mufti-infarct dementia and others), depression,
anxiety (including
without limitation manic-depressive illness, obsessive-compulsive disorders,
generalized anxiety
and others), different forms of epilepsy, schizophrenia and schizopphrenaform
disorders
(including without limitation schizoaffecto disorder), cerebral palsy and
hypertension (see, e.g.
U.S. Patent No. 5,861,283, incorporated herein by reference). The polypeptides
or
polynucleotides may provide treatment for the development of new neurons in
CNS/Spinal cord
injury.
3. SUMMARY OF THE INVENTION
This invention is based on the discovery of novel neurotrimin-like
polypeptides, novel
isolated polynucleotides encoding such polypeptides, including recombinant DNA
molecules,
cloned genes or degenerate variants thereof, especially naturally occurring
variants such as
allelic variants, antisense polynucleotide molecules, and antibodies that
specifically recognize
one or more epitopes present on such polypeptides, as well as hybridomas
producing such
antibodies. Specifically, the polynucleotides of the present invention are
based on a neurotrimin-
like polynucleotide isolated from a cDNA library prepared from human thalamus
(Hyseq clone
identification number 10468562).
The compositions of the present invention additionally include vectors such as
expression
vectors containing the polynucleotides of the invention, cells genetically
engineered to contain
such polynucleotides and cells genetically engineered to express such
polynucleotides.
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The compositions of the invention provide isolated polynucleotides that
include, but are not
limited to, a polynucleotide comprising the nucleotide sequence set forth in
the SEQ ID NO: 1-3, 5-
6, 8; or a fragment of SEQ ID NO: 1-3, 5-6, 8; a polynucleotide comprising the
full length protein
coding sequence of the SEQ ID NO: 3 or 6 (for example, SEQ ID NO: 4 or 7); and
a polynucleotide
comprising the nucleotide sequence of the mature protein coding sequence of
any of SEQ ID NO:
1-3, 5-6, 8. The polynucleotides of the present invention also include, but
are not limited to, a
polynucleotide that hybridizes under stringent hybridization conditions to (a)
the complement of any
of the nucleotide sequences set forth in SEQ ID NO: 1-3, 5-6, 8; (b) a
nucleotide sequence encoding
any one of the polypeptides of SEQ ID NO: 4, 7, 9-13; a polynucleotide which
is an allelic variant
of any polynucleotides recited above having at least 70% polynucleotide
sequence identity to the
polynucleotides; a polynucleotide which encodes a species homolog (e.g.
orthologs) of any of the
peptides recited above; or a polynucleotide that encodes a polypeptide
comprising a specific domain
or truncation of the polypeptide comprising SEQ ID NO: 4 or 7.
A collection as used in this application can be a collection of only one
polynucleotide. The
collection of sequence information or unique identifying information of each
sequence can be
provided on a nucleic acid array. In one embodiment, segments of sequence
information are
provided on a nucleic acid array to detect the polynucleotide that contains
the segment. The array
can be designed to detect full-match or mismatch to the polynucleotide that
contains the segment.
The collection can also be provided in a computer-readable format.
This invention further provides cloning or expression vectors comprising at
least a fragment
of the polynucleotides set forth above and host cells or organisms transformed
with these expression
vectors. Useful vectors include plasmids, cosmids, lambda phage derivatives,
phagemids, and the
like, that are well known in the art. Accordingly, the invention also provides
a vector including a
polynucleotide of the invention and a host cell containing the polynucleotide.
In general, the vector
contains an origin of replication functional in at least one organism,
convenient restriction
endonuclease sites, and a selectable marker for the host cell. Vectors
according to the invention
include expression vectors, replicationvectors, probe generationvectors, and
sequencing vectors. A
host cell according to the invention can be a prokaryotic or eukaryotic cell
and can be a unicellular
organism or part of a multicellular organism.
The compositions of the present invention include polypeptides comprising, but
not limited
to, an isolated polypeptide selected from the group comprising the amino acid
sequence of SEQ ID
NO: 4, 7, 9-13; or the corresponding full length or mature protein.
Polypeptides of the invention
also include polypeptides with biological activity that are encoded by (a) any
of the polynucleotides
having a nucleotide sequence set forth in the SEQ ID NO: 1-3, 5-6, 8; or (b)
polynucleotides that
hybridize to the complement of the polynucleotides of (a) under stringent
hybridization conditions.
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Biologically or immunologically active variants of any of the protein
sequences listed as SEQ ID
NO: 4, 7, 9-13 and substantial equivalents thereof that retain biological or
immunological activity
are also contemplated. The polypeptides of the invention may be wholly or
partially chemically
synthesized but are preferably produced by recombinant means using the
genetically engineered
cells (e.g. host cells) of the invention.
The invention also provides compositions comprising a polypeptide of the
invention.
Pharmaceutical compositions of the invention may comprise a polypeptide of the
invention and
an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically
acceptable, carrier.
The invention also relates to methods for producing a polypeptide of the
invention
comprising culturing host cells comprising an expression vector containing at
least a fragment of
a polynucleotide encoding the polypeptide of the invention in a suitable
culture medium under
conditions permitting expression of the desired polypeptide, and purifying the
protein or peptide
from the culture or from the host cells. Preferred embodiments include those
in which the
protein produced by such a process is a mature form of the protein.
Polynucleotides according to the invention have numerous applications in a
variety of
techniques known to those skilled in the art of molecular biology. These
techniques include use
as hybridization probes, use as oligomers, or primers, for PCR, use in an
array, use in computer-
readable media, use for chromosome and gene mapping, use in the recombinant
production of
protein, and use in generation of antisense DNA or RNA, their chemical analogs
and the like.
Fox example, when the expression of an mRNA is largely restricted to a
particular cell or tissue
type, polynucleotides of the invention can be used as hybridization probes to
detect the presence
of the particular cell or tissue mRNA in a sample using, e.g., in situ
hybridization.
In other exemplary embodiments, the polynucleotides are used in diagnostics as
expressed sequence tags for identifying expressed genes or, as well known in
the art and
exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed
sequence tags for
physical mapping of the human genome.
The polypeptides according to the invention can be used in a variety of
conventional
procedures and methods that are currently applied to other proteins. For
example, a polypeptide
of the invention can be used to generate an antibody that specifically binds
the polypeptide. Such
antibodies, particularly monoclonal antibodies, axe useful for detecting or
quantitating the
polypeptide in tissue. The polypeptides of the invention can also be used as
molecular weight
markers, and as a food supplement.
Methods are also provided for preventing, treating, or ameliorating a medical
condition
which comprises the step of administering to a mammalian subject a
therapeutically effective
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amount of a composition comprising a peptide of the present invention and a
pharmaceutically
acceptable carrier.
In particular, the polypeptides and polynucleotides of the invention can be
utilized, for
example, as part of methods for the prevention and/or treatment of
neurological disorders
involving , but not limited to cognition, memory and learning, mood, dementia
(including
without limitation Alzheimer's disease, dementia associated with Parkinson's
disease, multi-
infarct dementia and others), depression, anxiety (including without
limitation manic-depressive
illness, obsessive-compulsive disorders, generalized anxiety and others),
different forms of
epilepsy, schizophrenia and schizopphrenaform disorders (including without
limitation
schizoaffecto disorder), cerebral palsy and hypertension. The polypeptides or
polynucleotides
may also be used in treatment for the development of new neurons in CNS/Spinal
Cord injury
The methods of the invention also provides methods for the treatment of
disorders as
recited herein which comprise the administration of a therapeutically
effective amount of a
composition comprising a polynucleotide or polypeptide of the invention and a
pharmaceutically
acceptable carrier to a mammalian subject exhibiting symptoms or tendencies
related to disorders .
as recited herein. In addition, the invention encompasses methods for treating
diseases or
disorders as recited herein comprising the step of administering a composition
comprising
compounds and other substances that modulate the overall activity of the
target gene products
and a pharmaceutically acceptable carrier. Compounds and other substances can
effect such
modulation either on the level of target gene/protein expression or target
protein activity.
Specifically, methods are provided for preventing, treating or ameliorating a
medical condition,
including neurological diseases, which comprises administering to a mammalian
subject,
including but not limited to humans, a therapeutically effective amount of a
composition
comprising a polypeptide of the invention or a therapeutically effective
amount of a composition
comprising a binding partner of (e.g., antibody specifically reactive for)
neurotrimin-like
polypeptides of the invention. The mechanics of the particular condition or
pathology will
dictate whether the polypeptides of the invention or binding partners (or
inhibitors) of these
would be beneficial to the individual in need of treatment.
According to this method, polypeptides of the invention can be administered to
produce
an ih vitro or ih vivo inhibition of cellular function. A polypeptide of the
invention can be
administered in vivo alone or as an adjunct to other therapies. Conversely,
protein or other active
ingredients of the present invention may be included in formulations of a
particular agent to
minimize side effects of such an agent.
The invention further provides methods for manufacturing medicaments useful in
the
above-described methods.
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The present invention further relates to methods for detecting the presence of
the
polynucleotides or polypeptides of the invention in a sample (e.g., tissue or
sample). Such
methods can, for example, be utilized as part of prognostic and diagnostic
evaluation of disorders
as recited herein and for the identification of subjects exhibiting a
predisposition to such
conditions.
The invention provides a method for detecting a polypeptide of the invention
in a sample
comprising contacting the sample with a compound that binds to and forms a
complex with the
polypeptide under conditions and for a period sufficient to form the complex
and detecting
formation of the complex, so that if a complex is formed, the polypeptide is
detected.
The invention also provides kits comprising polynucleotide probes and/or
monoclonal
antibodies, and optionally quantitative standards, for carrying out methods of
the invention.
Furthermore, the invention provides methods for evaluating the efficacy of
drugs, and
monitoring the progress of patients, involved in clinical trials for the
treatment of disorders as
recited above.
The invention also provides methods for the identification of compounds that
modulate
(i.e., increase or decrease) the expression or activity of the polynucleotides
and/or polypeptides
of the invention. Such methods can be utilized, for example, for the
identification of compounds
that can ameliorate symptoms of disorders as recited herein. Such methods can
include, but axe
not limited to, assays for identifying compounds and other substances that
interact with (e.g.,
bind to) the polypeptides of the invention.
The invention provides a method for identifying a compound that binds to the
polypeptide of the present invention comprising contacting the compound with
the polypeptide
under conditions and for a time sufficient to form a polypeptide/compound
complex and
detecting the complex, so that if the polypeptide/compound complex is
detected, a compound
that binds to the polypeptide is identified.
Also provided is a method for identifying a compound that binds to the
polypeptide
comprising contacting the compound with the polypeptide in a cell for a time
sufficient to form a
polypeptide/compound complex wherein the complex drives expression of a
reporter gene
sequence in the cell and detecting the complex by detecting reporter gene
sequence expression so
that if the polypeptide/compound complex is detected a compound that binds to
the polypeptide
is identified.
4. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the BLASTX amino acid sequence alignment between SEQ ID NO: 4
(also identified as "Neurotrimin-like") and novel human protein with
immunoglobulin domain
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amino acids 1-200 of SEQ ID NO: 14 (identified as "Ig domain"), indicating
that the two
sequences share 100% similarity over 200 amino acid residues of SEQ ID NO: 4
and 100%
identity over 200 amino acid residues of SEQ ID NO: 4, wherein A=Alanine,
C=Cysteine,
D=Aspartic Acid, E= Glutamic Acid, F=Phenylalanine, G=Glycine, H=Histidine,
I=Isoleucine,
K=Lysine, L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,
R=Arginine,
S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented
as dashes.
Figure 2 shows the BLASTX amino acid sequence alignment between SEQ ID NO: 4
(also identified as "Neurotrimin-like") and rat neurotrimin precursor SEQ ID
NO: 15 (identified
as "neurotrimin"), indicating that the two sequences share 46% similarity over
270 amino acid
residues of SEQ ID NO: 4 and 29% identity over 270 amino acid residues of SEQ
ID NO: 4,
wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E= Glutamic Acid,
F=Phenylalanine,
G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,
N=Asparagine,
P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine, V=Valine,
W=Tryptophan,
Y=Tyrosine. Gaps are presented as dashes.
Figure 3 shows the BLASTX amino acid sequence alignment between SEQ ID NO: 7
(also identified as "Neurotrimin-like") and novel human protein with
immunoglobulin domain
SEQ ID NO: 14 (identified as "Ig domain"), indicating that the two sequences
share 99%
similarity over 299 amino acid residues of SEQ ID NO: 7 and 99% identity over
299 amino acid
residues of SEQ ID NO 7, wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=
Glutamic
Acid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,
L=Leucine,
M=Methionine, N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine,
T=Threonine,
V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.
Figure 4 shows the BLASTX amino acid sequence alignment between SEQ ID NO: 7
(also identified as "Neurotrimin-like") and rat neurotrimin precursor SEQ ID
NO:15 (identified
as "neurotrimin"), indicating that the two sequences share 46% similarity over
270 amino acid
residues of SEQ ID NO: 7 and 29% identity over 270 amino acid residues of SEQ
ID NO: 7,
wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E= Glutamic Acid,
F=Phenylalanine,
G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,
N=Asparagine,
P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine, V=Valine,
W=Tryptophan,
Y=Tyrosine. Gaps are presented as dashes.
5. DETAILED DESCRIPTION OF THE INVENTION
Soluble secreted neurotrimin-like (SEQ ID NO: 4) is an approximately 458-amino
acid
protein with a predicted molecular mass of approximately 51 kDa
unglycosylated. Protein
database searches with the BLASTX algorithm (Altschul S.F. et al., J. Mol.
Evol. 36:290-300
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(1993) and Altschul S.F. et al., J. Mol. Biol. 21:403-10 (1990), herein
incorporated by reference)
indicate that SEQ ID NO: 4 is homologous to novel human protein with
immunoglobulin
domains and rat neurotrimin. Protein database search with eMATRIX software
(Stanford
University, Stanford CA) further show that SEQ ID NO: 4 is homologous to
receptor Fc
immunoglobulin affinity and receptor tyrosine kinase class III proteins.
Figure 1 shows the
BLASTX amino acid sequence alignment between SEQ ID NO: 4 (also identified as
"neurotrimin-like") and the novel human protein with immunoglobulin domains
(SEQ ID NO:
14) indicating that the two sequences share 100% similarity over 200 amino
acid residues of
SEQ ID NO: 4 and 100% identity over 200 amino acid residues of SEQ ID NO: 4.
Figure 2
shows the BLASTX amino acid sequence alignment between SEQ ID NO: 4 (also
identified as
"neurotrimin-like") and the rat neurotrimin precursor (SEQ ID NO: 15)
indicating that the two
sequences share 46% similarity over 270 amino acid residues of SEQ ID NO: 4
and 29% identity
over 270 amino acid residues of SEQ ID NO: 4.
A splice variant of SEQ ID NO: 4 is SEQ ID NO: 7. The splice site occurs after
nucleotide 1481 of SEQ ID N0:3. The splice varient is an approximately 586
amino acid protein
with a predicted molecular mass of approximately 66 kDa unglycosylated.
Protein database
searches with the BLASTX algorithm (Altschul S.F. et al., J. Mol. Evol. 36:290-
300 (1993) and
Altschul S.F. et al., J. Mol. Biol. 21:403-10 (1990), herein incorporated by
reference) indicate
that SEQ ID NO: 7 is homologous to novel human protein with immunoglobulin
domains and rat
neurotrimin. Figure 3 and Figure 4 protein database search with eMATRIX
software (Stanford
University, Stanford CA) further show that SEQ ID NO: 7 is homologous to
receptor Fc
immunoglobulin affinity and receptor tyrosine kinase class III proteins.
Figure 3 shows the
BLASTX amino acid sequence alignment between SEQ ID NO: 7 (also identified as
"neurotrimin-like") and the novel human protein with immunoglobulin domains
(SEQ ID NO:
14) indicating that the two sequences share 99% similarity over 299 amino acid
residues of SEQ
ID NO: 7 and 99% identity over 299 amino acid residues of SEQ ID NO: 7. Figure
4 shows the
BLASTX amino acid sequence alignment between SEQ ID NO: 7 (also identified as
"neurotrimin-like") and the rat neurotrimin precursor (SEQ ID NO: 15)
indicating that the two
sequences share 46% similarity over 270 amino acid residues of SEQ ID NO: 7
and 29% identity
over 270 amino acid residues of SEQ ID NO: 7. The sequences of the present
invention are
expected to have soluble neurotrimin-like activity.
A predicted approximately sixteen residue signal peptide is encoded from
approximately
residue 1 to residue 16 of both SEQ ID NO: 4 and SEQ ID NO: 7 (SEQ ID NO: 11).
The
extracellular portion is useful on its own. This can be confirmed by
expression in mammalian
cells and sequencing of the cleaved product. The signal peptide region was
predicted using the
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Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol Biol, 157, pp. 105-
31 (1982),
incorporated herein by reference). One of skill in the art will recognize that
the actual cleavage
site may be different than that predicted by the computer program.
Using eMATRIX software package (Stanford University, Stanford, CA) (Wu et al.,
J.
Comp. Biol., vol. 6, pp. 219-235 (1999), herein incorporated by reference, the
soluble
neurotrimin-like polypeptide is expected to have receptor Fc immunoglobulin
affinity domain at
residues 292-328 of SEQ ID NO: 4 and at residues 291-327 of SEQ ID N0:7 (SEQ
ID NO: 9);
and a receptor tyrosine kinase class III protein domain at residues 155-179 of
SEQ ID N0:4 and
at residues 154-178 of SEQ ID N0:7 (SEQ ID NO: 10). The domains corresponding
to SEQ ID
NO: 4 are as follows wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=
Glutamic Acid,
F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine,
M=Methionine,
N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,
V=Valine,
W--Tryptophan, Y=Tyrosine:
Receptor Fc immunoglobulin affinity domain
(TLSIPSVQARDSGYYNCTATNNVGNPAKKTVNLLVR designated as SEQ ID NO: 9): p-
value of 5.378e-9, PD01270D (identification number correlating to signature),
located at
residues 292-328 of SEQ ID NO: 4 and located at residues 291-327 of SEQ ID NO:
7; and
Receptor tyrosine kinase class III proteins (LRCTVNSNPPARFIWKRGSDTLSH
designated as SEQ ID NO: 10): p-value of 9.809e-09, BL00240B (identification
number
correlating to signature), located at residues 155-179 of SEQ ID NO: 4 and
located at residues
154-178 of SEQ ID NO: 7. .
The polypeptides and polynucleotides of the invention can be utilized, for
example, as
part of methods for the prevention and/or treatment of disorders mediated by
loss or
overexpression of neurotrimin-like polypeptide. Such disorders include,
cognition, memory and
learning, mood, dementia (including without limitation Alzheimer's disease,
dementia associated
with Parkinson's disease, mufti-infarct dementia and others), depression,
anxiety (including
without limitation manic-depressive illness, obsessive-compulsive disorders,
generalized anxiety
and others), different forms of epilepsy, schizophrenia and schizopphrenaform
disorders
(including without limitation schizoaffecto disorder), cerebral palsy and
hypertension.
5.1 DEFINITTONS
It must be noted that as used herein and in the appended claims, the singular
forms "a",
"an" and "the" include plural references unless the context clearly dictates
otherwise.
The term "active" refers to those forms of the polypeptide which retain the
biologic
and/or immunologic activities of any naturally occurring polypeptide.
According to the
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invention, the terms "biologically active" or "biological activity" refer to a
protein or peptide
having structural, regulatory or biochemical fixnctions of a naturally
occurring molecule. The
term "neurotrimin-like activity" refers to biological activity that is similar
to the biological
activity of neurotrimin. Likewise "biologically active" or "biological
activity" refers to the
capability of the natural, recombinant or synthetic neurotrimin-like, or any
peptide thereof, to
induce a specific biological response in appropriate animals or cells and to
bind with specific
antibodies.
The term "activated cells" as used in this application are those cells which
are engaged in
extracellular or intracellular membrane trafficking, including the export of
secretory or
enzymatic molecules as part of a normal or disease process.
The terms "complementary" or "complementarity" refer to the natural binding of
polynucleotides by base pairing. For example, the sequence 5'-AGT-3' binds to
the
complementary sequence 3'-TCA-5'. Complementarity between two single-stranded
molecules
may be "partial" such that only some of the nucleic acids bind or it may be
"complete" such that
total complementarity exists between the single stranded molecules. The degree
of
complementarity between the nucleic acid strands has significant effects on
the efFciency and
strength of the hybridization between the nucleic acid strands.
The term "embryonic stem cells (ES)" refers to a cell that can give rise to
many
differentiated cell types in an embryo or an adult, including the germ cells.
The term "germ line
stem cells (GSCs)" refers to stem cells derived from primordial stem cells
that provide a steady
and continuous source of germ cells for the production of gametes. The term
"primordial germ
cells (PGCs)" refers to a small population of cells set aside from other cell
lineages particularly
from the yolk sac, mesenteries, or gonadal ridges during embryogenesis that
have the potential to
differentiate into germ cells and other cells. PGCs are the source from which
GSCs and ES cells
are derived The PGCs, the GSCs and the ES cells are capable of self renewal.
Thus these cells
not only populate the germ line and give rise to a plurality of terminally
differentiated cells that
comprise the adult specialized organs, but are able to regenerate themselves.
The term "expression modulating fragment," EMF, means a series of nucleotides
which
modulates the expression of an operably linked ORF or another EMF.
As used herein, a sequence is said to "modulate the expression of an operably
linked
sequence" when the expression of the sequence is altered by the presence of
the EMF. EMFs
include, but are not limited to, promoters, and promoter modulating sequences
(inducible
elements). One class of EMFs is nucleic acid fragments which induce the
expression of an
operably linked ORF~in response to a specific regulatory factor or
physiological event.
to
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The terms "nucleotide sequence" or "nucleic acid" or "polynucleotide" or
"oligonculeotide" are used interchangeably and refer to a heteropolymer of
nucleotides or the
sequence of these nucleotides. These phrases also refer to DNA or RNA of
genomic or synthetic
origin which may be single-stranded or double-stranded and may represent the
sense or the
antisense strand, to peptide nucleic acid (PNA) or to any DNA-like or RNA-like
material.
Generally, nucleic acid segments provided by this invention may be assembled
from fragments
of the genome and short oligonucleotide linkers, or from a series of
oligonucleotides, or from
individual nucleotides, to provide a synthetic nucleic acid which is capable
of being expressed in
a recombinant transcriptional unit comprising regulatory elements derived from
a microbial or
viral operon, or a eukaryotic gene.
The terms "oligonucleotide fragment" or a "polynucleotide fragment",
"portion," or
"segment" or "probe" or "primer" are used interchangeable and refer to a
sequence of nucleotide
residues which are at least about 5 nucleotides, more preferably at least
about 7 nucleotides,
more preferably at least about 9 nucleotides, more preferably at least about
11 nucleotides and
most preferably at least about 17 nucleotides. The fragment is preferably less
than about 500
nucleotides, preferably less than about 200 nucleotides, more preferably less
than about 100
nucleotides, more preferably less than about 50 nucleotides and most
preferably less than 30
nucleotides. Preferably the probe is from about 6 nucleotides to about 200
nucleotides,
preferably from about 15 to about 50 nucleotides, more preferably from about
17 to 30
nucleotides and most preferably from about 20 to 25 nucleotides. Preferably
the fragments can
be used in polymerase chain reaction (PCR), various hybridization procedures
or microarray
procedures to identify or amplify identical or related parts of mRNA or DNA
molecules. A
fragment or segment may uniquely identify each polynucleotide sequence of the
present
invention. Preferably the fragment comprises a sequence substantially similar
to a portion of
SEQ ID NO: 1-3, 5-6, 8. More preferably the fragment comprises a sequence
substantially
similar to a portion nucleotides 1-884 and 1485-1699 of SEQ ID NO: 3 and 1-881
and
nucleotides 1776-2196 of SEQ ID NO: 6.
Probes may, for example, be used to determine whether specific mRNA molecules
are
present in a cell or tissue or to isolate similar nucleic acid sequences from
chromosomal DNA as
described by Walsh et al. (Walsh, P.S. et al., 1992, PCR Methods Appl 1:241-
250). They may
be labeled by nick translation, Klenow fill-in reaction, PCR, or other methods
well known in the
art. Probes of the present invention, their preparation and/or labeling are
elaborated in
Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor
Laboratory, NY; or Ausubel, F.M. et al., 1989, Current Protocols in Molecular
Biology, John
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Wiley & Sons, New York NY, both of which are incorporated herein by reference
in their
entirety.
The nucleic acid sequences of the present invention also include the sequence
information from any of the nucleic acid sequences of SEQ ID NO: 1-3, 5-6, 8.
The sequence
information can be a segment of SEQ ID NO: 1-3, 5-6, 8 that uniquely
identifies or represents
the sequence information of SEQ ID NO: 1-3, 5-6, 8. One such segment can be a
twenty-mer
nucleic acid sequence because the probability that a twenty-mer is fully
matched in the human
genome is 1 in 300. In the human genome, there are three billion base pairs in
one set of
chromosomes. Because 42° possible twenty-mers exist, there are 300
times more twenty-mers
than there are base pairs in a set of human chromosome. Using the same
analysis, the probability
for a seventeen-mer to be fully matched in the human genome is approximately 1
in 5. When
these segments are used in arrays for expression studies, fifteen-mer segments
can be used. The
probability that the fifteen-mer is fully matched in the expressed sequences
is also approximately
one in five because expressed sequences comprise less than approximately 5% of
the entire
genome sequence.
Similarly, when using sequence information for detecting a single mismatch, a
segment can
be a twenty-five mer. The probability that the twenty-five mer would appear in
a human genome
with a single mismatch is calculated by multiplying the probability for a full
match ( 1-425) times the
increased probability for mismatch at each nucleotide position (3 x 25). The
probability that an
eighteen mer with a single mismatch can be detected in an array for expression
studies is
approximately one in five. The probability that a twenty-mer with a single
mismatch can be
detected in a human genome is approximately one in five.
The term "open reading frame," ORF, means a series of nucleotide triplets
coding for
amino acids without any termination codons and is a sequence translatable into
protein.
The terms "operably linked" or "operably associated" refer to functionally
related nucleic
acid sequences. For example, a promoter is operably associated or operably
linked with a coding
sequence if the promoter controls the transcription of the coding sequence.
While operably
linked nucleic acid sequences can be contiguous and in the same reading frame,
certain genetic
elements e.g. repressor genes are not contiguously linked to the coding
sequence but still control
transcription/translation of the coding sequence.
The term "pluripotent" refers to the capability of a cell to differentiate
into a number of
differentiated cell types that are present in an adult organism. A pluripotent
cell is restricted in its
differentiation capability in comparison to a totipotent cell.
The terms "polypeptide" or "peptide" or "amino acid sequence" refer to an
oligopeptide,
peptide, polypeptide or protein sequence or fragment thereof and to naturally
occurring or
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synthetic molecules. A polypeptide "fragment," "portion," or "segment" is a
stretch of amino
acid residues of at least about 5 amino acids, preferably at least about 7
amino acids, more
preferably at least about 9 amino acids and most preferably at least about 17
or more amino
acids. The peptide fragment preferably is not greater than about 500 amino
acids, more
preferably less than 200 amino acids and most preferably less than 100 amino
acids. Preferably
the peptide is from about 5 to about 200 amino acids. Preferably, the
polypeptide comprises a
contiguous sequence similar to a contiguous portion of SEQ ID NO: 4, 7, 9-13,
more preferably
a contiguous sequence from amino acids 1- 471 and amino acids 536-586 of SEQ
ID N0:7, and
amino acids 1 - 238 and amino acids 439 - 458 of SEQ ID N0:4. To be active,
any polypeptide
must have sufficient length to display biological and/or immunological
activity.
The term "naturally occurring polypeptide" refers to polypeptides produced by
cells that
have not been genetically engineered and specifically contemplates various
polypeptides arising
from post-translational modifications of the polypeptide including, but not
limited to,
acetylation, carboxylation, glycosylation, phosphorylation, lipidation and
acylation.
The term "translated protein coding portion" means a sequence which encodes
for the full
length protein which may include any leader sequence or a processing sequence.
The term "mature protein coding sequence" refers to a sequence which encodes a
peptide
or protein without any leader/signal sequence. The peptide may have the leader
sequences
removed during processing in the cell or the protein may have been produced
synthetically or
using a polynucleotide only encoding for the mature protein coding sequence.
The term "derivative" refers to polypeptides chemically modified by such
techniques as
ubiquitination, labeling (e.g., with radionuclides or various enzymes),
covalent polymer
attachment such as pegylation (derivatization with polyethylene glycol) and
insertion or
substitution by chemical synthesis of amino acids such as ornithine, which do
not normally occur
in human proteins.
The term "variant"(or "analog") refers to any polypeptide differing from
naturally
occurring polypeptides by amino acid insertions, deletions, and substitutions,
created using, a g.,
recombinant DNA techniques. Guidance in determining which amino acid residues
may be
replaced, added or deleted without abolishing activities of interest,~may be
found by comparing
the sequence of the particular polypeptide with that of homologous peptides
and minimizing the
number of amino acid sequence changes made in regions of high homology
(conserved regions)
or by replacing amino acids with consensus sequence.
Alternatively, recombinant variants encoding these same or similar
polypeptides may be
synthesized or selected by making use of the "redundancy" in the genetic code.
Various codon
substitutions, such as the silent changes which produce various restriction
sites, may be
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introduced to optimize cloning into a plasmid or viral vector or expression in
a particular
prokaryotic or eukaryotic system. Mutations in the polynucleotide sequence may
be reflected in
the polypeptide or domains of other peptides added to the polypeptide to
modify the properties of
any part of the polypeptide, to change characteristics such as ligand-binding
affinities, interchain
affinities, or degradation/turnover rate.
Preferably, amino acid "substitutions" are the result of replacing one amino
acid with
another amino acid having similar structural and/or chemical properties, i.e.,
conservative amino
acid replacements. "Conservative" 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
glutatnine; positively charged (basic) amino acids include arginine, lysine,
and histidine; and
negatively charged (acidic) amino acids include aspartic acid and glutamic
acid. "Insertions" or
"deletions" are preferably in the range of about 1 to 20 amino acids, more
preferably 1 to 10
amino acids. The variation allowed may be experimentally determined by
systematically making
insertions, deletions, or substitutions of amino acids in a polypeptide
molecule using
recombinant DNA techniques and assaying the resulting recombinant variants for
activity.
Alternatively, where alteration of function is desired, insertions, deletions
or non-
conservative alterations can be engineered to produce altered polypeptides.
Such alterations can,
for example, alter one or more of the biological functions or biochemical
characteristics of the
polypeptides of the invention. For example, such alterations may change
polypeptide
characteristics such as ligand-binding affinities, interchain affinities, or
degradation/turnover
rate. Further, such alterations can be selected so as to generate polypeptides
that are better suited
for expression, scale up and the like in the host cells chosen for expression.
For example,
cysteine residues can be deleted or substituted with another amino acid
residue in order to
eliminate disulfide bridges.
The terms "purified" or "substantially purified" as used herein denotes that
the indicated
nucleic acid or polypeptide is present in the substantial absence of other
biological
macromolecules, e.g., polynucleotides, proteins, and the like. In one
embodiment, the
polynucleotide or polypeptide is purified such that it constitutes at least
95% by weight, more
preferably at least 99% by weight, of the indicated biological macromolecules
present (but water,
buffers, and other small molecules, especially molecules having a molecular
weight of less than
1000 daltons, can be present).
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The term "isolated" as used herein refers to a nucleic acid or polypeptide
separated from
at least one other component (e.g., nucleic acid or polypeptide) present with
the nucleic acid or
polypeptide in its natural source. In one embodiment, the nucleic acid or
polypeptide is found in
the presence of (if anything) only a solvent, buffer, ion, or other components
normally present in
a solution of the same. The terms "isolated" and "purified" do not encompass
nucleic acids or
polypeptides present in their natural source.
The term "recombinant," when used herein to refer to a polypeptide or protein,
means
that a polypeptide or protein is derived from recombinant (e.g., microbial,
insect, or mammalian)
expression systems. "Microbial" refers to recombinant polypeptides or proteins
made in
bacterial or fungal (e.g., yeast) expression systems. As a product,
"recombinant microbial"
defines a polypeptide or protein essentially free of native endogenous
substances and
unaccompanied by associated native glycosylation. Polypeptides or proteins
expressed in most
bacterial cultures, e.g., E. coli, will be free of glycosylation
modifications; polypeptides or
proteins expressed in yeast will have a glycosylation pattern in general
different from those
expressed in mammalian cells.
The term "recombinant expression vehicle or vector" refers to a plasmid or
phage or virus
or vector, for expressing a polypeptide from a DNA (RNA) sequence. An
expression vehicle can
comprise a transcriptional unit comprising an assembly of (1) a genetic
element or elements
having a regulatory role in gene expression, for example, promoters or
enhancers, (2) a structural
or coding sequence which is transcribed into mRNA and translated into protein,
and (3)
appropriate transcription initiation and termination sequences. Structural
units intended for use
in yeast or eukaryotic expression systems preferably include a leader sequence
enabling
extracellular secretion of translated protein by a host cell. Alternatively,
where recombinant
protein is expressed without a leader or transport sequence, it may include an
amino terminal
methionine residue. This residue may or may not be subsequently cleaved from
the expressed
recombinant protein to provide a final product.
The term "recombinant expression system" means host cells which have stably
integrated
a recombinant transcriptional unit into chromosomal DNA or carry the
recombinant
transcriptional unit extrachromosomally. Recombinant expression systems as
defined herein will
express heterologous polypeptides or proteins upon induction of the regulatory
elements linked
to the DNA segment or synthetic gene to be expressed. This term also means
host cells which
have stably integrated a recombinant genetic element or elements having a
regulatory role in
gene expression, for example, promoters or enhancers. Recombinant expression
systems as
defined herein will express polypeptides or proteins endogenous to the cell
upon induction of the
CA 02399644 2002-08-02
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regulatory elements linked to the endogenous DNA segment or gene to be
expressed. The cells
can be prokaryotic or eukaryotic.
The term "secreted" includes a protein that is transported across or through a
membrane,
including transport as a result of signal sequences in its amino acid sequence
when it is
expressed in a suitable host cell. "Secreted" proteins include without
limitation proteins secreted
wholly (e.g., soluble proteins) or partially (e.g., receptors) from the cell
in which they are
expressed. "Secreted" proteins also include without limitation proteins that
are transported
across the membrane of the endoplasmic reticulum. "Secreted" proteins are also
intended to
include proteins containing non-typical signal sequences (e.g. Interleukin-1
Beta, see I~rasney,
P.A. and Young, P.R. (1992) Cytokine 4(2):134 -143) and factors released from
damaged cells
(e.g. Interleukin-1 Receptor Antagonist, see Arend, W.P. et. al. (1998) Annu.
Rev. Immunol.
16:27-55)
Where desired, an expression vector may be designed to contain a "signal or
leader
sequence" which will direct the polypeptide through the membrane of a cell.
Such a sequence
may be naturally present on the polypeptides of the present invention or
provided from
heterologous protein sources by recombinant DNA techniques.
The term "stringent" is used to refer to conditions that axe commonly
understood in the
art as stringent. Stringent conditions can include highly stringent conditions
(i.e., hybridization
to filter-bound DNA in 0.5 M NaHP04, 7% sodium dodecyl sulfate (SDS), 1 mM
EDTA at
65°C, and washing in O.1X SSC/0.1% SDS at 68°C), and moderately
stringent conditions (i.e.,
washing in 0.2X SSC/0.1% SDS at 42°C). Other exemplary hybridization
conditions are
described herein in the examples.
In instances of hybridization of deoxyoligonucleotides, additional exemplary
stringent
hybridization conditions include washing in 6X SSC/0.05% sodium pyrophosphate
at 37°C (for
14-base oligonucleotides), 48°C (for 17-base oligonucleotides),
55°C (for 20-base
oligonucleotides), and 60°C (for 23-base oligonucleotides).
As used herein, "substantially equivalent" or "substantially similar" can
refer both to
nucleotide and amino acid sequences, for example a mutant sequence, that
varies from a
reference sequence by one or more substitutions, deletions, or additions, the
net effect of
which does not result in an adverse functional dissimilarity between the
reference and subject
sequences. Typically, such a substantially equivalent sequence varies from one
of those listed
herein by no more than about 35 % (i. e. , the number of individual residue
substitutions,
additions, andlor deletions in a substantially equivalent sequence, as
compared to the
corresponding reference sequence, divided by the total number of residues in
the substantially
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equivalent sequence is about 0.35 or less). Such a sequence is said to have 65
% sequence
identity to the listed sequence. In one embodiment, a substantially
equivalent, e. g. , mutant,
sequence of the invention varies from a listed sequence by no more than 30 %
(70 % sequence
identity); in a variation of this embodiment, by no more than 25 % (75 %
sequence identity);
and in a further variation of this embodiment, by no more than 20 % (80 %
sequence identity)
and in a further variation of this embodiment, by no more than 10 % (90 %
sequence identity)
and in a further variation of this embodiment, by no more that 5 % (95 %
sequence identity).
Substantially equivalent, e. g. , mutant, amino acid sequences according to
the invention
preferably have at least 80 % sequence identity with a listed amino acid
sequence, more
preferably at least 85 % sequence identity, more preferably at least 90 %
sequence identity,
more preferably at least 95 % sequence identity, more preferably at least 98 %
sequence
identity, and most preferably at least 99% sequence identity. Substantially
equivalent
nucleotide sequence of the invention can have lower percent sequence
identities, taking into
account, for example, the redundancy or degeneracy of the genetic code.
Preferably, the
nucleotide sequence has at least about 65 % identity, more preferably at least
about 75
identity, more preferably at least about 80 % sequence identity, more
preferably at least 85
sequence identity, more preferably at least 90% sequence identity, more
preferably at least
about 95 % sequence identity, more preferably at least 98 % sequence identity,
and most
preferably at least 99 % sequence identity. For the purposes of the present
invention,
sequences having substantially equivalent biological activity and
substantially equivalent
expression characteristics are considered substantially equivalent. For the
purposes of
determining equivalence, truncation of the mature sequence (e. g. , via a
mutation which creates
a spurious stop codon) should be disregarded. Sequence identity may be
determined, e.g.,
using the Jotun Hein method (Rein, J. (1990) Methods Enzymol. 183:626-645).
Identity
between sequences can also be determined by other methods known in the art,
e.g. by varying
hybridization conditions.
The term "totipotent" refers to the capability of a cell to differentiate into
all of the cell
types of an adult organism.
The term "transformation" means introducing DNA into a suitable host cell so
that the
DNA is replicable, either as an extrachromosomal element, or by chromosomal
integration. The
term "transfection" refers to the taking up of an expression vector by a
suitable host cell, whether
or not any coding sequences are in fact expressed. The term "infection" refers
to the introduction
of nucleic acids into a suitable host cell by use of a virus or viral vector.
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As used herein, an "uptake modulating fragment," UMF, means a series of
nucleotides
which mediate the uptake of a linked DNA fragment into a cell. UMFs can be
readily identified
using known UMFs as a target sequence or target motif with the computer-based
systems
described below. The presence and activity of a UMF can be confirmed by
attaching the
suspected UMF to a marker sequence. The resulting nucleic acid molecule is
then incubated
with an appropriate host under appropriate conditions and the uptake of the
marker sequence is
determined. As described above, a UMF will increase the frequency of uptake of
a linked
marker sequence.
Each of the above terms is meant to encompass all that is described for each,
unless the
context dictates otherwise.
5.2 NUCLEIC ACIDS OF THE INVENTION
The invention is based on the discovery of a novel secreted neurotrimin-like
polypeptide,
the polynucleotides encoding the neurotrimin-like polypeptide and the use of
these compositions
for the diagnosis, treatment or prevention of neurological conditions and
disorders.
The isolated polynucleotides of the invention include, but are not limited to
a
polynucleotide comprising any of the nucleotide sequences of the SEQ ID NO: 1-
3, 5-6, 8; or a
fragment of SEQ ID NO: 1-3, S-6,8, a polynucleotide comprising a full length
protein coding
sequence of SEQ ID NO: 4, 7; and a polynucleotide comprising the nucleotide
sequence
encoding the mature protein coding sequence of the polynucleotides of any one
of SEQ ID NO:
1-3, 5-6, 8. The polynucleotides of the present invention also include, but
are not limited to, a
polynucleotide that hybridizes under stringent conditions to (a) the
complement of any of the
nucleotides sequences of the SEQ ID NO: 1-3, 5-6, 8; (b) a polynucleotide
encoding any one of
the polypeptides of SEQ ID NO: 4, 7, 9-13; (c) a polynucleotide which is an
allelic variant of
any polynucleotide recited above; (d) a polynucleotide which encodes a species
homolog of any
of the proteins recited above; or (e) a polynucleotide that encodes a
polypeptide comprising a
specific domain or truncation of the polypeptides of SEQ ID NO: 4, 7, 9-13.
Domains of interest
may depend on the nature of the encoded polypeptide; e.g., domains in receptor-
like
polypeptides include ligand-binding, extracellular, transmembrane, or
cytoplasmic domains, or
combinations thereof; domains in immunoglobulin-like proteins~include the
variable
immunoglobulin-like domains; domains in enzyme-like polypeptides include
catalytic and
substrate binding domains; and domains in ligand polypeptides include receptor-
binding
domains.
The polynucleotides of the invention include naturally occurring or wholly or
partially
synthetic DNA, e.g., cDNA and genomic DNA, and RNA, e.g., mRNA. The
polynucleotides
is
CA 02399644 2002-08-02
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may include all of the coding region of the cDNA or may represent a portion of
the coding
region of the cDNA.
The present invention also provides genes corresponding to the cDNA sequences
disclosed
herein. The corresponding genes can be isolated in accordance with known
methods using the
sequence information disclosed herein. Such methods include the preparation of
probes or primers
from the disclosed sequence information for identification andlor
amplification of genes in
appropriate genomic libraries or other sources of genomic materials. Further
5' and 3' sequence can
be obtained using methods known in the art. For example, full length cDNA or
genomic DNA that
corresponds to any of the polynucleotides of the SEQ ID NO: 1-3, 5-6, 8 can be
obtained by
screening appropriate cDNA or genomic DNA libraries under suitable
hybridization conditions
using any of the polynucleotides of the SEQ ID NO: 1-3, 5-6, 8 or a portion
thereof as a probe.
Alternatively, the polynucleotides of the SEQ ID NO: 1-3, 5-6, 8 may be used
as the basis for
suitable primers) that allow identification and/or amplification of genes in
appropriate genomic
DNA or cDNA libraries.
The nucleic acid sequences of the invention can be assembled from ESTs and
sequences
(including cDNA and genomic sequences) obtained from one or more public
databases, such as
dbEST, gbpri, and UniGene. The EST sequences can provide identifying sequence
information,
representative fragment or segment information, or novel segment information
for the full-length
gene.
The polynucleotides of the invention also provide polynucleotides including
nucleotide
sequences that are substantially equivalent to the polynucleotides recited
above.
Polynucleotides according to the invention can have, e. g. , at least about 65
% , at least about
70 % , at least about 75 % , at least about 80 % , 81 % , 82 % , 83 % , 84 % ,
more typically at least
about 85 % , 86 % , 87 % , 88 % , 89 % , more typically at least about 90 % ,
91 % , 92 % , 93 % ,
94 % , and even more typically at least about 95 % , 96 % , 97 % , 98 % , 99 %
sequence identity to
a polynucleotide recited above.
Included within the scope of the nucleic acid sequences of the invention are
nucleic acid
sequence fragments that hybridize under stringent conditions to any of the
nucleotide sequences
of the SEQ ID NO: 1-3, 5-6, 8, or complements thereof, which fragment is
greater than about 5
nucleotides, preferably 7 nucleotides, more preferably greater than 9
nucleotides and most
preferably greater than 17 nucleotides. Fragments of, e.g. 15, 17, or 20
nucleotides or more that
are selective for (i.e. specifically hybridize to any one of the
polynucleotides of the invention)
are contemplated. Probes capable of specifically hybridizing to a
polynucleotide can
differentiate polynucleotide sequences of the invention from other
polynucleotide sequences in
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the same family of genes or can differentiate human genes from genes of other
species, and are
preferably based on unique nucleotide sequences.
The sequences falling within the scope of the present invention are not
limited to these
specific sequences, but also include allelic and species variations thereof.
Allelic and species
variations can be routinely determined by comparing the sequence provided in
SEQ ID NO: 1-3, 5-
6, 8, a representative fragment thereof, or a nucleotide sequence at least 90%
identical, preferably
95% identical, to SEQ ID NO: 1-3, 5-6, 8 with a sequence from another isolate
of the same species.
Furthermore, to accommodate codon variability, the invention includes nucleic
acid molecules
coding for the same amino acid sequences as do the specific ORFs disclosed
herein. In other words,
in the coding region of an ORF, substitution of one codon for another codon
that encodes the same
amino acid is expressly contemplated.
The nearest neighbor result for the nucleic acids of the present invention,
including SEQ ID
NO: 1-3, 5-6, 8, can be obtained by searching a database using an algorithm or
a program.
Preferably, a BLAST which stands for Basic Local Alignment Search Tool is used
to search for
local sequence alignments (Altschul, S.F. J Mol. Evol. 36: 290-300 (1993) and
Altschul S.F. et al. J.
Mol. Biol. 21:403-410 (1990))
Species homologs (or orthologs) of the disclosed polynucleotides and proteins
are also
provided by the present invention. Species homologs may be isolated and
identified by making
suitable probes or primers from the sequences provided herein and screening a
suitable nucleic
acid source from the desired species.
The invention also encompasses allelic variants of the disclosed
polynucleotides or
proteins; that is, naturally-occurring alternative forms of the isolated
polynucleotide which also
encode proteins which are identical, homologous or related to that encoded by
the
polynucleotides.
The nucleic acid sequences of the invention are further directed to sequences
which
encode variants of the described nucleic acids. These amino acid sequence
variants may be
prepared by methods known in the art by introducing appropriate nucleotide
changes into a
native or variant polynucleotide. There axe two variables in the construction
of amino acid
sequence variants: the location of the mutation and the nature of the
mutation. Nucleic acids
encoding the amino acid sequence variants are preferably constructed by
mutating the
polynucleotide to encode an amino acid sequence that does not occur in nature.
These nucleic
acid alterations can be made at sites that differ in the nucleic acids from
different species
(variable positions) or in highly conserved regions (constant regions). Sites
at such locations
will typically be modified in series, e.g., by substituting first with
conservative choices (e.g.,
hydrophobic amino acid to a different hydrophobic amino acid) and then with
more distant
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choices (e.g., hydrophobic amino acid to a charged amino acid), and then
deletions or insertions
may be made at the target site. Amino acid sequence deletions generally range
from about 1 to
30 residues, preferably about 1 to 10 residues, and are typically contiguous.
Amino acid
insertions include amino- and/or carboxyl-terminal fusions ranging in length
from one to one
hundred or more residues, as well as intrasequence insertions of single or
multiple amino acid
residues. Intrasequence insertions may range generally from about 1 to 10
amino residues,
preferably from 1 to 5 residues. Examples of terminal insertions include the
heterologous signal
sequences necessary for secretion or for intracellular targeting in different
host cells and
sequences such as FLAG or poly-histidine sequences useful for purifying the
expressed protein.
In a preferred method, polynucleotides encoding the novel amino acid sequences
are
changed via site-directed mutagenesis. This method uses oligonucleotide
sequences to alter a
polynucleotide to encode the desired amino acid variant, as well as sufficient
adjacent
nucleotides on both sides of the changed amino acid to form a stable duplex on
either side of the
site of being changed. In general, the techniques of site-directed mutagenesis
are well known to
those of skill in the art and this technique is exemplified by publications
such as, Edelman et al.,
DNA 2:183 (1983). A versatile and efficient method for producing site-specific
changes in a
polynucleotide sequence was published by Zoller and Smith, Nucleic Acids Res.
10:6487-6500
(1982). PCR may also be used to create amino acid sequence variants of the
novel nucleic acids.
When small amounts of template DNA are used as starting material, primers)
that differs
slightly in sequence from the corresponding region in the template DNA can
generate the desired
amino acid variant. PCR amplification results in a population of product DNA
fragments that
differ from the polynucleotide template encoding the polypeptide at the
position specified by the
primer. The product DNA fragments replace the corresponding region in the
plasmid and this
gives a polynucleotide encoding the desired amino acid variant.
A further technique for generating amino acid variants is the cassette
mutagenesis
technique described in Wells et al., Gene 34:315 (1985); and other mutagenesis
techniques well
known in the art, such as, for example, the techniques in Sambrook et al.,
supra, and Current
Protocols i~c Molecular Biology, Ausubel et al. Due to the inherent degeneracy
of the genetic
code, other DNA sequences which encode substantially the same or a
functionally equivalent
amino acid sequence may be used in the practice of the invention for the
cloning and expression
of these novel nucleic acids. Such DNA sequences include those which are
capable of
hybridizing to the appropriate novel nucleic acid sequence under stringent
conditions.
Polynucleotides encoding preferred polypeptide truncations of the invention
can be used
to generate polynucleotides encoding chimeric or fusion proteins comprising
one or more
domains of the invention and heterologous protein sequences.
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The polynucleotides of the invention additionally include the complement of
any of the
polynucleotides recited above. The polynucleotide can be DNA (genomic, cDNA,
amplified, or
synthetic) or RNA. Methods and algorithms for obtaining such polynucleotides
are well known
to those of skill in the art and can include, for example, methods for
determining hybridization
conditions that can routinely isolate polynucleotides of the desired sequence
identities.
In accordance with the invention, polynucleotide sequences comprising the
mature
protein coding sequences corresponding to any one of SEQ ID NO: 3, 5, 6,8 or
functional
equivalents thereof, may be used to generate recombinant DNA molecules that
direct the
expression of that nucleic acid, or a functional equivalent thereof, in
appropriate host cells. Also
included are the cDNA inserts of any of the clones identified herein.
A polynucleotide according to the invention can be joined to any of a variety
of other
nucleotide sequences by well-established recombinant DNA techniques (see
Sambrook J et al.
(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,
NY). Useful
nucleotide sequences for joining to polynucleotides include an assortment of
vectors, e.g.,
plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are
well known in the
axt. Accordingly, the invention also provides a vector including a
polynucleotide of the
invention and a host cell containing the polynucleotide. In general, the
vector contains an origin
of replication functional in at least one organism, convenient restriction
endonuclease sites, and a
selectable maxker for the host cell. Vectors according to the invention
include expression
vectors, replication vectors, probe generation vectors, and sequencing
vectors. A host cell
according to the invention can be a prokaryotic or eukaryotic cell and can be
a unicellular
organism or part of a multicellular organism.
The present invention further provides recombinant constructs comprising a
nucleic acid
having any of the nucleotide sequences of the SEQ ID NO: 1-3, 5-6, 8 or a
fragment thereof or
any other polynucleotides of the invention. In one embodiment, the recombinant
constructs of
the present invention comprise a vector, such as a plasmid or viral vector,
into which a nucleic
acid having any of the nucleotide sequences of the SEQ ID NO: 1-3, 5-6, 8 or a
fragment thereof
is inserted, in a forward or reverse orientation. In the case of a vector
comprising one of the
ORFs of the present invention, the vector may further comprise regulatory
sequences, including
for example, a promoter, operably linked to the ORF. Laxge numbers of suitable
vectors and
promoters are known to those of skill in the art and are commercially
available for generating the
recombinant constructs of the present invention. The following vectors are
provided by way of
example. Bacterial: pBs, phagescript, PsiXl74, pBluescript SIB, pBs KS, pNH8a,
pNHl6a,
pNHl8a, pNH46a (Stratagene); pTrc99A, pI~K223-3, pKK233-3, pDR540, pRITS
(Pharmacia).
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Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV, pMSG,
pSVL
(Pharmacia).
The isolated polynucleotide of the invention may be operably linked to an
expression
control sequence such as the pMT2 or pED expression vectors disclosed in
Kaufinan et al.,
Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce the protein
recombinantly. Many
suitable expression control sequences are known in the art. General methods of
expressing
recombinant proteins are also known and are exemplified in R. Kaufman, Methods
in
Ehzymology 185, 537-566 (1990). As defined herein "operably linked" means that
the isolated
polynucleotide of the invention and an expression control sequence are
situated within a vector
or cell in such a way that the protein is expressed by a host cell which has
been transformed
(transfected) with the ligated polynucleotide/expression control sequence.
Promoter regions can be selected from any desired gene using CAT
(chloramphenicol
transferase) vectors or other vectors with selectable markers. Two appropriate
vectors are
pKK232-8 and pCM7. Particular named bacterial promoters include lacI, lacZ,
T3, T7; gpt,
lambda PR, and trc. Eukaryotic promoters include CMV immediate early, HSV
thymidine
kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-
I. Selection of
the appropriate vector and promoter is well within the level of ordinary skill
in the art.
Generally, recombinant expression vectors will include origins of replication
and selectable
markers permitting transformation of the host cell, e.g., the ampicillin
resistance gene of E. coli
and S. ce~evisiae TRP 1 gene, and a promoter derived from a highly expressed
gene to direct
transcription of a downstream structural sequence. Such promoters can be
derived from operons
encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor,
acid
phosphatase, or heat shock proteins, among others. The heterologous structural
sequence is
assembled in appropriate phase with translation initiation and termination
sequences, and
preferably, a leader sequence capable of directing secretion of translated
protein into the
periplasmic space or extracellular medium. Optionally, the heterologous
sequence can encode a
fusion protein including an amino terminal identification peptide imparting
desired
characteristics, e.g., stabilization or simplified purification of expressed
recombinant product.
Useful expression vectors for bacterial use are constructed by inserting a
structural DNA
sequence encoding a desired protein together with suitable translation
initiation and termination
signals in operable reading phase with a functional promoter. The vector will
comprise one or
more phenotypic selectable markers and an origin of replication to ensure
maintenance of the
vector and to, if desirable, provide amplification within the host. Suitable
prokaryotic hosts for
transformation include E. coli, Bacillus subtilis, Salmov~ella typhimurium and
various species
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within the genera Pseudomo~cas, Streptomyces, and Staphylococcus, although
others may also be
employed as a matter of choice.
As a representative but non-limiting example, useful expression vectors for
bacterial use
can comprise a selectable marker and bacterial origin of replication derived
from commercially
available plasmids comprising genetic elements of the well known cloning
vector pBR322
(ATCC 37017). Such commercial vectors include, for example, pKK223-3
(Pharmacia Fine
Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, WI, USA).
These
pBR322 "backbone" sections are combined with an appropriate promoter and the
structural
sequence to be expressed. Following transformation of a suitable host strain
and growth of the
host strain to an appropriate cell density, the selected promoter is induced
or derepressed by
appropriate means (e.g., temperature shift or chemical induction) and cells
are cultured for an
additional period. Cells are typically harvested by centrifugation, disrupted
by physical or
chemical means, and the resulting crude extract retained for further
purification.
Polynucleotides of the invention can also be used to induce immune responses.
For
example, as described in Fan et al., Nat. Biotech. 17:870-872 (1999),
incorporated herein by
reference, nucleic acid sequences encoding a polypeptide may be used to
generate antibodies
against the encoded polypeptide following topical administration of naked
plasmid DNA or
following injection, and preferably intramuscular injection of the DNA. The
nucleic acid
sequences are preferably inserted in a recombinant expression vector and may
be in the form of
naked DNA.
5.3 ANTISENSE
Another aspect of the invention pertains to isolated antisense nucleic acid
molecules that
are hybridizable to or complementary to the nucleic acid molecule comprising
the nucleotide
sequence of SEQ ID NO: 1 - 3, S - 6, or 8, or fragments, analogs or
derivatives thereof. An
"antisense" nucleic acid comprises a nucleotide sequence that is complementary
to a "sense"
nucleic acid encoding a protein, e. g. , complementary to the coding strand of
a double-stranded
cDNA molecule or complementary to an mRNA sequence. In specific aspects,
antisense
nucleic acid molecules are provided that comprise a sequence complementary to
at least about
10, 25, 50, 100, 250 or 500 nucleotides or an entire coding strand, or to only
a portion
thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and
analogs of a
protein of any of SEQ ID NO: 1 - 3, 5 - 6, or 8 or antisense nucleic acids
complementary to a
nucleic acid sequence of SEQ ID NO: 1 - 3, 5 - 6, or 8 are additionally
provided.
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In one embodiment, an antisense nucleic acid molecule is antisense to a
"coding region"
of the coding strand of a nucleotide sequence of the invention. The term
"coding region"
refers to the region of the nucleotide sequence comprising codons which are
translated into
amino acid residues. In another embodiment, the antisense nucleic acid
molecule is antisense to
a "noncoding region" of the coding strand of a nucleotide sequence of the
invention. The term
"noncoding region" refers to 5' and 3' sequences that flank the coding region
that are not
translated into amino acids (l. e. , also referred to as 5' and 3'
untranslated regions).
Given the coding strand sequences encoding a nucleic acid disclosed herein (e.
g. , SEQ
ID NO: 1 - 3, 5 - 6, or S, antisense nucleic acids of the invention can be
designed according to
the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic
acid molecule
can be complementary to the entire coding region of an mRNA, but more
preferably is an
oligonucleotide that is antisense to only a portion of the coding or noncoding
region of an
mRNA. For example, the antisense oligonucleotide can be complementary to the
region
surrounding the translation start site of an mRNA. An antisense
oligonucleotide can be, for
example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
An antisense
nucleic acid of the invention can be constructed using chemical synthesis or
enzymatic ligation
reactions using procedures known in the art. For example, an antisense nucleic
acid (e. g. , an
antisense oligonucleotide) can be chemically synthesized using naturally
occurring nucleotides
or variously modified nucleotides designed to increase the biological
stability of the molecules
or to increase the physical stability of the duplex formed between the
antisense and sense
nucleic acids, e. g. , phosphorothioate derivatives and acridine substituted
nucleotides can be
used. .
Examples of modified nucleotides that can be used to generate the antisense
nucleic acid
include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xanthine,
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.
Alternatively, the
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antisense nucleic acid can be produced biologically using an expression vector
into which a
nucleic acid has been subcloned in an antisense orientation (i. e. , RNA
transcribed from the
inserted nucleic acid will be of an antisense orientation to a target nucleic
acid of interest,
described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically
administered to a
subject or generated i~c situ such that they hybridize with or bind to
cellular mRNA and/or
genomic DNA encoding a protein according to the invention to thereby inhibit
expression of
the protein, e. g. , by inhibiting transcription and/or translation. The
hybridization can be by
conventional nucleotide complementarity to form a stable duplex, or, for
example, in the case
of an antisense nucleic acid molecule that binds to DNA duplexes, through
specific interactions
in the major groove of the double helix. An example of a route of
administration of antisense
nucleic acid molecules of the invention includes direct injection at a tissue
site. Alternatively,
antisense nucleic acid molecules can be modified to target selected cells and
then administered
systemically. For example, for systemic administration, antisense molecules
can be modified
I S such that they specifically bind to receptors or antigens expressed on a
selected cell surface,
e. g. , by linking the antisense nucleic acid molecules to peptides or
antibodies that bind to cell
surface receptors or antigens. The antisense nucleic acid molecules can also
be delivered to
cells using the vectors described herein. To achieve sufficient intracellular
concentrations of
antisense molecules, vector constructs in which the antisense nucleic acid
molecule is placed
under the control of a strong pol II or pol III promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the
invention is an
a-anomeric nucleic acid molecule. An oc -anomeric nucleic acid molecule forms
specific
double-stranded hybrids with complementary RNA in which, contrary to the usual
oc -units, the
strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res
15: 6625-6641). The
antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide
(moue et al.
(1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA -DNA analogue (Inoue
et al.
(1987) FEBS Lett 215: 327-330).
5.4 RIBOZYMES AND PNA MOIETIES
In still another embodiment, an antisense nucleic acid of the invention is a
ribozyme.
Ribozymes are catalytic RNA molecules with ribonuclease activity that are
capable of cleaving
a single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region.
Thus, ribozymes (e. g. , hammerhead ribozymes (described in Haselhoff and
Gerlach (1988)
Nature 334:585-591)) can be used to catalytically cleave mRNA transcripts to
thereby inhibit
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translation of an mRNA. A ribozyme having specificity for a nucleic acid of
the invention can
be designed based upon the nucleotide sequence of a DNA disclosed herein (i.
e. , SEQ ID NO:
1 - 3, 5 - 6, or 8). For example, a derivative of Tetrahymena L-19 IVS RNA can
be
constructed in which the nucleotide sequence of the active site is
complementary to the
nucleotide sequence to be cleaved in a SECX-encoding mRNA. See, e. g. , Cech
et al. U. S.
Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively,
SECX mRNA
can be used to select a catalytic RNA having a specific ribonuclease activity
from a pool of
RNA molecules. See, e.g., Bartel et al., (1993) Science 261:1411-1418.
Alternatively, gene expression can be inhibited by targeting nucleotide
sequences
complementary to the regulatory region (e. g. , promoter and/or enhancers) to
form triple
helical structures that prevent transcription of the gene in target cells. See
generally, Helene.
(1991) Anticancer Drug Des. 6: 569-84; Helene. et al. (1992) Ann. N. Y. Acad.
Sci.
660:27-36; and Maher (1992) Bioassays 14: 807-15.
In various embodiments, the nucleic acids of the invention can be modified at
the base
moiety, sugar moiety or phosphate backbone to improve, e. g. , the stability,
hybridization, or
solubility of the molecule. For example, the deoxyribose phosphate backbone of
the nucleic
acids can be modified to generate peptide nucleic acids (see Hyrup et al.
(1996) Bioorg Med
Chem 4: 5-23). As used herein, the terms "peptide nucleic acids" or "PNAs"
refer to nucleic
acid mimics, e. g. , DNA mimics, in which the deoxyribose phosphate backbone
is replaced by
a pseudopeptide backbone and only the four natural nucleobases are retained.
The neutral
backbone of PNAs has been shown to allow for specific hybridization to DNA and
RNA under
conditions of low ionic strength. The synthesis of PNA oligomers can be
performed using
standard solid phase peptide synthesis protocols as described in Hyrup et al.
(1996) above;
Perry-O'Keefe et al. (1996) PNAS 93: 14670-675.
PNAs of the invention can be used in therapeutic and diagnostic applications.
For
example, PNAs can be used as antisense or antigene agents for sequence-
specific modulation
of gene expression by, e. g.., inducing transcription or translation arrest or
inhibiting
replication. PNAs of the invention can also be used, e. g. , in the analysis
of single base pair
mutations in a gene by, e. g. , PNA directed PCR clamping; as artificial
restriction enzymes
when used in combination with other enzymes, e.g., S1 nucleases (Hyrup B.
(1996) above); or
as probes or primers for DNA sequence and hybridization (Hyrup et al. (1996),
above;
Perry-O'Keefe (1996), above).
In another embodiment, PNAs of the invention can be modified, e. g. , to
enhance their
stability or cellular uptake, by attaching lipophilic or other helper groups
to PNA, by the
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formation of PNA-DNA chimeras, or by the use of liposomes or other techniques
of drug
delivery known in the art. For example, PNA-DNA chimeras can be generated that
may
combine the advantageous properties of PNA and DNA. Such chimeras allow DNA
recognition enzymes, e. g. , RNase H and DNA polymerises, to interact with the
DNA portion
while the PNA portion would provide high binding affinity and specificity. PNA-
DNA
chimeras can be linked using linkers of appropriate lengths selected in terms
of base stacking,
number of bonds between the nucleobases, and orientation (Hyrup (1996) above).
The
synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996)
above and
Finn et al. (1996) Nucl Acids Res 24: 3357-63. For example, a DNA chain can be
synthesized
on a solid support using standard phosphoramidite coupling chemistry, and
modified
nucleoside analogs, e. g. , 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine
phosphoramidite, can
be used between the PNA and the 5' end of DNA (Mag et al. (1989) Nucl Acid Res
17:
5973-88). PNA monomers are then coupled in a stepwise manner to produce a
chimeric
molecule with a 5' PNA segment and a 3' DNA segment (Finn et al. (1996)
above).
Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and
a 3' PNA
segment. See, Petersen et al. (1975) Bioorg Med Chem Lett 5: 1119-11124.
In other embodiments, the oligonucleotide may include other appended groups
such as
peptides (e. g. , for targeting host cell receptors in vivo), or agents
facilitating transport across
the cell membrane (see, e. g. , Letsinger et al. , 1989, Proc. Natl. Acid.
Sci. U. S.A.
86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acid. Sci. 84:648-652; PCT
Publication No.
W088109810) or the blood-brain barrier (see, e.g., PCT Publication No.
W089110134). In
addition, oligonucleotides can be modified with hybridization triggered
cleavage agents (See,
e. g. , Krol et al. , 1988, BioTechniques 6:958-976) or intercalating agents.
(See, e. g. , Zon,
1988, Pha~n. Res. 5: 539-549). To this end, the oligonucleotide may be
conjugated to another
molecule, e. g. , a peptide, a hybridization triggered cross-linking agent, a
transport agent, a
hybridization-triggered cleavage agent, etc.
5.5 HOSTS
The present invention further provides host cells genetically engineered to
contain the
polynucleotides of the invention. For example, such host cells may contain
nucleic acids of the
invention introduced into the host cell using known transformation,
transfection or infection
methods. The present invention still further provides host cells genetically
engineered to express
the polynucleotides of the invention, wherein such polynucleotides are in
operative association
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with a regulatory sequence heterologous to the host cell which drives
expression of the
polynucleotides in the cell.
Knowledge of neurotrimin-like DNA sequences allows for modification of cells
to
permit, or increase, expression of neurotrimin-like polypeptide. Cells can be
modified (e.g., by
homologous recombination) to provide increased neurotrimin-like polypeptide
expression by
replacing, in whole or in part, the naturally occurring neurotrimin-like
promoter with all or part
of a heterologous promoter so that the cells express neurotrimin-like
polypeptide at higher levels.
The heterologous promoter is inserted in such a manner that it is operatively
linked to
neurotrimin-like encoding sequences. See, for example, PCT International
Publication No.
W094/12650, PCT International Publication No. W092/20808, and PCT
International
Publication No. W091/09955. It is also contemplated that, in addition to
heterologous promoter
DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene
which
encodes carbamyl phosphate synthase, aspartate transcarbamylase, and
dihydroorotase) and/or
intron DNA may be inserted along with the heterologous promoter DNA. If linked
to the
neurotrimin-like coding sequence, amplification of the marker DNA by standard
selection
methods results in co-amplification of the neurotrimin-like coding sequences
in the cells.
The host cell can be a higher eukaryotic host cell, such as a mammalian cell,
a lower
eukaryotic host cell, such as a yeast cell, or the host cell can be a
prokaryotic cell, such as a
bacterial cell. Introduction of the recombinant construct into the host cell
can be effected by
calcium phosphate transfection, DEAE, dextran-mediated transfection, or
electroporation (Davis,
L. et al., Basic Methods ih Molecular Biology (1986)). The host cells
containing one of the
polynucleotides of the invention, can be used in conventional manners to
produce the gene
product encoded by the isolated fragment (in the case of an ORF) or can be
used to produce a
heterologous protein under the control of the EMF.
Any host/vector system can be used to express one or more of the ORFs of the
present
invention. These include, but are not limited to, eukaryotic hosts such as
HeLa cells, Cv-1 cell,
COS cells, 293 cells, and S~ cells, as well as prokaryotic host such as E.
coli and B. subtilis.
The most preferred cells are those which do not normally express the
particular polypeptide or
protein or which expresses the polypeptide or protein at low natural level.
Mature proteins can
be expressed in mammalian cells, yeast, bacteria, or other cells under the
control of appropriate
promoters. Cell-free translation systems can also be employed to produce such
proteins using
RNAs derived from the DNA constructs of the present invention. Appropriate
cloning and
expression vectors for use with prokaryotic and eukaryotic hosts are described
by Sambrook, et
al., in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring
Harbor, New
York (1989), the disclosure of which is hereby incorporated by reference.
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Various mammalian cell culture systems can also be employed to express
recombinant
protein. Examples of mammalian expression systems include the COS-7 lines of
monkey kidney
fibroblasts, described by Gluzman, Cell 23:175 (1981). Other cell lines
capable of expressing a
compatible vector are, for example, the C127, monkey COS cells, Chinese
Hamster Ovary
(CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Co1o205
cells, 3T3
cells, CV-1 cells, other transformed primate cell lines, normal diploid cells,
cell strains derived
from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L
cells, BHK, HL-
60, U937, HaK or Jurkat cells. Mammalian expression vectors will comprise an
origin of
replication, a suitable promoter and also any necessary ribosome binding
sites, polyadenylation
site, splice donor and acceptor sites, transcriptional termination sequences,
and 5' flanking
nontranscribed sequences. DNA sequences derived from the SV40 viral genome,
for example,
SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may
be used to provide
the required nontranscribed genetic elements. Recombinant polypeptides and
proteins produced
in bacterial culture are usually isolated by initial extraction from cell
pellets, followed by one or
more salting-out, aqueous ion exchange or size exclusion chromatography steps.
Protein
refolding steps can be used, as necessary, in completing configuration of the
mature protein.
Finally, high performance liquid chromatography (HPLC) can be employed for
final purification
steps. Microbial cells employed in expression of proteins can be disrupted by
any convenient
method, including freeze-thaw cycling, sonication, mechanical disruption, or
use of cell lysing
agents.
Alternatively, it may be possible to produce the protein in lower eukaryotes
such as yeast
or insects or in prokaryotes such as bacteria. Potentially suitable yeast
strains include
Saccharomyces ce~evisiae, Schizosaccharomyces pombe, Kluyve~omyces strains,
Candida, or
any yeast strain capable of expressing heterologous proteins. Potentially
suitable bacterial
strains include Escherichia coli, Bacillus subtilis, Salmonella typhimu~ium,
or any bacterial
strain capable of expressing heterologous proteins. If the protein is made in
yeast or bacteria, it
may be necessary to modify the protein produced therein, for example by
phosphorylation or
glycosylation of the appropriate sites, in order to obtain the functional
protein. Such covalent
attachments may be accomplished using known chemical or enzymatic methods.
In another embodiment of the present invention, cells and tissues may be
engineered to
express an endogenous gene comprising the polynucleotides of the invention
under the control of
inducible regulatory elements, in which case the regulatory sequences of the
endogenous gene
may be replaced by homologous recombination. As described herein, gene
targeting can be used
to replace a gene's existing regulatory region with a regulatory sequence
isolated from a different
gene or a novel regulatory sequence synthesized by genetic engineering
methods. Such
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regulatory sequences may be comprised of promoters, enhancers, scaffold-
attachment regions,
negative regulatory elements, transcriptional initiation sites, regulatory
protein binding sites or
combinations of said sequences. Alternatively, sequences which affect the
structure or stability
of the RNA or protein produced may be replaced, removed, added, or otherwise
modified by
targeting. These sequence include polyadenylation signals, mRNA stability
elements, splice
sites, leader sequences for enhancing or modifying transport or secretion
properties of the
protein, or other sequences which alter or improve the function or stability
of protein or RNA
molecules.
The targeting event may be a simple insertion of the regulatory sequence,
placing the
gene under the control of the new regulatory sequence, e.g., inserting a new
promoter or
enhancer or both upstream of a gene. Alternatively, the targeting event may be
a simple deletion
of a regulatory element, such as the deletion of a tissue-specific negative
regulatory element.
Alternatively, the targeting event may replace an existing element; for
example, a tissue-specific
enhancer can be replaced by an enhancer that has broader or different cell-
type specificity than
the naturally occurring elements. Here, the naturally occurring sequences axe
deleted and new
sequences are added. In all cases, the identification of the targeting event
may be facilitated by
the use of one or more selectable marker genes that are contiguous with the
targeting DNA,
allowing for the selection of cells in which the exogenous DNA has integrated
into the host cell
genome. The identification of the targeting event may also be facilitated by
the use of one or
more marker genes exhibiting the property of negative selection, such that the
negatively
selectable marker is linked to the exogenous DNA, but configured such that the
negatively
selectable marker flanks the targeting sequence, and such that a correct
homologous
recombination event with sequences in the host cell genome does not result in
the stable
integration of the negatively selectable marker. Markers useful for this
purpose include the
Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-
guanine
phosphoribosyl-transferase (gpt) gene.
The gene targeting or gene activation techniques which can be used in
accordance with
this aspect of the invention axe more particularly described in U.S. Patent
No. 5,272,071 to
Chappel; U.S. Patent No. 5,578,461 to Sherwin et al.; International
Application No.
PCT/US92/09627 (W093/09222) by Selden et al.; and International Application
No.
PCT/LJS90/06436 (W091/06667) by Skoultchi et al., each of which is
incorporated by reference
herein in its entirety.
5.6 POLYPEPTIDES OF THE INVENTION
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The isolated polypeptides of the invention include, but are not limited to, a
polypeptide
comprising: the amino acid sequence set forth as any one of SEQ ID NO: 4, 7, 9-
13 or an amino
acid sequence encoded by any one of the nucleotide sequences SEQ ID NO: 1-3, 5-
6, 8 or the
corresponding full length or mature protein. Polypeptides of the invention
also include
polypeptides preferably with biological or immunological activity that are
encoded by: (a) a
polynucleotide having any one of the nucleotide sequences set forth in the SEQ
ID NO: 1-3, 5-6,
8 or (b) polynucleotides encoding any one of the amino acid sequences set
forth as SEQ ID NO:
4, 7, 9-13 or (c) polynucleotides that hybridize to the complement of the
polynucleotides of
either (a) or (b) under stringent hybridization conditions. The invention also
provides
biologically active or immunologically active variants of any of the amino
acid sequences set
forth as SEQ ID NO: 4, 7, 9-13 or the corresponding full length or mature
protein; and
"substantial equivalents" thereof (e.g. , with at least about 65 % , at least
about 70 % , at least
about 75 % , at least about 80 % , at least about 85 % , 86 % , 87 % , 88 % ,
89 % , at least about
90 % , 91 % , 92 % , 93 % , 94 % , typically at least about 95 % , 96 % , 97 %
, more typically at least
about 98 % , or most typically at least about 99 % amino acid identity) that
retain biological
activity. Polypeptides encoded by allelic variants may have a similar,
increased, or decreased
activity compared to polypeptides comprising SEQ ID NO: 4, 7, 9-13.
Fragments of the proteins of the present invention which are capable of
exhibiting
biological activity are also encompassed by the present invention. Fragments
of the protein may
be in linear form or they may be cyclized using known methods, for example, as
described in H.
U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S. McDowell,
et al., J. Amer.
Chem. Soc. 114, 9245-9253 (1992), both of which are incorporated herein by
reference. Such
fragments may be fused to carrier molecules such as immunoglobulins for many
purposes,
including increasing the valency of protein binding sites.
The present invention also provides both full-length and mature forms (for
example,
without a signal sequence or precursor sequence) of the disclosed proteins.
The protein coding
sequence is identified in the sequence listing by translation of the disclosed
nucleotide
sequences. The mature form of such protein may be obtained by expression of a
full-length
polynucleotide in a suitable mammalian cell or other host cell. The sequence
of the mature form
of the protein is also determinable from the amino acid sequence of the full-
length form. Where
proteins of the present invention are membrane bound, soluble forms of the
proteins are also
provided. In such forms, part or all of the regions causing the proteins to be
membrane bound
are deleted so that the proteins are fully secreted from the cell in which it
is expressed.
Protein compositions of the present invention may further comprise an
acceptable carrier,
such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.
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The present invention further provides isolated polypeptides encoded by the
nucleic acid
fragments of the present invention or by degenerate variants of the nucleic
acid fragments of the
present invention. By "degenerate variant" is intended nucleotide fragments
which differ from a
nucleic acid fragment of the present invention (e.g., an ORF) by nucleotide
sequence but, due to
the degeneracy of the genetic code, encode an identical polypeptide sequence.
Preferred nucleic
acid fragments of the present invention are the ORFs that encode proteins.
A variety of methodologies known in the art can be utilized to obtain any one
of the
isolated polypeptides or proteins of the present invention. At the simplest
level, the amino acid
sequence can be synthesized using commercially available peptide synthesizers.
The
synthetically-constructed protein sequences, by virtue of sharing primary,
secondary or tertiary
structural and/or conformational characteristics with proteins may possess
biological properties
in common therewith, including protein activity. This technique is
particularly useful in
producing small peptides and fragments of larger polypeptides. Fragments are
useful, for
example, in generating antibodies against the native polypeptide. Thus, they
may be employed
as biologically active or immunological substitutes for natural, purified
proteins in screening of
therapeutic compounds and in immunological processes for the development of
antibodies.
The polypeptides and proteins of the present invention can alternatively be
purified from
cells which have been altered to express the desired polypeptide or protein.
As used herein, a
cell is said to be altered to express a desired polypeptide or protein when
the cell, through genetic
manipulation, is made to produce a polypeptide or protein which it normally
does not produce or
which the cell normally produces at a lower level. One skilled in the art can
readily adapt
procedures for introducing and expressing either recombinant or synthetic
sequences into
eukaryotic or prokaryotic cells in order to generate a cell which produces one
of the polypeptides
or proteins of the present invention.
The invention also relates to methods for producing a polypeptide comprising
growing a
culture of host cells of the invention in a suitable culture medium, and
purifying the protein from
the cells or the culture in which the cells are grown. For example, the
methods of the invention
include a process for producing a polypeptide in which a host cell containing
a suitable
expression vector that includes a polynucleotide of the invention is cultured
under conditions that
allow expression of the encoded polypeptide. The polypeptide can be recovered
from the
culture, conveniently from the culture medium, or from a lysate prepared from
the host cells and
further purified. Preferred embodiments include those in which the protein
produced by such
process is a full length or mature form of the protein.
In an alternative method, the polypeptide or protein is purified from
bacterial cells which
naturally produce the polypeptide or protein. One skilled in the art can
readily follow known
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methods for isolating polypeptides and proteins in order to obtain one of the
isolated
polypeptides or proteins of the present invention. These include, but are not
limited to,
immunochromatography, HPLC, size-exclusion chromatography, ion-exchange
chromatography,
and immuno-affinity chromatography. See, e.g., Scopes, Protein Purification:
Principles and
Practice, Springer-Verlag (1994); Sambrook, et al., in Molecular Cloning: A
Laboratory
Manual; Ausubel et al., Curreht Protocols ire Molecular Biology. Polypeptide
fragments that
retain biological/immunological activity include fragments comprising greater
than about 100
amino acids, or greater than about 200 amino acids, and fragments that encode
specific protein
domains.
The purified polypeptides can be used in in vitro binding assays which are
well known in
the art to identify molecules which bind to the polypeptides. These molecules
include but are not
limited to, for e.g., small molecules, molecules from combinatorial libraries,
antibodies or other
proteins. The molecules identified in the binding assay are then tested for
antagonist or agonist
activity in ih vivo tissue culture or animal models that are well known in the
art. In brief, the
molecules are titrated into a plurality' of cell cultures or animals and then
tested for either
cell/animal death or prolonged survival of the animal/cells.
In addition, the peptides of the invention or molecules capable of binding to
the peptides
may be complexed with toxins, e.g., ricin or cholera, or with other compounds
that are toxic to
cells. The toxin-binding molecule complex is then targeted to a tumor or other
cell by the
specificity of the binding molecule for SEQ ID NO: 4, 7, 9-13.
The protein of the invention may also be expressed as a product of transgenic
animals,
e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep
which are characterized
by somatic or germ cells containing a nucleotide sequence encoding the
protein.
The proteins provided herein also include proteins characterized by amino acid
sequences
similar to those of purified proteins but into which modification are
naturally provided or
deliberately engineered. For example, modifications, in the peptide or DNA
sequence, can be
made by those skilled in the art using known techniques. Modifications of
interest in the protein
sequences may include the alteration, substitution, replacement, insertion or
deletion of a
selected amino acid residue in the coding sequence. For example, one or more
of the cysteine
residues may be deleted or replaced with another amino acid to alter the
conformation of the
molecule. Techniques for such alteration, substitution, replacement, insertion
or deletion are
well known to those skilled in the art (see, e.g., U.S. Pat. No. 4,518,584).
Preferably, such
alteration, substitution, replacement, insertion or deletion retains the
desired activity of the
protein. Regions of the protein that are important for the protein function
can be determined by
various methods known in the art including the alanine-scanning method which
involved
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systematic substitution of single or strings of amino acids with alanine,
followed by testing the
resulting alanine-containing variant for biological activity. This type of
analysis determines the
importance of the substituted amino acids) in biological activity. Regions of
the protein that are
important for protein function cay be determined by the eMATRIX program.
Other fragments and derivatives of the sequences of proteins which would be
expected to
retain protein activity in whole or in part and are useful for screening or
other immunological
methodologies may also be easily made by those skilled in the art given the
disclosures herein.
Such modifications are encompassed by the present invention.
The protein may also be produced by operably linking the isolated
polynucleotide of the
invention to suitable control sequences in one or more insect expression
vectors, and employing
an insect expression system. Materials and methods for baculovirus/insect cell
expression
systems are commercially available in kit form from, e.g., Invitrogen, San
Diego, Calif., U.S.A.
(the MaxBatTM kit), and such methods are well known in the art, as described
in Summers and
Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987),
incorporated herein by
reference. As used herein, an insect cell capable of expressing a
polynucleotide of the present
invention is "transformed."
The protein of the invention may be prepared by culturing transformed host
cells under
culture conditions suitable to express the recombinant protein. The resulting
expressed protein
may then be purified from such culture (i.e., from culture medium or cell
extracts) using known
purification processes, such as gel filtration and ion exchange
chromatography. The purification
of the protein may also include an affinity column containing agents which
will bind to the
protein; one or more column steps over such affinity resins as concanavalin A-
agarose, heparin-
toyopearlTM or Cibacrom blue 3GA SepharoseTM; one or more steps involving
hydrophobic
interaction chromatography using such resins as phenyl ether, butyl ether, or
propyl ether; or
immunoaffinity chromatography.
Alternatively, the protein of the invention may also be expressed in a form
which will
facilitate purification. For example, it may be expressed as a fusion protein,
such as those of
maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin
(TRX), or as a
His tag. Kits for expression and purification of such fusion proteins are
commercially available
from New England BioLab (Beverly, Mass.), Pharmacia (Piscataway, N.J.) and
Invitrogen,
respectively. The protein can also be tagged with an epitope and subsequently
purified by using
a specific antibody directed to such epitope. One such epitope ("FLAG~") is
commercially
available from Kodak (New Haven, Conn.).
Finally, one or more reverse-phase high performance liquid chromatography (RP-
HPLC)
steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant
methyl or other
CA 02399644 2002-08-02
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aliphatic groups, can be employed to further purify the protein. Some or all
of the foregoing
purification steps, in various combinations, can also be employed to provide a
substantially
homogeneous isolated recombinant protein. The protein thus purified is
substantially free of
other mammalian proteins and is defined in accordance with the present
invention as an "isolated
protein."
The polypeptides of the invention include analogs (variants). The polypeptides
of the
invention include neurotrimin-like analogs. This embraces fragments of
neurotrimin-like
polypeptide of the invention, as well neurotrimin-like polypeptide which
comprise one or more
amino acids deleted, inserted, or substituted. Also, analogs of the
neurotrimin-like polypeptide
of the invention embrace fusions of the neurotrimin-like polypeptide or
modifications of the
neurotrimin-like polypeptide, wherein the neurotrimin-like polypeptide or
analog is fused to
another moiety or moieties, e.g., targeting moiety or another therapeutic
agent. Such analogs
may exhibit improved properties such as activity and/or stability. Examples of
moieties which
may be fused to the neurotrimin-like polypeptide or an analog include, for
example, targeting
moieties which provide for the delivery of polypeptide to neurons, e.g.,
antibodies to central
nervous system, or antibodies to receptor and ligands expressed on neuronal
cells. Other
moieties which may be fused to neurotrimin-like polypeptide include
therapeutic agents which
are used for treatment, for example anti depressant drugs or other medications
for neurological
disorders. Also, neurotrimin-like polypeptide may be fused to neuron growth
modulators, and
other chemokines for targeted delivery.
5.6.1 DETERMINING POLYPEPTIDE AND POLYNUCLEOTIDE IDENTITY
AND SIMILARITY
Preferred identity and/or similarity are designed to give the largest match
between the
sequences tested. Methods to determine identity and similarity are codified in
computer
programs including, but are not limited to, the GCG program package, including
GAP
(Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984); Genetics
Computer Group,
University of Wisconsin, Madison, WI), BLASTP, BLASTN, BLASTX, FASTA
(Altschul, S.F.
et al., J. Molec. Biol. 215:403-410 (1990), PSI-BLAST (Altschul S.F. et al.,
Nucleic Acids Res.
vol. 25, pp. 3389-3402, herein incorporated by reference), eMatrix software
(Wu et al., J. Comp.
Biol., vol. 6, pp. 219-235 (1999), herein incorporated by reference), eMotif
software (Nevill-
Manning et al, ISMB-97, vol 4, pp. 202-209, herein incorporated by reference)
and the Kyte-
Doolittle hydrophobocity prediction algorithm (J. Mol Biol, 157, pp. 105-31
(1982),
incorporated herein by reference). The BLAST programs are publicly available
from the
National Center for Biotechnology Information (NCBI) and other sources (BLAST
Manual,
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Altschul, S., et al. NCB NLM NIH Bethesda, MD 20894; Altschul, S., et al., J.
Mol. Biol.
215:403-410 (1990).
5.7 CHIMERIC AND FUSION PROTEINS
The invention also provides chimeric or fusion proteins. As used herein, a
"chimeric
protein" or "fusion protein" comprises a polypeptide of the invention
operatively linked to
another polypeptide. Within a fusion protein the polypeptide according to the
invention can
correspond to all or a portion of a protein according to the invention. In one
embodiment, a
fusion protein comprises at least one biologically active portion of a protein
according to the
invention. In another embodiment, a fusion protein comprises at least two
biologically active
portions of a protein according to the invention. Within the fusion protein,
the term
"operatively linked" is intended to indicate that the polypeptide according to
the invention and
the other polypeptide are fused in-frame to each other. The polypeptide can be
fused to the
N-terminus or C-terminus, or to the middle.
For example, in one embodiment a fusion protein comprises a polypeptide
according to
the invention operably linked to the extracellular domain of a second protein.
In another embodiment, the fusion protein is a GST-fusion protein in which the
polypeptide sequences of the invention are fused to the C-terminus of the GST
(i.e.,
glutathione S-transferase) sequences.
In another embodiment, the fusion protein is an immunoglobulin fusion protein
in
which the polypeptide sequences according to the invention comprise one or
more domains
fused to sequences derived from a member of the immunoglobulin protein family.
The
immunoglobulin fusion proteins of the invention can be incorporated into
pharmaceutical
compositions and administered to a subject to inhibit an interaction between a
ligand and a
protein of the invention on the surface of a cell, to thereby suppress signal
transductionin vivo.
The immunoglobulin fusion proteins can be used to affect the bioavailability
of a cognate
Iigand. Inhibition of the Iigand/protein interaction may be useful
therapeutically for both the
treatment of proliferative and differentiative disorders, e.g., cancer as well
as modulating
(e. g. , promoting or inhibiting) cell survival. Moreover, the immunoglobulin
fusion proteins of
the invention can be used as immunogens to produce antibodies in a subject, to
purify ligands,
and in screening assays to identify molecules that inhibit the interaction of
a polypeptide of the
invention with a ligand.
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A chimeric or fusion protein of the invention can be produced by standard
recombinant
DNA techniques. For example, DNA fragments coding for the different
polypeptide
sequences are ligated together in-frame in accordance with conventional
techniques, e. g. , by
employing blunt-ended or stagger-ended termini for ligation, restriction
enzyme digestion to
provide for appropriate termini, filling-in of cohesive ends as appropriate,
alkaline phosphatase
treatment to avoid undesirable joining, and enzymatic ligation. In another
embodiment, the
fusion gene can be synthesized by conventional techniques including automated
DNA
synthesizers. Alternatively, PCR amplification of gene fragments can be
carried out using
anchor primers that give rise to complementary overhangs between two
consecutive gene
fragments that can subsequently be annealed and reamplified to generate a
chimeric gene
sequence (see, for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN
MOLECULAR
BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are
commercially
available that already encode a fusion moiety (e.g., a GST polypeptide). A
nucleic acid
encoding a polypeptide of the invention can be cloned into such an expression
vector such that
the fusion moiety is linked in-frame to the protein of the invention.
5.8 GENE THERAPY
Mutations in the polynucleotides of the invention gene may result in loss of
normal
function of the encoded protein. The invention thus provides gene therapy to
restore normal
activity of the polypeptides of the invention; or to treat disease states
involving polypeptides of
the invention. Delivery of a functional gene encoding polypeptides of the
invention to
appropriate cells is effected ex vivo, ih situ, or in vivo by use of vectors,
and more particularly
viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or
ex vivo by use of
physical DNA transfer methods (e.g., liposomes or chemical treatments). See,
for example,
Anderson, Nature, supplement to vol. 392, no. 6679, pp.25-20 (1998). For
additional reviews of
gene therapy technology see Friedmann, Science, 244: 1275-1281 (1989); Verma,
Scientific
American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992). Introduction
of any one of
the nucleotides of the present invention or a gene encoding the polypeptides
of the present
invention can also be accomplished with extrachromosomal substrates (transient
expression) or
artificial chromosomes (stable expression). Cells may also be cultured ex vivo
in the presence of
proteins of the present invention in order to proliferate or to produce a
desired effect on or
activity in such cells. Treated cells can then be introduced in vivo for
therapeutic purposes.
Alternatively, it is contemplated that in other human disease states,
preventing the expression of
or inhibiting the activity of polypeptides of the invention will be useful in
treating the disease
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states. It is contemplated that antisense therapy or gene therapy could be
applied to negatively
regulate the expression of polypeptides of the invention.
Other methods inhibiting expression of a protein include the introduction of
antisense
molecules to the nucleic acids of the present invention, their complements, or
their translated RNA
sequences, by methods known in the art. Further, the polypeptides of the
present invention can be
inhibited by using targeted deletion methods, or the insertion of a negative
regulatory element such
as a silencer, which is tissue specific.
The present invention still further provides cells genetically engineered i~c
vivo to express the
polynucleotides of the invention, wherein such polynucleotides are in
operative association with a
regulatory sequence heterologous to the host cell which drives expression of
the polynucleotides in
the cell. These methods can be used to increase or decrease the expression of
the polynucleotides of
the present invention.
Knowledge of DNA sequences provided by the invention allows for modification
of cells to
permit, increase, or decrease, expression of endogenous polypeptide. Cells can
be modified (e.g., by
homologous recombination) to provide increased polypeptide expression by
replacing, in whole or
in part, the naturally occurring promoter with all or part of a heterologous
promoter so that the cells
express the protein at higher levels. The heterologous promoter is inserted in
such a manner that it is
operatively linked to the desired protein encoding sequences. See, for
example, PCT International
PublicationNo. WO 94/12650, PCT International PublicationNo. WO 92/20808, and
PCT
InternationalPublicationNo. WO 91/09955. It is also contemplatedthat, in
addition to heterologous
promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional
CAD gene which
encodes carbamyl phosphate synthase, aspartate transcarbamylase, and
dihydroorotase) and/or
intron DNA may be inserted along with the heterologous promoter DNA. If linked
to the desired
protein coding sequence, amplification of the marker DNA by standard selection
methods results in
co-amplificationof the desired protein coding sequences in the cells.
In another embodiment of the present invention, cells and tissues may be
engineered to
express an endogenous gene comprising the polynucleotides of the invention
under the control of
inducible regulatory elements, in which case the regulatory sequences of the
endogenous gene may
be replaced by homologous recombination. As described herein, gene targeting
can be used to
replace a gene's existing regulatory region with a regulatory sequence
isolated from a different gene
or a novel regulatory sequence synthesized by genetic engineering methods.
Such regulatory
sequences may be comprised of promoters, enhancers, scaffold-
attachmentregions, negative
regulatory elements, transcriptional initiation sites, regulatory protein
binding sites or combinations
of said sequences. Alternatively, sequences which affect the structure or
stability of the RNA or
3 5 protein produced may be replaced, removed, added, or otherwise modified by
targeting. These
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sequences include polyadenylation signals, mRNA stability elements, splice
sites, leader sequences
for enhancing or modifying transport or secretion properties of the protein,
or other sequences
which alter or improve the function or stability of protein or RNA molecules.
The targeting event may be a simple insertion of the regulatory sequence,
placing the gene
under the control of the new regulatory sequence, e.g., inserting a new
promoter or enhancer or both
upstream of a gene. Alternatively, the targeting event may be a simple
deletion of a regulatory
element, such as the deletion of a tissue-specific negative regulatory
element. Alternatively, the
targeting event may replace an existing element; for example, a tissue-
specific enhancer can be
replaced by an enhancer that has broader or different cell-type specificity
than the naturally
occurring elements. Here, the naturally occurring sequences are deleted and
new sequences are
added. In all cases, the identification of the targeting event may be
facilitated by the use of one or
more selectable marker genes that are contiguous with the targeting DNA,
allowing for the selection
of cells in which the exogenous DNA has integrated into the cell genome. The
identification of the
targeting event may also be facilitated by the use of one or more marker genes
exhibiting the
property of negative selection, such that the negatively selectable marker is
linked to the exogenous
DNA, but configured such that the negatively selectable marker flanks the
targeting sequence, and
such that a correct homologous recombination event with sequences in the host
cell genome does
not result in the stable integration of the negatively selectable marker.
Markers useful for this
purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the
bacterial xanthine-
guanine phosphoribosyl-transferase(gpt) gene.
The gene targeting or gene activation techniques which can be used in
accordance with this
aspect of the invention are more particularly described in U.S. Patent No.
5,272,071 to Chappel;
U.S. Patent No. 5,578,461 to Sherwin et al.; International ApplicationNo.
PCT/US92/09627
(W093/09222)by Selden et al.; and InternationalApplicationNo. PCT/LJS90/06436
(W091 /06667) by Skoultchi et al., each of which is incorporated by reference
herein in its entirety.
5.9 TRANSGENIC ANIMALS
In preferred methods to determine biological functions of the polypeptides of
the
invention in vivo, one or more genes provided by the invention are either over
expressed or
inactivated in the germ line of animals using homologous recombination
[Capecchi, Science
244:1288-1292 (1989)]. Animals in which the gene is over expressed, under the
regulatory
control of exogenous or endogenous promoter elements, are known as transgenic
animals.
Animals in which an endogenous gene has been inactivated by homologous
recombination are
referred to as "knockout" animals. Knockout animals, preferably non-human
marmnals, can be
prepared as described in U.S. Patent No. 5,557,032, incorporated herein by
reference. Transgenic
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animals are useful to determine the roles polypeptides of the invention play
in biological
processes, and preferably in disease states. Transgenic animals are useful as
model systems to
identify compounds that modulate lipid metabolism. Transgenic animals,
preferably non-human
mammals, are produced using methods as described in U.S. Patent No 5,489,743
and PCT
S Publication No. W094/28122, incorporated herein by reference.
Transgenic animals can be prepared wherein all or part of a promoter of the
polynucleotides of the invention is either activated or inactivated to alter
the level of expression
of the polypeptides of the invention. Inactivation can be carried out using
homologous ,
recombination methods described above. Activation can be achieved by
supplementing or even
replacing the homologous promoter to provide for increased protein expression.
The homologous
promoter can be supplemented by insertion of one or more heterologous enhancer
elements
knomn to confer promoter activation in a particular tissue.
The polynucleotides of the present invention also make possible the
development,
through, e.g., homologous recombination or knock out strategies, of animals
that fail to express
functional neurotrimin-like polypeptide or that express a variant of
neurotrimin-like polypeptide.
Such animals are useful as models for studying the in vivo activities of
neurotrimin-like
polypeptide as well as for studying modulators of neurotrimin-like
polypeptide.
In preferred methods to determine biological functions of the polypeptides of
the
invention i~c vivo, one or more genes provided by the invention are either
over expressed or
inactivated in the germ line of animals using homologous recombination
[Capecchi, Science
244:1288-1292 (1989)]. Animals in which the gene is over expressed, under the
regulatory
control of exogenous or endogenous promoter elements, are known as transgenic
animals.
Animals in which an endogenous gene has been inactivated by homologous
recombination are
referred to as "knockout" animals. Knockout animals, preferably non-human
mammals, can be
prepared as described in U.S. Patent No. 5,557,032, incorporated herein by
reference. Transgenic
animals are useful to determine the roles polypeptides of the invention play
in biological
processes, and preferably in disease states. Transgenic animals are useful as
model systems to
identify compounds that modulate lipid metabolism. Transgenic animals,
preferably non-human
mammals, are produced using methods as described in U.S. Patent No 5,489,743
and PCT
Publication No. W094/28122, incorporated herein by reference.
Transgenic animals can be prepared wherein all or part of the polynucleotides
of the
invention promoter is either activated or inactivated to alter the level of
expression of the
polypeptides of the invention. Inactivation can be carried out using
homologous recombination
methods described above. Activation can be achieved by supplementing or even
replacing the
homologous promoter to provide for increased protein expression. The
homologous promoter
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can be supplemented by insertion of one or more heterologous enhancer elements
known to
confer promoter activation in a particular tissue.
5.10 USES AND BIOLOGICAL ACTIVITY OF HUMAN NEUROTRIMIN-LIKE
POLYPEPTIDE
The polynucleotides and proteins of the present invention are expected to
exhibit one or
more of the uses or biological activities (including those associated with
assays cited herein)
identified herein. Uses or activities described for proteins of the present
invention may be
provided by administration or use of such proteins or of polynucleotides
encoding such proteins
(such as, for example, in gene therapies or vectors suitable for introduction
of DNA). The
mechanism underlying the particular condition or pathology will dictate
whether the
polypeptides of the invention, the polynucleotides of the invention or
modulators (activators or
inhibitors) thereof would be beneficial to the subject in need of treatment.
Thus, "therapeutic
compositions of the invention" include compositions comprising isolated
polynucleotides
(including recombinant DNA molecules, cloned genes and degenerate variants
thereof) or
polypeptides of the invention (including full length protein, mature protein
and truncations or
domains thereof), or compounds and other substances that modulate the overall
activity of the
target gene products, either at the level of target gene/protein expression or
target protein
activity. Such modulators include polypeptides, analogs, (variants), including
fragments and
fusion proteins, antibodies and other binding proteins; chemical compounds
that directly or
indirectly activate or inhibit the polypeptides of the invention (identified,
e.g., via drug screening
assays as described herein); antisense polynucleotides and polynucleotides
suitable for triple
helix formation; and in particular antibodies or other binding partners that
specifically recognize
one or more epitopes of the polypeptides of the invention.
The polypeptides of the present invention may likewise be involved in cellular
activation
or in one of the other physiological pathways described herein.
5.10.1 RESEARCH USES AND UTILITIES
The polynucleotides provided by the present invention can be used by the
research
community for various purposes. The polynucleotides can be used to express
recombinant
protein for analysis, characterization or therapeutic use; as markers for
tissues in which the
corresponding protein is preferentially expressed (either constitutively or at
a particular stage of
tissue differentiation or development or in disease states); as molecular
weight markers on gels;
as chromosome markers or tags (when labeled) to identify chromosomes or to map
related gene
positions; to compare with endogenous DNA sequences in patients to identify
potential genetic
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disorders; as probes to hybridize and thus discover novel, related DNA
sequences; as a source of
information to derive PCR primers for genetic fingerprinting; as a probe to
"subtract-out" known
sequences in the process of discovering other, novel polynucleotides; for
selecting and making
oligomers for attachment to a "gene chip" or other support, including for
examination of
expression patterns; to raise anti-protein antibodies using DNA immunization
techniques; and as
an antigen to raise anti-DNA antibodies or elicit another immune response.
Where the
polynucleotide encodes a protein which binds or potentially binds to another
protein (such as, for
example, in a receptor-ligand interaction), the polynucleotide can also be
used in interaction trap
assays (such as, for example, that described in Gyuris et al., Cell 75:791-803
(1993)) to identify
polynucleotides encoding the other protein with which binding occurs or to
identify inhibitors of
the binding interaction.
The polypeptides provided by the present invention can similarly be used in
assays to
determine biological activity, including in a panel of multiple proteins for
high-throughput
screening; to raise antibodies or to elicit another immune response; as a
reagent (including the
labeled reagent) in assays designed to quantitatively determine levels of the
protein (or its
receptor) in biological fluids; as markers for tissues in which the
corresponding polypeptide is
preferentially expressed (either constitutively or at a particular stage of
tissue differentiation or
development or in a disease state); and, of course, to isolate correlative
receptors or ligands.
Proteins involved in these binding interactions can also be used to screen for
peptide or small
molecule inhibitors or agonists of the binding interaction.
The polypeptides of the invention are also useful for making antibody
substances that are
specifically immunoreactive with neurotrimin-like proteins. Antibodies and
portions thereof
(e.g., Fab fragments) which bind to the polypeptides of the invention can be
used to identify the
presence of such polypeptides in a sample. Such determinations are carried out
using any
suitable immunoassay format, and any polypeptide of the invention that is
specifically bound by
the antibody can be employed as a positive control.
Any or all of these research utilities are capable of being developed into
reagent grade or
kit format for commercialization as research products.
Methods for performing the uses listed above are well known to those skilled
in the art.
References disclosing such methods include without limitation "Molecular
Cloning: A
Laboratory Manual", 2d ed., Cold Spring Haxbor Laboratory Press, Sambrook, J.,
E. F. Fritsch
and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular
Cloning
Techniques", Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.
5.10.2 NUTRITIONAL USES
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Polynucleotides and polypeptides of the present invention can also be used as
nutritional
sources or supplements. Such uses include without limitation use as a protein
or amino acid
supplement, use as a carbon source, use as a nitrogen source and use as a
source of carbohydrate. In
such cases the polypeptide or polynucleotide of the invention can be added to
the feed of a
particular organism or can be administered as a separate solid or liquid
preparation, such as in the
form of powder, pills, solutions, suspensions or capsules. In the case of
microorganisms, the
polypeptide or polynucleotide of the invention can be added to the medium in
or on which the
microorganism is cultured. .
Additionally, the polypeptides of the invention can be used as molecular
weight markers,
and as a food supplement. A polypeptide consisting of SEQ ID NO: 4, for
example, has a
molecular mass of approximately 51 kDa in its unprocessed and unglycosylated
state. A
polypeptide consisting of SEQ ID NO: 7, for example has a predicted molecular
mass of
approximately 66 kDa. Protein food supplements are well known and the
formulation of suitable
food supplements including polypeptides of the invention is within the level
of skill in the food
preparation art
5.10.3 CYTOKINE AND CELL PROLIFERATION/DIFFERENTIATION
ACTIVITY
A polypeptide of the present invention may exhibit activity relating to
cytokine, cell
proliferation (either inducing or inhibiting) or cell differentiation (either
inducing or inhibiting)
activity or may induce production of other cytokines in certain cell
populations. A
polynucleotide of the invention can encode a polypeptide exhibiting such
attributes. Many
protein factors discovered to date, including all known cytokines, have
exhibited activity in one
or more factor-dependent cell proliferation assays, and hence the assays serve
as a convenient
confirmation of cytokine activity. The activity of therapeutic compositions of
the present
invention is evidenced by any one of a number of routine factor dependent cell
proliferation
assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9,
B9/11, BaF3,
MC9/G, M+(preB M+), 2E8, RBS, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e, CMI~,
HUVEC, and Caco. Therapeutic compositions of the invention can be used in the
following:
Assays for T-cell or thymocyte proliferation include without limitation those
described
in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D.
H. Margulies, E.
M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-
Interscience (Chapter 3,
Ih Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic
studies in
Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Bertagnolli et al., J.
Immunol.
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145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133:327-341,
1991; Bertagnolli,
et al., I. Immunol. 149:3778-3783, 1992; Bowman et al., I. Immunol. 152:1756-
1761, 1994.
Assays for cytokine production and/or proliferation of spleen cells, lymph
node cells or
thymocytes include, without limitation, those described in: Polyclonal T cell
stimulation,
Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in Immunology. J. E.
e.a. Coligan
eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and
Measurement of mouse
and human interleukin-~y, Schreiber, R. D. In Current Protocols in Immunology.
J. E. Coligan
eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.
Assays for proliferation and differentiation of hematopoietic and
lymphopoietic cells
include, without limitation, those described in: Measurement of Human and
Murine Interleukin 2
and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current
Protocols in
Immunology. J. E. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons,
Toronto. 1991;
deVries et al., J. Exp. Med. 173:1205-121 l, 1991; Moreau et al., Nature
336:690-692, 1988;
Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983;
Measurement of mouse
and human interleukin 6--Nordan, R. In Current Protocols in Immunology. J. E.
Coligan eds. Vol
1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc.
Natl. Aced. Sci.
U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11--Bennett, F.,
Giannotti, J.,
Clark, S. C. and Turner, K. J. In Current Protocols in Immunology. J. E.
Coligan eds. Vol 1 pp.
6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse and human
Interleukin 9-
Ciarletta, A., Giannotti, J., Clark, S. C. and Turner, K. J. In Current
Protocols in Immunology. J.
E. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991.
Assays for T-cell clone responses to antigens (which will identify, among
others, proteins
that affect APC-T cell interactions as well as direct T-cell effects by
measuring proliferation and
/
cytokine production) include, without limitation, those described in: Current
Protocols in
Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M.
Shevach, W Strober,
Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro
assays for Mouse
Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors;
Chapter 7,
Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA
77:6091-6095,
1980; Weinberger et al., Eur. J. Immun. 11:405-411, 1981; Takai et al., J.
Immunol. 137:3494-
3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988.
5.10.4 STEM CELL GROWTH FACTOR ACTIVITY
A polypeptide of the present invention may exhibit stem cell growth factor
activity and
be involved in the proliferation, differentiation and survival of pluripotent
and totipotent stem
cells including primordial germ cells, embryonic stem cells, hematopoietic
stem cells and/or
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germ line stem cells. Administration of the polypeptide of the invention to
stem cells in vivo or
ex vivo is expected to maintain and expand cell populations in a totipotential
or pluripotential
state which would be useful for re-engineering damaged or diseased tissues,
transplantation,
manufacture of bio-pharmaceuticals and the development of bio-sensors. The
ability to produce
large quantities of human cells has important working applications for the
production of human
proteins which currently must be obtained from non-human sources or donors,
implantation of
cells to treat diseases such as Parkinson's, Alzheimer's and other
neurodegenerative diseases;
tissues for grafting such as bone marrow, skin, cartilage, tendons, bone,
muscle (including
cardiac muscle), blood vessels, cornea, neural cells, gastrointestinal cells
and others; and organs
for transplantation such as kidney, liver, pancreas (including islet cells),
heart and lung.
It is contemplated that multiple different exogenous growth factors and/or
cytokines may
be administered in combination with the polypeptide of the invention to
achieve the desired
effect, including any of the growth factors listed herein, other stem cell
maintenance factors, and
specifically including stem cell factor (SCF), leukemia inhibitory factor
(LIF), Flt-3 ligand (Flt-
3L), any of the interleukins, recombinant soluble IL-6 receptor fused to IL-6,
macrophage
inflammatory protein 1-alpha (MIP-1-alpha), G-CSF, GM-CSF, thrombopoietin
(TPO), platelet
factor 4 (PF-4), platelet-derived growth factor (PDGF), neural growth factors
and basic
fibroblast growth factor (bFGF).
Since totipotent stem cells can give rise to virtually any mature cell type,
expansion of
these cells in culture will facilitate the production of laxge quantities of
mature cells. Techniques
for culturing stem cells are known in the art and administration of
polypeptides of the invention,
optionally with other growth factors and/or cytokines, is expected to enhance
the survival and
proliferation of the stem cell populations. This can be accomplished by direct
administration of
the polypeptide of the invention to the culture medium. Alternatively, stroma
cells transfected
with a polynucleotide that encodes for the polypeptide of the invention can be
used as a feeder
layer for the stem cell populations in culture or in vivo. Stromal support
cells for feeder layers
may include embryonic bone marrow fibroblasts, bone marrow stromal cells,
fetal liver cells, or
cultured embryonic fibroblasts (see U.S. Patent No. 5,690,926).
Stem cells themselves can be transfected with a polynucleotide of the
invention to induce
autocrine expression of the polypeptide of the invention. This will allow for
generation of
undifferentiated totipotential/pluripotential stem cell lines that axe useful
as is or that can then be
differentiated into the desired mature cell types. These stable cell lines can
also serve as a source
of undifferentiated totipotential/pluripotential mRNA to create cDNA libraries
and templates for
polymerase chain reaction experiments. These studies would allow for the
isolation and
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identification of differentially expressed genes in stem cell populations that
regulate stem cell
proliferation and/or maintenance.
Expansion and maintenance of totipotent stem cell populations will be useful
in the
treatment of many pathological conditions. For example, polypeptides of the
present invention
may be used to manipulate stem cells in culture to give rise to
neuroepithelial cells that can be
used to augment or replace cells damaged by illness, autoimmune disease,
accidental damage or
genetic disorders. The polypeptide of the invention may be useful for inducing
the proliferation
of neural cells and for the regeneration of nerve and brain tissue, i.e. for
the treatment of central
and peripheral nervous system diseases and neuropathies, as well as mechanical
and traumatic
disorders which involve degeneration, death or trauma to neural cells or nerve
tissue. In addition,
the expanded stem cell populations can also be genetically altered for gene
therapy purposes and
to decrease host rejection of replacement tissues after grafting or
implantation.
Expression of the polypeptide of the invention and its effect on stem cells
can also be
manipulated to achieve controlled differentiation of the stem cells into more
differentiated cell
types. A broadly applicable method of obtaining pure populations of a specific
differentiated
cell type from undifferentiated stem cell populations involves the use of a
cell-type specific
promoter driving a selectable marker. The selectable marker allows only cells
of the desired type
to survive. For example, stem cells can be induced to differentiate into
cardiomyocytes (Wobus
et al., Differentiation, 48: 173-182, (1991); Klug et al., J. Clin. Invest.,
98(1): 216-224, (1998))
or skeletal muscle cells (Browder, L. W. In: Principles of Tissue E~zgineering
eds. Lanza et al.,
Academic Press (1997)). Alternatively, directed differentiation of stem cells
can be
" accomplished by culturing the stem cells in the presence of a
differentiation factor such as
retinoic acid and an antagonist of the polypeptide of the invention which
would inhibit the
effects of endogenous stem cell factor activity and allow differentiation to
proceed.
In vitro cultures of stem cells can be used to determine if the polypeptide of
the invention
exhibits stem cell growth factor activity. Stem cells are isolated from any
one of various cell
sources (including hematopoietic stem cells and embryonic stem cells) and
cultured on a feeder
layer, as described by Thompson et al. Proc. Natl. Acad. Sci, U.S.A., 92: 7844-
7848 (1995), in
the presence of the polypeptide of the invention alone or in combination with
other growth
factors or cytokines. The ability of the polypeptide of the invention to
induce stem cells
proliferation is determined by colony formation on semi-solid support e.g. as
described by
Bernstein et al., Blood, 77: 2316-2321 (1991).
5.10.5 HEMATOPOIESIS REGULATING ACTIVITY
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A polypeptide of the present invention may be involved in regulation of
hematopoiesis
and, consequently, in the treatment of myeloid or lymphoid cell disorders.
Even marginal
biological activity in support of colony forming cells or of factor-dependent
cell lines indicates
involvement in regulating hematopoiesis, e.g. in supporting the growth and
proliferation of
erythroid progenitor cells alone or in combination with other cytokines,
thereby indicating
utility, for example, in treating various anemias or for use in conjunction
with
irradiation/chemotherapy to stimulate the production of erythroid precursors
and/or erythroid
cells; in supporting the growth and proliferation of myeloid cells such as
granulocytes and
monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in
conjunction with
chemotherapy to prevent or treat consequent myelo-suppression; in supporting
the growth and
proliferation of megakaryocytes and consequently of platelets thereby allowing
prevention or
treatment of various platelet disorders such as thrombocytopenia, and
generally for use in place
of or complimentary to platelet transfusions; and/or in supporting the growth
and proliferation of
hematopoietic stem cells which are capable of maturing to any and all of the
above-mentioned
hematopoietic cells and therefore find therapeutic utility in various stem
cell disorders (such as
those usually treated with transplantation, including, without limitation,
aplastic anemia and
paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell
compartment
post irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in conjunction
with bone marrow
transplantation or with peripheral progenitor cell transplantation (homologous
or heterologous))
as normal cells or genetically manipulated for gene therapy.
Therapeutic compositions of the invention can be used in the following:
Suitable assays for proliferation and differentiation of various hematopoietic
lines are
cited above.
Assays for embryonic stem cell differentiation (which will identify, among
others,
proteins that influence embryonic differentiation hematopoiesis) include,
without limitation,
those described in: Johansson et al. Cellular Biology 15:141-151, 1995; Keller
et al., Molecular
and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915,
1993.
Assays for stem cell survival and differentiation (which will identify, among
others,
proteins that regulate lympho-hematopoiesis) include, without limitation,
those described in:
Methylcellulose colony forming assays, Freshney, M. G. In Culture of
Hematopoietic Cells. R. I.
Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y. 1994;
Hirayama et al.,
Proc. Natl. Acad. Sci. IJSA 89:5907-5911, 1992; Primitive hematopoietic colony
forming cells
with high proliferative potential, McNiece, I. K. and Briddell, R. A. In
Culture of Hematopoietic
Cells. R. I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York,
N.Y. 1994; Neben et
al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell
assay,
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Ploemacher, R. E. In Culture of Hematopoietic Cells. R. I. Freshney, et al.
eds. Vol pp. 1-21,
Wiley-Liss, Inc., New York, N.Y. 1994; Long term bone marrow cultures in the
presence of
stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of
Hematopoietic Cells. R. I.
Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, N.Y. 1994;
Long term culture
initiating cell assay, Sutherland, H. J. In Culture of Hematopoietic Cells. R.
I. Freshney, et al.
eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, N.Y. 1994.
5.10.6 TISSUE GROWTH ACTIVITY
A polypeptide of the present invention also may be involved in bone,
cartilage, tendon,
ligament and/or nerve tissue growth or regeneration, as well as in wound
healing and tissue
repair and replacement, and in healing of burns, incisions and ulcers.
A polypeptide of the present invention which induces cartilage and/or bone
growth in
circumstances where bone is not normally formed, has application in the
healing of bone
fractures and cartilage damage or defects in humans and other animals.
Compositions of a
polypeptide, antibody, binding partner, or other modulator of the invention
may have
prophylactic use in closed as well as open fracture reduction and also in the
improved fixation of
artificial joints. De novo bone formation induced by an osteogenic agent
contributes to the
repair of congenital, trauma induced, or oncologic resection induced
craniofacial defects, and
also is useful in cosmetic plastic surgery.
A polypeptide of this invention may also be involved in attracting bone-
forming cells,
stimulating growth of bone-forming cells, or inducing differentiation of
progenitors of bone-
forming cells. Treatment of osteoporosis, osteoarthritis, bone degenerative
disorders, or
periodontal disease, such as through stimulation of bone and/or cartilage
repair or by blocking
inflammation or processes of tissue destruction (collagenase
activity,.osteoclast activity, etc.)
mediated by inflammatory processes may also be possible using the composition
of the
invention.
Another category of tissue regeneration activity that may involve the
polypeptide of the
present invention is tendon/ligament formation. Induction of tendon/ligament-
like tissue or
other tissue formation in circumstances where such tissue is not normally
formed, has
application in the healing of tendon or ligament tears, deformities and other
tendon or ligament
defects in humans and other animals. Such a preparation employing a
tendon/ligament-like
tissue inducing protein may have prophylactic use in preventing damage to
tendon or ligament
tissue, as well as use in the improved fixation of tendon or ligament to bone
or other tissues, and
in repairing defects to tendon or ligament tissue. De novo tendon/ligament-
like tissue formation
induced by a composition of the present invention contributes to the repair of
congenital, trauma
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induced, or other tendon or ligament defects of other origin, and is also
useful in cosmetic plastic
surgery for attachment or repair of tendons or ligaments. The compositions of
the present
invention may provide environment to attract tendon- or ligament-forming
cells, stimulate
growth of tendon- or ligament-forming cells, induce differentiation of
progenitors of tendon- or
ligament-forming cells, or induce growth of tendon/ligament cells or
progenitors ex vivo for
return in vivo to effect tissue repair. The compositions of the invention may
also be useful in the
treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament
defects. The
compositions may also include an appropriate matrix and/or sequestering agent
as a carrier as is
well known in the art.
The compositions of the present invention may also be useful for proliferation
of neural
cells and for regeneration of nerve and brain tissue, i.e. for the treatment
of central and peripheral
nervous system diseases and neuropathies, as well as mechanical and traumatic
disorders, which
involve degeneration, death or trauma to neural cells or nerve tissue. More
specifically, a
composition may be used in the treatment of diseases of the peripheral nervous
system, such as
peripheral nerve injuries, peripheral neuropathy and localized neuropathies,
and central nervous
system diseases, such as Alzheimer's, Paxkinson's disease, Huntington's
disease, amyotrophic
lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be
treated in
accordance with the present invention include mechanical and traumatic
disorders, such as spinal
cord disorders, head trauma and cerebrovascular diseases such as stroke.
Peripheral neuropathies
resulting from chemotherapy or other medical therapies may also be treatable
using a
composition of the invention.
Compositions of the invention may also be useful to promote better or faster
closure of
non-healing wounds, including without limitation pressure ulcers, ulcers
associated with vasculax
insufficiency, surgical and traumatic wounds, and the like.
Compositions of the present invention may also be involved in the generation
or
regeneration of other tissues, such as organs (including, for example,
pancreas, liver, intestine,
kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular
(including vascular
endothelium) tissue, or for promoting the growth of cells comprising such
tissues. Part of the
desired effects may be by inhibition or modulation of fibrotic scarring may
allow normal tissue
to regenerate. A polypeptide of the present invention may also exhibit
angiogenic activity.
A composition of the present invention may also be useful for gut protection
or
regeneration and treatment of lung or liver fibrosis, reperfusion injury in
various tissues, and
conditions resulting from systemic cytokine damage.
so
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A composition of the present invention may also be useful for promoting or
inhibiting
differentiation of tissues described above from precursor tissues or cells; or
for inhibiting the
growth of tissues described above.
Therapeutic compositions of the invention can be used in the following:
Assays for tissue generation activity include, without limitation, those
described in:
International Patent Publication No. W095/16035 (bone, cartilage, tendon);
International Patent
Publication No. W095/05846 (nerve, neuronal); International Patent Publication
No.
W091/07491 (skin, endothelium).
Assays for wound healing activity include, without limitation, those described
in: Winter,
Epidermal Wound Healing, pps. 71-112 (Maibach, H. I. and Rovee, D. T., eds.),
Year Book
Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J.
Invest. Dermatol
71:382-84 (1978).
5.10.7 IMMUNE STIMULATING OR SUPPRESSING ACTIVITY
A polypeptide of the present invention may also exhibit immune stimulating or
immune
suppressing activity, including without limitation the activities for which
assays are described
herein. A polynucleotide of the invention can encode a polypeptide exhibiting
such activities. A
protein may be useful in the treatment of various immune deficiencies and
disorders (including
severe combined immunodeficiency (SCID)), e.g., in regulating (up or down)
growth and
proliferation of T and/or B lymphocytes, as well as effecting the cytolytic
activity of NK cells
and other cell populations. These immune deficiencies may be genetic or be
caused by viral (e.g.,
HIV) as well as bacterial or fungal infections, or may result from autoimmune
disorders. More
specifically, infectious diseases causes by viral, bacterial, fungal or other
infection may be
treatable using a protein of the present invention, including infections by
HIV, hepatitis viruses,
herpes viruses, mycobacteria, Leishmania spp., malaria spp. and various fungal
infections such
as candidiasis. Of course, in this regard, proteins of the present invention
may also be useful
where a boost to the immune system generally may be desirable, i.e., in the
treatment of cancer.
Autoimmune disorders which may be treated using a protein of the present
invention
include, for example, connective tissue disease, multiple sclerosis, systemic
lupus erythematosus,
rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Bane
syndrome,
autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia
gravis, graft-versus-host
disease and autoimmune inflammatory eye disease. Such a protein (or
antagonists thereof,
including antibodies) of the present invention may also to be useful in the
treatment of allergic
reactions and conditions (e.g., anaphylaxis, serum sickness, drug reactions,
food allergies, insect
venom allergies, mastocytosis, allergic rhinitis, hypersensitivity
pneumonitis, urticaxia,
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angioedema, eczema, atopic dermatitis, allergic contact dermatitis, erythema
multiforme,
Stevens-Johnson syndrome, allergic conjunctivitis, atopic
keratoconjunctivitis, venereal
keratoconjunctivitis, giant papillary conjunctivitis and contact allergies),
such as asthma
(particularly allergic asthma) or other respiratory problems. Other
conditions, in which immune
suppression is desired (including, fox example, organ transplantation), may
also be treatable
using a protein (or antagonists thereof) of the present invention. The
therapeutic effects of the
polypeptides or antagonists thereof on allergic reactions can be evaluated by
in vivo animals
models such as the cumulative contact enhancement test (Lastbom et al.,
Toxicology 125: 59-66,
1998), skin prick test (Hoffmann et al., Allergy 54: 446-54, 1999), guinea pig
skin sensitization
test (Voter et al., Arch. Toxocol. 73: 501-9), and marine local lymph node
assay (Kimber et al.,
J. Toxicol. Environ. Health 53: 563-79).
Using the proteins of the invention it may also be possible to modulate immune
responses, in a number of ways. Down regulation may be in the form of
inhibiting or blocking an
immune response already in progress or may involve preventing the induction of
an immune
response. The functions of activated T cells may be inhibited by suppressing T
cell responses or
by inducing specific tolerance in T cells, or both. Immunosuppression of T
cell responses is
generally an active, non-antigen-specific, process which requires continuous
exposure of the T
cells to the suppressive agent. Tolerance, which involves inducing non-
responsiveness or anergy
in T cells, is distinguishable from immunosuppression in that it is generally
antigen-specific and
persists after exposure to the tolerizing agent has ceased. Operationally,
tolerance can be
demonstrated by the lack of a T cell response upon reexposure to specific
antigen in the absence
of the tolerizing agent.
Down regulating or preventing one or more antigen functions (including without
limitation B lymphocyte antigen functions (such as, for example, B7)), e.g.,
preventing high
level lymphokine synthesis by activated T cells, will be useful in situations
of tissue, skin and
organ transplantation and in graft-versus-host disease (GVHD). For example,
blockage of T cell
function should result in reduced tissue destruction in tissue
transplantation. Typically, in tissue
transplants, rejection of the transplant is initiated through its recognition
as foreign by T cells,
followed by an immune reaction that destroys the transplant. The
administration of a therapeutic
composition of the invention may prevent cytokine synthesis by immune cells,
such as T cells,
and thus acts as an immunosuppressant. Moreover, a lack of costimulation may
also be sufficient
to anergize the T cells, thereby inducing tolerance in a subject. Induction of
long-term tolerance
by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated
administration
of these blocking reagents. To achieve sufficient immunosuppression or
tolerance in a subject, it
may also be necessary to block the function of a combination of B lymphocyte
antigens.
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The efficacy of particular therapeutic compositions in preventing organ
transplant
rejection or GVHD can be assessed using animal models that are predictive of
efFcacy in
humans. Examples of appropriate systems which can be used include allogeneic
cardiac grafts in
rats and xenogeneic pancreatic islet cell grafts in mice, both of which have
been used to examine
the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described
in Lenschow et
al., Science 257:789-792 (1992) and Turka et al., Proc. Natl. Acad. Sci USA,
89:11102-11105
(1992). In addition, marine models of GVHD (see Paul ed., Fundamental
Immunology, Raven
Press, New York, 1989, pp. 846-847) can be used to determine the effect of
therapeutic
compositions of the invention on the development of that disease.
Blocking antigen function may also be therapeutically useful for treating
autoimmune
diseases. Many autoimmune disorders are the result of inappropriate
activation~of T cells that are
reactive against self tissue and which promote the production of cytokines and
autoantibodies
involved in the pathology of the diseases. Preventing the activation of
autoreactive T cells may
reduce or eliminate disease symptoms. Administration of reagents which block
stimulation of T
cells can be used to inhibit T cell activation and prevent production of
autoantibodies or T cell-
derived cytokines which may be involved in the disease process. Additionally,
blocking reagents
may induce antigen-specific tolerance of autoreactive T cells which could lead
to long-term
relief from the disease. The efficacy of blocking reagents in preventing or
alleviating
autoimmune disorders can be determined using a number of well-characterized
animal models of
human autoimmune diseases. Examples include marine experimental autoimmune
encephalitis,
systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, marine
autoimmune
collagen arthritis, diabetes mellitus in NOD mice and BB rats, and marine
experimental
myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New
York, 1989, pp.
840-856).
Upregulation of an antigen function (e.g., a B lymphocyte antigen function),
as a means
of up regulating immune responses, may also be useful in therapy. Upregulation
of immune
responses may be in the form of enhancing an existing immune response or
eliciting an initial
immune response. For example, enhancing an immune response may be useful in
cases of viral
infection, including systemic viral diseases such as influenza, the common
cold, and
encephalitis.
Alternatively, anti-viral immune responses may be enhanced in an infected
patient by
removing T cells from the patient, costimulating the T cells in vitro with
viral antigen-pulsed
APCs either expressing a peptide of the present invention or together with a
stimulatory form of
a soluble peptide of the present invention and reintroducing the in vitro
activated T cells into the
patient. Another method of enhancing anti-viral immune responses would be to
isolate infected
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cells from a patient, transfect them with a nucleic acid encoding a protein of
the present
invention as described herein such that the cells express all or a portion of
the protein on their
surface, and reintroduce the transfected cells into the patient. The infected
cells would now be
capable of delivering a costimulatory signal to, and thereby activate, T cells
in vivo.
A polypeptide of the present invention may provide the necessary stimulation
signal to T
cells to induce a T cell mediated immune response against the transfected
tumor cells. In
addition, tumor cells which lack MHC class I or MHC class II molecules, or
which fail to
reexpress sufficient mounts of MHC class I or MHC class II molecules, can be
transfected with
nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain
truncated portion) of an
MHC class I alpha chain protein and [32 microglobulin protein or an MHC class
II alpha chain
protein and an MHC class II beta chain protein to thereby express MHC class I
or MHC class II
proteins on the cell surface. Expression of the appropriate class I or class
II MHC in conjunction
with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-
2, B7-3) induces a T
cell mediated immune response against the transfected tumor cell. Optionally,
a gene encoding
1 S an antisense construct which blocks expression of an MHC class II
associated protein, such as
the invariant chain, can also be cotransfected with a DNA encoding a peptide
having the activity
of a B lymphocyte antigen to promote presentation of tumor associated antigens
and induce
tumor specific immunity. Thus, the induction of a T cell mediated immune
response in a human
subject may be sufficient to overcome tumor-specific tolerance in the subject.
The activity of a protein of the invention may, among other means, be measured
by the
following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without
limitation,
those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.
M. Kruisbeek, D.
H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and
Wiley-
2S Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-
3.19; Chapter 7,
Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492,
1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J.
Immunol. 13S:1S64-
1572, 1985; Takai et al., I. Immunol. 137:3494-3500, 1986; Takai et al., J.
Immunol. 140:508-
512, 1988; Hemnann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981;
Herrmann et al., J.
Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol. 13S:1S64-1572, 1985;
Takai et al., J.
Immunol. 137:3494-3500, 1986; Bowmanet al., J. Virology 61:1992-1998; Takai et
al., J.
Immunol. 140:508-512, 1988; Bertagnolli et al., Cellular Immunology 133:327-
341, 1991;
Brown et al., J. Immunol. 153:3079-3092, 1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching
(which
3S will identify, among others, proteins that modulate T-cell dependent
antibody responses and that
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affect Thl/Th2 profiles) include, without limitation, those described in:
Maliszewski, J.
Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro
antibody production,
Mond, J. J. and Brunswick, M. In Current Protocols in Immunology. J. E. e.a.
Coligan eds. Vol 1
pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.
Mixed lymphocyte reaction (MLR) assays (which will identify, among others,
proteins
that generate predominantly Thl and CTL responses) include, without
limitation, those described
in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. I~ruisbeek, D.
H. Margulies, E.
M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-
Interscience (Chapter 3,
In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic
studies in
Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J.
Immunol. 140:508-512,
1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.
Dendritic cell-dependent assays (which will identify, among others, proteins
expressed
by dendritic cells that activate naive T-cells) include, without limitation,
those described in:
Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of
Experimental Medicine
173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079,
1995; Porgador et
al., Journal of Experimental Medicine 182:255-260, 1995; Nair et al., Journal
of Virology
67:4062-4069, 1993; Huang et al., Science 264:961-965, 1994; Macatonia et al.,
Journal of
Experimental Medicine 169:1255-1264, 1989; Bhaxdwaj et al., Journal of
Clinical Investigation
94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631-
640, 1990.
Assays for lymphocyte survival/apoptosis (which will identify, among others,
proteins
that prevent apoptosis after superantigen induction and proteins that regulate
lymphocyte
homeostasis) include, without. limitation, those described in: Darzynkiewicz
et al., Cytometry
13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al.,
Cancer Research
53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of
Immunology
145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et
al., International
Journal of Oncology 1:639-648, 1992.
Assays for proteins that influence early steps of T-cell commitment and
development
include, without limitation, those described in: Antica et al., Blood 84:111-
117, 1994; Fine et al.,
Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995;
Toki et al.,
Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
5.10.8 ACTIVIN/INHIBIN ACTIVITY
A polypeptide of the present invention may also exhibit activin- or inhibin-
related
activities. A polynucleotide of the invention may encode a polypeptide
exhibiting such
characteristics. Inhibins are characterized by their ability to inhibit the
release of follicle
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stimulating hormone (FSH), while activins and are characterized by their
ability to stimulate the
release of follicle stimulating hormone (FSH). Thus, a polypeptide of the
present invention,
alone or in heterodimers with a member of the inhibin family, may be useful as
a contraceptive
based on the ability of inhibins to decrease fertility in female mammals and
decrease
spermatogenesis in male mammals. Administration of sufficient amounts of other
inhibins can
induce infertility in these mammals. Alternatively, the polypeptide of the
invention, as a
homodimer or as a heterodimer with other protein subunits of the inhibin
group, may be useful as
a fertility inducing therapeutic, based upon the ability of activin molecules
in stimulating FSH
release from cells of the anterior pituitary. See, for example, U.S. Pat. No.
4,798,885. A
polypeptide of the invention may also be useful for advancement of the onset
of fertility in
sexually immature mammals, so as to increase the lifetime reproductive
performance of domestic
animals such as, but not limited to, cows, sheep and pigs.
The activity of a polypeptide of the invention may, among other means, be
measured by
the following methods.
Assays for activin/inhibin activity include, without limitation, those
described in: Vale et
al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986;
Vale et al., Nature
321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al.,
Proc. Natl. Acad. Sci.
USA 83:3091-3095, 1986.
5.10.9 CHEMOTACTIC/CHEMOKINETIC ACTIVITY
A polypeptide of the present invention may be involved in chemotactic or
chemokinetic
activity for mammalian cells, including, for example, monocytes, fibroblasts,
neutrophils, T-
cells, mast cells, eosinophils, epithelial and/or endothelial cells. A
polynucleotide of the
invention can encode a polypeptide exhibiting such attributes. Chemotactic and
chemokinetic
receptor activation can be used to mobilize or attract a desired cell
population to a desired site of
action. Chemotactic or chemokinetic compositions (e.g. proteins, antibodies,
binding partners, or
modulators of the invention) provide particular advantages in treatment of
wounds and other
trauma to tissues, as well as in treatment of localized infections. For
example, attraction of
lymphocytes, monocytes or neutrophils to tumors or sites of infection may
result in improved
immune responses against the tumor or infecting agent.
A protein or peptide has chemotactic activity for a particular cell population
if it can
stimulate, directly or indirectly, the directed orientation or movement of
such cell population.
Preferably, the protein or peptide has the ability to directly stimulate
directed movement of cells.
Whether a parEicular protein has chemotactic activity for a population of
cells can be readily
determined by employing such protein or peptide in any known assay for cell
chemotaxis.
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Therapeutic compositions of the invention can be used in the following:
Assays for chemotactic activity (which will identify proteins that induce or
prevent
chemotaxis) consist of assays that measure the ability of a protein to induce
the migration of
cells across a membrane as well as the ability of a protein to induce the
adhesion of one cell
population to another cell population. Suitable assays for movement and
adhesion include,
without limitation, those described in: Current Protocols in Immunology, Ed by
J. E. Coligan, A.
M. Kruisbeek, D. H. Marguiles, E. M. Shevach, W. Strober, Pub. Greene
Publishing Associates
and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines
6.12.1-
6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376, 1995; Lind et al. APMIS
103:140-146, 1995;
Muller et al Eur. J. Immunol. 25:1744-1748; Gruber et al. J. of Immunol.
152:5860-5867, 1994;
Johnston et al. J. of Immunol. 153:1762-1768, 1994.
5.10.10 HEMOSTATIC AND THROMBOLYTIC ACTIVITY
A polypeptide of the invention may also be involved in hemostatis or
thrombolysis or
thrombosis. A polynucleotide of the invention can encode a polypeptide
exhibiting such
attributes. Compositions may be useful in treatment of various coagulation
disorders (including
hereditary disorders, such as hemophiliac) or to enhance coagulation and other
hemostatic events
in treating wounds resulting from trauma, surgery or other causes. A
composition of the
invention may also be useful for dissolving or inhibiting formation of
thromboses and for
treatment and prevention of conditions resulting therefrom (such as, for
example, infarction of
cardiac and central nervous system vessels (e.g., stroke).
Therapeutic compositions of the invention can be used in the following:
Assay for hemostatic and thrombolytic activity include, without limitation,
those
described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et
al., Thrombosis Res.
45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub,
Prostaglandins 35:467-
474, 1988.
5.10.11 CANCER DIAGNOSIS AND THERAPY
Polypeptides of the invention may be involved in cancer cell generation,
proliferation or
metastasis. Detection of the presence or amount of polynucleotides or
polypeptides of the
invention may be useful for the diagnosis and/or prognosis of one or more
types of cancer. For
example, the presence or increased expression of a polynucleotide/polypeptide
of the invention
may indicate a hereditary risk of cancer, a precancerous condition, or an
ongoing malignancy.
Conversely, a defect in the gene or absence of the polypeptide may be
associated with a cancer
condition. Identification of single nucleotide polymorphisms associated with
cancer or a
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predisposition to cancer may also be useful for diagnosis or prognosis.
Cancer treatments promote tumor regression by inhibiting tumor cell
proliferation,
inhibiting angiogenesis (growth of new blood vessels that is necessary to
support tumor growth)
and/or prohibiting metastasis by reducing tumor cell motility or invasiveness.
Therapeutic
compositions of the invention may be effective in adult and pediatric oncology
including in solid
phase tumors/malignancies, locally advanced tumors, human soft tissue
sarcomas, metastatic
cancer, including lymphatic metastases, blood cell malignancies including
multiple myeloma,
acute and chronic leukemias, and lymphomas, head and neck cancers including
mouth cancer,
larynx cancer and thyroid cancer, lung cancers including small cell carcinoma
and non-small cell
cancers, breast cancers including small cell carcinoma and ductal carcinoma,
gastrointestinal
cancers including esophageal cancer, stomach cancer, colon cancer, colorectal
cancer and polyps
associated with colorectal neoplasia, pancreatic cancers, liver cancer,
urologic cancers including
bladder cancer and prostate cancer, malignancies of the female genital tract
including ovarian
carcinoma, uterine (including endometrial) cancers, and solid tumor in the
ovarian follicle,
kidney cancers including renal cell carcinoma, brain cancers including
intrinsic brain tumors,
neuroblastoma, astrocytic brain tumors, gliomas, metastatic tumor cell
invasion in the central
nervous system, bone cancers including osteomas, skin cancers including
malignant melanoma,
tumor progression of human skin keratinocytes, squamous cell carcinoma, basal
cell carcinoma,
hemangiopericytoma and I~arposi's sarcoma.
Polypeptides, polynucleotides, or modulators of polypeptides of the invention
(including
inhibitors and stimulators of the biological activity of the polypeptide of
the invention) may be
administered to treat cancer. Therapeutic compositions can be administered in
therapeutically
effective dosages alone or in combination with adjuvant cancer therapy such as
surgery,
chemotherapy, radiotherapy, thermotherapy, and laser therapy, and may provide
a beneficial
effect, e.g. reducing tumor size, slowing rate of tumor growth, inhibiting
metastasis, or otherwise
improving overall clinical condition, without necessarily eradicating the
cancer.
The composition can also be administered in therapeutically effective amounts
as a
portion of an anti-cancer cocktail. An anti-cancer cocktail is a mixture of
the polypeptide or
modulator of the invention with one or more anti-cancer drugs in addition to a
pharmaceutically
acceptable carrier for delivery. The use of anti-cancer cocktails as a cancer
treatment is routine.
Anti-cancer drugs that are well known in the art and can be used as a
treatment in combination
with the polypeptide or modulator of the invention include: Actinomycin D,
Aminoglutethimide,
Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine, Chlorambucil,
Cisplatin (cis-
DDP), Cyclophosphamide, Cytarabine HCl (Cytosine arabinoside), Dacarbazine,
Dactinomycin,
Daunorubicin HCI, Doxorubicin HCI, Estramustine phosphate sodium, Etoposide (V
16-213),
ss
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Floxuridine, 5-Fluorouracil (5-Fu), Flutamide, Hydroxyurea (hydroxycarbamide),
Ifosfamide,
Interferon Alpha-2a, Interferon Alpha-2b, Leuprolide acetate (LHRH-releasing
factor analog),
Lomustine, Mechlorethamine HCl (nitrogen mustard), Melphalan, Mercaptopurine,
Mesna,
Methotrexate (MTX), Mitomycin, Mitoxantrone HCI, Octreotide, Plicamycin,
Procarbazine HCI,
Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate,
Vincristine sulfate,
Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2, Mitoguazone,
Pentostatin,
Semustine, Teniposide, and Vindesine sulfate.
In addition, therapeutic compositions of the invention may be used for
prophylactic
treatment of cancer. There are hereditary conditions and/or enviromnental
situations (e.g.
exposure to carcinogens) known in the art that predispose an individual to
developing cancers.
Under these circumstances, it may be beneficial to treat these individuals
with therapeutically
effective doses of the polypeptide of the invention to reduce the risk of
developing cancers.
In vitro models can be used to determine the effective doses of the
polypeptide of the
invention as a potential cancer treatment. These ih vitro models include
proliferation assays of
cultured tumor cells, growth of cultured tumor cells in soft agar (see
Freshney, (1987) Culture of
Animal Cells: A Manual of Basic Technique, Wily-Liss, New York, NY Ch 18 and
Ch 21),
tumor systems in nude mice as described in Giovanella et al., J. Natl. Can.
Inst., 52: 921-30
(1974), mobility and invasive potential of tumor cells in Boyden Chamber
assays as described in
Pilkington et al., Anticancer Res., 17: 4107-9 (1997), and angiogenesis assays
such as induction
of vascularization of the chick chorioallantoic membrane or induction of
vascular endothelial
cell migration as described in Ribatta et al., Intl. J. Dev. Biol., 40: 1189-
97 (1999) and Li et al.,
Clin. Exp. Metastasis, 17:423-9 (1999), respectively. Suitable tumor cells
lines are available,
e.g. from American Type Tissue Culture Collection catalogs.
5.10.12 RECEPTOR/LIGAND ACTIVITY
A polypeptide of the present invention may also demonstrate activity as
receptor,
receptor ligand or inhibitor or agonist of receptor/ligand interactions. A
polynucleotide of the
invention can encode a polypeptide exhibiting such characteristics. Examples
of such receptors
and ligands include, without limitation, cytokine receptors and their ligands,
receptor kinases and
their ligands, receptor phosphatases and their ligands, receptors involved in
cell-cell interactions
and their ligands (including without limitation, cellular adhesion molecules
(such as selectins,
integrins and their ligands) and receptor/ligand pairs involved in antigen
presentation, antigen
recognition and development of cellular and humoral immune responses.
Receptors and ligands
are also useful for screening of potential peptide or small molecule
inhibitors of the relevant
receptor/ligand interaction. A protein of the present invention (including,
without limitation,
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fragments of receptors and ligands) may themselves be useful as inhibitors of
receptor/ligand
interactions.
The activity of a polypeptide of the invention may, among other means, be
measured by
the following methods:
Suitable assays for receptor-ligand activity include without limitation those
described in:
Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.
Margulies, E. M.
Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley- Interscience
(Chapter 7.28,
Measurement of Cellular Adhesion under static conditions 7.28.1- 7.28.22),
Takai et al., Proc.
Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med. 168:1145-
1156, 1988;
Rosenstein et al., J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J.
Immunol. Methods
175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.
By way of example, the polypeptides of the invention may be used as a receptor
for a
ligand(s) thereby transmitting the biological activity of that ligand(s).
Ligands may be identified
through binding assays, affinity chromatography, dihybrid screening assays,
BIAcore assays, gel
overlay assays, or other methods known in the art.
Studies characterizing drugs or proteins as agonist or antagonist or partial
agonists or a
partial antagonist require the use of other proteins as competing ligands. The
polypeptides of the
present invention or ligand(s) thereof may be labeled by being coupled to
radioisotopes,
colorimetric molecules or a toxin molecules by conventional methods. ("Guide
to Protein
Purification" Murray P. Deutscher (ed) Methods in Enzymology Vol. 182 (1990)
Academic
Press, Inc. San Diego). Examples of radioisotopes include, but are not limited
to, tritium and
carbon-14 . Examples of colorimetric molecules include, but axe not limited
to, fluorescent
molecules such as fluorescamine, or rhodamine or other colorimetric molecules.
Examples of
toxins include, but axe not limited, to ricin.
5.10.13 DRUG SCREENING
This invention is particularly useful for screening chemical compounds by
using the
novel polypeptides or binding fragments thereof in any of a variety of drug
screening techniques.
The polypeptides or fragments employed in such a test may either be free in
solution, affixed to a
solid support, borne on a cell surface or located intracellularly. One method
of drug screening
utilizes eukaryotic or prokaryotic host cells which are stably transformed
with recombinant
nucleic acids expressing the polypeptide or a fragment thereof. Drugs are
screened against such
transformed cells in competitive binding assays. Such cells, either in viable
or fixed form, can
be used for standard binding assays. One may measure, for example, the
formation of
complexes between polypeptides of the invention or fragments and the agent
being tested or
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examine the diminution in complex formation between the novel polypeptides and
an
appropriate cell line, which are well known in the art.
Sources for test compounds that may be screened for ability to bind to or
modulate (i.e.,
increase or decrease) the activity of polypeptides of the invention include
(1) inorganic and
organic chemical libraries, (2) natural product libraries, and (3)
combinatorial libraries
comprised of either random or mimetic peptides, oligonucleotides or organic
molecules.
Chemical libraries may be readily synthesized or purchased from a number of
commercial sources, and may include structural analogs of known compounds or
compounds
that are identified as "hits" or "leads" via natural product screening.
The sources of natural product libraries are microorganisms (including
bacteria and
fungi), animals, plants or other vegetation, or marine organisms, and
libraries of mixtures for
screening may be created by: (1) fermentation and extraction of broths from
soil, plant or marine
microorganisms or (2) extraction of the organisms themselves. Natural product
libraries include
polyketides, non-ribosomal peptides, and (non-naturally occurring) variants
thereof. For a
review, see Science 22:63-68 (1998).
Combinatorial libraries are composed of large numbers of peptides,
oligonucleotides or
organic compounds and can be readily prepared by traditional automated
synthesis methods,
PCR, cloning or proprietary synthetic methods. Of particular interest are
peptide and
oligonucleotide combinatorial libraries. Still other libraries of interest
include peptide, protein,
peptidomimetic, multiparallel synthetic collection, recombinatorial, and
polypeptide libraries.
For a review of combinatorial chemistry and libraries created therefrom, see
Myers, Curr. Opin.
Biotechhol. 8:701-707 (1997). For reviews and examples of peptidomimetic
libraries, see Al-
Obeidi et al., Mol. Biotech~col, 9(3):205-23 (1998); Hruby et al., Cu~~ Opih
Chem Biol,
1(1):114-19 (1997); Dorner et al., Bioo~gMed Chem, 4(5):709-15 (1996)
(alkylated dipeptides).
Identification of modulators through use of the various libraries described
herein permits
modification of the candidate "hit" (or "lead") to optimize the capacity of
the "hit" to bind a
polypeptide of the invention. The molecules identified in the binding assay
are then tested for
antagonist or agonist activity in in vivo tissue culture or animal models that
are well known in the
art. In brief, the molecules are titrated into a plurality of cell cultures or
animals and then tested
for either celllanimal death or prolonged survival of the animal/cells.
The binding molecules thus identified may be complexed with toxins, e.g.,
ricin or
cholera, or with other compounds that are toxic to cells such as
radioisotopes. The toxin-binding
molecule complex is then targeted to a tumor or other cell by the specificity
of the binding
molecule for a polypeptide of the invention. Alternatively, the binding
molecules may be
complexed with imaging agents for targeting and imaging purposes.
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5.10.14 ASSAY FOR RECEPTOR ACTIVITY
The invention also provides methods to detect specific binding of a
polypeptide e.g. a
ligand or a receptor. The art provides numerous assays particularly useful for
identifying
previously unknown binding partners for receptor polypeptides of the
invention. For example,
expression cloning using mammalian or bacterial cells, or dihybrid screening
assays can be used
to identify polynucleotides encoding binding partners. As another example,
affinity
chromatography with the appropriate immobilized polypeptide of the invention
can be used to
isolate polypeptides that recognize and bind polypeptides of the invention.
There are a number
of different libraries used for the identification of compounds, and in
particular small molecule,
that modulate (i. e., increase or decrease) biological activity of a
polypeptide of the invention.
Ligands for receptor polypeptides of the invention can also be identified by
adding exogenous
ligands, or cocktails of ligands to two cells populations that are genetically
identical except for
the expression of the receptor of the invention: one cell population expresses
the receptor of the
invention whereas the other does not. The response of the two cell populations
to the addition of
ligands(s) are then compared. Alternatively, an expression library can be co-
expressed with the
polypeptide of the invention in cells and assayed for an autocrine response to
identify potential
ligand(s). As still another example, BIAcore assays, gel overlay assays, or
other methods known
in the art can be used to identify binding partner polypeptides, including,
(1) organic and
inorganic chemical libraries, (2) natural product libraries, and (3)
combinatorial libraries
comprised of random peptides, oligonucleotides or organic molecules.
The role of downstream intracellular signaling molecules in the signaling
cascade of the
polypeptide of the invention can be determined. For example, a chimeric
protein in which the
cytoplasmic domain of the polypeptide of the invention is fused to the
extracellular portion of a
protein, whose ligand has been identified, is produced in a host cell. The
cell is then incubated
with the ligand specific for the extracellular portion of the chimeric
protein, thereby activating
the chimeric receptor. Known downstream proteins involved in intracellular
signaling can then
be assayed for expected modifications i.e. phosphorylation. Other methods
known to those in the
art can also be used to identify signaling molecules involved in receptor
activity.
5.10.15 ANTI-INFLAMMATORY ACTIVITY
Compositions of the present invention may also exhibit anti-inflammatory
activity. The
anti-inflammatory activity may be achieved by providing a stimulus to cells
involved in the
inflammatory response, by inhibiting or promoting cell-cell interactions (such
as, for example,
cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the
inflammatory
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process, inhibiting or promoting cell extravasation, or by stimulating or
suppressing production
of other factors which more directly inhibit or promote an inflammatory
response. Compositions
with such activities can be used to treat inflammatory conditions including
chronic or acute
conditions), including without limitation intimation associated with infection
(such as septic
shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-
reperfusion injury,
endotoxin lethality, arthritis, complement-mediated hyperacute rejection,
nephritis, cytokine or
chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or
resulting from
over production of cytokines such as TNF or IL-1. Compositions of the
invention may also be
useful to treat anaphylaxis and hypersensitivity to an antigenic substance or
material.
Compositions of this invention may be utilized to prevent or treat conditions
such as, but not
limited to, sepsis, acute pancreatitis, endotoxin shock, cytokine induced
shock, rheumatoid
arthritis, chronic inflammatory arthritis, pancreatic cell damage from
diabetes mellitus type 1,
graft versus host disease, inflammatory bowel disease, inflamation associated
with pulmonary
disease, other autoimmune disease or inflammatory disease, an
antiproliferative agent such as for
acute or chronic myelogenous leukemia or in the prevention of premature labor
secondary to
intrauterine infections.
5.10.16 LEUKEMIAS
Leukemias and related disorders may be treated or prevented by administration
of a
therapeutic that promotes or inhibits function of the polynucleotides and/or
polypeptides of the
invention. Such leukemias and related disorders include. but are not limited
to acute leukemia,
acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic,
promyelocytic,
myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic
myelocytic
(granulocytic) leukemia and chronic lymphocytic leukemia (for a review of such
disorders, see
Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia).
5.10.17 NERVOUS SYSTEM DISORDERS
Nervous system disorders, involving cell types which can be tested for
efficacy of
intervention with compounds that modulate the activity of the polynucleotides
and/or
polypeptides of the invention, and which can be treated upon thus observing an
indication of
therapeutic utility, include but are not limited to nervous system injuries,
and diseases or
disorders which result in either a disconnection of axons, a diminution or
degeneration of
neurons, or demyelination. Nervous system lesions which may be treated in a
patient (including
human and non-human mammalian patients) according to the invention include but
are not
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limited to the following lesions of either the central (including spinal cord,
brain) or peripheral
nervous systems:
(i) traumatic lesions, including lesions caused by physical injury or
associated with
surgery, for example, lesions which sever a portion of the nervous system, or
compression
injuries;
(ii) ischemic lesions, in which a lack of oxygen in a portion of the nervous
system
results in neuronal injury or death, including cerebral infarction or
ischemia, or spinal cord
infarction or ischemia;
(iii) infectious lesions, in which a portion of the nervous system is
destroyed or
injured as a result of infection, for example, by an abscess or associated
with infection by human
immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme
disease,
tuberculosis, syphilis;
(iv) degenerative lesions, in which a portion of the nervous system is
destroyed or
injured as a result of a degenerative process including but not limited to
degeneration associated
with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or
amyotrophic lateral
sclerosis;
(v) lesions associated with nutritional diseases or disorders, in which a
portion of the
nervous system is destroyed or injured by a nutritional disorder or disorder
of metabolism
including but not limited to, vitamin B 12 deficiency, folic acid deficiency,
Wernicke disease,
tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration
of the corpus
callosum), and alcoholic cerebellar degeneration;
(vi) neurological lesions associated with systemic diseases including but not
limited to
diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus,
carcinoma, or
sarcoidosis;
(vii) lesions caused by toxic substances including alcohol, lead, or
particular
neurotoxins; and
(viii) demyelinated lesions in which a portion of the nervous system is
destroyed or
injured by a demyelinating disease including but not limited to multiple
sclerosis, human
immunodeficiency virus-associated myelopathy, transverse myelopathy or various
etiologies,
progressive multifocal leukoencephalopathy, and central pontine myelinolysis.
Therapeutics which are useful according to the invention for treatment of a
nervous
system disorder may be selected by testing for biological activity in
promoting the survival or
differentiation of neurons. For example, and not by way of limitation,
therapeutics which elicit
any of the following effects may be useful according to the invention:
(i) increased survival time of neurons in culture;
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(ii) increased sprouting of neurons in culture or in vivo;
(iii) increased production of a neuron-associated molecule in culture or ivy
vivo, e.g.,
choline acetyltransferase or acetylcholinesterase with respect to motor
neurons; or
(iv) decreased symptoms of neuron dysfunction in vivo.
Such effects may be measured by any method known in the art. In preferred, non-
limiting embodiments, increased survival of neurons may be measured by the
method set forth in
Arakawa et al. (1990, J. Neurosci. 10:3507-3515); increased sprouting of
neurons may be
detected by methods set forth in Pestronk et al. (1980, Exp. Neurol. 70:65-82)
or Brown et al.
(1981, Ann. Rev. Neurosci. 4:17-42); increased production of neuron-associated
molecules may
be measured by bioassay, enzymatic assay, antibody binding, Northern blot
assay, etc.,
depending on the molecule to be measured; and motor neuron dysfunction may be
measured by
assessing the physical manifestation of motor neuron disorder, e.g., weakness,
motor neuron
conduction velocity, or functional disability.
In specific embodiments, motor neuron disorders that may be treated according
to the
invention include but are not limited to disorders such as infarction,
infection, exposure to toxin,
trauma, surgical damage, degenerative disease or malignancy that may affect
motor neurons as
well as other components of the nervous system, as well as disorders that
selectively affect
neurons such as amyotrophic lateral sclerosis, and including but not limited
to progressive spinal
muscular atrophy, progressive bulbar palsy, primary lateral sclerosis,
infantile and juvenile
muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe
syndrome),
poliomyelitis and the post polio syndrome, and Hereditary Motorsensory
Neuropathy (Charcot-
Maxie-Tooth Disease). .
5.10.18 OTHER ACTIVITIES
A polypeptide of the invention may also exhibit one or more of the following
additional
activities or effects: inhibiting the growth, infection or function of, or
killing, infectious agents,
including, without limitation, bacteria, viruses, fungi and other parasites;
effecting (suppressing
or enhancing) bodily characteristics, including, without limitation, height,
weight, hair color, eye
color, skin, fat to lean ratio or other tissue pigmentation, or organ or body
part size or shape
(such as, for example, breast augmentation or diminution, change in bone form
or shape);
effecting biorhythms or circadian cycles or rhythms; effecting the fertility
of male or female
subjects; effecting the metabolism, catabolism, anabolism, processing,
utilization, storage or
elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals,
co-factors or other
nutritional factors or component(s); effecting behavioral characteristics,
including, without
limitation, appetite, libido, stress, cognition (including cognitive
disorders), depression
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(including depressive disorders) and violent behaviors; providing analgesic
effects or other pain
reducing effects; promoting differentiation and growth of embryonic stem cells
in lineages other
than hematopoietic lineages; hormonal or endocrine activity; in the case of
enzymes, correcting
deficiencies of the enzyme and treating deficiency-related diseases; treatment
of
hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-
like activity (such
as, for example, the ability to bind antigens or complement); and the ability
to act as an antigen
in a vaccine composition to raise an immune response against such protein or
another material or
entity which is cross-reactive with such protein.
5.10.19 IDENTIFICATION OF POLYMORPHISMS
The demonstration of polymorphisms makes possible the identification of such
polymorphisms in human subjects and the pharmacogenetic use of this
information for diagnosis
and treatment. Such polymorphisms may be associated with, e.g., differential
predisposition or
susceptibility to various disease states (such as disorders involving
inflammation or immune
response) or a differential response to drug administration, and this genetic
information can be
used to tailor preventive or therapeutic treatment appropriately. For example,
the existence of a
polymorphism associated with a predisposition to inflammation or autoimmune
disease makes
possible the diagnosis of this condition in humans by identifying the presence
of the
polymorphism.
Polyinorphisms can be identified in a vaxiety of ways known in the art which
all
generally involve obtaining a sample from a patient, analyzing DNA from the
sample, optionally
involving isolation or amplification of the DNA, and identifying the presence
of the
polymorphism in the DNA. For example, PCR may be used to amplify an
appropriate fragment
of genomic DNA which may then be sequenced. Alternatively, the DNA may be
subjected to
allele-specific oligonucleotide hybridization (in which appropriate
oligonucleotides are
hybridized to the DNA under conditions permitting detection of a single base
mismatch) or to a
single nucleotide extension assay (in which an oligonucleotide that hybridizes
immediately
adjacent to the position of the polymorphism is extended with one or more
labeled nucleotides).
In addition, traditional restriction fragment length polymorphism analysis
(using restriction
enzymes that provide differential digestion of the genomic DNA depending on
the presence or
absence of the polymorphism) may be performed. Arrays with nucleotide
sequences of the
present invention can be used to detect polymorphisms. The array can comprise
modified
nucleotide sequences of the present invention in order to detect the
nucleotide sequences of the
present invention. In the alternative, any one of the nucleotide sequences of
the present
invention can be placed on the array to detect changes from those sequences.
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Alternatively a polymorphism resulting in a change in the amino acid sequence
could
also be detected by detecting a corresponding change in amino acid sequence of
the protein, e.g.,
by an antibody specific to the variant sequence.
5.10.20 ARTHRITIS AND INFLAMMATION
The immunosuppressive effects of the compositions of the invention against
rheumatoid
arthritis is determined in an experimental animal model system. The
experimental model system
is adjuvant induced arthritis in rats, and the protocol is described by J.
Holoshitz, et at., 1983,
Science, 219:56, or by B. Waksman et al., 1963, Int. Arch. Allergy Appl.
Immunol., 23:129.
Induction of the disease can be caused by a single injection, generally
intradermally, of a
suspension of killed Mycobacterium tuberculosis in complete Freund's adjuvant
(CFA). The
route of injection can vary, but rats may be injected at the base of the tail
with an adjuvant
mixture. The polypeptide is administered in phosphate buffered solution (PBS)
at a dose of about
1-5 mg/kg. The control consists of administering PBS only.
The procedure for testing the effects of the test compound would consist of
intradermally
injecting killed Mycobacterium tuberculosis in CFA followed by immediately
administering the
test compound and subsequent treatment every other day until day 24. At 14,
15, 18, 20, 22, and
24 days after injection of Mycobacterium CFA, an overall arthritis score may
be obtained as
described by J. Holoskitz above. An analysis of the data would reveal that the
test compound
would have a dramatic affect on the swelling of the joints as measured by a
decrease of the
arthritis score.
5.11 THERAPEUTIC METHODS
The compositions (including polypeptide fragments, analogs, variants and
antibodies or
other binding partners or modulators including antisense polynucleotides) of
the invention have
numerous applications in a variety of therapeutic methods. Examples of
therapeutic applications
include, but are not limited to, those exemplified herein.
5.11.1 EXAMPLE
One embodiment of the invention is the administration of an effective amount
of the
neurotrimin-like polypeptides or other composition of the invention to
individuals affected by a
disease or disorder that can be modulated by regulating the soluble
neurotrimin-like polypeptides
of the invention. While the mode of administration is not particularly
important, parenteral
administration is preferred. An exemplary mode of administration is to deliver
an intravenous
bolus. The dosage of neurotrimin-like polypeptides or other composition of the
invention will
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normally be determined by the prescribing physician. It is to be expected that
the dosage will
vary according to the age, weight, condition and response of the individual
patient. Typically,
the amount of polypeptide administered per dose will be in the range of about
0.01 ~,g/kg to 100
mg/kg of body weight, with the preferred dose being about 0.1 ~g/kg to 10
mg/kg of patient body
weight. For parenteral administration, neurotrimin-like polypeptides of the
invention will be
formulated in an injectable form combined with a pharmaceutically acceptable
parenteral
vehicle. Such vehicles are well known in the art and examples include water,
saline, Ringer's
solution, dextrose solution, and solutions consisting of small amounts of the
human serum
albumin. The vehicle may contain minor amounts of additives that maintain the
isotonicity and
stability of the polypeptide or other active ingredient. The preparation of
such solutions is within
the skill of the art.
5.12 PHARMACEUTICAL FORMULATIONS AND ROUTES OF
ADMINISTRATION
A protein or other composition of the present invention (from whatever source
derived,
including without limitation from recombinant and non-recombinant sources and
including
antibodies and other binding partners of the polypeptides of the invention)
may be administered
to a patient in need, by itself, or in pharmaceutical compositions where it is
mixed with suitable
carriers or excipient(s) at doses to treat or ameliorate a variety of
disorders. Such a composition
may optionally contain (in addition to protein or other active ingredient and
a carrier) diluents,
fillers, salts, buffers, stabilizers, solubilizers, and other materials well
known in the art. The term
"pharmaceutically acceptable" means a non-toxic material that does not
interfere with the
effectiveness of the biological activity of the active ingredient(s). The
characteristics of the
carrier will depend on the route of administration. The pharmaceutical
composition of the
invention may also contain cytokines, lymphokines, or other hematopoietic
factors such as M-
CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,
IL-11, IL-12,
IL-13, IL-14, IL-15, IFN, TNFO, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin,
stem cell
factor, and erythropoietin. In further compositions, proteins of the invention
may be combined
with other agents beneficial to the treatment of the disease or disorder in
question. These agents
include various growth factors such as epidermal growth factor (EGF), platelet-
derived growth
factor (PDGF), transforming growth factors (TGF-a and TGF-(3), insulin-like
growth factor
(IGF), as well as cytokines described herein.
The pharmaceutical composition may further contain other agents which either
enhance
the activity of the protein or other active ingredient or complement its
activity or use in
treatment. Such additional factors andlor agents may be included in the
pharmaceutical
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composition to produce a synergistic effect with protein or other active
ingredient of the
invention, or to minimize side effects. Conversely, protein or other active
ingredient of the
present invention may be included in formulations of the particular clotting
factor, cytokine,
lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic
factor, or anti-
s inflammatory agent to minimize side effects of the clotting factor,
cytokine, lymphokine, other
hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-
inflammatory agent (such as
IL-lRa, IL-1 Hyl, IL-1 Hy2, anti-TNF, corticosteroids, immunosuppressive
agents). A protein
of the present invention may be active in multirners (e.g., heterodimers or
homodimers) or
complexes with itself or other proteins. As a result, pharmaceutical
compositions of the
invention may comprise a protein of the invention in such multimeric or
complexed form.
As an alternative to being included in a pharmaceutical composition of the
invention
including a first protein, a second protein or a therapeutic agent may be
concurrently
administered with the first protein (e.g., at the same time, or at differing
times provided that
therapeutic concentrations of the combination of agents is achieved at the
treatment site)..
Techniques for formulation and administration of the compounds of the instant
application may
be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co.,
Easton, PA, latest
edition. A therapeutically effective dose further refers to that amount of the
compound sufficient
to result in amelioration of symptoms, e.g., treatment, healing, prevention or
amelioration of the
relevant medical condition, or an increase in rate of treatment, healing,
prevention or
amelioration of such conditions. When applied to an individual active
ingredient, administered
alone, a therapeutically effective dose refers to that ingredient alone. When
applied to a
combination, a therapeutically effective dose refers to combined amounts of
the active
ingredients that result in the therapeutic effect, whether administered in
combination, serially or
simultaneously.
In practicing the method of treatment or use of the present invention, a
therapeutically
effective amount of protein or other active ingredient of the present
invention is administered to
a mammal having a condition to be treated. Protein or other active ingredient
of the present
invention may be administered in accordance with the method of the invention
eithex alone or in
combination with other therapies such as treatments employing cytokines,
lymphokines or other
hematopoietic factors. When co- administered with one or more cytokines,
lymphokines or other
hematopoietic factors, protein or other active ingredient of the present
invention may be
administered either simultaneously with the cytokine(s), lymphokine(s), other
hematopoietic
factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If
administered sequentially,
the attending physician will decide on the appropriate sequence of
administering protein or other
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active ingredient of the present invention in combination with cytokine(s),
lymphokine(s), other
hematopoietic factor(s), thrombolytic or anti-thrombotic factors.
5.12.1 ROUTES OF ADMINISTRATION
Suitable routes of administration may, for example, include oral, rectal,
transmucosal, or
intestinal administration; parenteral delivery, including intramuscular,
subcutaneous,
intramedullary injections, as well as intrathecal, direct intraventricular,
intravenous,
intraperitoneal, intranasal, or intraocular injections. Administration of
protein or other active
ingredient of the present invention used in the pharmaceutical composition or
to practice the
method of the present invention can be carried out in a variety of
conventional ways, such as oral
ingestion, inhalation, topical application or cutaneous, subcutaneous,
intraperitoneal, parenteral
or intravenous injection. Intravenous administration to the patient is
preferred.
Alternately, one may administer the compound in a local rather than systemic
manner, for
example, via injection of the compound directly into a arthritic joints or in
fibrotic tissue, often
in a depot or sustained release formulation. In order to prevent the scarring
process frequently
occurring as complication of glaucoma surgery, the compounds may be
administered topically,
for example, as eye drops. Furthermore, one may administer the drug in a
targeted drug delivery
system, for example, in a liposome coated with a specific antibody, targeting,
for example,
arthritic or fibrotic tissue. The liposomes will be targeted to and taken up
selectively by the
afflicted tissue.
The polypeptides of the invention are administered by any route that delivers
an effective
dosage to the desired site of action. The determination of a suitable route of
administration and
an effective dosage for a particular indication is within the level of skill
in the art. Preferably for
wound treatment, one administers the therapeutic compound directly to the
site. Suitable dosage
ranges for the polypeptides of the invention can be extrapolated from these
dosages or from
similar studies in appropriate animal models. Dosages can then be adjusted as
necessary by the
clinician to provide maximal therapeutic benefit.
5.12.2 COMPOSITIONS/FORMULATIONS
Pharmaceutical compositions for use in accordance with the present invention
thus may
be formulated in a conventional manner using one or more physiologically
acceptable carriers
comprising excipients and auxiliaries which facilitate processing of the
active compounds into
preparations which can be used pharmaceutically. These pharmaceutical
compositions may be
manufactured in a manner that is itself known, e.g., by means of conventional
mixing,
dissolving, granulating, dragee-making, levigating, emulsifying,
encapsulating, entrapping or
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lyophilizing processes. Proper formulation is dependent upon the route of
administration
chosen. When a therapeutically effective amount of protein or other active
ingredient of the
present invention is administered orally, protein or other active ingredient
of the present
invention will be in the form of a tablet, capsule, powder, solution or
elixir. When administered
in tablet form, the pharmaceutical composition of the invention may
additionally contain a solid
carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder
contain from about 5 to
95% protein or other active ingredient of the present invention, and
preferably from about 25 to
90% protein or other active ingredient of the present invention. When
administered in liquid
form, a liquid carrier such as water, petroleum, oils of animal or plant
origin such as peanut oil,
mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The
liquid form of the
pharmaceutical composition may further contain physiological saline solution,
dextrose or other
saccharide solution, or glycols such as ethylene glycol, propylene glycol or
polyethylene glycol.
When administered in liquid form, the pharmaceutical composition contains from
about 0.5 to
90% by weight of protein or other active ingredient of the present invention,
and preferably from
about I to 50% protein or other active ingredient of the present invention.
When a therapeutically effective amount of protein or other active ingredient
of the
present invention is administered by intravenous, cutaneous or subcutaneous
injection, protein or
other active ingredient of the present invention will be in the form of a
pyrogen-free, parenterally
acceptable aqueous solution. The preparation of such parenterally acceptable
protein or other
active ingredient solutions, having due regard to pH, isotonicity, stability,
and the like, is within
the skill in the art. A preferred pharmaceutical composition for intravenous,
cutaneous, or
subcutaneous injection should contain, in addition to protein or other active
ingredient of the
present invention, an isotonic vehicle such as Sodium Chloride Injection,
Ringer's Injection,
Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's
Injection, or
other vehicle as known in the art. The pharmaceutical composition of the
present invention may
also contain stabilizers, preservatives, buffers, antioxidants, or other
additives known to those of
skill in the art. For injection, the agents of the invention may be formulated
in aqueous
solutions, preferably in physiologically compatible buffers such as Hanks's
solution, Ringer's
solution, or physiological saline buffer. For transmucosal administration,
penetrants appropriate
to the barrier to be permeated are used in the formulation. Such penetrants
are generally known
in the art.
For oral administration, the compounds can be formulated readily by combining
the
active compounds with pharmaceutically acceptable Garners well known in the
art. Such carriers
enable the compounds of the invention to be formulated as tablets, pills,
dragees, capsules,
liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion
by a patient to be
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treated. Pharmaceutical preparations for oral use can be obtained solid
excipient, optionally
grinding a resulting mixture, and processing the mixture of granules, after
adding suitable
auxiliaries, if desired, to obtain tablets or dragee cores. Suitable
excipients are, in particular,
fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol;
cellulose preparations
such as, for example, maize starch, wheat starch, rice starch, potato starch,
gelatin, gum
tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethylcellulose,
and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be
added, such as the
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof
such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose,
concentrated sugar solutions
may be used, which may optionally contain gum arabic, talc, polyvinyl
pyrrolidone, carbopol
gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and
suitable organic solvents
or solvent mixtures. Dyestuffs or pigments may be added to the tablets or
dragee coatings for
identification or to characterize different combinations of active compound
doses.
Pharmaceutical preparations which can be used orally include push-fit capsules
made of
gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer,
such as glycerol or
sorbitol. The push-fit capsules can contain the active ingredients in
admixture with filler such as
lactose, binders such as starches, and/or lubricants such as talc or magnesium
stearate and,
optionally, stabilizers. In soft capsules, the active compounds may be
dissolved or suspended in
suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene
glycols. In addition,
stabilizers may be added. All formulations for oral administration should be
in dosages suitable
for such administration. Fox buccal administration, the compositions may take
the form of
tablets or lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the
present
invention are conveniently delivered in the form of an aerosol spray
presentation from
pressurized packs or a nebuliser, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane,
carbon dioxide or
other suitable gas. In the case of a pressurized aerosol the dosage unit may
be determined by
providing a valve to deliver a metered amount. Capsules and cartridges of,
e.g., gelatin for use
in an inhaler or insufflator may be formulated containing a powder mix of the
compound and a
suitable powder base such as lactose or starch. The compounds may be
formulated for parenteral
administration by injection, e.g., by bolus injection or continuous infusion.
Formulations for
injection may be presented in unit dosage form, e.g., in ampules or in mufti-
dose containers, with
an added preservative. The compositions may take such forms as suspensions,
solutions or
emulsions in oily or aqueous vehicles, and may contain formulatory agents such
as suspending,
stabilizing and/or dispersing agents.
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Pharmaceutical formulations for parenteral administration include aqueous
solutions of
the active compounds in water-soluble form. Additionally, suspensions of the
active compounds
may be prepared as appropriate oily injection suspensions. Suitable lipophilic
solvents or
vehicles include fatty oils such as sesame oil, or synthetic fatty acid
esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may contain
substances which
increase the viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or
dextran. Optionally, the suspension may also contain suitable stabilizers or
agents which
increase the solubility of the compounds to allow for the preparation of
highly concentrated
solutions. Alternatively, the active ingredient may be in powder form for
constitution with a
suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal compositions such as
suppositories or
retention enemas, e.g., containing conventional suppository bases such as
cocoa butter or other
glycerides. In addition to the formulations described previously, the
compounds may also be
formulated as a depot preparation. Such long acting formulations may be
administered by
implantation (for example subcutaneously or intramuscularly) or by
intramuscular injection.
Thus, for example, the compounds may be formulated with suitable polymeric or
hydrophobic
materials (for example as an emulsion in an acceptable oil) or ion exchange
resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble salt.
A pharmaceutical carrier for the hydrophobic compounds of the invention is a
co-solvent
system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible
organic polymer, and
an aqueous phase. The co-solvent system may be the VPD co-solvent system. VPD
is a solution
of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80,
and 65% w/v
polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-
solvent system
(VPD:SV~ consists of VPD diluted 1:1 with a 5% dextrose in water solution.
This co-solvent
system dissolves hydrophobic compounds well, and itself produces low toxicity
upon systemic
administration. Naturally, the proportions of a co-solvent system may be
varied considerably
without destroying its solubility and toxicity characteristics. Furthermore,
the identity of the co-
solvent components may be varied: for example, other low-toxicity nonpolar
surfactants may be
used instead of polysorbate 80; the fraction size of polyethylene glycol may
be varied; other
biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl
pyrrolidone; and other
sugars or polysaccharides may substitute for dextrose. Alternatively, other
delivery systems for
hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions
are well
known examples of delivery vehicles or carriers for hydrophobic drugs. Certain
organic solvents
such as dimethylsulfoxide also may be employed, although usually at the cost
of greater toxicity.
Additionally, the compounds may be delivered using a sustained-release system,
such as
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semipermeable matrices of solid hydrophobic polymers containing the
therapeutic agent.
Various types of sustained-release materials have been established and are
well known by those
skilled in the art. Sustained-release capsules may, depending on their
chemical nature, release the
compounds for a few weeks up to over 100 days. Depending on the chemical
nature and the
biological stability of the therapeutic reagent, additional strategies for
protein or other active
ingredient stabilization may be employed.
The pharmaceutical compositions also may comprise suitable solid or gel phase
carriers
or excipients. Examples of such carriers or excipients include but are not
limited to calcium
carbonate, calcium phosphate, various sugars, starches, cellulose derivatives,
gelatin, and
polymers such as polyethylene glycols. Many of the active ingredients of the
invention may be
provided as salts with pharmaceutically compatible counter ions. Such
pharmaceutically
acceptable base addition salts are those salts which retain the biological
effectiveness and
properties of the free acids and which are obtained by reaction with inorganic
or organic bases
such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamine,
dialkylaxnine,
monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate,
triethanol amine and
the like.
The pharmaceutical composition of the invention may be in the form of a
complex of the
proteins) or other active ingredient of present invention along with protein
or peptide antigens.
The protein and/or peptide antigen will deliver a stimulatory signal to both B
and T lymphocytes.
B lymphocytes will respond to antigen through their surface immunoglobulin
receptor. T
lymphocytes will respond to antigen through the T cell receptor (TCR)
following presentation of
the antigen by MHC proteins. MHC and structurally related proteins including
those encoded by
class I and class II MHC genes on host cells will serve to present the peptide
antigens) to T
lymphocytes. The antigen components could also be supplied as purified MHC-
peptide
complexes alone or with co-stimulatory molecules that can directly signal T
cells. Alternatively
antibodies able to bind surface immunoglobulin and other molecules on B cells
as well as
antibodies able to bind the TCR and other molecules on T cells can be combined
with the
pharmaceutical composition of the invention.
The pharmaceutical composition of the invention may be in the form of a
liposome in
which protein of the present invention is combined, in addition to other
pharmaceutically
acceptable carriers, with amphipathic agents such as lipids which exist in
aggregated form as
micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous
solution. Suitable
lipids for liposomal formulation include, without limitation, monoglycerides,
diglycerides,
sulfatides, lysolecithins, phospholipids, saponin, bile acids, and the like.
Preparation of such
liposomal formulations is within the level of skill in the art, as disclosed,
for example, in U.S.
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Patent Nos. 4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are
incorporated
herein by reference.
The amount of protein or other active ingredient of the present invention in
the
pharmaceutical composition of the present invention will depend upon the
nature and severity of
the condition being treated, and on the nature of prior treatments which the
patient has
undergone. Ultimately, the attending physician will decide the amount of
protein or other active
ingredient of the present invention with which to treat each individual
patient. Initially, the
attending physician will administer low doses of protein or other active
ingredient of the present
invention and observe the patient's response. Larger doses of protein or other
active ingredient
of the present invention may be administered until the optimal therapeutic
effect is obtained for
the patient, and at that point the dosage is not increased further. It is
contemplated that the
various pharmaceutical compositions used to practice the method of the present
invention should
contain about 0.01 ~.g to about 100 mg (preferably about 0.1 ~,g to about 1.0
mg, more preferably
about 0.1 ~,g to about I mg) of protein or other active ingredient of the
present invention per kg
body weight. For compositions of the present invention which are useful for
bone, cartilage,
tendon or ligament regeneration, the therapeutic method includes administering
the composition
topically, systematically, or locally as an implant or device. When
administered, the therapeutic
composition for use in this invention is, of course, in a pyrogen-free,
physiologically acceptable
form. Further, the composition may desirably be encapsulated or injected in a
viscous form for
delivery to the site of bone, cartilage or tissue damage. Topical
administration maybe suitable
for wound healing and tissue repair. Therapeutically useful agents other than
a protein or other
active ingredient of the invention which may also optionally be included in
the composition as
described above, may alternatively or additionally, be administered
simultaneously or
sequentially with the composition in the methods of the invention. Preferably
for bone and/or
cartilage formation, the composition would include a matrix capable of
delivering the protein-
containing or other active ingredient-containing composition to the site of
bone and/or cartilage
damage, providing a structure for the developing bone and cartilage and
optimally capable of
being resorbed into the body. Such matrices may be formed of materials
presently in use for
other implanted medical applications.
The choice of matrix material is based on biocompatibility, biodegradability,
mechanical
properties, cosmetic appearance and interface properties. The particular
application of the
compositions will define the appropriate formulation. Potential matrices for
the compositions
may be biodegradable and chemically defined calcium sulfate, tricalcium
phosphate,
hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides. Other
potential materials
are biodegradable and biologically well-defined, such as bone or dermal
collagen. Further
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matrices are comprised of pure proteins or extracellular matrix components.
Other potential
matrices are nonbiodegradable and chemically defined, such as sintered
hydroxyapatite, bioglass,
aluminates, or other ceramics. Matrices may be comprised of combinations of
any of the above
mentioned types of material, such as polylactic acid and hydroxyapatite or
collagen and
tricalcium phosphate. The bioceramics may be altered in composition, such as
in calcium-
aluminate-phosphate and processing to alter pore size, particle size, particle
shape, and
biodegradability. Presently preferred is a 50:50 (mole weight) copolymer of
lactic acid and
glycolic acid in the form of porous particles having diameters ranging from
150 to 800 microns.
In some applications, it will be useful to utilize a sequestering agent, such
as carboxymethyl
cellulose or autologous blood clot, to prevent the protein compositions from
disassociating from
the matrix.
A preferred family of sequestering agents is cellulosic materials such as
alkylcelluloses
(including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose,
hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose,
and
carboxymethylcellulose, the most preferred being cationic salts of
carboxymethylcellulose
(CMC). Other preferred sequestering agents include hyaluronic acid, sodium
alginate,
polyethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and
polyvinyl alcohol).
The amount of sequestering agent useful herein is 0.5-20 wt %, preferably 1-10
wt % based on
total formulation weight, which represents the amount necessary to prevent
desorption of the
protein from the polymer matrix and to provide appropriate handling of the
composition, yet not
so much that the progenitor cells are prevented from infiltrating the matrix,
thereby providing the
protein the opportunity to assist the osteogenic activity of the progenitor
cells. In further
compositions, proteins or other active ingredient of the invention may be
combined with other
agents beneficial to the treatment of the bone and/or cartilage defect, wound,
or tissue in
question. These agents include various growth factors such as epidermal growth
factor (EGF),
platelet derived growth factor (PDGF), transforming growth factors (TGF-a and
TGF-(3), and
insulin-like growth factor (IGF).
The therapeutic compositions are also presently valuable for veterinary
applications.
Particularly domestic animals and thoroughbred horses, in addition to humans,
are desired
patients for such treatment with proteins or other active ingredient of the
present invention. The
dosage regimen of a protein-containing pharmaceutical composition to be used
in tissue
regeneration will be determined by the attending physician considering various
factors which
modify the action of the proteins, e.g., amount of tissue weight desired to be
formed, the site of
damage, the condition of the damaged tissue, the size of a wound, type of
damaged tissue (e.g.,
bone), the patient's age, sex, and diet, the severity of any infection, time
of administration and
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other clinical factors. The dosage may vary with the type of matrix used in
the reconstitution
and with inclusion of other proteins in the pharmaceutical composition. For
example, the
addition of other known growth factors, such as IGF I (insulin like growth
factor I), to the final
composition, may also effect the dosage. Progress can be monitored by periodic
assessment of
tissue/bone growth and/or repair, for example, X-rays, histomoxphometric
determinations and
tetracycline labeling.
Polynucleotides of the present invention can also be used for gene therapy.
Such
polynucleotides can be introduced either in vivo or ex vivo into cells for
expression in a
mammalian subject. Polynucleotides of the invention may also be administered
by other known
methods for introduction of nucleic acid into a cell or organism (including,
without limitation, in
the form of viral vectors or naked DNA). Cells may also be cultured ex vivo in
the presence of
proteins of the present invention in order to proliferate or to produce a
desired effect on or
activity in such cells. Treated cells can then be introduced in vivo for
therapeutic purposes.
5.12.3 EFFECTIVE DOSAGE
Pharmaceutical compositions suitable for use in the present invention include
compositions wherein the active ingredients are contained in an effective
amount to achieve its
intended purpose. More specifically, a therapeutically effective amount means
an amount
effective to prevent development of or to alleviate the existing symptoms of
the subject being
treated. Determination of the effective amount is well within the capability
of those skilled in
the axt, especially in light of the detailed disclosure provided herein. For
any compound used in
the method of the invention, the therapeutically effective dose can be
estimated initially from
appropriate in vitro assays. For example, a dose can be formulated in animal
models to achieve a
circulating concentration range that can be used to more accurately determine
useful doses in
humans. For example, a dose can be formulated in animal models to achieve a
circulating
concentration range that includes the ICSO as determined in cell culture
(i.e., the concentration of
the test compound which achieves a half maximal inhibition of the protein's
biological activity).
Such information can be used to more accurately determine useful doses in
humans.
A therapeutically effective dose refers to that amount of the compound that
results in
amelioration of symptoms or a prolongation of survival in a patient. Toxicity
and therapeutic
efficacy of such compounds can be determined by standard pharmaceutical
procedures in cell
cultures or experimental animals, e.g., for determining the LDSO (the dose
lethal to 50% of the
population) and the EDSO (the dose therapeutically effective in 50% of the
population). The dose
ratio between toxic and therapeutic effects is the therapeutic index and it
can be expressed as the
ratio between LDSO and EDSO. Compounds which exhibit high therapeutic indices
are preferred.
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The data obtained from these cell culture assays and animal studies can be
used in formulating a
range of dosage for use in human. The dosage of such compounds lies preferably
within a range
of circulating concentrations that include the EDSO with little or no
toxicity. The dosage may
vary within this range depending upon the dosage form employed and the route
of administration
utilized. The exact formulation, route of administration and dosage can be
chosen by the
individual physician in view of the patient's condition. See, e.g., Fingl et
al., 1975, in "The
Pharmacological Basis of Therapeutics", Ch. 1 p.1 . Dosage amount and interval
may be adjusted
individually to provide plasma levels of the active moiety which are
sufficient to maintain the
desired effects, or minimal effective concentration (MEC). The MEC will vary
for each
compound but can be estimated from ivy vitro data. Dosages necessary to
achieve the MEC will
depend on individual characteristics and route of administration. However,
HPLC assays or
bioassays can be used to determine plasma concentrations.
Dosage intervals can also be determined using MEC value. Compounds should be
administered using a regimen which maintains plasma levels above the MEC for
10-90% of the
time, preferably between 30-90% and most preferably between 50-90%. In cases
of local
administration or selective uptake, the effective local concentration of the
drug may not be
related to plasma concentration.
An exemplary dosage regimen for polypeptides or other compositions of the
invention
will be in the range of about 0.01 p,g/kg to 100 mglkg of body weight daily,
with the preferred
dose being about 0.1 ~,g/kg to 25 mg/kg of patient body weight daily, varying
in adults and
children. Dosing may be once daily, or equivalent doses may be delivered at
longer or shorter
intervals.
The amount of composition administered will, of course, be dependent on the
subject
being treated, on the subject's age and weight, the severity of the
affliction, the manner of
administration and the judgment of the prescribing physician.
5.12.4 PACKAGING
The compositions may, if desired, be presented in a pack or dispenser device
which may
contain one or more unit dosage forms containing the active ingredient. The
pack may, for
example, comprise metal or plastic foil, such as a blister pack. The pack or
dispenser device may
be accompanied by instructions fox administration. Compositions comprising a
compound of the
invention formulated in a compatible pharmaceutical carrier may also be
prepared, placed in an
appropriate container, and labeled for treatment of an indicated condition.
5.13 ANTIBODIES
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Also included in the invention are antibodies to proteins, or fragments of
proteins of the
invention. The term "antibody" as used herein refers to immunoglobulin
molecules and
immunologically active portions of immunoglobulin (Ig) molecules, i.e.,
molecules that contain
an antigen-binding site that specifically binds (immunoreacts with) an
antigen. Such antibodies
include, but are not limited to, polyclonal, monoclonal, chimeric, single
chain, Fab, Fab° and
Fcab~>a fragments, and an Fab expression library. In general, an antibody
molecule obtained from
humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ
from one
another by the nature of the heavy chain present in the molecule. Certain
classes have
subclasses as well, such as IgG~, IgGa, and others. Furthermore, in humans,
the light chain
may be a kappa chain or a lambda chain. Reference herein to antibodies
includes a reference
to all such classes, subclasses and types of human antibody species.
An isolated related protein of the invention may be intended to serve as an
antigen, or a
portion or fragment thereof, and additionally can be used as an immunogen to
generate
antibodies that immunospecifically bind the antigen, using standard techniques
for polyclonal
and monoclonal antibody preparation. The full-length protein can be used or,
alternatively, the
invention provides antigenic peptide fragments of the antigen for use as
immunogens. An
antigenic peptide fragment comprises at least 6 amino acid residues of the
amino acid sequence
of the full length protein, such as an amino acid sequence shown in SEQ ID NO:
4, 7, or 9 -
13, and encompasses an epitope thereof such that an antibody raised against
the peptide forms
a specific immune complex with the full length protein or with any fragment
that contains the
epitope. Preferably, the antigenic peptide comprises at least 10 amino acid
residues, or at least
IS amino acid residues, or at least 20 amino acid residues, or at least 30
amino acid residues.
Preferred epitopes encompassed by the antigenic peptide are regions of the
protein that are
located on its surface; commonly these are hydrophilic regions.
In certain embodiments of the invention, at least one epitope encompassed by
the
antigenic peptide is a region of neurotrimin-like protein that is located on
the surface of the
protein, e.g., a hydrophilic region. A hydrophobicity analysis of the human
related protein
sequence will indicate which regions of a related protein are particularly
hydrophilic and,
therefore, are likely to encode surface residues useful for targeting antibody
production. As a
means for targeting antibody production, hydropathy plots showing regions of
hydrophilicity
and hydrophobicity may be generated by any method well known in the art,
including, for
example, the Kyte Doolittle or the Hopp Woods methods, either with or without
Fourier
transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78:
3824-3828;
Kyte and Doolittle 1982, J. Mol. Biol. 157: 105-142, each of which is
incorporated herein by
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reference in its entirety. Antibodies that are specific for one or more
domains within an
antigenic protein, or derivatives, fragments, analogs or homologs thereof, are
also provided
herein.
A protein of the invention, or a derivative, fragment, analog, homolog or
ortholog
thereof, may be utilized as an immunogen in the generation of antibodies that
immunospecifically bind these protein components.
The term "specific for" indicates that the variable regions of the antibodies
of the
invention recognize and bind polypeptides of the invention exclusively (i. e.
, able to distinguish
the polypeptide of the invention from other similar polypeptides despite
sequence identity,
homology, or similarity found in the family of polypeptides), but may also
interact with other
proteins (for example, S. aureus protein A or other antibodies in ELISA
techniques) through
interactions with sequences outside the variable region of the antibodies, and
in particular, in
the constant region of the molecule. Screening assays to determine binding
specificity of an
antibody of the invention are well known and routinely practiced in the art.
For a
comprehensive discussion of such assays, see Harlow et al. (Eds), Antibodies A
Laboratory
Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, NY (1988), Chapter
6.
Antibodies that recognize and bind fragments of the polypeptides of the
invention are also
contemplated, provided that the antibodies are first and foremost specific
for, as defined above,
full-length polypeptides of the invention. As with antibodies that are
specific for full length
polypeptides of the invention, antibodies of the invention that recognize
fragments are those
which can distinguish polypeptides from the same family of polypeptides
despite inherent
sequence identity, homology, or similarity found in the family of proteins.
Antibodies of the invention are useful for, for example, therapeutic purposes
(by
modulating activity of a polypeptide of the invention), diagnostic purposes to
detect or
quantitate a polypeptide of the invention, as well as purification of a
polypeptide of the
invention. Kits comprising an antibody of the invention for any of the
purposes described
herein are also comprehended. In general, a kit of the invention also includes
a control antigen
for which the antibody is immunospecific. The invention further provides a
hybridoma that
produces an antibody according to the invention. Antibodies of the invention
are useful for
detection and/or purification of the polypeptides of the invention.
Monoclonal antibodies binding to the protein of the invention may be useful
diagnostic
agents for the immunodetection of the protein. Neutralizing monoclonal
antibodies binding to
the protein may also be useful therapeutics for both conditions associated
with the protein and
also in the treatment of some forms of cancer where abnormal expression of the
protein is
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involved. In the case of cancerous cells or leukemic cells, neutralizing
monoclonal antibodies
against the protein may be useful in detecting and preventing the metastatic
spread of the
cancerous cells, which may be mediated by the protein.
The labeled antibodies of the present invention can be used for ih vitro, in
vivo, and ih
situ assays to identify cells or tissues in which a fragment of the
polypeptide of interest is
expressed. The antibodies may also be used directly in therapies or other
diagnostics. The
present invention further provides the above-described antibodies immobilized
on a solid
support. Examples of such solid supports include plastics such as
polycarbonate, complex
carbohydrates such as agarose and Sepharose~, acrylic resins and such as
polyacrylamide and
latex beads. Techniques for coupling antibodies to such solid supports are
well known in the
art (Weir, D.M. et al., "Handbook of Experimental Immunology" 4th Ed.,
Blackwell
Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby, W.D. et
al., Meth.
Enzym. 34 Academic Press, N.Y. (1974)). The immobilized antibodies of the
present
invention can be used for i~c vitro, in vivo, and in situ assays as well as
for immuno-affinity
purification of the proteins of the present invention.
Various procedures known within the art may be used for the production of
polyclonal
or monoclonal antibodies directed against a protein of the invention, or
against derivatives,
fragments, analogs homologs or orthologs thereof (see, for example,
Antibodies: A Laboratory
Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold
Spring
Harbor, NY, incorporated herein by reference). Some of these antibodies are
discussed
below.
5.13.1 POLYCLONAL ANTIBODIES
For the production of polyclonal antibodies, various suitable host animals
(e.g., rabbit,
goat, mouse or other mammal) may be immunized by one or more injections with
the native
protein, a synthetic variant thereof, or a derivative of the foregoing. An
appropriate
immunogenic preparation can contain, fox example, the naturally occurring
immunogenic
protein, a chemically synthesized polypeptide representing the immunogenic
protein, or a
recombinantly expressed immunogenic protein. Furthermore, the protein may be
conjugated
to a second protein known to be immunogenic in the mammal being immunized.
Examples of
such immunogenic proteins include but axe not limited to keyhole limpet
hemocyanin, serum
albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation
can further
include an adjuvant. Various adjuvants used to increase the immunological
response include,
but are not limited to, Freund's (complete and incomplete), mineral gels
(e.g., aluminum
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hydroxide), surface-active substances (e.g., lysolecithin, pluronic polyols,
polyanions,
peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such
as Bacille
Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory
agents.
Additional examples of adjuvants that can be employed include MPL-TDM adjuvant
(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
The polyclonal antibody molecules directed against the immunogenic protein can
be
isolated from the mammal (e.g., from the blood) and further purified by well
known
techniques, such as affinity chromatography using protein A or protein G,
which provide
primarily the IgG fraction of immune serum. Subsequently, or alternatively,
the specific
antigen which is the target of the immunoglobulin sought, or an epitope
thereof, may be
immobilized on a column to purify the immune specific antibody by
immunoaffinity
chromatography. Purification of immunoglobulins is discussed, fox example, by
D. Wilkinson
(The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14,
No. 8 (April 17,
2000), pp. 25-28).
5.13.2 MONOCLONAL ANTIBODIES
The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as
used herein, refers to a population of antibody molecules that contain only
one molecular
species of antibody molecule consisting of a unique light chain gene product
and a unique
heavy chain gene product. In particular, the complementarity determining
regions (CDRs) of
the monoclonal antibody are identical in all the molecules of the population.
MAbs thus
contain an antigen-binding site capable of immunoreacting with a particular
epitope of the
antigen characterized by a unique binding affinity for it.
Monoclonal antibodies can be prepared using hybridoma methods, such as those
described by I~ohler and Milstein, Nature, 256:495 (1975). In a hybridoma
method, a mouse,
hamster, or other appropriate host animal, is typically immunized with an
immunizing agent to
elicit lymphocytes that produce or are capable of producing antibodies that
will specifically
bind to the immunizing agent. Alternatively, the lymphocytes cari be immunized
in vitro.
The immunizing agent will typically include the protein antigen, a fragment
thereof or a
fusion protein thereof. Generally, either peripheral blood lymphocytes are
used if cells of
human origin are desired, or spleen cells or lymph node cells are used if non-
human
mammalian sources are desired. The lymphocytes are then fused with an
immortalized cell
line using a suitable fusing agent, such as polyethylene glycol, to form a
hybridoma cell
(Goding, Monoclonal Antibodies: Principles and Practice, Academic Press,
(1986) pp. 59-
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103). Immortalized cell lines are usually transformed mammalian cells,
particularly myeloma
cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell
lines are
employed. The hybridoma cells can be cultured in a suitable culture medium
that preferably
contains one or more substances that inhibit the growth or survival of the
unfused,
immortalized cells. For example, if the parental cells lack the enzyme
hypoxanthine guanine
phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the
hybridomas
typically will include hypoxanthine, aminopterin, and thymidine ("HAT
medium"), which
substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support
stable high
level expression of antibody by the selected antibody-producing cells, and are
sensitive to a
medium such as HAT medium. More preferred immortalized cell lines are murine
myeloma
lines, which can be obtained, for instance, from the Salk Institute Cell
Distribution Center, San
Diego, California and the American Type Culture Collection, Manassas,
Virginia. Human
myeloma and mouse-human heteromyeloma cell lines also have been described for
the
production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001
(1984); Brodeur
et al., Monoclonal Antibody Production Technielues and Applications, Marcel
Dekker, Inc.,
New York, (1987) pp. 51-63).
The culture medium in which the hybridoma cells are cultured can then be
assayed for
the presence of monoclonal antibodies directed against the antigen.
Preferably, the binding
specificity of monoclonal antibodies produced by the hybridoma cells is
determined by
immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay
(RIA) or
enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are
known in the
art. The binding affinity of the monoclonal antibody can, for example, be
determined by the
Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
Preferably,
antibodies having a high degree of specificity and a high binding affinity for
the target antigen
are isolated.
After the desired hybridoma cells are identified, the clones can be subcloned
by limiting
dilution procedures and grown by standard' methods. Suitable culture media for
this purpose
include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.
Alternatively, the hybridoma cells can be grown in vivo as ascites in a
mammal.
The monoclonal antibodies secreted by the subclones can be isolated or
purified from
the culture medium or ascites fluid by conventional immunoglobulin
purification procedures
such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
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The monoclonal antibodies can also be made by recombinant DNA methods, such as
those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal
antibodies of
the invention can be readily isolated and sequenced using conventional
procedures (e.g., by
using oligonucleotide probes that are capable of binding specifically to genes
encoding the
S heavy and light chains of murine antibodies). The hybridoma cells of the
invention serve as a
preferred source of such DNA. Once isolated, the DNA can be placed into
expression vectors,
which are then transfected into host cells such as simian COS cells, Chinese
hamster ovary
(CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin
protein, to
obtain the synthesis of monoclonal antibodies in the recombinant host cells.
The DNA also can
be modified, for example, by substituting the coding sequence for human heavy
and light chain
constant domains in place of the homologous murine sequences (U.S. Patent No.
4,816,567;
Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the
immunoglobulin coding
sequence all or part of the coding sequence for a non-immunoglobulin
polypeptide. Such a
non-immunoglobulin polypeptide can be substituted for the constant domains of
an antibody of
1 S the invention, or can be substituted for the variable domains of one
antigen-combining site of
an antibody of the invention to create a chimeric bivalent antibody.
5.13.3 HUMANIZED ANTIBODIES
The antibodies directed against the protein antigens of the invention can
further
comprise humanized antibodies or human antibodies. These antibodies are
suitable for
administration to humans without engendering an immune response by the human
against the
administered immunoglobulin. Humanized forms of antibodies are chimeric
immunoglobulins,
immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab' , Flab' )a
or other antigen-
binding subsequences of antibodies) that are principally comprised of the
sequence of a human
2S immunoglobulin, and contain minimal sequence derived from a non-human
immunoglobulin.
Humanization can be performed following the method of Winter and co-workers
(Jones et al.,
Nature, 321:522-S2S (1986); Riechmann et al., Nature, 332:323-327 (1988);
Verhoeyen et al.,
Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences
for the
corresponding sequences of a human antibody. (See also U.S. Patent No.
S,22S,S39). In
some instances, Fv framework residues of the human immunoglobulin are replaced
by
corresponding non-human residues. Humanized antibodies can also comprise
residues that are
found neither in the recipient antibody nor in the imported CDR or framework
sequences. In
general, the humanized antibody will comprise substantially all of at least
one, and typically
two, variable domains, in which all or substantially all of the CDR regions
correspond to those
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of a non-human immunoglobulin and all or substantially all of the framework
regions are those
of a human immunoglobulin consensus sequence. The humanized antibody optimally
also will
comprise at least a portion of an immunoglobulin constant region (Fc),
typically that of a
human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta,
Curr. Op.
Struct. Biol., 2:593-596 (1992)).
5.13.4 HUMAN ANTIBODIES
Fully human antibodies relate to antibody molecules in which essentially the
entire
sequences of both the light chain and the heavy chain, including the CDRs,
arise from human
genes. Such antibodies are termed "human antibodies", or "fully human
antibodies" herein.
Human monoclonal antibodies can be prepared by the trioma technique; the human
B-cell
hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV
hybridoma
technique to produce human monoclonal antibodies (see Cole, et al. , 1985 In:
MONOCLONAL
ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human
monoclonal
antibodies may be utilized in the practice of the present invention and may be
produced by
using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:
2026-2030) or
by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et
al., 1985 In:
MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
In addition, human antibodies can also be produced using additional
techniques,
including phage display libraries (Hoogenboom and Winter, J. Mol. Biol.,
227:381 (1991);
Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can
be made by
introducing human immunoglobulin loci into transgenic animals, e.g., rnice in
which the
endogenous immunoglobulin genes have been partially or completely inactivated.
Upon
challenge, human antibody production is observed, which closely resembles that
seen in
humans in all respects, including gene rearrangement, assembly, and antibody
repertoire. This
approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806;
5,569,825;
5,625,126; 5,633,425; 5,661,016, and in Marks et al. (Bio/Technology 10, 779-
783 (1992));
Lonberg et al. Nature 368 856-859 (1994)); Morrison (Nature 368, 812-13
(1994)); Fishwild
et al, (Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature
Biotechnology 14, 826
(I996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13 65-93 (1995)).
Human antibodies may additionally be produced using transgenic nonhuman
animals
that are modified so as to produce fully human antibodies rather than the
animal's endogenous
antibodies in response to challenge by an antigen. (See PCT publication
W094/02602). The
endogenous genes encoding the heavy and light immunoglobulin chains in the
nonhuman host
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have been incapacitated, and active loci encoding human heavy and light chain
immunoglobulins are inserted into the host's genome. The human genes are
incorporated, for
example, using yeast artificial chromosomes containing the requisite human DNA
segments.
An animal which provides all the desired modifications is then obtained as
progeny by
crossbreeding intermediate transgenic animals containing fewer than the full
complement of the
modifications. The preferred embodiment of such a nonhuman animal is a mouse,
and is
termed the XenomouseTM as disclosed in PCT publications WO 96/33735 and WO
96/34096.
This animal produces B cells that secrete fully human immunoglobulins. The
antibodies can be
obtained directly from the animal after immunization with an immunogen of
interest, as, for
example, a preparation of a polyclonal antibody, or alternatively from
immortalized B cells
derived from the animal, such as hybridomas producing monoclonal antibodies.
Additionally,
the genes encoding the immunoglobulins with human variable regions can be
recovered and
expressed to obtain the antibodies directly, or can be further modified to
obtain analogs of
antibodies such as, for example, single chain Fv molecules.
An example of a method of producing a nonhuman host, exemplified as a mouse,
lacking expression of an endogenous immunoglobulin heavy chain is disclosed in
U.S. Patent
No. 5,939,598. It can be obtained by a method including deleting the J segment
genes from at
least one endogenous heavy chain locus in an embryonic stem cell to prevent
rearrangement of
the locus and to prevent formation of a transcript of a rearranged
immunoglobulin heavy chain
locus, the deletion being effected by a targeting vector containing a gene
encoding a selectable
marker; and producing from the embryonic stem cell a transgenic mouse whose
somatic and
germ cells contain the gene encoding the selectable marker.
A method for producing an antibody of interest, such as a human antibody, is
disclosed
in U.S. Patent No. 5,916,771. It includes introducing an expression vector
that contains a
nucleotide sequence encoding a heavy chain into one mammalian host cell in
culture,
introducing an expression vector containing a nucleotide sequence encoding a
light chain into
another mammalian host cell, and fusing the two cells to form a hybrid cell.
The hybrid cell
expresses an antibody containing the heavy chain and the light chain.
In a further improvement on this procedure, a method for identifying a
clinically
relevant epitope on an immunogen, and a correlative method for selecting an
antibody that
binds immunospecifically to the relevant epitope with high affinity, are
disclosed in PCT
publication WO 99/53049.
5.13.5 FAB FRAGMENTS AND SINGLE CHAIN ANTIBODIES
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According to the invention, techniques can be adapted for the production of
single-chain antibodies specific to an antigenic protein of the invention (see
e.g., U.S. Patent
No. 4,946,778). In addition, methods can be adapted for the construction of
Fab expression
libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid
and effective
identification of monoclonal Fab fragments with the desired specificity for a
protein or
derivatives, fragments, analogs or homologs thereof. Antibody fragments that
contain the
idiotypes to a protein antigen may be produced by techniques known in the art
including, but
not limited to: (i) an F(ab')2 fragment produced by pepsin digestion of an
antibody molecule; (ii)
an Fab fragment generated by reducing the disulfide bridges of an F~ab~>z
fragment; (iii) an Fab
fragment generated by the treatment of the antibody molecule with papain and a
reducing agent
and (iv) F~ fragments.
5.13.6 BISPECIFIC ANTIBODIES
Bispecific antibodies are monoclonal, preferably human or humanized,
antibodies that
have binding specificities for at least two different antigens. In the present
case, one of the
binding specificities is for an antigenic protein of the invention. The second
binding target is
any other antigen, and advantageously is a cell-surface protein or receptor or
receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally,
the
recombinant production of bispecific antibodies is based on the co-expression
of two
immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have
different
specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of
the random
assortment of immunoglobulin heavy and light chains, these hybridomas
(quadromas) produce
a potential mixture of ten different antibody molecules, of which only one has
the correct
bispecific structure. The purification of the correct molecule is usually
accomplished by
affinity chromatography steps. Similar procedures are disclosed in WO
93/08829, published
13 May 1993, and in Traunecker et al., 1991 EMBO J., 10:3655-3659.
Antibody variable domains with the desired binding specificities (antibody-
antigen
combining sites) can be fused to immunoglobulin constant domain sequences. The
fusion
preferably is with an immunoglobulin heavy-chain constant domain, comprising
at least part of
the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain
constant region
(CH1) containing the site necessary for light-chain binding present in at
least one of the
fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired,
the
immunoglobulin light chain, are inserted into separate expression vectors, and
are co-
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transfected into a suitable host organism. For further details of generating
bispecific antibodies
see, for example, Suresh et al., Methods in Enzymolo~y, 121:210 (1986).
According to another approach described in WO 96/27011, the interface between
a pair
of antibody molecules can be engineered to maximize the percentage of
heterodimers that are
recovered from recombinant cell culture. The preferred interface comprises at
least a part of
the CH3 region of an antibody constant domain. In this method, one or more
small amino acid
side chains from the interface of the first antibody molecule are replaced
with larger side
chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or
similar size to
the large side chains) are created on the interface of the second antibody
molecule by
replacing large amino acid side chains with smaller ones (e.g. alanine or
threonine). This
provides a mechanism for increasing the yield of the heterodimer over other
unwanted
end-products such as homodimers.
Bispecific antibodies can be prepared as full length antibodies or antibody
fragments
(e.g. F(ab')z bispecific antibodies). Techniques for generating bispecific
antibodies from
antibody fragments have been described in the literature. For example,
bispecific antibodies
can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985)
describe a
procedure wherein intact antibodies are proteolytically cleaved to generate
F(ab')z fragments.
These fragments are reduced in the presence of the dithiol complexing agent
sodium arsenite to
stabilize vicinal dithiols and prevent intermolecular disulfide formation. The
Fab' fragments
generated are then converted to thionitrobenzoate (TNB) derivatives. One of
the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is
mixed with an equimolar amount of the other Fab'-TNB derivative to form the
bispecific
antibody. The bispecific antibodies produced can be used as agents for the
selective
immobilization of enzymes.
Additionally, Fab' fragments can be directly recovered from E. coli and
chemically
coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-
225 (1992)
describe the production of a fully humanized bispecific antibody F(ab')z
molecule. Each Fab'
fragment was separately secreted from E. coli and subjected to directed
chemical coupling in
vitro to form the bispecific antibody. The bispecific antibody thus formed was
able to bind to
cells overexpressing the ErbB2 receptor and normal human T cells, as well as
trigger the lytic
activity of human cytotoxic lymphocytes against human breast tumor targets.
Various techniques for making and isolating bispecific antibody fragments
directly from
recombinant cell culture have also been described. For example, bispecific
antibodies have
been produced using leucine zippers. Kostelny et al., J. Irnmunol. 148(5):1547-
1553 (1992).
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The leucine zipper peptides from the Fos and Jun proteins were linked to the
Fab' portions of
two different antibodies by gene fusion. The antibody homodimers were reduced
at the hinge
region to form monomers and then re-oxidized to form the antibody
heterodimers. This
method can also be utilized for the production of antibody homodirners. The
"diabody"
technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-
6448 (1993)
has provided an alternative mechanism for making bispecific antibody
fragments. The
fragments comprise a heavy-chain variable domain (VH) connected to a light-
chain variable
domain (VL) by a linker which is too short to allow pairing between the two
domains on the
same chain. Accordingly, the VH and VL domains of one fragment axe forced to
pair with the
complementary Vr. and VH domains of another fragment, thereby forming two
antigen-binding
sites. Another strategy for making bispecific antibody fragments by the use of
single-chain Fv
(sFv) dimers has also been reported. See, Gruber et al., J. Immunol. 152:5368
(1994).
Antibodies with more than two valencies are contemplated. For example,
trispecific
antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).
Exemplary bispecific antibodies can bind to two different epitopes, at Least
one of
which originates in the protein antigen of the invention. Alternatively, an
anti-antigenic arm of
an immunoglobulin molecule can be combined with an arm which binds to a
triggering
molecule on a leukocyte such as'a T-cell receptor molecule (e.g. CD2, CD3,
CD28, or B7), or
Fc receptors for IgG (Fc R), such as Fc RI (CD64), Fc RII (CD32) and Fc RIII
(CD16) so as
to focus cellular defense mechanisms to the cell expressing the particular
antigen. Bispecific
antibodies can also be used to direct cytotoxic agents to cells which express
a particular
antigen. These antibodies possess an antigen-binding arm and an arm which
binds a cytotoxic
agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another
bispecific antibody of interest binds the protein antigen described herein and
further binds
tissue factor (TF).
5.13.7 HETEROCONJUGATE ANTIBODIES
Heteroconjugate antibodies are also within the scope of the present invention.
Heteroconjugate antibodies are composed of two covalently joined antibodies.
Such antibodies
have, for example, been proposed to target immune system cells to unwanted
cells (U.S.
Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360; WO
92/200373; EP
03089). It is contemplated that the antibodies can be prepared in vitro using
known methods in
synthetic protein chemistry, including those involving crosslinking agents.
For example,
immunotoxins can be constructed using a disulfide exchange reaction or by
forming a thioether
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bond. Examples of suitable reagents for this purpose include iminothiolate and
methyl-4-
mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No.
4,676,980.
5.13.8 EFFECTOR FUNCTION ENGINEERING
It can be desirable to modify the antibody of the invention with respect to
effector
function, so as to enhance, e.g., the effectiveness of the antibody in
treating cancer. For
example, cysteine residues) can be introduced into the Fc region, thereby
allowing interchain
disulfide bond formation in this region. The homodimeric antibody thus
generated can have
improved internalization capability and/or increased complement-mediated cell
killing and
antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp
Med., 176: 1191-
1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric
antibodies with
enhanced anti-tumor activity can also be prepared using heterobifunctional
cross-linkers as
described in Wolff et al. Cancer Research, 53: 2560-2565 (1993).
Alternatively, an antibody
can be engineered that has dual Fc regions and can thereby have enhanced
complement lysis
and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-
230 (1989).
5.13.9 IMMLTNOCONJUGATES
The invention also pertains to immunoconjugates comprising an antibody
conjugated to
a cytotoxic agent such as a chemotherapeutic agent, toxin (e. g. , an
enzymatically active toxin
of bacterial, fungal, plant, or animal origin, or fragments thereof), or a
radioactive isotope
(i.e., a radioconjugate).
' Chemotherapeutic agents useful in the generation of such immunoconjugates
have been
described above. Enzymatically active toxins and fragments thereof that can be
used include
diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin
A chain (from
Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-
sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins
(PAPI, PAPII, and
PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis
inhibitor,
gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the
tricothecenes. A variety of
radionuclides are available for the production of radioconjugated antibodies.
Examples include
212$i' 131I' 131In, soY, and ls6Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of
bifunctional
protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate
(SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl
adipimidate
HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as
glutareldehyde), bis-
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azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives
(such as bis-(p-diazoniumbenzoyl)-ethylenediarnine), diisocyanates (such as
tolyene 2,6-
diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-
dinitrobenzene).
For example, a ricin immunotoxin can be prepared as described in Vitetta et
al., Science, 238:
1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene
triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for
conjugation of
radionucleotide to the antibody. See W094/11026.
In another embodiment, the antibody can be conjugated to a "receptor" (such
streptavidin) for utilization in tumor pretargeting wherein the antibody-
receptor conjugate is
administered to the patient, followed by removal of unbound conjugate from the
circulation
using a clearing agent and then administration of a "ligand" (e.g., avidin)
that is in turn
conjugated to a cytotoxic agent.
5.14 COMPUTER READABLE SEQUENCES
In one application of this embodiment, a nucleotide sequence of the present
invention can
be recorded on computer readable media. As used herein; "computer readable
media" refers to
any medium which can be read and accessed directly by a computer. Such media
include, but
are not limited to: magnetic storage media, such as floppy discs, hard disc
storage medium, and
magnetic tape; optical storage media such as CD-ROM; electrical storage media
such as RAM
and ROM; and hybrids of these categories such as magneticloptical storage
media. A skilled
artisan can readily appreciate how any of the presently known computer
readable mediums can
be used to create a manufacture comprising computer readable medium having
recorded thereon
a nucleotide sequence of the present invention. As used herein, "recorded"
refers to a process for
storing information on computer readable medium. A skilled artisan can readily
adopt any of the
presently known methods for recording information on computer readable medium
to generate
manufactures comprising the nucleotide sequence information of the present
invention.
A variety of data storage structures are available to a skilled artisan for
creating a
computer readable medium having recorded thereon a nucleotide sequence of the
present
invention. The choice of the data storage structure will generally be based on
the means chosen
to access the stored information. In addition, a variety of data processor
programs and formats
can be used to store the nucleotide sequence information of the present
invention on computer
readable medium. The sequence information can be represented in a word
processing text file,
formatted in commercially-available software such as WordPerfect and Microsoft
Word, or
represented in the form of an ASCII file, stored in a database application,
such as DB2, Sybase,
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Oracle, or the Like. A skilled artisan can readily adapt any number of data
processor structuring
formats (e.g. text file or database) in order to obtain computer readable
medium having recorded
thereon the nucleotide sequence information of the present invention.
By providing any of the nucleotide sequences SEQ ID NO: 1-3, 5-6, 8 or a
representative
S fragment thereof; or a nucleotide sequence at least 95% identical to any of
the nucleotide
sequences of the SEQ ID NO: 1-3, 5-6, 8 in computer readable form, a skilled
artisan can
routinely access the sequence information for a variety of purposes. Computer
software is
publicly available which allows a skilled artisan to access sequence
information provided in a
computer readable medium. The examples which follow demonstrate how software
which
implements the BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) and
BLAZE (Brutlag
et al., Comp. Chem. 17:203-207 (1993)) search algorithms on a Sybase system is
used to identify
open reading frames (ORFs) within a nucleic acid sequence. Such ORFs may be
protein
encoding fragments and may be useful in producing commercially important
proteins such as
enzymes used in fermentation reactions and in the production of commercially
useful
5 metabolites.
As used herein, "a computer-based system" refers to the hardware means,
software
means, and data storage means used to analyze the nucleotide sequence
information of the
present invention. The minimum hardware means of the computer-based systems of
the present
invention comprises a central processing unit (CPS, input means, output means,
and data
storage means. A skilled artisan can readily appreciate that any one of the
currently available
computer-based systems are suitable for use in the present invention. As
stated above, the
computer-based systems of the present invention comprise a data storage means
having stored
therein a nucleotide sequence of the present invention and the necessary
hardware means and
software means for supporting and implementing a search means. As used herein,
"data storage
means" refers to memory which can store nucleotide sequence information of the
present
invention, or a memory access means which can access manufactures having
recorded thereon
the nucleotide sequence information of the present invention.
As used herein, "search means" refers to one or more programs which are
implemented
on the computer-based system to compare a target sequence or target structural
motif with the
sequence information stored within the data storage means. Search means are
used to identify
fragments or regions of a known sequence which match a particular target
sequence or target
motif. A variety of known algorithms are disclosed publicly and a variety of
commercially
available software for conducting search means are and can be used in the
computer-based
systems of the present invention. Examples of such software includes, but is
not limited to,
Smith-Waterman, MacPattern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA). A
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skilled artisan can readily recognize that any one of the available algorithms
or implementing
software packages for conducting homology searches can be adapted for use in
the present
computer-based systems. As used herein, a "target sequence" can be any nucleic
acid or amino
acid sequence of six or more nucleotides or two or more amino acids. A skilled
artisan can
S readily recognize that the longer a target sequence is, the less likely a
target sequence will be
present as a random occurrence in the database. The most preferred sequence
length of a target
sequence is from about 10 to 100 amino acids, or from about 30 to 300
nucleotide residues.
However, it is well recognized that searches for commercially important
fragments, such as
sequence fragments involved in gene expression and protein processing, may be
of shorter
length.
As used herein, "a target structural motif," or "target motif," refers to any
rationally
selected sequence or combination of sequences in which the sequences) are
chosen based on a
three-dimensional configuration which is formed upon the folding of the target
motif. There are
a variety of target motifs known in the art. Protein target motifs include,
but are not limited to,
1 S enzyme active sites and signal sequences. Nucleic acid target motifs
include, but are not limited
to, promoter sequences, hairpin structures and inducible expression elements
(protein binding
sequences).
5.15 TRIPLE HELIX FORMATION
In addition, the fragments of the present invention, as broadly described, can
be used to
control gene expression through triple helix formation or antisense DNA or
RNA, both of which
methods are based on the binding of a polynucleotide sequence to DNA or RNA.
Polynucleotides suitable for use in these methods are usually 20 to 40 bases
in length and are
designed to be complementary to a region of the gene involved in transcription
(triple helix - see
2S Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science
15241:456 (1988); and Dervan
et al., Science 251:1360 (1991)) or to the mRNA itself (antisense - Olmno, J.
Neurochem.
S6:S60 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene
Expression, CRC Press,
Boca Raton, FL (1988)). Triple helix-formation optimally results in a shut-off
of RNA
transcription from DNA, while antisense RNA hybridization blocks translation
of an mRNA
molecule into polypeptide. Both techniques have been demonstrated to be
effective in model
systems. Information contained in the sequences of the present invention is
necessary fox the
design of an antisense or triple helix oligonucleotide.
5.I6 DIAGNOSTIC ASSAYS AND KITS
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The present invention further provides methods to identify the presence or
expression of
one of the ORFs of the present invention, or homolog thereof, in a test
sample, using a nucleic
acid probe or antibodies of the present invention, optionally conjugated or
otherwise associated
with a suitable label.
In general, methods for detecting a polynucleotide of the invention can
comprise
contacting a sample with a compound that binds to and forms a complex with the
polynucleotide
for a period sufficient to form the complex, and detecting the complex, so
that if a complex is
detected, a polynucleotide of the invention is detected in the sample. Such
methods can also
comprise contacting a sample under stringent hybridization conditions with
nucleic acid primers
that anneal to a polynucleotide of the invention under such conditions, and
amplifying annealed
polynucleotides, so that if a polynucleotide is amplified, a polynucleotide of
the invention is
detected in the sample.
In general, methods for detecting a polypeptide of the invention can comprise
contacting
a sample with a compound that binds to and forms a complex with the
polypeptide for a period
sufficient to form the complex, and detecting the complex, so that if a
complex is detected, a
polypeptide of the invention is detected in the sample.
In detail, such methods comprise incubating a test sample with one or more of
the
antibodies or one or more of the nucleic acid probes of the present invention
and assaying for
binding of the nucleic acid probes or antibodies to components within the test
sample.
Conditions for incubating a nucleic acid probe or antibody with a test sample
vary.
Incubation conditions depend on the format employed in the assay, the
detection methods
employed, and the type and nature of the nucleic acid probe or antibody used
in the assay. One
skilled in the art will recognize that any one of the commonly available
hybridization,
amplification or immunological assay formats can readily be adapted to employ
the nucleic acid
probes or antibodies of the present invention. Examples of such assays can be
found in Chard,
T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier
Science Publishers,
Amsterdam, The Netherlands (1986); Bullock, G.R. et al., Techniques in
Immunocytochemistry,
Academic Press, Orlando, FL Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985);
Tijssen, P., Practice
and Theory of immunoassays: Laboratory Techniques in Biochemistry and
Molecular Biology,
Elsevier Science Publishers, Amsterdam, The Netherlands (1985). The test
samples of the
present invention include cells, protein or membrane extracts of cells, or
biological fluids such as
sputum, blood, serum, plasma, or urine. The test sample used in the above-
described method
will vary based on the assay format, nature of the detection method and the
tissues, cells or
extxacts used as the sample to be assayed. Methods for preparing protein
extracts or membrane
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extracts of cells are well known in the art and can be readily be adapted in
order to obtain a
sample which is compatible with the system utilized.
In another embodiment of the present invention, kits are provided which
contain the
necessary reagents to carry out the assays of the present invention.
Specifically, the invention
provides a compartment kit to receive, in close confinement, one or more
containers which
comprises: (a) a first container comprising one of the probes or antibodies of
the present
invention; and (b) one or more other containers comprising one or more of the
following: wash
reagents, reagents capable of detecting presence of a bound probe or antibody.
In detail, a compartment kit includes any kit in which reagents are contained
in separate
containers. Such containers include small glass containers, plastic containers
or strips of plastic
or paper. Such containers allows one to efficiently transfer reagents from one
compartment to
another compartment such that the samples and reagents are not cross-
contaminated, and the
agents or solutions of each container can be added in a quantitative fashion
from one
compartment to another. Such containers will include a container which will
accept the test
sample, a container which contains the antibodies used in the assay,
containers which contain
wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and
containers which
contain the reagents used to detect the bound antibody or probe. Types of
detection reagents
include labeled nucleic acid probes, labeled secondary antibodies, or in the
alternative, if the
primary antibody is labeled, the enzymatic, or antibody binding reagents which
are capable of
reacting with the labeled antibody. One skilled in the art will readily
recognize that the disclosed
probes and antibodies of the present invention can be readily incorporated
into one of the
established kit formats which are well known in the art.
5.17 MEDICAL IMAGING
The novel polypeptides and binding partners of the invention are useful in
medical
imaging of sites expressing the molecules of the invention (e.g., where the
polypeptide of the
invention is involved in the immune response, for imaging sites of
inflammation or infection).
See, e.g., Kunkel et al., U.S. Pat. NO. 5,413,778. Such methods involve
chemical attachment of
a labeling or imaging agent, administration of the labeled polypeptide to a
subject in a
pharmaceutically acceptable carrier, and imaging the labeled polypeptide in
vivo at the target
site.
5.18 SCREENING ASSAYS
Using the isolated proteins and polynucleotides of the invention, the present
invention
fiuther provides methods of obtaining and identifying agents which bind to a
polypeptide
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encoded by an ORF corresponding to any of the nucleotide sequences set forth
in the SEQ ID
NO: 1-3, 5-6, 8, or bind to a specific domain of the polypeptide encoded by
the nucleic acid. In
detail, said method comprises the steps of
(a) contacting an agent with an isolated protein encoded by an ORF of the
present
invention, or nucleic acid of the invention; and
(b) determining whether the agent binds to said protein or said nucleic acid.
In general, therefore, such methods for identifying compounds that bind to a
polynucleotide of the invention can comprise contacting a compound with a
polynucleotide of
the invention for a time sufficient to form a polynucleotide/compound complex,
and detecting
the complex, so that if a polynucleotide/compound complex is detected, a
compound that binds
to a polynucleotide of the invention is identified.
Likewise, in general, therefore, such methods for identifying compounds that
bind to a
polypeptide of the invention can comprise contacting a compound with a
polypeptide of the
invention for a time sufficient to form a polypeptide/compound complex, and
detecting the
complex, so that if a polypeptide/compound complex is detected, a compound
that binds to a
polynucleotide of the invention is identified.
Methods for identifying compounds that bind to a polypeptide of the invention
can also
comprise contacting a compound with a polypeptide of the invention in a cell
for a time
sufficient to form a polypeptide/compound complex, wherein the complex drives
expression of a
receptor gene sequence in the cell, and detecting the complex by detecting
reporter gene
sequence expression, so that if a polypeptide/compound complex is detected, a
compound that
binds a polypeptide of the invention is identified.
Compounds identified via such methods can include compounds which modulate the
activity of a polypeptide of the invention (that is, increase or decrease its
activity, relative to
activity observed in the absence of the compound). Alternatively, compounds
identified via such
methods can include compounds which modulate the expression of a
polynucleotide of the
invention (that is, increase or decrease expression relative to expression
levels observed in the
absence of the compound). Compounds, such as compounds identified via the
methods of the
invention, can be tested using standard assays well known to those of skill in
the art for their
ability to modulate activity/expression.
The agents screened in the above assay can be, but are not limited to,
peptides,
carbohydrates, vitamin derivatives, or other pharmaceutical agents. The agents
can be selected
and screened at random or rationally selected or designed using protein
modeling techniques.
For random screening, agents such as peptides, carbohydrates, pharmaceutical
agents and
the like are selected at random and are assayed for their ability to bind to
the protein encoded by
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the ORF of the present invention. Alternatively, agents may be rationally
selected or designed.
As used herein, an agent is said to be "rationally selected or designed" when
the agent is chosen
based on the configuration of the particular protein. For example, one skilled
in the art can
readily adapt currently available procedures to generate peptides,
pharmaceutical agents and the
like, capable of binding to a specific peptide sequence, in order to generate
rationally designed
antipeptide peptides, for example see Hurby et al., Application of Synthetic
Peptides: Antisense
Peptides," In Synthetic Peptides, A User's Guide, W.H. Freeman, NY (1992), pp.
289-307, and
I~aspczak et al., Biochemistry 28:9230-8 (1989), or pharmaceutical agents, or
the like.
In addition to the foregoing, one class of agents of the present invention, as
broadly
described, can be used to control gene expression through binding to one of
the ORFs or EMFs
of the present invention. As described above, such agents can be randomly
screened or
rationally designed/selected. Targeting the ORF or EMF allows a skilled
artisan to design
sequence specific or element specific agents, modulating the expression of
either a single ORF or
multiple ORFs which rely on the same EMF for expression control. One class of
DNA binding
agents are agents which contain base residues which hybridize or form a triple
helix formation
by binding to DNA or RNA. Such agents can be based on the classic
phosphodiester,
ribonucleic acid backbone, or can be a variety of sulfhydryl or polymeric
derivatives which have
base attachment capacity.
Agents suitable for use in these methods usually contain 20 to 40 bases and
are designed
to be complementary to a region of the gene involved in transcription (triple
helix - see Lee et al.,
Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and
Dervan et al.,
Science 251:1360 (1991)) or to the mRNA itself (antisense - Okano, J.
Neurochem. 56:560
(1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC
Press, Boca
Raton, FL (1988)). Triple helix-formation optimally results in a shut-off of
RNA transcription
from DNA, while antisense RNA hybridization blocks translation of an mRNA
molecule into
polypeptide. Both techniques have been demonstrated to be effective in model
systems.
Information contained in the sequences of the present invention is necessary
for the design of an
antisense or triple helix oligonucleotide and other DNA binding agents.
Agents which bind to a protein encoded by one of the ORFs of the present
invention can
be used as a diagnostic agent. Agents which bind to a protein encoded by one
of the ORFs of the
present invention can be formulated using known techniques to generate a
pharmaceutical
composition.
5.19 USE OF NUCLEIC ACIDS AS PROBES
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Another aspect of the subject invention is to provide for polypeptide-specific
nucleic acid
hybridization probes capable of hybridizing with naturally occurring
nucleotide sequences. The
hybridization probes of the subject invention may be derived from any of the
nucleotide
sequences SEQ ID NO: 1-3, 5-6, 8. Because the corresponding gene is only
expressed in a
limited number of tissues, a hybridization probe derived from of any of the
nucleotide sequences
SEQ ID NO: 1-3, 5-6, 8 can be used as an indicator of the presence of RNA of
cell type of such a
tissue in a sample.
Any suitable hybridization technique can be employed, such as, for example, in
situ
hybridization. PCR as described in US Patents Nos. 4,683,195 and 4,965,188
provides
additional uses for oligonucleotides based upon the nucleotide sequences. Such
probes used in
PCR may be of recombinant origin, may be chemically synthesized, or a mixture
of both. The
probe will comprise a discrete nucleotide sequence for the detection of
identical sequences or a
degenerate pool of possible sequences for identification of closely related
genomic sequences.
Other means for producing specific hybridization probes for nucleic acids
include the
cloning of nucleic acid sequences into vectors for the production of mRNA
probes. Such vectors
are known in the art and are commercially available and may be used to
synthesize RNA probes
in vitro by means of the addition of the appropriate RNA polymerase as T7 or
SP6 RNA
polymerase and the appropriate xadioactively labeled nucleotides. The
nucleotide sequences may
be used to construct hybridization probes for mapping their respective genomic
sequences. The
nucleotide sequence provided herein may be mapped to a chromosome or specific
regions of a
chromosome using well known genetic and/or chromosomal mapping techniques.
These
techniques include in situ hybridization, linkage analysis against known
chromosomal markers,
hybridization screening with libraries or flow-sorted chromosomal preparations
specific to
known chromosomes, and the like. The technique of fluorescent in situ
hybridization of
chromosome spreads has been described, among other places, in Verma et al
(1988) Human
Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York NY.
Fluorescent in situ hybridization of chromosomal preparations and other
physical
chromosome mapping techniques may be correlated with additional genetic map
data. Examples
of genetic map data can be found in the 1994 Genome Issue of Science
(265:1981f). Correlation
between the location of a nucleic acid on a physical chromosomal map and a
specific disease (or
predisposition to a specific disease) may help delimit the region of DNA
associated with that
genetic disease. The nucleotide sequences of the subject invention may be used
to detect
differences in gene sequences between normal, carrier or affected individuals.
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5.20 PREPARATION OF SUPPORT BOUND OLIGONUCLEOTIDES
Oligonucleotides, i.e., small nucleic acid segments, may be readily prepared
by, for
example, directly synthesizing the oligonucleotide by chemical means, as is
commonly practiced
using an automated oligonucleotide synthesizer.
Support bound oligonucleotides may be prepared by any of the methods known to
those of
skill in the art using any suitable support such as glass, polystyrene or
Teflon. One strategy is to
precisely spot oligonucleotides synthesized by standard synthesizers.
Immobilization can be
achieved using passive adsorption (Inouye & Hondo,1990 J. Clin Microbiol 28(6)
1462-72); using
UV light (Nagata et al., 1985; Dahlen et al., 1987; Morrissey & Collins, Mol.
Cell Probes 1989 3(2)
189-207) or by covalent binding of base modified DNA (Keller et al.,
1988;1989); all references
being specificallyincorporatedherein.
Another strategy that may be employed is the use of the strong biotin-
streptavidin
interaction as a linker. For example, Broude et al. (1994) Proc. Natl. Acad.
Sci USA 91 (8) 3072-6
describe the use of biotinylated probes, although these are duplex probes,
that are immobilized on
streptavidin-coatedmagnetic beads. Streptavidin-coatedbeads may be purchased
from Dynal,
Oslo. Of course, this same linking chemistry is applicable to coating any
surface with streptavidin.
Biotinylated probes may be purchased from various sources, such as, e.g.,
Operon Technologies
(Alameda, CA).
Nunc Laboratories (Naperville, IL) is also selling suitable material that
could be used. Nunc
Laboratories have developed a method by which DNA can be covalently bound to
the microwell
surface termed Covalink NH. CovaLink NH is a polystyrene surface grafted with
secondary amino
groups (>NH) that serve as bridge-heads for further covalent coupling.
CovaLink Modules may be
purchased from Nunc Laboratories. DNA molecules may be bound to CovaLink
exclusively at the
5'-end by a phosphoramidate bond, allowing immobilization of more than 1 pmol
of DNA
(Rasmussenetal., (1991)AnalBiochem198(1)138-42.
The use of CovaLink NH strips for covalent binding of DNA molecules at the 5'-
end has
been described (Rasmussen et a1.,1991). In this technology, a
phosphoramidatebond is employed
(Chu et a1.,1983 Nucleic Acids 11(18) 6513-29). This is beneficial as
immobilizationusing only a,
single covalentbond is preferred. The phosphoramidatebond joins the DNA to the
CovaLinkNH
secondary amino groups that are positioned at the end of spacer arms
covalently grafted onto the
polystyrene surface through a 2 nm long spacer arm. To link an oligonucleotide
to CovaLink NH
via an phosphoramidatebond, the oligonucleotideterminus must have a 5'-end
phosphate group. It
is, perhaps, even possible for biotin to be covalently bound to CovaLinle and
then streptavidin used
to bind the probes.
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More specifically, the linkage method includes dissolving DNA in water (7.5
ng/ul) and
denaturing for 10 min. at 95°C and cooling on ice for 10 min. Ice-cold
0.1 M 1-methylimidazole,
pH 7.0 (1-MeIm~), is then added to a final concentrationof 10 xnM 1-MeIm~. A
ss DNA solution is
then dispensed into CovaLink NH strips (75 ul/well) standing on ice.
Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide(EDC),
dissolved in
mM 1-MeIm~, is made fresh and 25 u1 added per well. The strips are incubated
for 5 hours at
50°C. After incubation the strips are washed using, e.g., Nunc-Immuno
Wash; first the wells are
washed 3 times, then they are soaked with washing solution for 5 min., and
finally they are washed
3 times (where in the washing solution is 0.4 N NaOH, 0.25% SDS heated to
50°C).
10 It is contemplated that a further suitable method for use with the present
invention is that
described in PCT Patent Application WO 90/03382 (Southern & Maskos),
incorporatedherein by
reference. This method of preparing an oligonucleotide bound to a support
involves attaching a
nucleoside 3'-reagent through the phosphate group by a covalent
phosphodiesterlink to aliphatic
hydroxyl groups carried by the support. The oligonucleotide is then
synthesized on the supported
nucleoside and protecting groups removed from the synthetic oligonucleotide
chain under standard
conditions that do not cleave the oligonucleotide from the support. Suitable
reagents include
nucleoside phosphoramidite and nucleoside hydrogen phosphorate.
An on-chip strategy for the preparation of DNA probe for the preparation of
DNA probe
arrays may be employed. For example, addressable laser-
activatedphotodeprotectionmay be
employed in the chemical synthesis of oligonucleotides directly on a glass
surface, as described by
Fodor et al. (1991) Science 251(4995) 767-73, incorporatedherein by reference.
Probes may also
be immobilized on nylon supports as described by Van Ness et al. ( 1991)
Nucleic Acids Res.
19(12) 3345-50; or linked to Teflon using the method of Duncan & Cavalier
(1988) Anal Biochem
169(1) 104-8; all referencesbeing specificallyincorporatedherein.
To link an oligonucleotide to a nylon support, as described by Van Ness et al.
(1991),
requires activation of the nylon surface via alkylation and selective
activation of the 5'-amine of
oligonucleotides with cyanuric chloride.
One particular way to prepare support bound oligonucleotides is to utilize the
light-
generated synthesis described by Pease et al., (1994) Proc. Natl. Acad. Sci
USA 91 (11) 5022-6.
These authors used currentphotolithographictechniques to generate arrays of
immobilized
oligonucleotide probes (DNA chips). These methods, in which light is used to
direct the synthesis
of oligonucleotideprobes in high-density, miniaturized arrays, utilize
photolabile 5'-protectedN
acyl-deoxynucleosidephosphoramidites, surface linker chemistry and versatile
combinatorial
synthesis strategies. A matrix of 256 spatially defined oligonucleotideprobes
may be generated in
3 5 this manner.
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5.21 PREPARATION OF NUCLEIC ACID FRAGMENTS
The nucleic acids may be obtained from any appropriate source, such as cDNAs,
genomic
DNA, chromosomal DNA, microdissectedchromosome bands, cosmid or YAC inserts,
and RNA,
including mRNA without any amplification steps. For example, Sambrook et al.
(1989) describes
three protocols for the isolation of high molecular weight DNA from mammalian
cells (p. 9.14-
9.23).
DNA fragments may be prepared as clones in M 13, plasmid or lambda vectors
and/or
prepared directly from genomic DNA or cDNA by PCR or other
amplificationmethods. Samples
may be prepared or dispensed in multiwell plates. About 100-1000 ng of DNA
samples may be
prepared in 2-500 ml of final volume.
The nucleic acids would then be fragmented by any of the methods known to
those of skill
in the art including, for example, using restriction enzymes as described at
9.24-9.28 of Sambrook et
al. (1989), shearing by ultrasound and NaOH treatment.
Low pressure shearing is also appropriate, as described by Schriefer et al.
(1990) Nucleic
Acids Res. 18(24) 7455-6. In this method, DNA samples are passed through a
small French
pressure cell at a variety of low to intermediate pressures. A lever device
allows controlled
application of low to intermediate pressures to the cell. The results of these
studies indicate that
low-pressure shearing is a useful alternative to sonic and enzymatic DNA
fragmentation methods.
One particularly suitable way for fragmenting DNA is contemplated to be that
using the two
base recognition endonuclease, CviJI, described by Fitzgerald et al. (1992)
Nucleic Acids Res.
20(14) 3753-62. These authors described an approach for the rapid
fragmentation and fractionation
of DNA into particular sizes that they contemplated to be suitable for shotgun
cloning and
sequencing.
The restriction endonuclease CviJI normally cleaves the recognition sequence
PuGCPy
between the G and C to leave blunt ends. Atypical reaction conditions, which
alter the specificity of
this enzyme (CviJI* *), yield a quasi-random distribution of DNA fragments
form the small
moleculepUCl9 (2688 base pairs). Fitzgerald et al. (1992)
quantitativelyevaluatedthe
randomness of this fragmentation strategy, using a CviJI* * digest of pUC 19
that was size
fractionated by a rapid gel filtration method and directly ligated, without
end repair, to a lac Z minus
M13 cloning vector. Sequence analysis of 76 clones showed that CviJI* *
restricts pyGCPy and
PuGCPu, in addition to PuGCPy sites, and that new sequence data is accumulated
at a rate
consistent with random fragmentation.
As reported in the literature, advantages of this approach compared to
sonication and
agarose gel fractionation include: smaller amounts of DNA are required (0.2-
0.5 ug instead of 2-S
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ug); and fewer steps are involved (no preligation, end repair, chemical
extraction, or agaxose geI
electrophoresis and elution are needed).
Irrespective of the manner in which the nucleic acid fragments are obtained or
prepared, it is
important to denature the DNA to give single stranded pieces available for
hybridization. This is
achieved by incubating the DNA solution for 2-5 minutes at 80-90°C. The
solution is then cooled
quickly to 2°C to prevent renaturation of the DNA fragments before they
are contacted with the
chip. Phosphate groups must also be removed from genomic DNA by methods known
in the art.
5.22 PREPARATION OF DNA ARRAYS
Arrays may be prepared by spotting DNA samples on a support such as a nylon
membrane.
Spotting may be performedby using arrays of metal pins (the positions of which
correspondto an
array of wells in a microtiter plate) to repeated by transfer of about 20 n1
of a DNA solution to a
nylon membrane. By offset printing, a density of dots higher than the density
of the wells is
achieved. One to 25 dots may be accommodated in 1 mm2, depending on the type
of label used. By
avoiding spotting in some preselected number of rows and columns, separate
subsets (subarrays)
1 S may be formed. Samples in one subarray may be the same genomic segment of
DNA (or the same
gene) from different individuals, or may be different, overlapped genomic
clones. Each of the
subarrays may represent replica spotting of the same samples. In one example,
a selected gene
segment may be amplified from 64 patients. For each patient, the amplified
gene segment may be in
one 96-well plate (all 96 wells containing the same sample). A plate for each
of the 64 patients is
prepared. By using a 96-pin device, all samples may be spotted on one 8 x 12
cm membrane.
Subarrays may contain 64 samples, one from each patient. Where the 96
subarrays are identical, the
dot span may be 1 mm2 and there may be a 1 mm space between subarrays.
Another approach is to use membranes or plates (available from NUNC,
Naperville, Illinois)
which may be partitioned by physical spacers e.g. a plastic grid molded over
the membrane, the grid
being similar to the sort of membrane applied to the bottom of multiwell
plates, or hydrophobic
strips. A fixed physical spacer is not preferred for imaging by exposure to
flat phosphor-storage
screens or x-ray films.
The present invention is illustrated in the following examples. Upon
consideration of the
present disclosure, one of skill in the art will appreciate that many other
embodiments and variations
may be made in the scope of the present invention. Accordingly, it is intended
that the broader
aspects of the present invention not be limited to the disclosure of the
following examples. The
present invention is not to be limited in scope by the exemplified embodiments
which are intended
as illustrations of single aspects of the invention, and compositions and
methods which are
functionally equivalent are within the scope of the invention. Indeed,
numerous modifications and
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variations in the practice of the invention are expected to occur to those
skilled in the art upon
consideration of the present preferred embodiments. Consequently, the only
limitations which
should be placed upon the scope of the invention are those which appear in the
appended claims.
All references cited within the body of the instant specification are hereby
incorporated by
reference in their entirety.
6.0 EXAMPLES
EXAMPLE 1
Isolation of SEO ID NO: 1 from a cDNA Library of Human Thalamus
A plurality of novel nucleic acids were obtained from a cDNA library prepared
from
human thalamus (Hyseq clone identification number 10468562) using standard
PCR, sequencing
by hybridization sequence signature analysis, and Sanger sequencing
techniques. The inserts of
the library were amplified with PCR using primers specif c for vector
sequences flanking the
inserts. These samples were spotted onto nylon membranes and interrogated with
oligonucleotide probes to give sequence signatures. The clones were clustered
into groups of
similar or identical sequences, and single representative clones were selected
from each group
for gel sequencing. The 5' sequence of the amplified inserts was then deduced
using the reverse
M13 sequencing primer in a typical Sanger sequencing protocol. PCR products
were purified
and subjected to fluorescent dye terminator cycle sequencing. Single-pass gel
sequencing was
done using a 377 Applied Biosystems (ABI) sequencer. The insert was identified
as a novel
sequence not previously obtained from this library and not previously reported
in public
databases. The sequence is designated as SEQ ID NO: 1.
EXAMPLE 2
ASSEMBLAGE OF SEQ ID NO: 2
The nucleic acid of the present invention, designated as SEQ ID NO: 2 was
assembled using
an EST sequence as a seed. Then a recursive algorithm was used to extend the
seed EST into an
extended assemblage, by pulling additional sequences from different databases
(i.e., Hyseq's
database containing EST sequences, dbEST version 114, gb pri 114, and UniGene
version 101) that
belong to this assemblage. The algorithm terminated when there was no
additional sequences from
the above databases that would extend the assemblage. Inclusion of component
sequences into the
assemblage was based on a BLASTN hit to the extending assemblage with BLAST
score greater
than 300 and percent identity greater than 95%.
3 5 The nearest neighbor result for the assembled contig was obtained by a
FASTA version 3
search against Genpept release 114, using FASTXY algorithm. FASTXY is an
improved version of
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FASTA alignment which allows in-codon frame shifts. The nearest neighbor
result showed the
closest homologue for each assemblage from Genpept (and contains the
translated amino acid
sequences for which the assemblage encodes). The nearest neighbor results is
set forth below:
AccessionNo. Description Smith- % Identity
Waterman
Score
U16845 Rattusnorvegicusneurotrimin321 28.467
A polypeptide was predicted to be encoded by SEQ ID NO: 2 as set forth below.
The
polypeptide was predicted using a software program called FASTY (available
from
htttr//fasta.bioch.virain.ia.edu) which selects a polypeptide based on a
comparison of translated
novel polynucleotide to known polypeptides (W.R. Pearson, Methods in
Enzymology, l 83: 63-98
(1990), herein incorporatedby reference). See U.S. Serial No. 09/496,914
incorporatedby
reference herein.
PredictedPredicted AMINO ACID ENCODED BY SEQ ID NO: 2
beginningend (A=Alanine, C=Cysteine, D=Aspartic Acid,
E=
nucleotidenucleotide Glutamic Acid, F=Phenylalanine, G=Glycine,
location location H=Histidine, I=Isoleucine, K=Lysine,
L=Leucine,
correspond-correspond-M=Methionine, N=Asparagine, P=Proline,
ing to ing to lastQ=Glutarnine, R=Arginine, S=Serine, T=Threonine,
first
amino amino acid V=Valine, W=Tryptophan, Y=Tyrosine,
acid
residue residue X=Unknown, *=Stop Codon, /=possible nucleotide
of of
amino amino acid deletion, \=possible nucleotide insertion)
acid
segment segment
2 1125 PLSRTSDRPLLLQAETAWGCRLKSIRVDVQYLDEPMLT
VHQTVSDVRGNFYQEKTVFLRCTVNSNPPARFIWKRGS
DTLSHSQDNGVDIYEPLYTQGETKVLKLKNLRPQDYAS
YTCQVSVRNVCGIPDKAITFRLTNTTAPPALKLSVNET
LWNPGENVTVQCLLTGGDPLPQLQWSHGPGPLPLGAL
AQGGTLSIPSVQARDSGYYNCTATNNVGNPAKKTVNLL
VRSMKNATFQITPDVIKESENIQLGQDLKLSCHVDAVP
QEKVTYQWFKNGKPARMSKRLLVTRNDPELPAVTSSLE
LIDLHFSDYGTYLCMASFPGAPVPDLSVEVNTSSETVP
PTISVPKGRAWTVREGSPAELQCEVRGKPRP (SEQ
ID NO: 16)
EXAMPLE 3
ASSEMBLAGE OF SEO ID NO: 3
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Assembly of novel nucleotide sequence of SEQ ID NO: 3 was accomplished by
using an
EST sequence SEQ ID NO: 1 as a seed. The seed was extended by gel sequencing
(377 Applied
Biosystems (ABI) sequences) and RACE (Rapid Amplification of cDNA ends) using
primers to
extend both 5' and 3' ends.
A polypeptide (SEQ ID NO: 4) was predicted to be encoded by SEQ ID NO: 3 as
set forth
below. The polypeptide was predicted using a software program called BLASTX
which selects a
polypeptide based on a comparison of translated novel polynucleotide to known
polypeptides. The
initial methionine, ATG, starts at position 171 of the nucleotide sequence and
the putative stop
codon, TAG, begins at position 1545.
SEQ ID NO: 3 was determined to be present in the following tissues: adult
brain
(Clontech) (Hyseq library names ABR006 and ABR008), adrenal gland (Clontech)
(Hyseq library
name ADR002), adult ovary (Invitrogen) (Hyseq library name AOV001), fetal
brain (Clontech)
(Hyseq library names FBR001, FBR004, and FBR006), fetal skin (Invitrogen)
(Hyseq library
name FSK001), mammary gland (Invitrogen) (Hyseq library name MMG001), and
thalamus
(Clontech) (Hyseq library name THA002). The tissue expression information was
determined
using the tissue source of the ESTs that comprise SEQ ID NO: 3 and the tissue
sources of the
other ESTs of the cluster to which those ESTs belong. Clusters were made
depending on the
sequence signature of each sequence as described in Example 1.
EXAMPLE 4
ASSEMBLAGE OF SEQ ID NO: 6
The nucleic acid of the present invention, designated as SEQ ID NO: 6 was
assembled using
an EST sequence as a seed. Then a recursive algorithm was used to extend the
seed EST into an
extended assemblage, by pulling additional sequences from different databases
(i.e., Hyseq's
database containing EST sequences, dbEST version 118, gb pri I 18, and UniGene
version 118) that
belong to this assemblage. The algoritlun terminated when there was no
additional sequences from
the above databases that would extend the assemblage. Inclusion of component
sequences into the
assemblage was based on a BLASTN hit to the extending assemblage with BLAST
score greater
than 300 and percent identity greater than 95%.
Using PHRAP (Univ. of Washington) or CAP4 (Paracel), a full length gene cDNA
sequence and its corresponding protein sequence were generated from the
assemblage. Any
frame shifts and incorrect stop codons were corrected by hand editing. During
editing, the
sequence was checked using FASTY and/or BLAST against Genbank. (.e. dbEST
version 1 I8,
gb pri 118, UniGeneversion 118, Genepeptrelease 118). Other computer programs
which rnay
105
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have been used in the editing process were phredPhrap and Consed (University
of Washington)
and ed-ready, ed-ext and gc-zip-2 (Hyseq, Inc.). The full-length nucleotide
sequence is shown as
SEQ ID NO: 6.
A polypeptide (SEQ ID NO: 7) was predictedto be encoded by SEQ ID NO: 6 as set
forth
below. The polypeptide was predicted using a software program called BLASTX
which selects a
polypeptide based on a comparison of translated novel polynucleotide to known
polypeptides. The
initial methionine ATG starts at position I7I of the nucleotide sequence and
the putative stop
codon, TAG, begins at position 1929.
SEQ ID NO: 6 was determined to be present in the following tissues: adult
brain
(Clontech) (Hyseq library names ABR006 and ABR008), adrenal gland (Clontech)
(Hyseq library
name ADR002), adult ovary (Invitrogen) (Hyseq library name AOVOOI), fetal
brain (Clontech)
(Hyseq library names FBR001, FBR004, and FBR006), fetal skin (Invitrogen)
(Hyseq library
name FSI~001), mammary gland (Invitrogen) (Hyseq library name MMG001), and
thalamus
(Clontech) (Hyseq library name THA002). The tissue expression information was
determined
using the tissue source of the ESTs that comprise SEQ ID NO: 6 and the tissue
sources of the
other ESTs of the cluster to which those ESTs belong. Clusters were made
depending on the
sequence signature of each sequence as described in Example 1.
EXAMPLE 5
A. Expression of SEO ID NO: 4, 7, 9-13 in cells
Chinese Hamster Ovary (CHO) cells or other suitable cell types are grown in
DMEM
(ATCC) and 10% fetal bovine serum (FBS) (Gibco) to 70% confluence. Prior to
transfection the
media is changed to DMEM and 0.5% FCS. Cells are transfected with cDNAs for
SEQ ID NO:
3 or Swith pBGal vector by the FuGENE-6 transfection reagent (Boehringer). In
summary, 4 ~.1
of FuGENE-6 is diluted in 100 ~1 of DMEM and incubated for 5 minutes. Then,
this is added to
1 ~,g of DNA and incubated for 15 minutes before adding it to a 35 mm dish of
CHO cells. The
CHO cells are incubated at 37°C with 5% CO2. After 24 hours, media and
cell lysates are
collected, centrifuged and dialyzed against assay buffer (15 mM Tris pH 7.6,
134 mM NaCI, 5
mM glucose, 3 mM CaCl2 and MgCl2.
B. Expression Study Using SEQ ID NO: 3 and 6
The expression of SEQ ID NO: 3 and 6 in various tissues is analyzed using a
semi-
quantitative polymerase chain reaction-based technique. Human cDNA libraries
are used as
sources of expressed genes from tissues of interest (adult bladder, adult
brain, adult heart, adult
kidney, adult Iymph node, adult liver, adult lung, adult ovary, adult
placenta, adult rectum, adult
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spleen, adult testis, bone marrow, thymus, thyroid gland, fetal kidney, fetal
liver, fetal liver-
spleen, fetal skin, fetal brain, fetal leukocyte and macrophage). Gene-
specific primers are used
to amplify portions of the SEQ ID NO: 3 or 6 sequence from the samples.
Amplified products
are separated on an agarose gel, transferred and chemically linked to a nylon
filter. The filter is
then hybridized with a radioactively labeled (33P-dCTP) double-stranded probe
generated from
SEQ ID NO: 3 or 6 using a I~lenow polymerase, random-prime method. The f hers
are washed
(high stringency) and used to expose a phosphorimaging screen for several
hours. Bands
indicate the presence of cDNA including SEQ ID NO: 3 and 6 sequences in a
specific library,
and thus mRNA expression in the corresponding cell type or tissue.
l07
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SEQUENCE LISTTNG
<110> Hyseq, Inc.
Boyle, Bryan J et al
<120> METHODS AND MATERIALS RELATING TO NEUROTRIMIN-LIKE
POLYPEPTIDES AND POLYNUCLEOTIDES
<130> HYS-17/21272-031
<140> Not Yet Assigned
<141> 2001-02-02
<150> 09/632,085
<151> 2000-08-02
<150> 09/560,875
<151> 2000-04-27
<150> 09/496,914
<151> 2000-02-03
<160> 16
<170> PatentIn Ver. 2.1
<210> 1
<211> 398
<212> DNA
<213> Homo Sapiens
<400> 1
tggggttgac atctatgagc ccctctacac tcagggggag accaaggtcc tgaagctgaa 60
gaacctgegg ccccaggact atgccageta cacctgccag gtgtctgtgc gtaacgtgtg 120
cggcatccca gacaaggcca tcaccttccg gctcaccaac accacggcac caccagccct 180
gaagctgtct gtgaacgaaa ctctggtggt gaaccctggg gagaatgtga cggtgcagtg 240
tctgctgaca ggcggtgatc ccctccccca gctgcagtgg tcccatgggc ctggcccact 300
gcecctgggt gctctggecc agggtggcac cctcagcatc ccttcagtgc aggcccggga 360
ctctggctac tacaactgca cagccaccaa caatgtgg 398
<210> 2
<211> 1124
<212> DNA
<213> Homo Sapiens
<400> 2
cccactaagt aggacatcag aceggccgct actactgcag gctgaaacgg cgtgggggtg 60
tcgtcttaag tccatccgcg tggacgtgca gtacctggat gagecaatgc tgacggtgca 120
ccagacggtg agcgatgtgc gaggcaactt ctaccaggag aagacggtgt tcctgcgctg 180
tactgtcaac tCCaaCCCaC CtgCCCgCtt catctggaag cggggttccg ataccetatc 240
ccacagccag gacaatgggg ttgacatcta tgagcccctc tacactcagg gggagaccaa 300
ggtcctgaag ctgaagaacc tgcggcccca ggactatgcc agctacacct gccaggtgtc 360
tgtgcgtaac gtgtgcggca tcccagacaa ggccatcacc ttccggctca ccaacaccac 420
ggcaccacca gccctgaagc tgtctgtgaa cgaaactctg gtggtgaacc ctggggagaa 480
tgtgacggtg cagtgtctgc tgacaggcgg tgatcccctc ccccagctgc agtggtccca 540
tgggcctggc ccactgcccc tgggtgctct ggcccagggt ggcaccctca gcatccettc 600
agtgcaggcc cgggactctg gctactacaa ctgcacagec accaacaatg tgggcaacec 660
tgccaagaag actgtcaacc tgctggtgcg atccatgaag aacgctacat tccagatcac 720
tcctgacgtg atcaaagaga gtgagaacat ccagctgggc caggacctga agctatcgtg 780
ccacgtggat gcagtgccec aggagaaggt gacctaccag tggttcaaga atggcaagcc 840
ggcacgcatg tccaagcggc tgctggtgac ccgcaatgat cctgagctgc ccgcagtcac 900
cagcagccta gagctcattg acctgcactt cagtgactat ggcacctacc tgtgcatggc 960
ttctttccca ggggcacccg tgcccgacct cagcgtcgag gtcaacatet cctctgagac 1020
1
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agtgccgccc accatcagtg tgcccaaggg tagggccgtg gtgaccgtgc gcgagggatc 1080
gcctgccgag ctgcaatgcg aggtgcgggg caagccgcgg ccgc 1124
<210> 3
<211> 1699
<212> DNA
<213> Homo Sapiens
<220>
<221> CDS
<222> (171)..(1547)
<400> 3
tcttcgtcgc cgctctctct ctcacctctc agggaaaggg ggggacatag gggcgtcgcg 60
gggccccggc gaatgCgCCC CCCgCCgCCt CtCgggCtgC gccgcctcac tatagggctc 120
gagcggccgc cgccggcagg tctcgcgggg atgaagcacc ggccgtgaag atg gag 176
Met Glu
1
gtg acc tgc ctt cta ctt ctg gcg ctg atc ccc ttc cac tgc cgg gga 224
Val Thr Cys Leu Leu Leu Leu Ala Leu Ile Pro Phe His Cys Arg Gly
~10 15
caa gga gtc tac get cca gcc cag gcg cag atc gtg cat gcg ggc cag 272
Gln Gly Val Tyr Ala Pro Ala Gln Ala Gln Ile Val His Ala Gly Gln
20 25 30
gca tgt gtg gtg aaa gag gac aat atc agc gag cgt gtc tac acc atc 320
Ala Cys Val Val Lys Glu Asp Asn Ile Ser Glu Arg Val Tyr Thr Ile
35 40 45 50
cgg gag ggg gac acc ctc atg ctg cag tgc ctt gta aca ggg cac cct 368
Arg Glu Gly Asp Thr Leu Met Leu Gln Cys Leu Val Thr Gly His Pro
55 60 65
cga ccc cag gta cgg tgg acc aag acg gca ggt agc gcc tcg gac aag 416
Arg Pro Gln Val Arg Trp Thr Lys Thr Ala Gly Ser Ala Ser Asp Lys
70 75 80
ttc cag gag aca tcg gtg ttc aac gag acg ctg cgc atc gag cgt att 464
Phe Gln Glu Thr Ser Val Phe Asn Glu Thr Leu Arg Ile Glu Arg Ile
85 90 95
gca cgc acg cag ggc ggc cgc tac tac tgc aag get gag aac ggc gtg 512
Ala Arg Thr Gln Gly Gly Arg Tyr Tyr Cys Lys Ala Glu Asn Gly Val
100 105 110
ggg gtg ccg gcc atc aag tcc atc cgc gtg gac gtg cag tac ctg gat 560
Gly Val Pro Ala Ile Lys Ser Ile Arg Val Asp Val Gln Tyr Leu Asp
115 120 125 130
gag cca atg ctg acg gtg cac cag acg gtg agc gat gtg cga ggc aac 608
Glu Pro Met Leu Thr Val His Gln Thr Val Ser Asp Val Arg Gly Asn
135 140 145
ttc tac cag gag aag acg gtg ttc ctg cgc tgt act gtc aac tcc aac 656
Phe Tyr Gln Glu Lys Thr Val Phe Leu Arg Cys Thr Val Asn Ser Asn
150 155 160
cca cct gcc cgc ttc atc tgg aag cgg ggt tcc gat acc cta tcc cac 704
Pro Pro Ala Arg Phe Ile Trp Lys Arg Gly Ser Asp Thr Leu Ser His
CA 02399644 2002-08-02
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165 170 175
agc cag gac aat ggg gtt gac ate tat gag ccc etc tac act cag ggg 752
Ser Gln Asp Asn Gly Val Asp Ile Tyr Glu Pro Leu Tyr Thr Gln Gly
180 185 190
gag acc aag gtc ctg aag ctg aag aac ctg cgg ccc cag gac tat gcc 800
Glu Thr Lys Val Leu Lys Leu Lys Asn Leu Arg Pro Gln Asp Tyr Ala
195 200 205 210
agc tac acc tgc cag gtg tct gtg cgt aac gtg tgc ggc atc cca gac 848
Ser Tyr Thr Cys Gln Val Ser Val Arg Asn Val Cys Gly Ile Pro Asp
215 220 225
aag gcc atc acc ttc cgg ctc acc aac acc acg gca cca cca gcc ctg 896
Lys Ala Ile Thr Phe Arg Leu Thr Asn Thr Thr Ala Pro Pro Ala Leu
230 235 240
aag ctg tct gtg aac gaa act ctg gtg gtg aac cct ggg gag aat gtg 944
Lys Leu Ser Val Asn Glu Thr Leu Val Val Asn Pro Gly Glu Asn Val
245 250 255
acg gtg cag tgt ctg ctg aca ggc ggt gat CCC CtC CCC Cag ctg cag 992
Thr Val Gln Cys Leu Leu Thr Gly Gly Asp Pro Leu Pro Gln Leu Gln
260 265 270
tgg tcc cat ggg cct ggc cca ctg ccc ctg ggt get ctg gcc cag ggt 1040
Trp Ser His Gly Pro Gly Pro Leu Pro Leu Gly Ala Leu Ala Gln Gly
275 280 285 290
ggc acc ctc agc atc cct tca gtg cag gcc cgg gac tct ggc tac tac 1088
Gly Thr Leu Ser Ile Pro Ser Val Gln Ala Arg Asp Ser Gly Tyr Tyr
295 300 305
aac tgc aca gcc acc aac aat gtg ggc aac cct gcc aag aag act gtc 1136
Asn Cys Thr Ala Thr Asn Asn Val Gly Asn Pro Ala Lys Lys Thr Val
310 315 320
aac ctg ctg gtg cga tcc atg aag aac get aca ttc cag ate act cct 1184
Asn Leu Leu Val Arg Ser Met Lys Asn Ala Thr Phe Gln Ile Thr Pro
325 330 335
gac gtg atc aaa gag agt gag aac atc cag ctg ggc cag gac ctg aag 1232
Asp Val Ile Lys Glu Ser Glu Asn Ile Gln Leu Gly Gln Asp Leu Lys
340 345 350
cta tcg tgc cac gtg gat gca gtg ccc cag gag aag gtg acc tac cag 1280
Leu Ser Cys His Val Asp Ala Val Pro Gln Glu Lys Val Thr Tyr Gln
355 360 365 370
tgg ttc aag aat ggc aag ccg gca cgc atg tcc aag cgg ctg ctg gtg 1328
Trp Phe Lys Asn Gly Lys Pro Ala Arg Met Sex Lys Arg Leu Leu Val
375 380 385
acc cgc aat gat cct gag ctg ccc gca gtc acc agc agc cta gag etc 1376
Thr Arg Asn Asp Pro Glu Leu Pro Ala Val Thr Ser Ser Leu Glu Leu
390 395 400
att gac ctg cac ttc agt gac tat ggc acc tac ctg tgc atg get tet 1424
Ile Asp Leu His Phe Ser Asp Tyr Gly Thr Tyr Leu Cys Met Ala Ser
405 410 415
ttc cca ggg gca ccc gtg ccc gac ctc agc gtc gag gtc aac atc tcc 1472
Phe Pro Gly Ala Pro Val Pro Asp Leu Ser Val Glu Val Asn Ile Ser
3
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420 425 430
tct gag aca ggt ggg cag ctg gag ggg cac tgc gga aat gtg agg ggg 1520
Ser Glu Thr Gly Gly Gln Leu Glu Gly His Cys Gly Asn Val Arg GIy
435 440 445 450
gtg gag agg ccc aca gac agg agg tag tggggaggca acggcttggg 1567
Val Glu Arg Pro Thr Asp Arg Arg
455
aaagggaagc cagagtgagg aggccaccac ttgttcatgc cacgtgcttt tattgagtcc 1627
ctgttaatat gccaggcact gtgctaggtc ctggggtaga gtggtgggca aaacaggctc 1687
agaacaacga ca 1699
<210> 4
<211> 458
<212> PRT
<213> Homo sapiens
<400> 4
Met Glu Val Thr Cys Leu Leu Leu Leu Ala Leu Ile Pro Phe His Cys
1 5 10 15
Arg Gly Gln Gly Val Tyr Ala Pro Ala Gln Ala Gln Ile Val His Ala
20 25 30
Gly Gln Ala Cys Val Val Lys Glu Asp Asn Ile Ser Glu Arg Val Tyr
35 40 45
Thr Ile Arg Glu Gly Asp Thr Leu Met Leu Gln Cys Leu Val Thr Gly
50 55 60
His Pro Arg Pro Gln Val Arg Trp Thr Lys Thr Ala Gly Ser Ala Ser
65 70 75 80
Asp Lys Phe Gln Glu Thr Ser Val Phe Asn Glu Thr Leu Arg Ile Glu
85 90 95
Arg Ile Ala Arg Thr Gln GIy Gly Arg Tyr Tyr Cys Lys Ala Glu Asn
100 105 110
Gly Val Gly Val Pro Ala Ile Lys Ser Ile Arg Val Asp Val Gln Tyr
115 120 125
Leu Asp Glu Pro Met Leu Thr Val His Gln Thr Val Ser Asp Val Arg
130 135 140
Gly Asn Phe Tyr Gln Glu Lys Thr Val Phe Leu Arg Cys Thr Val Asn
145 150 155 160
Ser Asn Pro Pro Ala Arg Phe Ile Trp Lys Arg Gly Ser Asp Thr Leu
165 170 175
Ser His Ser Gln Asp Asn Gly Val Asp Ile Tyr Glu Pro Leu Tyr Thr
180 185 I90
Gln Gly Glu Thr Lys Val Leu Lys Leu Lys Asn Leu Arg Pro Gln Asp
195 200 205
Tyr Ala Ser Tyr Thr Cys Gln Val Ser Val Arg Asn Val Cys Gly Ile
210 215 220
Pro Asp Lys Ala Ile Thr Phe Arg Leu Thr Asn Thr Thr Ala Pro Pro
225 230 235 240
Ala Leu Lys Leu Ser Val Asn Glu Thr Leu Val Val Asn Pro Gly Glu
245 250 255
Asn Val Thr Va1 Gln Cys Leu Leu Thr Gly Gly Asp Pro Leu Pro Gln
260 265 270
Leu Gln Trp Ser His Gly Pro Gly Pro Leu Pro Leu Gly Ala Leu Ala
275 280 285
Gln Gly Gly Thr Leu Ser Ile Pro Ser Val Gln Ala Arg Asp Ser Gly
290 295 300
Tyr Tyr Asn Cys Thr Ala Thr Asn Asn Val Gly Asn Pro Ala Lys Lys
305 310 315 320
Thr Val Asn Leu Leu Val Arg Ser Met Lys Asn Ala Thr Phe Gln Ile
4
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325 330 335
Thr Pro Asp Val Ile Lys Glu Ser Glu Asn Ile Gln Leu Gly Gln Asp
340 345 350
Leu Lys Leu Ser Cys His Val Asp Ala Val Pro Gln Glu Lys Val Thr
355 360 365
Tyr Gln Trp Phe Lys Asn Gly Lys Pro Ala Arg Met Ser Lys Arg Leu
370 375 380
Leu Val Thr Arg Asn Asp Pro Glu Leu Pro Ala Val Thr Ser Ser Leu
385 390 395 400
Glu Leu Ile Asp Leu His Phe Ser Asp Tyr Gly Thr Tyr Leu Cys Met
405 410 415
Ala Ser Phe Pro Gly Ala Pro Val Pro Asp Leu Ser Val Glu Val Asn
420 425 430
Ile Ser Ser Glu Thr Gly Gly Gln Leu Glu Gly His Cys Gly Asn Val
435 440 445
Arg Gly Val Glu Arg Pro Thr Asp Arg Arg
450 455
<210> 5
<211> 1377
<212> DNA
<213> Homo Sapiens
<400> 5
atggaggtga cctgccttct acttctggcg ctgatcccct tccactgccg gggacaagga 60
gtctacgctc cagcccaggc gcagatcgtg catgcgggcc aggcatgtgt ggtgaaagag 120
gacaatatca gcgagcgtgt ctacaccatc cgggaggggg acaccctcat gctgcagtgc 180
cttgtaacag ggcaccctcg accccaggta cggtggacca agacggcagg tagcgcctcg 240
gacaagttcc aggagacatc ggtgttcaac gagacgctgc gcatcgagcg tattgcacgc 300
acgcagggcg gccgctacta ctgcaaggct gagaacggcg tgggggtgcc ggccatcaag 360
tccatccgcg tggacgtgca gtacctggat gagccaatgc tgacggtgca ccagacggtg 420
agcgatgtgc gaggcaactt ctaccaggag aagacggtgt tcctgcgctg tactgtcaac 480
tCCaaCCCaC CtgCCCgCtt CatCtggaag cggggttccg ataccctatc ccacagccag 540
gacaatgggg ttgacatcta tgagcccctc tacactcagg gggagaccaa ggtcctgaag 600
ctgaagaacc tgcggcccca ggactatgcc agctacacct gccaggtgtc tgtgcgtaac 660
gtgtgeggca tcccagacaa ggccatcacc ttccggctca ccaacaccac ggcaccacca 720
gccctgaagc tgtctgtgaa cgaaactctg gtggtgaacc ctggggagaa tgtgacggtg 780
cagtgtctgc tgaCaggCgg tgatCCCCtC CCCCagCtgC agtggtCCCa tgggcctggc 840
CCaCtgCCCC tgggtgctct ggcccagggt ggcaccctca gcatcccttc agtgcaggcc 900
cgggactctg gctactacaa ctgcacagcc accaacaatg tgggcaaccc tgccaagaag 960
actgtcaacc tgctggtgcg atccatgaag aacgctacat tccagatcac tcctgacgtg 1020
atcaaagaga gtgagaacat ccagctgggc caggacctga agctatcgtg ccacgtggat 1080
gcagtgcccc aggagaaggt gacctaccag tggttcaaga atggcaagcc ggcacgcatg 1140
tccaagcggc tgctggtgac ccgcaatgat cctgagctgc ccgcagtcac cagcagccta 1200
gagctcattg acctgcactt cagtgactat ggcacctacc tgtgcatggc ttctttccca 1260
ggggcacccg tgcccgacct cagcgtcgag gtcaacatct cctctgagac aggtgggcag 1320
ctggaggggc actgcggaaa tgtgaggggg gtggagaggc ccacagacag gaggtag 1377
<210> 6
<21I> 2196
<212> DNA
<213> Homo Sapiens
<220>
<221> CDS
<222> (171)..(1931)
<400> 6
tCttCgtCgC CgC'tCCCtCt ctcacctctc agggaaaggg ggggacatag gggcgtcgcg 60
gggccccggc gaatgcgccc cccgccgcct ctcgggctgc gccgcctcac tatagggctc 120
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gagcggccgc cgccggcagg tctcgcgggg atgaagcacc ggccgtgaag atg gag 176
Met Glu
1
gtg acc tgc ctt cta ctt ctg gcg ctg atc ccc ttc cac tgc cgg gga 224
Val Thr Cys Leu Leu Leu Leu Ala Leu Ile Pro Phe His Cys Arg Gly
10 15-
caa gga gtc tac get cca gec cag gcg cag atc gtg cat gcg ggc cag 272
Gln Gly Val Tyr Ala Pro Ala Gln Ala Gln Ile Val His Ala Gly Gln
20 25 30
gca tgt gtg gtg aaa gag gac aat atc agc gag cgt gtc tac acc atc 320
Ala Cys Val Val Lys Glu Asp Asn Ile Ser Glu Arg Val Tyr Thr Ile
35 40 45 50
cgg gag ggg gac acc ctc atg ctg cag tgc ctt gta aca ggg cac cct 368
Arg Glu Gly Asp Thr Leu Met Leu Gln Cys Leu Val Thr Gly His Pro
55 60 65
cga ccc cag gta cgg tgg acc aag acg gca ggt agc gcc tcg gac aag 416
Arg Pro Gln Val Arg Trp Thr Lys Thr Ala Gly Ser Ala Ser Asp Lys
70 75 80
ttc cag gag aca tcg gtg ttc aac gag acg ctg cgc ate gag cgt att 464
Phe Gln Glu Thr Ser Val Phe Asn Glu Thr Leu Arg Ile Glu Arg Ile
85 90 95
gca cgc acg cag ggc ggc cgc tac tac tgc aag get gag aac ggc gtg 512
Ala Arg Thr Gln Gly Gly Arg Tyr Tyr Cys Lys Ala Glu Asn Gly Val
100 105 110
ggg gtg ccg atc aag tcc atc cgc gtg gac gtg cag tac ctg gat gag 560
Gly Val Pro Ile Lys Ser Ile Arg Val Asp Val Gln Tyr Leu Asp Glu
115 120 225 I30
cca atg ctg acg gtg cac cag acg gtg agc gat gtg cga ggc aac ttc 608
Pro Met Leu Thr Val His Gln Thr Val Ser Asp Val Arg Gly Asn Phe
135 140 145
tac cag gag aag acg gtg ttc ctg cgc tgt act gtc aac tcc aac cca 656
Tyr Gln Glu Lys Thr Val Phe Leu Arg Cys Thr Val Asn Ser Asn Pro
150 155 160
cet gcc cgc ttc atc tgg aag cgg ggt tcc gat acc cta tcc cac agc 704
Pro Ala Arg Phe Ile Trp Lys Arg Gly Ser Asp Thr Leu Ser His Ser
165 170 175
cag gac aat ggg gtt gac atc tat gag ccc ctc tac act cag ggg gag 752
Gln Asp Asn Gly Val Asp Ile Tyr Glu Pro Leu Tyr Thr Gln Gly Glu
180 185 190
acc aag gtc ctg aag ctg aag aac ctg cgg ccc cag gac tat gcc agc 800
Thr Lys Val Leu Lys Leu Lys Asn Leu Arg Pro Gln Asp Tyr Ala Ser
195 200 205 210
tac acc tgc cag gtg tct gtg cgt aac gtg tgc ggc atc cca gac aag 848
Tyr Thr Cys Gln Val Ser Val Arg Asn Val Cys Gly Ile Pro Asp Lys
215 220 225
gcc atc acc ttc cgg ctc acc aac acc acg gca cca cca gcc ctg aag 896
Ala Ile Thr Phe Arg Leu Thr Asn Thr Thr Ala Pro Pro Ala Leu Lys
230 235 240
6
CA 02399644 2002-08-02
WO 01/57175 PCT/USO1/03651
ctg tct gtg aac gaa act ctg gtg gtg aac cct ggg gag aat gtg acg 944
Leu Ser Val Asn Glu Thr Leu Val Val Asn Pro Gly Glu Asn Val Thr
245 250 255
gtg cag tgt ctg ctg aca ggc ggt gat CCC Ct C CCC Cag ctg cag tgg 992
Val Gln Cys Leu Leu Thr Gly Gly Asp Pro Leu Pro Gln Leu Gln Trp
260 265 270
tcc cat ggg cct ggc cca ctg ccc ctg ggt get ctg gcc cag ggt ggc 1040
Ser His Gly Pro Gly Pro Leu Pro Leu Gly Ala Leu Ala Gln Gly Gly
275 280 285 290
acc ctc age atc cct tea gtg cag gcc cgg gac tct ggc tae tac aac 1088
Thr Leu Ser Ile Pro Ser Val Gln Ala Arg Asp Ser Gly Tyr Tyr Asn ,
295 300 305
tgc aca gcc acc aac aat gtg ggc aac cct gec aag aag act gtc aac 1136
Cys Thr Ala Thr Asn Asn Val Gly Asn Pro Ala Lys Lys Thr Val Asn
310 315 320
ctg ctg gtg cga tcc atg aag aac get aca ttc cag atc act cct gac 1184
Leu Leu Val Arg Ser Met Lys Asn Ala Thr Phe Gln Ile Thr Pro Asp
325 330 335
gtg atc aaa gag agt gag aac atc cag ctg ggc cag gac ctg aag cta 1232
Val Ile Lys Glu Ser Glu Asn Ile Gln Leu Gly Gln Asp Leu Lys Leu
340 345 350
tcg tgc cac gtg gat gca gtg ccc cag gag aag gtg acc tac cag tgg 1280
Ser Cys His Val Asp Ala Val Pro Gln Glu Lys Val Thr Tyr Gln Trp
355 360 365 370
ttc aag aat ggc aag ccg gca cgc atg tcc aag cgg ctg ctg gtg acc 1328
Phe Lys Asn Gly Lys Pro Ala Arg Met Ser Lys Arg Leu Leu Val Thr
375 380 385
cgc aat gat cct gag ctg ccc gca gtc acc agc agc cta gag ctc att 1376
Arg Asn Asp Pro Glu Leu Pro Ala Val Thr Ser Ser Leu Glu Leu Ile
390 395 400
gac ctg cac ttc agt gac tat ggc acc tac ctg tgc atg get tct ttc 1424
Asp Leu His Phe Ser Asp Tyr Gly Thr Tyr Leu Cys Met Ala Ser Phe
405 410 415
cca ggg gca ccc gtg CCC gac ctc agc gtc gag gtc aac atc tcc tct 1472
Pro Gly Ala Pro Val Pro Asp Leu Ser Val Glu Val Asn Ile Ser Ser
420 425 430
gag aca gtg ccg ccc acc atc agt gtg ccc aag ggt agg gcc gtg gtg 1520
Glu Thr Val Pro Pro Thr Ile Ser Val Pro Lys Gly Arg Ala Val Val
435 440 445 450
acc gtg cgc gag gga tcg ect gcc gag ctg caa tgc gag gtg cgg ggc 1568
Thr Val Arg Glu Gly Ser Pro Ala Glu Leu Gln Cys Glu Val Arg Gly
455 460 465
aag ccg egg ccg cca gtg ctc tgg tcc cgc gtg gac aag gag get gca 1616
Lys Pro Arg Pro Pro Val Leu Trp Ser Arg Val Asp Lys Glu Ala Ala
470 475 480
ctg ctg ccc tcg ggg ctg ccc ctg gag gag act ccg gac ggg aag ctg 1664
Leu Leu Pro Ser Gly Leu Pro Leu Glu Glu Thr Pro Asp Gly Lys Leu
485 490 495
7
CA 02399644 2002-08-02
WO 01/57175 PCT/USO1/03651
cgg ctg gag cga gtg agc cga gac atg agc ggg acc tac cgc tgc cag 1712
Arg Leu Glu Arg Val Ser Arg Asp Met Ser Gly Thr Tyr Arg Cys Gln
500 505 510
acg gcc cgc tat aat ggc ttc aac gtg cgc ccc cgt gag gcc cag gtg 1760
Thr Ala Arg Tyr Asn Gly Phe Asn Val Arg Pro Arg Glu Ala Gln VaI
515 520 525 530
cag ctg aac gtg cag tgt gag tcc acc acg cca ccc gga cta cct tcc 1808
Gln Leu Asn Val Gln Cys Glu Ser Thr Thr Pro Pro Gly Leu Pro Ser
535 540 545
Cag Ctt tCC ttC tgg CtC CCa gCC ggc ctg tCC CCt Ctt Cat CCt C'tC 1856
Gln Leu Ser Phe Trp Leu Pro Ala Gly Leu Ser Pro Leu His Pro Leu
550 555 560
aCC Ca.C CCC agt CCa aCC aaa CCC CaC Cag Cta CtC Ctg ata CCC Cta 1904
Thr His Pro Ser Pro Thr Lys Pro His Gln Leu Leu Leu Ile Pro Leu
565 570 575
atg ctc ctt gac agc ccc atg ctg tag gacacggagg tctgtgttct 1951
Met Leu Leu Asp Ser Pro Met Leu
580 585
ttgctccatt tcatcaagta tctcccatct tcattttcag ggacccggtg aacaaaaaaa 2011
aaattacttg attttgccag gtggaaatgg gattttgaag gaggaggaag gacactcaag~2071
ggaaacactg tctgataaca agatgatgaa aattaccacc atgtgccaaa cactgttcta 2131
ggagttttaa ggcactaact cattgaactt cccctcgtgc cgaattcttg gcctcgaggg 2191
gccaa 2196
<210> 7
<211> 586
<212> PRT
<213> Homo Sapiens
<400> 7
Met Glu Val Thr Cys Leu Leu Leu Leu Ala Leu Ile Pro Phe His Cys
1 5 10 15
Arg Gly Gln Gly Val Tyr Ala Pro Ala Gln Ala Gln Ile Val His Ala
20 25 30
Gly Gln Ala Cys Val Val Lys Glu Asp Asn Ile Ser Glu Arg Val Tyr
35 40 45
Thr Ile Arg Glu Gly Asp Thr Leu Met Leu Gln Cys Leu Val Thr Gly
50 55 60
His Pro Arg Pro GIn Val Arg Trp Thr Lys Thr Ala Gly Ser Ala Ser
65 70 75 80
Asp Lys Phe Gln Glu Thr Ser Val Phe Asn Glu Thr Leu Arg Ile Glu
85 90 95
Arg Ile Ala Arg Thr Gln Gly Gly Arg Tyr Tyr Cys Lys Ala Glu Asn
100 105 110
Gly Val Gly Val Pro Ile Lys Ser Ile Arg Val Asp Val Gln Tyr Leu
115 120 125
Asp Glu Pro Met Leu Thr Val His Gln Thr Val Ser Asp Val Arg Gly
130 135 140
Asn Phe Tyr Gln Glu Lys Thr Val Phe Leu Arg Cys Thr Val Asn Ser
145 150 155 160
Asn Pro Pro Ala Arg Phe Ile Trp Lys Arg Gly Ser Asp Thr Leu Ser
165 170 175
g
CA 02399644 2002-08-02
WO 01/57175 PCT/USO1/03651
His Ser Gln Asp Asn Gly Val Asp Ile Tyr Glu Pro Leu Tyr Thr Gln
180 185 190
Gly Glu Thr Lys Val Leu Lys Leu Lys Asn Leu Arg Pro Gln Asp Tyr
195 200 205
Ala Ser Tyr Thr Cys Gln Val Ser Val Arg Asn Val Cys Gly Ile Pro
210 215 220
Asp Lys Ala Ile Thr Phe Arg Leu Thr Asn Thr Thr Ala Pro Pro Ala
225 230 235 240
Leu Lys Leu Ser Val Asn Glu Thr Leu Val Val Asn Pro Gly Glu Asn
245 250 255
Val Thr Val Gln Cys Leu Leu Thr Gly Gly Asp Pro Leu Pro Gln Leu
260 265 270
Gln Trp Ser His Gly Pro Gly Pro Leu Pro Leu Gly Ala Leu Ala Gln
275 280 285
Gly Gly Thr Leu Ser Ile Pro Ser Val Gln Ala Arg Asp Ser Gly Tyr
290 295 300
Tyr Asn Cys Thr Ala Thr Asn Asn Val Gly Asn Pro Ala Lys Lys Thr
305 310 315 320
Val Asn Leu Leu Val Arg Ser Met Lys Asn Ala Thr Phe Gln Ile Thr
325 330 335
Pro Asp Val Ile Lys Glu Ser Glu Asn Ile Gln Leu Gly Gln Asp Leu
340 345 350
Lys Leu Ser Cys His Val Asp Ala Val Pro Gln Glu Lys Val Thr Tyr
355 360 365
Gln Trp Phe Lys Asn Gly Lys Pro Ala Arg Met Ser Lys Arg Leu Leu
370 375 380
Val Thr Arg Asn Asp Pro Glu Leu Pro Ala Val Thr Ser Ser Leu Glu
385 390 395 400
Leu Ile Asp Leu His Phe Ser Asp Tyr Gly Thr Tyr Leu Cys Met Ala
405 410 415
Ser Phe Pro Gly Ala Pro Val Pro Asp Leu Ser Val Glu Val Asn Ile
420 425 430
Ser Ser Glu Thr Val~Pro Pro Thr Ile Ser Val Pro Lys Gly Arg Ala
435 440 445
Val Val Thr Val Arg Glu Gly Ser Pro Ala Glu Leu Gln Cys Glu Val
450 455 460
Arg Gly Lys Pro Arg Pro Pro Val Leu Trp Ser Arg Val Asp Lys Glu
465 470 475 480
Ala Ala Leu Leu Pro Ser Gly Leu Pro Leu Glu Glu Thr Pro Asp Gly
485 490 495
Lys Leu Arg Leu Glu Arg Val Ser Arg Asp Met Ser Gly Thr Tyr Arg
500 505 510
Cys Gln Thr Ala Arg Tyr Asn Gly Phe Asn Val Arg Pro Arg Glu Ala
515 520 525
Gln Val Gln Leu Asn Val Gln Cys Glu Ser Thr Thr Pro Pro Gly Leu
530 535 540
Pro Ser Gln Leu Ser Phe Trp Leu Pro Ala Gly Leu Ser Pro Leu His
545 550 555 560
Pro Leu Thr His Pro Ser Pro Thr Lys Pro His Gln Leu Leu Leu Ile
565 570 575
Pro Leu Met Leu Leu Asp Ser Pro Met Leu
580 585
<210> 8
<211> 1761
<212> DNA
<213> Homo Sapiens
<400> 8
atggaggtga cctgccttct acttctggcg ctgatcccct tccactgccg gggacaagga 60
gtctacgctc cagcccaggc gcagatcgtg catgcgggcc aggcatgtgt ggtgaaagag 120
gacaatatca gcgagcgtgt ctacaccatc cgggaggggg acaccctcat gctgcagtgc 180
9
CA 02399644 2002-08-02
WO 01/57175 PCT/USO1/03651
cttgtaacag ggcacceteg accccaggta cggtggacca agacggeagg tagegcctcg 240
gacaagttcc aggagacatc ggtgttcaac gagacgctgc gcatcgagcg tattgcacgc 300
acgcagggcg gccgctacta'ctgcaaggct gagaacggcg tgggggtgcc gatcaagtcc 360
atccgcgtgg acgtgcagta cctggatgag ccaatgctga cggtgcacca gacggtgagc 420
gatgtgcgag gcaacttcta ccaggagaag acggtgttcc tgcgctgtac tgtcaactcc 480
aacccacctg cecgcttcat ctggaagcgg ggttcegata ccctatecca cagccaggac 540
aatggggttg acatctatga gcccctctac actcaggggg agaccaaggt cctgaagctg 600
aagaacctgc ggccccagga ctatgccagc tacacctgcc aggtgtctgt gcgtaacgtg 660
tgcggcatcc cagacaaggc catCaCCttC cggctcacca acaccacggc accaccagcc 720
ctgaagctgt ctgtgaacga aactctggtg gtgaaccctg gggagaatgt gacggtgcag 780
tgtctgctga caggcggtga tcccctcccc cagctgcagt ggtcccatgg gcctggccca 840
ctgcccctgg gtgctctggc ccagggtggc accctcagca tcccttcagt gcaggcccgg 900
gactctggct actacaactg cacagccacc aacaatgtgg gcaaccctgc caagaagact 960
gtcaacctgc tggtgcgatc catgaagaac gctacattcc agatcactcc tgacgtgatc 1020
aaagagagtg agaacatcca gctgggccag gacctgaagc tatcgtgcca cgtggatgca 1080
gtgccccagg agaaggtgac ctaccagtgg ttcaagaatg gcaagccggc acgcatgtcc 1140
aagcggctgc tggtgacccg caatgatcct gagctgcccg cagtcaccag cagcctagag 1200
CtCattgaCC tgCaCttCag tgactatggc acctacctgt gcatggcttc tttcccaggg 1260
gCaCCCgtgC CCgaCCtCag cgtcgaggtc aacatCtCCt ctgagacagt gccgcccacc 1320
atcagtgtgc ccaagggtag ggccgtggtg accgtgcgcg agggatcgcc tgccgagctg 1380
caatgcgagg tgcggggcaa gccgcggccg ccagtgctct ggtcccgcgt ggacaaggag 1440
gCtgCaCtgC tgccctcggg gctgcccctg gaggagactc cggacgggaa gctgcggctg 1500
gagcgagtga gccgagacat gagcgggacc taccgctgcc agacggcccg ctataatggc 1560
ttcaacgtgc gcccccgtga ggcccaggtg cagctgaacg tgcagtgtga gtccaccacg 1620
CCaCCCggaC taCCttCCCa gCtttCCttC tggctcccag CCggCCtgtC CCCtCttCat 1680
CCtCtCaCCC aCCCCagtCC aaCCaaaCCC CaCCagCtaC tCCtgataCC CCtaatgCtC 1740
cttgacagcc ccatgctgta g 1761
<210> 9
<211> 36
<212> PRT
<213> Homo sapiens
<400> 9
Thr Leu Ser Ile Pro Ser Val Gln Ala Arg Asp Ser Gly Tyr Tyr Asn
1 5 10 15
Cys Thr Ala Thr Asn Asn Val Gly Asn Pro Ala Lys Lys Thr Val Asn
20 25 30
Leu Leu Val Arg
<210> 10
<211> 24
<212> PRT
<2l3> Homo sapiens
<400> 10
Leu Arg Cys Thr VaI Asn Ser Asn Pro Pro Ala Arg Phe Ile Trp Lys
1 5 10 15
Arg Gly Ser Asp Thr Leu Ser His
<210> 11
<211> 16
<212> PRT
<213> Homo sapiens
<400> 11
CA 02399644 2002-08-02
WO 01/57175 PCT/USO1/03651
Met Glu Val Thr Cys Leu Leu Leu Leu Ala Leu Ile Pro Phe His Cys
1 5 10 15
<210> 12
<211> 442
<212> PRT
<213> Homo sapiens
<400> 12
Arg Gly Gln Gly Val Tyr Ala Pro Ala Gln Ala Gln Ile Val His Ala
1 5 10 15
Gly Gln Ala Cys Val Val Lys Glu Asp Asn Ile Ser Glu Arg Val Tyr
20 25 30
Thr Ile Arg Glu Gly Asp Thr Leu Met Leu Gln Cys Leu Val Thr Gly
35 40 45
His Pro Arg Pro Gln Val Arg Trp Thr Lys Thr Ala Gly Ser Ala Ser
50 55 60
Asp Lys Phe Gln Glu Thr Ser Val Phe Asn Glu Thr Leu Arg Ile Glu
65 70 75 80
Arg Ile Ala Arg Thr Gln Gly Gly Arg Tyr Tyr Cys Lys Ala Glu Asn
85 90 95
Gly Val Gly Val Pro Ala Ile Lys Ser Ile Arg Val Asp Val Gln Tyr
100 105 1l0
Leu Asp Glu Pro Met Leu Thr Val His Gln Thr Val Ser Asp Val Arg
1l5 120 125
Gly Asn Phe Tyr Gln Glu Lys Thr Val Phe Leu Arg Cys Thr Val Asn
130 135 140
Ser Asn Pro Pro Ala Arg Phe Ile Trp Lys Arg Gly Ser Asp Thr Leu
145 - 150 155 160
Ser His Ser Gln Asp Asn Gly Val Asp Ile Tyr Glu Pro Leu Tyr Thr
165 170 175
Gln Gly Glu Thr Lys Val Leu Lys Leu Lys Asn Leu Arg Pro Gln Asp
180 185 I90
Tyr Ala Ser Tyr Thr Cys Gln Val Ser Val Arg Asn Val Cys Gly Ile
195 200 205
Pro Asp Lys Ala Ile Thr Phe Arg Leu Thr Asn Thr Thr Ala Pro Pro
210 215 220
Ala Leu Lys Leu Ser Val Asn Glu Thr Leu Val Val Asn Pro Gly Glu
225 230 235 240
Asn Val Thr Val Gln Cys Leu Leu Thr Gly Gly Asp Pro Leu Pro Gln
245 250 255
Leu Gln Trp Ser His Gly Pro Gly Pro Leu Pro Leu Gly Ala Leu Ala
260 265 270
Gln Gly Gly Thr Leu Ser Ile Pro Ser Val Gln Ala Arg Asp Ser Gly
275 280 285
11
CA 02399644 2002-08-02
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Tyr Tyr Asn Cys Thr Ala Thr Asn Asn Val Gly Asn Pro Ala Lys Lys
290 295 300
Thr Val Asn Leu Leu Val Arg Ser Met Lys Asn Ala Thr Phe Gln Ile
305 310 315 320
Thr Pro Asp Val Ile Lys Glu Ser Glu Asn Ile Gln Leu Gly Gln Asp
325 330 335
Leu Lys Leu Ser Cys His Val Asp Ala Val Pro Gln Glu Lys Val Thr
340 345 350
Tyr Gln Trp Phe Lys Asn Gly Lys Pro Ala Arg Met Ser Lys Arg Leu
355 360 365
Leu Val Thr Arg Asn Asp Pro Glu Leu Pro Ala Val Thr Ser Ser Leu
370 375 380
Glu Leu Ile Asp Leu His Phe Ser Asp Tyr Gly Thr Tyr Leu Cys Met
385 390 395 400
Ala Ser Phe Pro Gly Ala Pro Val Pro Asp Leu Ser Val Glu Val Asn
405 410 415
Ile Ser Sex Glu Thr Gly Gly Gln Leu Glu Gly His Cys Gly Asn Val
420 425 430
Arg Gly Val Glu Arg Pro Thr Asp Arg Arg
435 440
<210> 13
<211> 570
<212> PRT
<213> Homo Sapiens
<400> 13
Arg Gly Gln Gly Val Tyr Ala Pro Ala Gln Ala Gln Ile Val His Ala
1 5 10 15
Gly Gln Ala Cys Val Val Lys Glu Asp Asn Ile Ser Glu Arg Val Tyr
20 25 30
Thr Ile Arg Glu Gly Asp Thr Leu Met Leu Gln Cys Leu Val Thr Gly
35 40 45
His Pro Arg Pro Gln Val Arg Trp Thr Lys Thr Ala Gly Ser Ala Ser
50 55 60
Asp Lys Phe Gln Glu Thr Ser Val Phe Asn Glu Thr Leu Arg Tle Glu
65 70 75 80
Arg Ile Ala Arg Thr Gln Gly Gly Arg Tyr Tyr Cys Lys Ala Glu Asn
85 90 95
Gly Val Gly Val Pro Tle Lys Ser Ile Arg Val Asp Val Gln Tyr Leu
100 105 110
Asp Glu Pro Met Leu Thr Val His Gln Thr Val Ser Asp Val Arg Gly
115 120 125
Asn Phe Tyr Gln Glu Lys Thr Val Phe Leu Arg Cys Thr Val Asn Ser
130 135 140
12
CA 02399644 2002-08-02
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Asn Pro Pro Ala Arg Phe Ile Trp Lys Arg Gly Ser Asp Thr Leu Ser
145 150 155 160
His Ser Gln Asp Asn Gly Val Asp Ile Tyr Glu Pro Leu Tyr Thr Gln
165 170 175
Gly Glu Thr Lys Val Leu Lys Leu Lys Asn Leu Arg Pro Gln Asp Tyr
180 185 190
Ala Ser Tyr Thr Cys Gln Val Ser Val Arg Asn Val Cys Gly Ile Pro
195 200 205
Asp Lys Ala Ile Thr Phe Arg Leu Thr Asn Thr Thr Ala Pro Pro Ala
210 215 220
Leu Lys Leu Ser Val Asn Glu Thr Leu Val Val Asn Pro Gly Glu Asn
225 230 235 240
Val Thr Val Gln Cys Leu Leu Thr Gly Gly Asp Pro Leu Pro Gln Leu
245 250 255
Gln Trp Ser His Gly Pro Gly Pro Leu Pro Leu Gly Ala Leu Ala Gln
260 265 270
Gly Gly Thr Leu Ser Ile Pro Ser Val Gln Ala Arg Asp Ser Gly Tyr
275 280 285
Tyr Asn Cys Thr Ala Thr Asn Asn Val Gly Asn Pro Ala Lys Lys Thr
290 295 300
Val Asn Leu Leu Val Arg Ser Met Lys Asn Ala Thr Phe Gln Ile Thr
305 310 315 320
Pro Asp Val Ile Lys Glu Ser Glu Asn Ile Gln Leu Gly Gln Asp Leu
325 330 335
Lys Leu Ser Cys His Val Asp Ala Val Pro Gln Glu Lys Val Thr Tyr
340 345 350
Gln Trp Phe Lys Asn Gly Lys Pro Ala Arg Met Ser Lys Arg Leu Leu
355 360 365
Val Thr Arg Asn Asp Pro Glu Leu Pro Ala Val Thr Ser Ser Leu Glu
370 375' 380
Leu Ile Asp Leu His Phe Ser Asp Tyr Gly Thr Tyr Leu Cys Met Ala
385 390 395 400
Ser Phe Pro Gly Ala Pro Val Pro Asp Leu Ser Val Glu Val Asn Ile
405 410 415
Ser Ser Glu Thr Val Pro Pro Thr Ile Ser Val Pro Lys Gly Arg Ala
420 425 430
Val Val Thr Val Arg Glu Gly Ser Pro Ala Glu Leu Gln Cys Glu Val
435 440 445
Arg Gly Lys Pro Arg Pro Pro Val Leu Trp Ser Arg Val Asp Lys Glu
450 455 460
Ala Ala Leu Leu Pro Ser Gly Leu Pro Leu Glu Glu Thr Pro Asp Gly
465 470 475 480
Lys Leu Arg Leu Glu Arg Val Ser Arg Asp Met Ser Gly Thr Tyr Arg
13
CA 02399644 2002-08-02
WO 01/57175 PCT/USO1/03651
485 490 495
Cys Gln Thr Ala Arg Tyr Asn Gly Phe Asn Val Arg Pro Arg Glu Ala
500 505 510
Gln Val Gln Leu Asn Val Gln Cys Glu Ser Thr Thr Pro Pro Gly Leu
515 520 525
Pro Ser Gln Leu Ser Phe Trp Leu Pro Ala Gly Leu Ser Pro Leu His
530 535 540
Pro Leu Thr His Pro Ser Pro Thr Lys Pro His Gln Leu Leu Leu Ile
545 550 555 560
Pro Leu Met Leu Leu Asp Ser Pro Met Leu
565 570
<210> 14
<211> 299
<212> PRT
<213> Homo Sapiens
<400> 14
Pro Pro Ala Leu Lys Leu Ser Val Asn Glu Thr Leu Val Val Asn Pro
1 5 10 15
Gly Glu Asn Val Thr Val Gln Cys Leu Leu Thr Gly Gly Asp Pro Leu
20 25 30
Pro Gln Leu Gln Trp Ser His Gly Pro Gly Pro Leu Pro Leu Gly Ala
35 40 45
Leu Ala Gln Gly Gly Thr Leu Ser Ile Pro Ser Val Gln Ala Arg Asp
50 55 60
Ser Gly Tyr Tyr Asn Cys Thr Ala Thr Asn Asn Val Gly Asn Pro Ala
65 70 75 80
Lys Lys Thr Val Asn Leu Leu Val Arg Ser Met Lys Asn Ala Thr Phe
85 90 95
Gln Ile Thr Pro Asp Val Ile Lys Glu Ser Glu Asn Ile Gln Leu Gly
100 105 110
Gln Asp Leu Lys Leu Ser Cys His Val Asp Ala Val Pro Gln Glu Lys
115 120 125
Val Thr Tyr Gln Trp Phe Lys Asn Gly Lys Pro Ala Arg Met Ser Lys
130 135 140
Arg Leu Leu Val Thr Arg Asn Asp Pro Glu Leu Pro Ala Val Thr Ser
145 150 155 160
Ser Leu Glu Leu Ile Asp Leu His Phe Ser Asp Tyr Gly Thr Tyr Leu
165 170 175
Cys Met Ala Ser Phe Pro Gly Ala Pro Val Pro Asp Leu Ser Val Glu
180 185 190
Val Asn Ile Ser Ser Glu Thr Gly Val Pro Pro Thr Ile Ser Val Pro
195 200 205
Lys Gly Arg Ala Val Val Thr Val Arg Glu Gly Ser Pro Ala Glu Leu
14
CA 02399644 2002-08-02
WO 01/57175 PCT/USO1/03651
210 215 220
Gln Cys Glu Val Arg Gly Lys Pro Arg Pro Pro Val Leu Trp Ser Arg
225 230 235 240
Val Asp Lys Glu Ala Ala Leu Leu Pro Ser Gly Leu Pro Leu Glu Glu
245 250 255
Thr Pro Asp Gly Lys Leu Arg Leu Glu Arg Val Ser Arg Asp Met Ser
260 265 270
Gly Thr Tyr Arg Cys Gln Thr Ala Arg Tyr Asn Gly Phe Asn Val Arg
275 280 285
Pro Arg Glu Ala Gln Val Gln Leu Asn Val Gln
290 295
<210> 15
<211> 250
<212> PRT
<213> Rattus norvegicus
<400> 15
Gln Gly Glu Ser Ala Thr Leu Arg Cys Thr Ile Asp Asn Arg Val Thr
1 5 ZO 15
Arg Val Ala Trp Leu Asn Arg Ser Thr Ile Leu Tyr Ala Gly Asn Asp
20 25 30
Lys Trp Cys Leu Asp Pro Arg Val Val Leu Leu Ser Asn Thr Gln Thr
35 40 45
Gln Tyr Ser Ile Glu Ile Gln Asn Val Asp Val Tyr Asp Glu Gly Pro
50 55 60
Tyr Thr Cys Ser Val Gln Thr Asp Asn His Pro Lys Thr Ser Arg Val
65 70 75 80
His Leu Ile Val Gln Val Ser Pro Lys Ile Val Glu Ile Ser Ser Asp
85 90 95
Ile Ser Ile Asn Glu Gly Asn Asn Ile Ser Leu Thr Cys Ile Ala Thr
100 105 110
Gly Arg Pro Glu Pro Thr Val Thr Trp Arg His Ile Ser Pro Lys Ala
115 120 125
Val Gly Phe Val Ser Glu Asp Glu Tyr Leu Glu Ile Gln Gly Ile Thr
130 135 140
Arg Glu Gln Ser Gly Glu Tyr Glu Cys Ser Ala Ser Asn Asp Val Ala
l45 150 155 160
Ala Pro Val Val Arg Arg Val Asn Val Thr Val Asn Tyr Pro Pro Tyr
165 170 175
Ile Ser Glu Ala Lys Gly Thr Gly Val Pro Val Gly Gln Lys Gly Thr
180 185 190
Leu Gln Cys Glu Ala Ser Ala Val Pro Ser Ala Glu Phe Gln Trp Phe
195 200 205
Lys Asp Asp Lys Arg Leu Val Glu Gly Lys Lys Gly Val Lys Val Glu
CA 02399644 2002-08-02
WO 01/57175 PCT/USO1/03651
210 215 220
Asn Arg Pro Phe Leu Ser Arg Leu Thr Phe Phe Asn Val Ser Glu His
225 230 235 240
Asp Tyr Gly Asn Tyr Thr Cys Val Ala Ser
245 250
<210> 16
<211> 374
<212> PRT
<213> Homo Sapiens
<400> 16
Pro Leu Ser Arg Thr Ser Asp Arg Pro Leu Leu Leu Gln Ala Glu Thr
1 5 10 15
Ala Trp Gly Cys Arg Leu Lys Ser Ile Arg Val Asp Val Gln Tyr Leu
20 25 30
Asp Glu Pro Met Leu Thr Val His Gln Thr Val Ser Asp Val Arg Gly
35 40 45
Asn Phe Tyr Gln Glu Lys Thr Val Phe Leu Arg Cys Thr Val Asn Ser
50 55 60
Asn Pro Pro Ala Arg Phe Ile Trp Lys Arg Gly Ser Asp Thr Leu Ser
65 70 75 80
His Ser Gln Asp Asn Gly Val Asp Ile Tyr Glu Pro Leu Tyr Thr Gln
85 90 95
Gly Glu Thr Lys Val Leu Lys Leu Lys Asn Leu Arg Pro Gln Asp Tyr
100 105 110
Ala Ser Tyr Thr Cys Gln Val Ser Val Arg Asn Val Cys Gly Ile Pro
115 120 125
Asp Lys Ala Ile Thr Phe Arg Leu Thr Asn Thr Thr Ala Pro Pro Ala
130 135 140
Leu Lys Leu Ser Val Asn Glu Thr Leu Val Val Asn Pro Gly Glu Asn
145 150 155 160
Val Thr Val Gln Cys Leu Leu Thr Gly Gly Asp Pro Leu Pro Gln Leu
165 170 175
Gln Trp Ser His Gly Pro Gly Pro Leu Pro Leu Gly Ala Leu Ala Gln
180 185 190
Gly Gly Thr Leu Ser Ile Pro Ser Val Gln Ala Arg Asp Ser Gly Tyr
195 200 205
Tyr Asn Cys Thr Ala Thr Asn Asn Val Gly Asn Pro Ala Lys Lys Thr
210 215 220
Val Asn Leu Leu Val Arg Ser Met Lys Asn Ala Thr Phe Gln Ile Thr
225 230 235 240
Pro Asp Val Ile Lys Glu Ser Glu Asn Ile Gln Leu Gly Gln Asp Leu
245 250 255
Lys Leu Ser Cys His Val Asp Ala Val Pro Gln Glu Lys Val Thr Tyr
16
CA 02399644 2002-08-02
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260 265 270
Gln Trp Phe Lys Asn Gly Lys Pro Ala Arg Met Ser Lys Arg Leu Leu
275 280 285
Val Thr Arg Asn Asp Pro Glu Leu Pro Ala Val Thr Ser Ser Leu Glu
290 295 300
Leu Ile Asp Leu His Phe Ser Asp Tyr Gly Thr Tyr Leu Cys Met Ala
305 310 315 320
Ser Phe Pro Gly Ala Pro Val Pro Asp Leu Ser Val Glu Val Asn Ile
325 330 335
Ser Ser Glu Thr Val Pro Pro Thr Ile Ser Val Pro Lys Gly Arg AIa
340 345 350
Val Val Thr Val Arg Glu Gly Ser Pro Ala Glu Leu Gln Cys Glu Val
355 360 365
Arg Gly Lys Pro Arg Pro
370
17
