U.S. patent application number 12/046076 was filed with the patent office on 2008-08-21 for il-8 like protein.
Invention is credited to Mark Douglas Davies, Richard Fagan, Christopher Benjamin Phelps, Christine Power.
Application Number | 20080199435 12/046076 |
Document ID | / |
Family ID | 9942592 |
Filed Date | 2008-08-21 |
United States Patent
Application |
20080199435 |
Kind Code |
A1 |
Fagan; Richard ; et
al. |
August 21, 2008 |
IL-8 Like Protein
Abstract
This present invention relates to a novel protein, termed
INSP085, herein identified as an IL-8 like protein and to the use
of this protein and nucleic acid sequence from the encoding genes
in the diagnosis, prevention and treatment of disease.
Inventors: |
Fagan; Richard; (London,
GB) ; Phelps; Christopher Benjamin; (London, GB)
; Davies; Mark Douglas; (London, GB) ; Power;
Christine; (Thoiry, FR) |
Correspondence
Address: |
FROMMER LAWRENCE & HAUG
745 FIFTH AVENUE- 10TH FL.
NEW YORK
NY
10151
US
|
Family ID: |
9942592 |
Appl. No.: |
12/046076 |
Filed: |
March 11, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11062093 |
Feb 18, 2005 |
7341851 |
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12046076 |
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PCT/IB02/05490 |
Dec 19, 2002 |
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11062093 |
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Current U.S.
Class: |
424/93.2 ;
435/243; 435/320.1; 514/44R; 536/23.5; 536/24.3 |
Current CPC
Class: |
A61P 3/00 20180101; A61P
19/02 20180101; A61P 1/00 20180101; A61P 37/00 20180101; A61P 9/00
20180101; A61P 15/00 20180101; A61P 25/00 20180101; A61P 35/00
20180101; A61P 31/00 20180101; A61P 29/00 20180101; C07K 14/521
20130101 |
Class at
Publication: |
424/93.2 ;
536/23.5; 435/320.1; 435/243; 514/44; 536/24.3 |
International
Class: |
A61K 35/12 20060101
A61K035/12; C07H 21/00 20060101 C07H021/00; C12N 15/63 20060101
C12N015/63; A61P 35/00 20060101 A61P035/00; A61P 29/00 20060101
A61P029/00; A61P 1/00 20060101 A61P001/00; A61P 9/00 20060101
A61P009/00; A61P 31/00 20060101 A61P031/00; A61P 19/02 20060101
A61P019/02; A61P 3/00 20060101 A61P003/00; A61P 25/00 20060101
A61P025/00; A61P 37/00 20060101 A61P037/00; A61P 15/00 20060101
A61P015/00; A61K 31/7052 20060101 A61K031/7052; C12N 1/00 20060101
C12N001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 19, 2002 |
GB |
0219303.5 |
Claims
1-2. (canceled)
3. A purified nucleic acid molecule which encodes a polypeptide,
wherein the polypeptide: (i) comprises the amino acid sequence as
recited in SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ
ID NO:10 and/or SEQ ID NO:12; (ii) is a fragment thereof which
functions as a member of the IL-8 like chemokine family, or having
an antigenic determinant in common with the polypeptide of (i): or
(iii) is a functional equivalent of (i) or (ii).
4. The purified nucleic acid molecule according to claim 3, which
comprises the nucleic acid sequence as recited in SEQ ID NO: 1, SEQ
ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, and/or SEQ ID
NO:11, or is a redundant equivalent or fragment thereof; or wherein
the nucleic acid molecule hybridizes under high stringency
conditions with a nucleic acid molecule that encodes the
polypeptide.
5. A vector comprising a nucleic acid molecule as recited in claim
3.
6. A host cell transformed with a vector according to claim 5.
7-8. (canceled)
9. A nucleic acid molecule according to claim 3, a vector
comprising a nucleic acid molecule according to claim 5, or a host
cell transformed with a vector according to claim 6, for use in
therapy or diagnosis of disease.
10-12. (canceled)
13. A pharmaceutical or a vaccine composition comprising a nucleic
acid molecule according to claim 3, a vector comprising a nucleic
acid molecule according to claim 5, or a host cell transformed with
a vector according to claim 6.
14. A nucleic acid molecule according to claim 3, a vector
comprising a nucleic acid molecule according to claim 1 according
to claim 5, or a host cell transformed with a vector according to
claim 6, or a pharmaceutical composition comprising any of the
above, for use in the manufacture of a medicament for the treatment
of reproductive disorders, including infertility, cell
proliferative disorders, including neoplasm, melanoma, lung,
colorectal, breast, pancreas, head and neck and other solid
tumours; myeloproliferative disorders, such as leukemia,
non-Hodgkin lymphoma, leukopenia, thrombocytopenia, angiogenesis
disorder, Kaposis' sarcoma; autoimmune/inflammatory disorders,
including allergy, inflammatory bowel disease, arthritis, psoriasis
and respiratory tract inflammation, asthma, and organ transplant
rejection; cardiovascular disorders, including hypertension,
oedema, angina, atherosclerosis, thrombosis, sepsis, shock,
reperfusion injury, and ischemia; neurological disorders including
central nervous system disease, Alzheimer's disease, brain injury,
amyotrophic lateral sclerosis, and pain; developmental disorders;
metabolic disorders including diabetes mellitus, osteoporosis, and
obesity, AIDS and renal disease; infections including viral
infection, bacterial infection, fungal infection, parasitic
infection, rheumatoid arthritis (RA), psoriatic arthritis,
osteoarthritis, systemic lupus erythematosus (SLE), systemic
sclerosis, scleroderma, polymyositis, glomerulonephritis, fibrosis,
lung fibrosis and inflammation, allergic, or hypersensitivity
diseases, dermatitis, asthma, chronic obstructive pulmonary
disease, (COPD), Crohn's disease, ulcerative colitis, multiple
sclerosis, septic shock, HIV infection, transplant rejection, wound
healing, metastasis, endometriosis, hepatitis, liver fibrosis,
cancer, analgesia, and vascular inflammation related to
atherosclerosis, wherein said disease is a disease in which IL-8
like chemokines are implicated, and other pathological
conditions.
15-17. (canceled)
18. A kit, wherein: a) the kit is useful for diagnosing disease
comprising a first container containing a nucleic acid probe that
hybridises under stringent conditions with a nucleic acid molecule
according to claim 3; a second container containing primers useful
for amplifying said nucleic acid molecule; and instructions for
using the probe and primers for facilitating the diagnosis of
disease, wherein the kit optionally further comprises a third
container holding an agent for digesting unhybridized RNA; or b)
the kit comprises an array of nucleic acid molecules, at least one
of which is a nucleic acid molecule according to claim 3.
19-20. (canceled)
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of International
Patent Application PCT/GB2003/003646 filed Aug. 19, 2003 and
published as WO 2004/016654 on Feb. 26, 2004, which claims priority
from Great Britain Application 0219303.5 filed Aug. 19, 2002. Each
of the above referenced applications, and each document cited in
this text ("application cited documents") and each document cited
or referenced in each of the application cited documents, and any
manufacturer's specifications or instructions for any products
mentioned in this text and in any document incorporated into this
text, are hereby incorporated herein by reference; and, technology
in each of the documents incorporated herein by reference can be
used in the practice of this invention.
[0002] It is noted that in this disclosure, terms such as
"comprises", "comprised", "comprising", "contains", "containing"
and the like can have the meaning attributed to them in U.S. patent
law; e.g., they can mean "includes", "included", "including" and
the like. Terms such as "consisting essentially of" and "consists
essentially of" have the meaning attributed to them in U.S. patent
law, e.g., they allow for the inclusion of additional ingredients
or steps that do not detract from the novel or basic
characteristics of the invention, i.e., they exclude additional
unrecited ingredients or steps that detract from novel or basic
characteristics of the invention, and they exclude ingredients or
steps of the prior art, such as documents in the art that are cited
herein or are incorporated by reference herein, especially as it is
a goal of this document to define embodiments that are patentable,
e.g., novel, nonobvious, inventive, over the prior art, e.g., over
documents cited herein or incorporated by reference herein. And,
the terms "consists of" and "consisting of" have the meaning
ascribed to them in U.S. patent law; namely, that these terms are
closed ended.
[0003] This invention relates to a novel protein, termed INSP085,
herein identified as a secreted protein, in particular, as a member
of the Interleukin (IL) 8-like chemokine family and to the use of
this protein and nucleic acid sequence from the encoding gene in
the diagnosis, prevention and treatment of disease.
[0004] All publications, patents and patent applications cited
herein are incorporated in full by reference.
BACKGROUND
[0005] The process of drug discovery is presently undergoing a
fundamental revolution as the era of functional genomics comes of
age. The term "functional genomics" applies to as approach
utilising bioinformatics tools to ascribe function to protein
sequences of interest. Such tools are becoming increasingly
necessary as the speed of generation of sequence data is rapidly
outpacing the ability of research laboratories to assign functions
to these protein sequences.
[0006] As bioinformatics tools increase in potency and in accuracy,
these tools are rapidly replacing the conventional techniques of
biochemical characterisation. Indeed, the advanced bioinformatics
tools used in identifying the present invention are now capable of
outputting results in which a high degree of confidence can be
placed.
[0007] Various institutions and commercial organizations are
examining sequence data as they become available and significant
discoveries are being made on an on-going basis. However, there
remains a continuing need to identify and characterise further
genes and the polypeptides that they encode, as targets for
research and for drug discovery.
INTRODUCTION
Secreted Proteins
[0008] The ability for cells to make and secrete extracellular
proteins is central to many biological processes. Enzymes, growth
factors, extracellular matrix proteins and signalling molecules are
all secreted by cells. This is through fission of a secretory
vesicle with the plasmas membrane. In most cases, but not all,
proteins are directed to the endoplasmic reticulum and into
secretory vesicles by a signal peptide. Signal peptides are
cis-acting sequences that affect the transport of polypeptide
chains from the cytoplasm to a membrane bound compartment such as a
secretory vesicle. Polypeptides that are targeted to the secretory
vesicles are either secreted into the extracellular matrix or are
retained in the plasma membrane. The polypeptides that are retained
in the plasma membrane will have one or more transmembrane domains.
Examples of secreted proteins that play a central role in the
functioning of a cell are cytokines, hormones, extracellular matrix
proteins (adhesion molecules), proteases, and growth and
differentiation factors. Description of some of the properties of
these proteins follows.
Chemokines
[0009] These signalling molecules are distinct from cytokines and
are responsible for inducing chemotaxis or directed migration. They
are highly specific, a fact which is illustrated by the fact that
IL-8 is chemotactic to granulocytes but not monocytes. Chemokines
contain four conserved cysteine residues and are divided into three
families, .alpha.(CXC), .beta.(CC) and .gamma.(C), based on the
position of conserved cysteine residues. If the first two cysteines
are separated by another amino acid, then the chemokine is a member
of the .alpha. family, while the first two cysteine residues are
next to each other in the .beta. family members. Members of the
.gamma. family only have one cysteine residue, rather than two, in
their N-terminus. In the .alpha. and .beta. families, disulphide
bonds are formed between the first and third and the second and
fourth residues.
[0010] Specificity of chemokines depends on the presence of
specific receptors on cell surfaces. Chemokines have been shown to
play a role in the migration of leukocytes. Upon activation,
remodeling of the cytoskeleton of leukocytes is induced allowing
the cell to flatten and pass from an intravascular space into a
tissue space. Interaction of chemokines with seven-transmembrane
G-protein coupled receptors leads to rapid accumulation of
intracellular free calcium in the responding cells. This
mobilization is critical for chemotaxis, respiratory burst and
upregulation of adhesive interactions of leukocytes. Chemokines
have also been shown to regulate the expression of adhesion
molecules on neutrophils, monocytes, lymphocytes and eosinophils.
For example, MIP-1.alpha. and RANTES cause adhesion of monocytes to
endothelium while MIP-1.beta. induces CD8.sup.+ T-cell adhesion to
endothelium.
[0011] Increasing knowledge of these domains is therefore of
extreme importance in increasing the understanding of the
underlying pathways that lead to the disease states and associated
disease states mentioned above, and in developing more effective
gene and/or drug therapies to treat these disorders.
THE INVENTION
[0012] The invention is based on the discovery that the INSP085
polypeptide is an IL-8 like chemokine.
[0013] In one embodiment of the first aspect of the invention,
there is provided a polypeptide which: [0014] (i) comprises the
amino acid sequence as recited in SEQ ID NO:2, SEQ ID NO:4, SEQ ID
NO:6, SEQ ID NO:8, SEQ ID NO10 and/or SEQ ID NO:12; [0015] (ii) is
a fragment thereof which functions as a member IL-8 like chemokine
family, or has an antigenic determinant in common with the
polypeptides of (i); or [0016] (iii) is a functional equivalent of
(i) or (ii).
[0017] Preferably, the polypeptide according to this first aspect
of the invention: [0018] (i) comprises the amino acid sequence as
recited in SEQ ID NO:8, SEQ ID NO:10 and/or SEQ ID NO:12; [0019]
(ii) is a fragment thereof which functions as a member of the IL-8
like chemokine family, or has an antigenic determinant in common
with the polypeptides of (i); or [0020] (iii) is a functional
equivalent of (i) or (ii).
[0021] According to a second embodiment of this first aspect of the
invention, there is provided a polypeptide which: [0022] (i)
consists of the amino acid sequence as recited in SEQ ID NO:2, SEQ
ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and/or SEQ ID
NO:12; [0023] (ii) is a fragment thereof which functions as a
member of the IL-8 like chemokine family, or having an antigenic
determinant in common with the polypeptides of (i); or [0024] (iii)
is a functional equivalent of (i) or (ii).
[0025] The polypeptide having the sequence recited in SEQ ID NO:2
is referred to hereafter as "INSP085 exon 1 polypeptide". The
polypeptide having the sequence recited in SEQ ID NO:4 is referred
to hereafter as "INSP085 exon 2 polypeptide". The polypeptide
having the sequence recited in SEQ ID NO:6 is referred to hereafter
as "INSP085 exon 3 polypeptide". The polypeptide having the
sequence recited in SEQ ID NO:8 is referred to hereafter as the
"INSP085 polypeptide".
[0026] The term "INSP085 polypeptides" as used herein includes
polypeptides comprising the INSP085 exon 1 polypeptide, the INSP085
exon 2 polypeptide, the INSP085 exon 3 polypeptide and the NSP085
polypeptide.
[0027] Although the Applicant does not wish to be bound by this
theory, it is postulated that the first 16 amino acids of the
INSP085 exon 1 polypeptide form a signal peptide.
[0028] The INSP085 exon 1 and full length INSP085 polypeptide
sequences without this postulated signal sequence are recited in
SEQ ID NO:10 and SEQ ID NO:12 respectively.
[0029] The polypeptide having the sequence recited in SEQ ID NO:10
is referred to hereafter as "the INSP085 exon 1 mature
polypeptide". The polypeptide having the sequence recited in SEQ ID
NO:12 is referred to hereafter as "the INSP085 mature
polypeptide".
[0030] By "functions as a member of the IL 8 like chemokine family"
we refer to polypeptides that comprise amino acid sequence or
structural features that can be identified as conserved features
within the polypeptides of the IL-8 like chemokine family, such
that the polypeptide's interaction with ligand is not substantially
affected detrimentally in comparison to the function of the full
length wild type polypeptide. In particular, we refer to the
presence of cysteine residues in specific positions within the
polypeptide that allow the formation of intra-domain disulphide
bonds.
[0031] Studies on structure-activity relationships indicate that
chemokines bind and activate receptors by making use of the
amino-terminal region. Proteolytic digestion, mutagenesis, or
chemical modifications directed to amino acids in this region can
generate compounds having antagonistic activity (Loetscher P and
Clark-Lewis I, J Leukoc Biol, 69: 881-884, 2001 Lambeir A, et al. J
Biol Chem, 276: 29839-29845, 2001, Proost P, et al. Blood, 98
(13):3554-3561, 2001). Thus, antagonistic molecules resulting from
specific modifications (deletions, non-conservative substitutions)
of on-e or more residues in the amino-terminal region or in other
regions of the corresponding chemokine are considered having
therapeutic potential for inflammatory and autoimmune diseases (WO
02/28419; WO 00/27880; WO 99/33989; Schwarz M K and Wells T N, Curr
Opin Chem Biol, 3: 407-17, 1999). Therefore, a further object of
the present patent application is represented by such kind of
antagonists generated by modifying the polypeptides of the
invention.
[0032] The therapeutic applications of the polypeptides of the
invention and of the related reagents can be evaluated (in terms of
safety, pharmacokinetics and efficacy) by the means of the in
vivo/in vitro assays making use of animal cell, tissues and models
(Coleman R A et al., Drug Discov Today, 6: 1116-1126, 2001; Li AP,
Drug Discov Today, 6: 357-366, 2001; Methods Mol. Biol. vol. 138,
"Chemokines Protocols", edited by Proudfoot A I et al., Humana
Press Inc., 2000; Methods Enzymol, vol. 287 and 288, Academic
Press, 1997), or by the means of in silico/computational approaches
(Johnson D E and Wolfgang G H, Drug Discov Today, 5: 445-454,
2000), known for the validation of chemokines and other biological
products during drug discovery and preclinical development.
[0033] The present application discloses novel chemokine-like
polypeptides ad a series of related reagents that may be useful, as
active ingredients in pharmaceutical compositions appropriately
formulated, in the treatment or prevention of diseases such as cell
proliferative disorders, autoimmune/inflammatory disorders,
cardiovascular disorders, neurological disorders, developmental
disorders, metabolic disorder, infections and other pathological
conditions. In particular, given the known properties of
chemokines, the disclosed polypeptides and reagents should address
conditions involving abnormal or defective cell migration.
Non-limitative examples of such conditions are the following:
arthritis, rheumatoid arthritis (RA), psoriatic arthritis,
osteoarthritis, systemic lupus erythematosus (SLE), systemic
sclerosis, scleroderma, polymyositis, glomerulonephritis, fibrosis,
lung fibrosis and inflammation, allergic or hypersensitivity
diseases, dermatitis, asthma, chronic obstructive pulmonary disease
(COPD), inflammatory bowel disease (IBD), Crohn's disease,
ulcerative colitis, multiple sclerosis, septic shock, HIV
infection, transplant rejection, wound healing, metastasis,
endometriosis, hepatitis, liver fibrosis, cancer, analgesia, and
vascular inflammation related to atherosclerosis.
[0034] Several assays have been developed for testing specificity,
potency, and efficacy of chemokines using cell cultures or animal
models, for example in vitro chemotaxis assays (Proudfoot A, et al.
J Biol Chem 276: 10620-10626, 2001; Lusti-Naurasimhan M et al., J
Biol Chem, 270: 2716-21, 1995), or mouse ear swelling (Garrigue J L
et al., Contact Dermatitis, 30: 231-7, 1994). Many other assays and
technologies for generating useful tools and products (antibodies,
transgenic animals, radiolabeled proteins, etc.) have been
described in reviews and books dedicated to chemokines (Methods
Mol. Biol vol. 138, "Chemokines Protocols", edited by Proudfoot A I
et al., Humana Press Inc., 2000; Methods Enzymol, vol. 287 and 288,
Academic Press, 1997), and can be used to verify, in a more precise
manner, the biological activities of the chemokine-like
polypeptides of the invention and related reagents in connection
with possible therapeutic or diagnostic methods and uses.
[0035] The following in vitro cell-based tri-replicas assays
measure the effects of the protein of the invention on cytokine
secretion induced by Concanavalin A (Con A) acting on different
human peripheral blood mononuclear cells (hPBMC) cells as measured
by a cytokine bead array (CBA) assay for IL-2, IFN-.gamma.,
TNF-.alpha., IL-5, IL-4 and IL-10 such as the Human Th1/Th2
Cytokine CBA kit (Becton-Dickinson).
[0036] The optimal conditions are 100000 cells/well in 96-well
plates and 100 .mu.l final in 2% glycerol. The optimal
concentration of mitogen (ConA) is 5 ng/ml. The optimal time for
the assay is 48 h. The read-out choice is the CBA.
1 Purification of Human PBMC from a Buffy Coat
[0037] The buffy coat 1 to 2 is diluted with DMEM. 25 ml of diluted
blood was thereafter slowly added onto a 15 ml layer of Ficoll in a
50 ml Falcon tube, and tubes are centrifuged (2000 rpm, 20 min, at
RT without brake). The interphase (ring) is then collected and the
cells are washed with 25 ml of DMEM followed by a centrifuge step
(1200 rpm, 5 min). This procedure is repeated three times. A but
coat gives approximately 600.times.10.sup.6 total cells.
2 Screening
[0038] 80 .mu.l of 1.25.times.10.sup.6 cells/ml are diluted in
DMEM+2.5% Human Serum+1% L-Glutamine+1% Penicillin-Streptomycin and
thereafter added to a 96 well microtiter plate.
[0039] 10 .mu.l are added per well (one condition per well):
Proteins were diluted in PBS+20% Glycerol (the final dilution of
the proteins is 1/10).
[0040] 10 .mu.l of the ConA Stimulant (50 .mu.g/ml) are then added
per well (one condition per well--the final concentration of ConA
is 5 .mu.g/ml)
[0041] After 48 h, cell supernatants are collected and human
cytokines are measured by Human Th1/Th2 Cytokine CBA Kit
Becton-Dickinson.
3 CBA Analysis
[0042] (for more details, refer to the manufacturer's instructions
in the CBA kit) i) Preparation of mixed Human Th1/Th2 Capture
Beads
[0043] The number of assay tubes that are required for the
experiment are determined.
[0044] Each capture bead suspension is vigorously vortexed for a
few seconds before mixing. For each assay to be analysed, 10 .mu.l
aliquot of each capture bead are added into a single tube labelled
"mixed capture beads". The Bead mixture is thoroughly vortexed.
ii) Preparation of Test Samples
[0045] Supernatants are diluted (1:4) using the Assay Diluent (20
.mu.l of supernatants+60 .mu.l of Assay Diluent) The sample
dilution is then mixed before transferring samples into a 96 well
conical bottomed microtiter plate (Nunc).
iii) Human Th1/Th2 Cytokine CBA Assay Procedure
[0046] 50 .mu.l of the diluted supernatants are added into a 96
well conical bottomed microtiter plate (Nunc). 50 .mu.l of the
mixed capture beads are added followed by 50 .mu.l addition of the
Human Th1/Th2 PE Detection Reagent. The plate is then incubated for
3 hours at RT and protected from direct exposure to light followed
by centrifugation at 1500 rpm for 5 minutes. The supernatant is
then carefully discarded. In a subsequent step, 200 .mu.l of wash
buffer are twice added to each well, centrifuged at 1500 rpm for 5
minutes and supernatant carefully discarded. 130 .mu.l of wash
buffer are thereafter added to each well to resuspended the bead
pellet. The samples are finally analysed on a flow cytometer. The
data are then analysed using the CBA Application Software, Activity
Base and Microsoft Excel software.
[0047] From the read-out of the assay it can be evaluated whether
in vitro, the protein of the invention has a consistent inhibitory
effect on all cytokines tested (IFN-.gamma., TNF-.alpha., IL-2,
IL-4, IL-S, IL-10).
[0048] Moreover, based on the EC50 value, it can be easily
evaluated which cytokine is inhibited the most and then derive the
specific auto-immune/inflammatory disease, which is known to be
particularly linked to that cytokine.
[0049] In a second aspect, the invention provides a purified
nucleic acid molecule which encodes a polypeptide of the first
aspect of the invention.
[0050] Preferably, the purified nucleic acid molecule comprises the
nucleic acid sequence as recited in SEQ ID NO:1 (encoding the
INSP085 exon 1 polypeptide), SEQ ID NO:3 (encoding the INSP085 exon
2 polypeptide), SEQ ID NO:5 (encoding the INSP085 exon 3
polypeptide) and/or SEQ ID NO:7 (encoding the INSP085 polypeptide)
or is a redundant equivalent or fragment of any one of these
sequences.
[0051] The invention further provides that the purified nucleic
acid molecule consists of the nucleic acid sequence as recited in
SEQ ID NO:1 (encoding the INSP085 exon 1 polypeptide), SEQ ID NO:3
(encoding the INSP085 exon 2 polypeptide), SEQ ID NO:5 (encoding
the INSP085 exon 3 polypeptide) and/or SEQ ID NO:7 (encoding the
INSP085 polypeptide) or is a redundant equivalent or fragment of
any one of these sequences.
[0052] The polypeptide having the sequence recited in SEQ ID NO:9
is referred to hereafter as "the INSP085 exon 1 mature nucleotide
sequence" and encodes the INSP085 exon 1 mature polypeptide. The
polypeptide having the sequence recited in SEQ ID NO:11 is referred
to hereafter as "the INSP08-5 mature nucleotide sequence" and
encodes the INSP085 mature polypeptide.
[0053] In a third aspect, the invention provides a purified nucleic
acids molecule which hybridizes under high stringency conditions
with a nucleic acid molecule of the second aspect of the
invention.
[0054] In a fourth aspect, the invention provides a vector, such as
an expression vector, that contains a nucleic acid molecule of the
second or third aspect of the invention.
[0055] In a fifth aspect, the invention provides a host cell
transformed with a vector of the fourth aspect of the
invention.
[0056] In a sixth aspect, the invention provides a ligand which
binds specifically to protein members of the IL-8 like chemokine
family of the first aspect of the invention. Preferably, the ligand
inhibits the function of a polypeptide of the first aspect of the
invention which is a member of the IL-8 like chemokine family or
proteins Ligands to a polypeptide according to the invention may
come in various forms, including natural or modified substrates,
enzymes, receptors, small organic molecules such as small natural
or synthetic organic molecules of up to 2000 Da, preferably 800 Da
or less, peptidomimetics, inorganic molecules, peptides,
polypeptides, antibodies, structural or functional mimetics of the
aforementioned.
[0057] In a seventh aspect, the invention provides a compound that
is effective to alter the expression of a natural gene which
encodes a polypeptide of the first aspect of the invention or to
regulate the activity of a polypeptide of the first aspect of the
invention.
[0058] A compound of the seventh aspect of the invention Hay either
increase (agonize) or decrease (antagonize) the level of expression
of the gene or the activity of the polypeptide.
[0059] Importantly, the identification of the function of the
INSP085 polypeptides allows for the design of screening methods
capable of identifying compounds that are effective in the
treatment and/or diagnosis of disease. Ligands and compounds
according to the sixth and seventh aspects of the invention may be
identified using such methods. These methods are included as
aspects of the present invention.
[0060] In an eighth aspect, the invention provides a polypeptide of
the first aspect of the invention, or a nucleic acid molecule of
the second or bird aspect of the invention, or a vector of the
fourth aspect of the invention, or a host cell of the fifth aspect
of the invention, or a ligand of the sixth aspect of the invention,
or a compound of the seventh aspect of the invention, for use in
therapy or diagnosis of diseases in which members of the IL-8 like
chemokine family are implicated. Such diseases may include cell
proliferative disorders, including neoplasm, melanoma, lung,
colorectal, breast, pancreas, head and neck and other solid
tumours; myeloproliferative disorders, such as leukemia,
non-Hodgkin lymphoma, leukopenia, thrombocytopenia, angiogenesis
disorder, Kaposis' sarcoma, autoimmune/inflammatory disorders,
including allergy, inflammatory bowel disease, arthritis, psoriasis
and respiratory tract inflammation, asthma, and organ transplant
rejection; cardiovascular disorders, including hypertension,
oedema, angina, atherosclerosis, thrombosis, sepsis, shock,
reperfusion injury, and ischemia; neurological disorders including
central nervous system disease, Alzheimer's disease, brain injury,
amyotrophic lateral sclerosis, and pain, developmental disorders;
metabolic disorders including diabetes mellitus, osteoporosis, and
obesity, AIDS and renal disease; infections including viral
infection, bacterial infection, fungal infection and parasitic
infection and other pathological conditions. Preferably, the
disease is one in which the IL-8 like chemokine family is
implicated, such as arthritis, rheumatoid arthritis (RA), psoriatic
arthritis, osteoarthritis, systemic lupus erythematosus (SLE),
systemic sclerosis, scleroderma, polymyositis, glomerulonephritis,
fibrosis, lung fibrosis and inflammation, allergic or
hypersensitivity diseases, dermatitis, asthma, chronic obstructive
pulmonary disease (COPD), inflammatory bowel disease (IBD), Crohn's
diseases, ulcerative colitis, multiple sclerosis, septic shock, HIV
infection, transplant rejection, wound healing, metastasis,
endometriosis, hepatitis, liver fibrosis, cancer, analgesia, and
vascular inflammation related to atherosclerosis. These molecules
may also be used in the manufacture of a medicament for the
treatment of such diseases. These molecules may also be used in
contraception or for the treatment of reproductive disorders
including infertility.
[0061] In a ninth aspect, the invention provides a method of
diagnosing a disease in a patient, comprising assessing the level
of expression of a natural gene encoding a polypeptide of the first
aspect of the invention or the activity of a polypeptide of the
first aspect of the invention in tissue from said patient and
comparing said level of expression or activity to a control level,
wherein a level that is different to said control level is
indicative of disease. Such a method will preferably be carried out
in vitro. Similar methods may be used for monitoring the
therapeutic treatment of disease in a patient, wherein altering the
level of expression or activity of a polypeptide or nucleic acid
molecule over the period of time towards a control level is
indicative of regression of disease.
[0062] A preferred method for detecting polypeptides of the first
aspect of the invention comprises the steps of: (a) contacting a
ligand, such as an antibody, of the sixth aspect of the invention
with a biological sample under conditions suitable for the
formation of a ligand-polypeptide complex; and (b) detecting said
complex.
[0063] A number of different such methods according to the ninth
aspect of the invention exist, as the skilled reader will be aware,
such as methods of nucleic acid hybridization with short probes,
point mutation analysis, polymerase chain reaction (PCR)
amplification and methods using antibodies to detect aberrant
protein levels. Similar methods may be used on a short or long term
basis to allow therapeutic treatment of a disease to be monitored
in a patient. The invention also provides kits that are useful in
these methods for diagnosing disease.
[0064] In a tenth aspect, the invention provides for the use of a
polypeptide of the first aspect of the invention as an IL-8 like
chemokine. Suitable uses of the polypeptides of the invention as
IL-8 like chemokine proteins include use as a regulator of cellular
growth, metabolism or differentiation, use as part of a
receptor/ligand pair and use as a diagnostic marker for a
physiological or pathological condition selected from the list
given above.
[0065] In an eleventh aspect, the invention provides a
pharmaceutical composition comprising a polypeptide of the first
aspect of the invention, or a nucleic acid molecule of the second
or third aspect of the invention, or a vector of the fourth aspect
of the invention, or a host cell of the fifth aspect of the
invention, or a ligand of the sixth aspect of the invention, or a
compound of the seventh aspect of the invention, in conjunction
with a pharmaceutically-acceptable carrier.
[0066] In a twelfth aspect, the present invention provides a
polypeptide of the first aspect of the invention, or a nucleic acid
molecule of the second or third aspect of the inventions or a
vector of the fourth aspect of the invention, or a host cell of the
fifth aspect of the invention, or a ligand of the sixth aspect of
the invention, or a compound of the seventh aspect of the
invention, for use in the manufacture of a medicament for the
diagnosis or treatment of a disease.
[0067] In a thirteenth aspect, the invention provides a method of
treating a disease in a patient comprising administering to the
patient a polypeptide of the first aspect of the invention, or a
nucleic acid molecule of the second or third aspect of the
invention, or a vector of the fourth aspect of the invention, or a
host cell of the fifth aspect of the invention, or a ligand of the
sixth aspect of the invention, or a compound of the seventh aspect
of the invention.
[0068] For diseases in which the expression of a natural gene
encoding a polypeptide of the first aspect of the invention, or in
which the activity of a polypeptide of the first aspect of the
invention, is lower in a diseased patient when compared to the
level of expression or activity in a healthy patient, the
polypeptide, nucleic acid molecule, ligand or compound administered
to the patient should be an agonist. Conversely, for diseases in
which the expression of the natural gene or activity of the
polypeptide is higher in a diseased patient when compared to the
level of expression or activity in a healthy patient, the
polypeptide, nucleic acid molecule, ligand or compound administered
to the patient should be an antagonist Examples of such antagonists
include antisense nucleic acid molecules, ribozymes and ligands,
such as antibodies.
[0069] In a fourteenth aspect, the invention provides transgenic or
knockout non-human animals that have been transformed to express
higher, lower or absent Levels of a polypeptide of the first aspect
of the invention. Such transgenic animals are very useful models
for the study of disease and may also be used in screening requires
for the identification of compounds that are effective in the
treatment or diagnosis of such a disease.
[0070] A summary of standard techniques and procedures which may be
employed in order to utilise the invention is given below. It will
be understood that this invention is not limited to the particular
methodology, protocols, cell lines, vectors and reagents described.
It is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments only and it is not
intended that this terminology should limit the scope of the
present invention. The extent of the invention is limited only by
the terms of the appended claims.
[0071] Standard abbreviations for nucleotides and amino acids are
used in this specification.
[0072] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology,
microbiology, recombinant DNA technology and immunology, which are
within the skill of those working in the art.
[0073] Such techniques are explained fully in the literature.
Examples of particularly suitable texts for consultation include
the following: Sambrook Molecular Cloning; A Laboratory Manual,
Second Edition (1989); DNA Cloning, Volumes I and II (D. N Glover
ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic
Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984);
Transcription and Translation (B. D. Hames & S. J. Higgins eds.
1984); Animal Cell Culture (R. I. Freshney ed. 1986); Immobilized
Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide
to Molecular Cloning (1984); the Methods in Enzymology series
(Academic Press, Inc.), especially volumes 154 & 155; Gene
Transfer Vectors for Mammalian Cells (J. H. Miller and M. P. Calos
eds. 1987, Cold Spring Harbor Laboratory); Immunochemical Methods
in Cell and Molecular Biology (Mayer and Walker, eds. 1987,
Academic Press, London); Scopes, (1987) Protein Purification:
Principles and Practice, Second Edition (Springer Verlag, N.Y.);
and Handbook of Experimental Immunology, Volumes I-IV (D. M. Weir
and C. C. Blackwell eds. 1986).
[0074] As used herein, the term "polypeptide" includes any peptide
or protein comprising two or more amino acids joined to each other
by peptide bonds or modified peptide bonds, i.e. peptide isosteres.
This term refers both to short chains (peptides and oligopeptides)
and to longer chains (proteins).
[0075] The polypeptide of the present invention may be in the form
of a mature protein or may be a pre-, pro- or prepro-protein that
can be activated by cleavage of the pre-, pro- or prepro-portion to
produce an active mature polypeptide. In such polypeptides, the
pre-, pro- or prepro-sequence may be a leader or secretory sequence
or may be a sequence that is employed for purification of the
mature polypeptide sequence.
[0076] The polypeptide of the first aspect of the invention may
form part of a fusion protein. For example, it is often
advantageous to include one or more additional amino acid sequences
which may contain secretory or leader sequences, pro-sequences,
sequences which aid in purification, or sequences that confer
higher protein stability, for example during recombinant
production. Alternatively or additionally, the mature polypeptide
may be fused with another compound, such as a compound to increase
the half-life of the polypeptide (for example, polyethylene
glycol).
[0077] Polypeptides may contain amino acids other to the 20
gene-encoded amino acids, modified either by natural processes,
such as by post-translational processing or by chemical
modification techniques which are well known in the art. Among the
known modifications which may commonly be present in polypeptides
of the present invention are glycosylation, lipid attachment,
sulphation, gamma-carboxylation, for instance of glutamic acid
residues, hydroxylation and ADP-ribosylation. Other potential
modifications include acetylation, acylation, amidation, covalent
attachment of flavin, covalent attachment of a haeme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid derivative, covalent attachment of
phosphatidylinositol, cross-lining, cyclization, disulphide bond
formation, demethylation, formation of covalent cross-links,
formation of cysteine, formation of pyroglutamate, formation, GPI
anchor formation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, transfer-RNA mediated addition of
amino acids to proteins such as arginylation, and
ubiquitination.
[0078] Modifications can occur anywhere in a polypeptide, including
the peptide backbone, the amino acid side-chains and the amino or
carboxyl terminal. In fact, blockage of the amino or carboxyl
terminus in a polypeptide, or both by a covalent modification is
common in naturally-occurring and synthetic polypeptides and such
modifications may be present in polypeptides of the present
invention.
[0079] The modifications that occur in a polypeptide often will be
a function of how the polypeptide is made. For polypeptides that
are made recombinantly, the nature and extent of the modifications
in large part will be determined by the post-translational
modification capacity of the particular host cell and the
modification signals that are present in the ammo acid sequence of
the polypeptide in question. For instance, glycosylation patterns
vary between different types of host cell.
[0080] The polypeptides of the present invention can be prepared in
any suitable manner. Such polypeptides include isolated
naturally-occurring polypeptides (for example purified from cell
culture), recombinantly produced polypeptides including fusion
proteins), synthetically-produced polypeptides or polypeptides that
are produced by a combination of these methods.
[0081] The functionally-equivalent polypeptides of the first aspect
of the invention may be polypeptides that are homologous to the
INSP085 polypeptides. Two poly peptides are said to be
"homologous", as the term is used herein, if the sequence of one of
the polypeptides has a high enough degree of identity or similarity
to the sequence of the other polypeptide. "Identity" indicates that
at any particular position in the aligned sequences, the amino acid
residue is identical between the sequences. "Similarity" indicates
that, at any particular position in the aligned sequences, the
amino acid residue is of a similar type between the sequences.
Degrees of identity and similarity can be readily calculated
(Computational Molecular Biology, Lesk, A. M., ed., Oxford
University Press, New York, 1988; Biocomputing. Informatics and
Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;
Computer Analysis of Sequence Data, Part 1, Griffin, A. M., ad
Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence
Analysis in Molecular Biology, von Heinje, G., Academic Press,
1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J.,
eds., M Stockton Press, New York, 1991).
[0082] Homologous polypeptides therefore include natural biological
variants (for example, allelic variants or geographical variations
within the species from which the polypeptides are derived) and
mutants (such as mutants containing amino acid substitutions,
insertions or deletions) of the INSP085 polypeptides. Such mutants
may include polypeptides in which one or more of the amino acid
residues are substituted with a conserved or non-conserved amino
acid residue (preferably a conserved amino acid residue) and such
substituted amino acid residue may or may not be one encoded by the
genetic code. Typical such substitutions are among Ala, Val, Leu
and Ile; among Ser and Thr; among the acidic residues Asp and Glu;
among Asn and Gln; among the basic residues Lys and Arg; or among
the aromatic residues Phe and Tyr. Particularly preferred are
variants in which several, i.e. between 5 and 10, 1 and 5, 1 and 3,
1 and 2 or just 1 amino acids are substituted, deleted or added in
any combination Especially preferred are silent substitutions,
additions and deletions, which do mot alter the properties and
activities of the protein. Also especially preferred in this regard
are conservative substitutions. Such mutants also include
polypeptides in which one or more of the amino acid residues
includes a substituent group.
[0083] Typically, greater than 30% identity between two
polypeptides is considered to be an indication of functional
equivalence. Preferably, functionally equivalent polypeptides of
the first aspect of the invention have a degree of sequence
identity with the INSP085 polypeptide, or with active fragments
thereof, of greater than 80%. More preferred polypeptides have
degrees of identity of greater than 85%, 90%, 95%, 98% or 99%,
respectively.
[0084] The functionally-equivalent polypeptides of the first aspect
of the intention may also be polypeptides which have been
identified using one or more techniques of structural alignment.
For example, the Inpharmatica Genome Threader technology that forms
one aspect of the search tools used to generate the Biopendium.TM.
search database may be used (see PCT application WO 01/69507) to
identify polypeptides of presently-unknown function which, while
having low sequence identity as compared to the INSP085
polypeptides, are predicted to be members of the IL-8 like
chemokine family, by virtue of sharing significant structural
homology with the INSP085 polypeptide sequence. By "significant
structural homology" is meant that the Inpharmatica Genome Threader
predicts two proteins to share structural homology with a certainty
of 10% and above.
[0085] The polypeptides of the first aspect of the invention also
include fragments of the INSP085 polypeptides and fragments of the
functional equivalents of the INSP085 polypeptides, provided that
those fragments are members of the IL-8 like chemokine family or
have an antigenic determinant in common with the INSP085
polypeptides.
[0086] As used herein, the term "fragment" refers to a polypeptide
having an amino acid sequence that is the same as part, but not
all, of the amino acid sequence of the INSP085 polypeptide or one
of their functional equivalents. The fragments should comprise at
least n consecutive amino acids from the sequence and, depending on
the particular sequence, n preferably is 7 or more (for example, 8,
10, 12, 14, 16, 18, 20 or mere). Small fragments may form an
antigenic determinant.
[0087] Fragments of the full length INSP085 polypeptides may
consist of combinations of 2 or 3 of neighbouring exon sequences in
the INSP085 polypeptide sequences, respectively. For example, such
combinations include exons 1 and 2, 2 and 3 or 1, 2 and 3. Such
fragments are included in the present invention.
[0088] Such rents may be "free-standing", i.e. not part of or fused
to other amino acids or polypeptides, or they may be comprised
within a larger polypeptide of which they form a part or region.
When comprised within a larger polypeptide, the fragment of the
invention most preferably forms a single continuous region. For
instance, certain preferred embodiments relate to a fragment having
a pre- and/or pro-polypeptide region fused to the amino terminus of
the fragment and/or an additional region fused to the carboxyl
terminus of the fragment. However, several fragments may be
comprised within a single larger polypeptide.
[0089] The polypeptides of the present invention or their
immunogenic fragments (comprising at least one antigenic
determinant) can be used to generate ligands, such as polyclonal or
monoclonal antibodies, that are immunospecific for the
polypeptides. Such antibodies may be employed to isolate or to
identify clones expressing the polypeptides of the invention or to
purity the polypeptides by affinity chromatography. The antibodies
may also be employed as diagnostic or therapeutic aids, amongst
other applications, as will be apparent to the skilled reader.
[0090] The term "immunospecific" means that the antibodies have
substantially greater affinity for the polypeptides of the
invention than their affinity for other related polypeptides in the
prior art. As used herein, the term "antibody" refers to intact
molecules as well as to fragments thereof, such as Fab, F(ab)2 and
Fv, which are capable of binding to the antigenic determinant in
question. Such anti-bodies thus bind to the polypeptides of the
first aspect of the invention.
[0091] By "substantially greater affinity" we mean that there is a
measurable increase in the affinity for a polypeptide of the
invention as compared with the affinity for known secreted
proteins.
[0092] Preferably, the affinity is at least 1.5-fold, 2-fold,
5-fold 10-fold, 100-fold, 10.sup.3-fold, 104-fold, 10.sup.5-fold,
10.sup.6-fold or greater for a polypeptide of the invention than
for known secreted proteins such as members of the IL-8 chemokine
family of proteins.
[0093] If polyclonal antibodies are desired, a selected mammal,
such as a mouse, rabbit, goat or horse, may be immunised with a
polypeptide of the first aspect of the invention. The polypeptide
used to immunize the animal cam be derived by recombinant DNA
technology or can be synthesized chemically. If desired, the
polypeptide can be conjugated to a carrier protein. Commonly used
carriers to which the polypeptides may be chemically coupled
include bovine serum albumin, thyrglobulin and keyhole limpet
haemocyanin. The coupled polypeptide is then used to immunize the
animal. Serum from the immunized animal is collected and treated
according to known procedures, for example by immunoaffinity
chromatography.
[0094] Monoclonal antibodies to the polypeptides of the first
aspect of the invention can also be readily produced by one skilled
in the art. The general methodology for making monoclonal
antibodies using hybridoma technology is well known (see, for
example, Kohler, G. and Milstein, C., Nature 256: 495-497 (1975);
Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., 77-96 in
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.
(1985).
[0095] Panels of monoclonal antibodies produced against the
polypeptides of the first aspect of the invention can be screened
for various properties, i.e., for isotype, epitope, affinity, etc.
Monoclonal antibodies are particularly useful in purification of
the individual polypeptides against which they are directed.
Alternatively, genes encoding the monoclonal antibodies of interest
may be isolated from hybridomas, for instance by PCR techniques
known in the art, and cloned and expressed in appropriate
vectors.
[0096] Chimeric antibodies, in which non-human variable regions are
joined or fused to human constant regions (see, for example, Liu et
al., Proc. Natl. Acad. Sci. USA, 84, 3439 (1987)), may also be of
use.
[0097] The antibody may be modified to make it less immunogenic in
an individual, for example by humanization (see Jones et al.,
Nature, 321, 522 (1986); Verhoeyen et al., Science, 2390, 1534
(1988); Kabat et al., J. Immunol., 147, 1709 (1991); Queen et al.,
Proc. Natl. Acad. Sci. USA, 86, 10029 (1989); Gorman et al., Proc.
Natl. Acad. Sci-. USA, 88, 34181 (1991>; and Hodgson et al.,
Bio/Technology, 9, 421 (1991)). The term "humanized antibody", as
used herein, refers to antibody molecules in which the CDR amino
acids and selected other amino acids in the variable domains of the
heavy and/or light chains of a non-human donor antibody have been
substituted in place of the equivalent amino acids in a human
antibody. The humanized antibody thus closely resembles a human
antibody but has the binding ability of the donor antibody.
[0098] In a further alternative, the antibody may be a "bispecific"
antibody, that is an antibody having two different antigen binding
domains, each domain being directed against a different
epitope.
[0099] Phage display technology may be utilised to select genes
which encode antibodies pith binding activities towards the
polypeptides of the invention either from repertoires of PCR
amplified V-genes of lymphocytes from humans screened for
possessing the relevant antibodies, or from naive libraries
(McCafferty, J. et al., (1990), Nature 348, 552-554; Marks, J. et
al., (1992) Biotechnology 10, 779-783). The affinity of these
antibodies can also be improved by chain shuffling (Clackson, T. et
al., (1991) Nature 352, 624628).
[0100] Antibodies generated by the above techniques, whether
polyclonal or monoclonal, have additional utility in that they may
be employed as reagents in immunoassays, radioimmunoassays (RIA) or
enzyme-linked immunosorbent assays (ELISA). In these applications,
the antibodies can be labelled with an analytically-detectable
reagent such as a radioisotope, a fluorescent molecule or an
enzyme.
[0101] Preferred nucleic acid molecules of the second and third
aspects of the invention are those which encode a polypeptide
sequence as recited in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ
ID NO:8, SEQ ID NO:10 and SEQ ID NO:12 and functionally equivalent
polypeptides. These nucleic acid molecules may be used in the
methods and applications described herein. The nucleic acid
molecules of the invention preferably comprise at least n
consecutive nucleotides from the sequences disclosed herein where,
depending on the particular sequence, n is 10 or more (for example,
12, 14, 15, 18, 20, 25, 30, 35, 40 or more).
[0102] The nucleic acid molecules of the invention also include
sequences that are complementary to nucleic acid molecules
described above (for example, for antisense or probing
purposes).
[0103] Nucleic acid molecules of the present invention may be in
the form of RNA, such as mRNA, or in the form of DNA, including,
for instance cDNA, synthetic DNA or genomic DNA. Such nucleic acid
molecules may be obtained by cloning, by chemical synthetic
techniques or by a combination thereof. The nucleic acid Molecules
can be prepared, for example, by chemical synthesis using
techniques such as solid phase phosphoramidite chemical synthesis,
from genomic or cDNA libraries or by separation from an organism.
RNA molecules may generally be generated by the in vitro or in vivo
transcription of DNA sequences.
[0104] The nucleic acid molecules may be double-stranded or
single-stranded. Single-stranded DNA may be the coding strand, also
known as the sense strand, or it may be the non-coding strand, also
referred to as the anti-sense strand.
[0105] The term "nucleic acid molecule" also includes analogues of
DNA and RNA, such as those containing modified backbones, and
peptide nucleic acids (PNA). The term "PNA", as used herein, refers
to an antisense molecule or an anti-gene agent which comprises an
oligonucleotide of at least five nucleotides in length linked to a
peptide backbone of amino acid residues, which preferably ends in
lysine. The terminal lysine confers solubility to the composition.
PNAs may be pegylated to extend their lifespan in a cell, where
they preferentially bind complementary single stranded DNA and RNA
and stop transcript elongation (Nielsen, P. E. et al. (1993)
Anticancer Drug Des. 8:53-63).
[0106] A nucleic acid molecule which encodes a polypeptide of this
invention may be identical to the coding sequence of one or more of
the nucleic acid molecules disclosed herein.
[0107] These molecules also may have a different sequence which, as
a result of the degeneracy of the genetic code, encodes a
polypeptide SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ
ID NO:10 or SEQ ID NO:12. Such nucleic acid molecules may include,
but are not limited to, the coding sequence for the mature
polypeptide by itself; the coding sequence for the mature
polypeptide and additional coding sequences, such as those encoding
a leader or secretory sequence, such as a pro-, pre- or
prepro-polypeptide sequence; the coding sequence of the mature
polypeptide, with or without the aforementioned additional coding
sequences, together with further additional, non-coding sequences,
including non-coding 5' and 3' sequences, such as the transcribed,
non-translated sequences that play a role in transcription
(including termination signals), ribosome binding and mRNA
stability. The nucleic acid molecules may also include additional
sequences which encode additional amino acids, such as those which
provide additional functionalities.
[0108] The nucleic acid molecules of the second and third aspects
of the invention may also encode the fragments or the functional
equivalents of the polypeptides and fragments of the first aspect
of the invention. Such a nucleic acid molecule may be a
naturally-o-occurring variant such as a naturally-occurring allelic
variant, or the molecule may be a variant that is not known to
occur naturally. Such non-naturally occurring variants of the
nucleic acid molecule may be made by mutagenesis techniques,
including those applied to nucleic acid molecules, cells or
organisms.
[0109] Among variants in this regard are variants that differ from
the aforementioned nucleic acid molecules by nucleotide
substitutions, deletions or insertions. The substitutions,
deletions or insertions may involve one or more nucleotides. The
variants may be altered in coding or non-coding regions or both.
Alterations in the coding regions may produce conservative or
non-conservative amino acid substitutions, deletions or
insertions.
[0110] The nucleic acid molecules of the invention can also be
engineered, using methods generally known in the art, for a variety
of reason, including modifying the cloning, processing, and/or
expression of the gene product (the polypeptide). DNA shuffling by
random fragmentation and PCR reassembly of gene fragments and
synthetic oligonucleotides are included as techniques which may be
used to engineer the nucleotide sequences. Site-directed
mutagenesis may be used to insert new restriction sites, alter
glycosylation patterns, change codon preference, produce splice
variants, introduce mutations and so forth.
[0111] Nucleic acid molecules which encode a polypeptide of the
first aspect of the invention may be ligated to a heterologous
sequence so that the combined nucleic acid molecule encodes a
fusion protein. Such combined nucleic acid molecules are included
within the second or third aspects of the invention. For example,
to screen peptide libraries for inhibitors of the activity of the
polypeptide, it may be useful to express, using such a combined
nucleic acid molecule, a fusion protein that can be recognised by a
commercially-available antibody. A fusion protein may also be
engineered to contain a cleavage site located between the sequence
of the polypeptide of the invention and the sequence of a
heterologous protein so that the polypeptide may be cleaved and
purified away from the heterologous protein.
[0112] The nucleic acid molecules of the invention also include
antisense molecules that are partially complementary to nucleic
acid molecules encoding polypeptides of the present invention and
that therefore hybridize to the encoding nucleic acid molecules
(hybridization). Such antisense molecules, such as
oligonucleotides, can be designed to recognise, specifically bind
to and prevent transcription of a target nucleic acid encoding a
polypeptide of the inventions as will be known by those of ordinary
skill in the art (see, for example, Cohen, J. S., Trends in Pharm.
Sci., 10, 435 (1989), Okano, J. Neurochem. 56, 560 (1991);
O'Connor, J. Neurochem 56, 560 (1991); Lee et al., Nucleic Acids
Res 6, 3073 (1979); Cooney et at., Science 241, 456 (1988); Dervan
et al., Science 251, 1360 (1991).
[0113] The term "hybridization" as used here refers to the
association of two nucleic acid molecules with one another by
hydrogen bonding. Typically, one molecule sill be fixed to a solid
support and the other will be free in solution. Then, the two
molecules may be placed in contact with one another under
conditions that favour hydrogen bonding. Factors that affect this
bonding include: the type and volume of solvent; reaction
temperature; time of hybridization; agitation; agents to block the
non-specific attachment of the liquid phase molecule to the solid
support (Denhardt's reagent or BLOTTO); the concentration of the
molecules; use of compounds to increase the rate of association of
molecules (dextran sulphate or polyethylene glycol); and the
stringency of the washing conditions following hybridization (see
Sambrook et al. [supra]).
[0114] The inhibition of hybridization of a completely
complementary molecule to a target molecule may be examined using a
hybridization assay, as known in the art (see, for example,
Sambrook et al. [supra]). A substantially homologous molecule will
then compete for and inhibit the binding of a completely homologous
molecule to the target molecule under various conditions of
stringency, as taught in Wahl, G. M. and S. L. Berger (1987;
Methods Enzymol. 152:399-407) and Kimmel, A. R (1987; Methods
Enzymol. 152:507-511).
[0115] "Stringency" refers to conditions in a hybridization
reaction that favour the association of very similar molecules over
association of molecules that differ. High stringency hybridisation
conditions are defined as overnight incubation at 42.degree. C. in
a solution comprising 50% foramide, 5.times.SSC (150 mM NaCl, 15 mM
trisodium citrate), 50 mM sodium phosphate (pH7.6),
5.times.Denhardts solution, 10% dextran sulphate, and 20
microgram/ml denatured, sheared salmon sperm DNA, followed by
washing the filters in 0.1.times.SSC at approximately 65.degree. C.
Low stringency conditions involve the hybridisation reaction being
carried out at 35.degree. C. (see Sambrook et al. [supra]).
Preferably, the conditions used for hybridization are those of high
stringency.
[0116] Preferred embodiments of this aspect of the invention are
nucleic acid molecules that are at least 70% identical over their
entire length to a nucleic acid molecule encoding the INSP085
polypeptides and nucleic acid molecules that are substantially
complementary to such nucleic acid molecules. Preferably, a nucleic
acid molecule according to this aspect of the invention comprises a
region that is at least 80% identical over its entire length to
such coding sequences, or is a nucleic acid molecule that is
complementary thereto. In this regard, nucleic acid molecules at
least 90%, preferably at least 95%, more preferably at least 98%,
99% or more identical over their entire length to the same are
particularly preferred. Preferred embodiments in this respect are
nucleic acid molecules that encode polypeptides which retain
substantially the same biological function or activity as the
INSP085 polypeptides.
[0117] The invention also provides a process for detecting a
nucleic acid molecule of the invention, comprising the steps of:
(a) contacting a nucleic probe according to the invention with a
biological sample under hybridizing conditions to form duplexes;
and (b) detecting any such duplexes that are formed.
[0118] As discussed additionally below in connection with assays
that may be utilised according to the invention, a nucleic acid
molecule as described above may be used as a hybridization probe
for RNA, cDNA or genomic DNA, in order to isolate full-length cDNAs
and genomic clones encoding the INSP085 polypeptides and to isolate
cDNA and genomic clones of homologous or orthologous genes that
have a high sequence similarity to the gene encoding this
polypeptide.
[0119] In this regard, the following techniques, among others known
in the art, may be utilised and are discussed below for purposes of
illustration. Methods fox DNA sequencing and analysis are well
known and are generally available in the art and may, indeed, be
used to practice many of the embodiments of the invention discussed
herein. Such methods may employ such enzymes as the Klenow fragment
of DNA polymerase I, Sequenase (US Biochemical Corp, Cleveland,
Ohio), Taq polymerase (Perkin Elmer), thermostable T7 polymerase
(Amersham, Chicago, Ill.), or combinations of polymerases and
proof-reading exonucleases such as those found in the ELONGASE
Amplification System marketed by Gibco/BRL (Gaithersburg, Md.).
Preferably, the sequencing process may be automated using machines
such as the Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.), the
Peltier Thermal Cycler PTC200; MJ Research, Watertown, Mass.) and
the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
[0120] One method for isolating a nucleic acid molecule encoding a
polypeptide with an equivalent function to that of the INSP085
polypeptide is to probe a genomic or cDNA library with a natural or
artificially-designed probe using standard procedures that are
recognised in the art (see, for example, "Current Protocols in
Molecular Biology", Ausubel et al. (eds). Greene Publishing
Association and John Wiley Interscience, New York, 1989, 1992).
Probes comprising at least 15, preferably at least 30, and more
preferably at least 50, contiguous bases that correspond to, or are
complementary to, nucleic acid sequences from the appropriate
encoding gene (SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,
SEQ ID NO:9 and SEQ ID NO:11), are particularly useful probe. Such
probes may be labelled with an analytically-detectable reagent to
facilitate their identification. Useful reagents include, but are
not limited to, radioisotopes, fluorescent dyes and enzymes that
are capable of catalysing the formation of a detectable product.
Using these probes, the ordinarily skilled artisan will be capable
of isolating complementary copies of genomic DNA, cDNA or RNA
polynucleotides encoding proteins of interest from human, mammalian
or other animal sources and screening such sources for related
sequences, for example, for additional members of the family, type
and/or subtype.
[0121] In many cases, isolated cDNA sequences will be incomplete,
in that the region encoding the polypeptide Mill be cut short,
normally at the 5' end. Several methods are available to obtain
full length cDNAs, or to extend short cDNAs. Such sequences may be
extended utilising a partial nucleotide sequence and employing
various methods known in the art to detect upstream sequences such
as promoters and regulatory elements. For example, one method which
may be employed is based on the method of Rapid Amplification of
cDNA Ends (RACE; see, for example, Frohman et al., PNAS USA 85,
8998-9002, 1988) Recent modifications of this technique,
exemplified by the Marathon.TM. technology (Clontech Laboratories
Inc.), for example, have significantly simplified the search for
longer cDNAs. A slightly different technique, termed
"restriction-site" PCR, uses universal primers to retrieve unknown
nucleic acid sequence adjacent a known locus (Sarkar, G. (1993) PCR
Methods Applic. 2:318-322). Inverse PCR may also be used to amplify
or to extend sequences using divergent primers based on a known
region (Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186).
Another method which may be used is capture PCR which involves PCR
amplification of DNA fragments adjacent a known sequence in human
and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991)
PCR Methods Applic., 1, 111-119). Another method which may be used
to retrieve unknown sequences is that of Parker, J. D. et al.
(1991); Nucleic Acids Res. 19:3055-3060). Additionally, one may use
PCR, nested primers, and PromoterFinder.TM. libraries to walk
genomic DNA (Clontech, Palo Alto, Calif.) This process avoids the
need to screen libraries and is useful in finding intron/exon
junctions.
[0122] When screening for full-length cDNAs, it is preferable to
use Libraries that have been size-selected to include larger cDNAs.
Also, random-primed libraries are preferable, in that they will
contain more sequences that contain the 5' regions of genes. Use of
a randomly primed library may be especially preferable for
situations in which an oligo d(T) library does not yield a
full-length cDNA. Genomic libraries may be useful for extension of
sequence into 5' non-transcribed regulatory regions.
[0123] In one embodiment of the invention, the nucleic acid
molecules of the present invention may be used for chromosome
localisation. In this technique, a nucleic acid molecule is
specifically targeted to, and can hybridize with, a particular
location on an individual human chromosome. The mapping of relevant
sequences to chromosomes according to the present invention is an
important step in the confirmatory correlation of those sequences
with the gene-associated disease. Once a sequence has been mapped
to a precise chromosomal location, the physical position of the
sequence on the chromosome can be correlated with genetic map data.
Such data are found in, for example, V. McKusick, Mendelian
Inheritance in Man (available on-line through Johns Hopkins
University Welch Medical Library). The relationships between genes
and diseases that have been mapped to the same chromosomal region
are then identified through linkage analysis (coinheritance of
physically adjacent genes). This provides valuable information to
investigators searching for disease genes using positional cloning
or other gene discovery techniques. Once the disease or syndrome
has been crudely localised by genetic linkage to a particular
genomic region, any sequences mapping to that area may represent
associated or regulatory genes for finer investigation. The nucleic
acid molecule may also be used to detect differences in the
chromosomal location due to translocation, inversion, etc. among
normal, carrier, or affected individuals.
[0124] The nucleic acid molecules of the present invention are also
valuable for tissue localisation. Such techniques allow the
determination of expression patterns of the polypeptide in tissues
by detection of the mRNAs that encode them. These techniques
include in situ hybridization techniques and nucleotide
amplification techniques, such as PCR. Results from these studies
provide an indication of the normal functions of the polypeptide in
the organism. In addition, comparative studies of the normal
expression pattern of mRNAs with that of mRNAs encoded by a mutant
gene provide valuable insights into the role of mutant polypeptides
in disease. Suck inappropriate expression may be of a temporal,
spatial or quantitative nature.
[0125] Gene silencing approaches may also be undertaken to
down-regulate endogenous expression of a gene encoding a
polypeptide of the invention. RNA interference (RNAi) (Elbashir, S
M et al., Nature 2001, 411, 494-498) is one Method of sequence
specific post-transcriptional gene silencing that may be employed.
Short dsRNA oligonucleotides are synthesized in vitro and
introduced into a cell. The sequence specific binding of these
dsRNA oligonucleotides triggers the degradation of target mRNA,
reducing or ablating target protein expression.
[0126] Efficacy of the gene silencing approaches assessed above may
be assessed through the measurement of polypeptide expression (for
example, by Western blotting), and at the RNA level using
TaqMan-based methodologies.
[0127] The vectors of the present invention comprise nucleic acid
molecules of the invention and may be cloning or expression
vectors. The host cells of the invention, which may be transformed,
transfected or transduced with the vectors of the invention may be
prokaryotic or eukaryotic.
[0128] The polypeptides of the invention may be prepared in
recombinant form by expression of their encoding nucleic acid
molecules in vectors contained within a host cell. Such expression
methods are well known to those of skill in the art and many are
described in detail by Sambrook et al. (supra) and Fernandez &
Hoeffler (1998, eds. "Gene expression systems. Using nature for the
art of expression". Academic Press, San Diego, London, Boston, New
York, Sydney, Tokyo, Toronto).
[0129] Generally, any system or vector that is suitable to
maintain, propagate or express nucleic acid molecules to produce a
polypeptide in the required host may be used. The appropriate
nucleotide sequence may be inserted into an expression system by
any of a variety of well-known and routine techniques, such as, for
example, those described in Sambrook et al., (supra). Generally,
the encoding gene can be placed under the control of a control
element such as a promoter, ribosome binding site (for bacterial
expression) and, optionally, an operator, so that the DNA sequence
encoding the desired polypeptide is transcribed into RNA in the
transformed host cell.
[0130] Examples of suitable expression systems include, for
example, chromosomal, episomal and virus-derived systems,
including, for example, vectors derived from: bacterial plasmids,
bacteriophage, transposons, yeast episomes, insertion elements,
yeast chromosomal elements, viruses such as baculoviruses, papova
viruses such as SV40, vaccinia viruses, adenoviruses, fowl pox
viruses, pseudorabies viruses and retroviruses, or combinations
thereof, such as those derived from plasmid and bacteriophage
genetic elements, including cosmids and phagemids. Human artificial
chromosome s (HACs) may also be employed to deliver larger
fragments of DNA than can be contained and expressed in a
plasmid.
[0131] Particularly suitable expression systems include
microorganisms such as bacteria transformed with recombinant
bacteriophage, plasmid or cosmid DNA expression vectors; yeast
transformed with yeast expression vectors; insect cell systems
infected with virus expression vectors (for example, baculovirus);
plant cell systems transformed with virus expression vectors (for
example, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV)
or with bacterial expression vectors (for example, Ti or pBR322
plasmids); or animal cell systems. Cell-free translation systems
can also be employed to produce the polypeptides of the
invention.
[0132] Introduction of nucleic acid molecules encoding a
polypeptide of the present invention into host cells can be
effected by methods described in many standard laboratory manuals,
such as Davis et al., Basic Methods in Molecular Biology (1986) and
Sambrook et al., (supra). Particularly suitable methods include
calcium phosphate transfection, DEAE-dextran mediated transfection,
transfection, microinjection, cationic lipid-mediated transfection,
electroporation, transduction, scrape loading, ballistic
introduction or infection (see Sambrook et al, 1989 [supra];
Ausubel et al., 1991 [supra]; Spector, Goldman & Leinwald,
1998). In eukaryotic cells, expression systems may either be
transient (for example, episomal) or permanent (chromosomal
integration) according to the needs of the system.
[0133] The encoding nucleic acid molecule may or may not include a
sequence encoding a control sequence, such as a signal peptide or
leader sequence, as desired, for example, for secretion of the
translated polypeptide into the lumen of the endoplasmic reticulum,
into the periplasmic space or into the extracellular environment.
These signals may be endogenous to the polypeptide or they may be
heterologous signals. Leader sequences can be removed by the
bacterial host in post-translational processing.
[0134] In addition to control sequences, it may be desirable to add
regulatory sequences that allow for regulation of the expression of
the polypeptide relative to the growth of the host cell. Examples
of regulatory sequences are those which use the expression of a
gene to be increased or decreased in response to a chemical or
physical stimulus, including the presence of a regulatory compound
or to various temperature or metabolic conditions. Regulatory
sequences are those non-translated regions of the vector, such as
enhancers, promoters and 5' and 3' untranslated regions. These
interact with host cellular proteins to carry out transcription and
translation. Such regulatory sequences may vary in their strength
and specificity. Depending on the vector system and host utilised,
any number of suitable transcription and translation elements,
including constitutive and inducible promoters, may be used. For
example, when cloning in bacterial systems, inducible promoters
such as the hybrid lacZ promoter of the Bluescript phagemid
(Stratagene, LaJolla, Calif.) or pSport.TM. plasmid (Gibco BRL) and
the like may be used. The baculovirus polyhedrin promoter may be
used in insect cells. Promoters or enhancers derived from the
genomes of plant cells (for example, heat shock, RUBISCO and
storage protein genes) or from plant viruses (for example, viral
promoters or leader sequences) may be cloned into the vector. In
mammalian cell systems, promoters from mammalian genes or from
mammalian viruses are preferable. If it is necessary to generate a
cell line that contains multiple copies of the sequence, vectors
based on SV40 or EBV may be used with an appropriate selectable
marker.
[0135] An expression vector is constructed so that the particular
nucleic acid coding sequence is located in the vector with the
appropriate regulatory sequences, the positioning and orientation
of the coding sequence with respect to the regulatory sequences
being such that the coding sequence is transcribed under the
"control" of the regulatory sequences, i.e., RNA polymerase which
binds to the DNA molecule at the control sequences transcribes the
coding sequence. In some cases it may be necessary to modify the
sequence so that it may be attached to the control sequences with
the appropriate orientation; i.e., to maintain the reading
frame.
[0136] The control sequences and other regulatory sequences may be
ligated to the nucleic acid coding sequence prior to insertion into
a vector. Alternatively, the coding sequence can be cloned directly
into an expression vector that already contains the control
sequences and an appropriate restriction site.
[0137] For long-term, high-yield production of a recombinant
polypeptide, stable expression is preferred. For example, cell
lines which stably express the polypeptide of interest may be
transformed using expression vectors which may contain viral
origins of replication and/or endogenous expression elements and a
selectable marker gene on the same or on a separate vector.
Following the introduction of the vector, cells may be allowed to
grow for 1-2 days in an enriched media before they are switched to
selective media. The purpose of the selectable marker is to confer
resistance to selection, and its presence allows growth and
recovery of cells that successfully express the introduced
sequences. Resistant clones of stably transformed cells may be
proliferated using tissue culture techniques appropriate to the
cell type.
[0138] Mammalian cell lines available as hosts for expression are
known in the art and include many immortalised cell lines available
from the American Type Culture Collection (ATCC) including, but not
limited to, Chinese hamster ovary (CHO)), HeLa, baby hamster kidney
(BWH, monkey kidney (COS), C127, 3T3, BHK, HEK293, Bowes melanoma
and human hepatocellular carcinoma (for example Hep G2) cells and a
number of other cell lines.
[0139] In the baculovirus system, the materials for
baculovirus/insect cell expression systems are commercially
available in kit form, inter alia, Invitrogen, San Diego Calif.
(the "MaxBac" kit). These techniques are generally known to those
skilled in the art and are described fully in Summers and Smith
Texas Agricultural Experiment Station Bulletin No. 1555 (1987).
Particularly suitable host cells for use in this system include
insect cells such as Drosophila 82 and Spodoptera Sf9 cells.
[0140] There are many plant cell culture and whole plant genetic
expression systems known in the art. Examples of suitable plant
cellular genetic expression systems include those described in U.S.
Pat. No. 5,693,506; U.S. Pat. No. 5,659,122; and U.S. Pat. No.
5,608,143. Additional examples of genetic expression in plant cell
culture has been described by Zenk, Phytochemistry 30, 3861-3863
(1991).
[0141] In particular, all plants from which protoplasts can be
isolated and cultured to give whole regenerated plants can be
utilised, so that whole plants are recovered which contain the
transferred gene. Practically all plants can be regenerated from
cultured cells or tissues, including but not limited to all major
species of sugar cane, sugar beet, cotton, fruit and other trees,
legumes and vegetables.
[0142] Examples of particularly preferred bacterial host cells
include streptococci staphylococci, E. coli, Streptomyces and
Bacillus subtilis cells.
[0143] Examples of particularly suitable host cells for fungal
expression include yeast cells (for example, S. cerevisiae) and
Aspergillus cells.
[0144] Any number of selection systems are known in the art that
may be used to recover transformed cell lines. Examples include the
herpes simpler virus thymidine kinase (Wigler, M. et al. (1977)
Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et
al. (1980) Cell 22:817-23) genes that can be employed in tk.sup.-
or apr.sup..+-. cells, respectively.
[0145] Also, antimetabolite, antibiotic or herbicide resistance can
be used as the basis for selection; for example, dihydrofolate
reductase (DHFR) that confers resistance to methotrexate (Wigler,
M. et al (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which
confers resistance to the aminoglycosides neomycin and G418
(Colbere-(Garapin, F. et al. (1981) J. Mol. Diol. 150:1-14) and
also or pat, which confer resistance to chlorsulfuron and
phosphinothricin acetyltransferase, respectively. Additional
selectable genes have bean described, examples of which will be
clear to those of skill in the art.
[0146] Although the presence or absence of marker gene expression
suggests that the gene C of interest is also present, its presence
and expression may need to be confirmed. For example, if the
relevant sequence is inserted within a marker gene sequence,
transformed cells containing the appropriate sequences can be
identified by the absence of marker gene function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding a
polypeptide of the invention under the control of a single
promoter. Expression of be marker gene in response to induction or
selection usually indicates expression of the tandem gene as
well.
[0147] Alternatively, host cells that contain a nucleic acid
sequence encoding a polypeptide of the invention and which express
said polypeptide may be identified by a variety of procedures known
to those of skill in the art. These procedures include, but are not
limited to, DNA-DNA or DNA-RNA hybridizations and protein
bioassays, for example, fluorescence activated cell sorting (FACS)
or immunoassay techniques (such as the enzyme-linked immunosorbent
assay [ELISA] and radioimmunoassay [RIA]), that include membrane,
solution, or chip based technologies for the detection and/or
quantification of nucleic acid or protein (see Hampton, R. et al.
(1990) Serological Methods, a Laboratory Manual, APS Press, St
Paul, Minn.) and Maddox, D Y et al. (1983) J. Exp. Med. 158,
1211-1216).
[0148] A wide variety of labels and conjugation techniques are
known by those skilled in the art and may be used in various
nucleic acid and amino acid assays. Means for producing labelled
hybridization or PCR probes for detecting sequences related to
nucleic acid molecules encoding polypeptides of the present
invention include oligolabelling, nick translation, end-labelling
or PCR amplification using a labelled polynucleotide.
Alternatively, the sequences encoding the polypeptide of the
invention may be cloned into a vector for the production of an mRNA
probe. Such vectors are known in the art, are commercially
available, and may be used to synthesise RNA probes in vitro by
addition of an appropriate RNA polymerase such as T7, T3 or SP6 and
labelled nucleotides. These procedures may be conducted using a
variety of commercially available kits (Pharmacia & Upjohn,
(Kalamazoo, Mich.); Promega (Madison Wis.); and U.S. Biochemical
Corp., Cleveland, Ohio)).
[0149] Suitable reporter molecules or labels, which may be used for
ease of detection, include radionuclides, enzymes and fluorescent,
chemiluminescent or chromogenic agents as well as substrates,
cofactors, inhibitors, magnetic particles, and the like.
[0150] Nucleic acid molecules according to the present invention
may also be used to create transgenic animals, particularly rodent
animals. Such transgenic animals form a further aspect of the
present invention. This may be done locally by modification of
somatic cells, or by germ line therapy to incorporate heritable
modifications. Such transgenic animals may be particularly useful
in the generation of animal models for drug molecules effective as
modulators of the polypeptides of the present invention.
[0151] The polypeptide can be recovered and purified from
recombinant cell cultures by well-known methods including ammonium
sulphate or ethanol precipitation, acid extraction, anion or cation
exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromotography,
hydroxylapatite chromatography and lectin chromatography. High
performance liquid chromatography is particularly useful for
purification. Well known techniques for refolding proteins may be
employed to regenerate an active conformation when the polypeptide
is denatured during isolation and or purification.
[0152] Specialised vector constructions may also be used to
facilitate purification of protein, as desired, by joining
sequences encoding the polypeptides of the invention to a
nucleotide sequence encoding a polypeptide domain that will
facilitate purification of soluble proteins. Examples of such
purification-facilitating domains include metal chelating peptides
such as histidine-tryptophan modules that allow purification on
immobilised metals, protein A domains that allow purification on
immobilised immunoglobulin, and the domain utilised in the FLAGS
extension/affinity purification system (Immunex Corp., Seattle,
Wash.). The inclusion of cleavable linker sequences such as those
specific for Factor XA or enterokinase (Invitrogen, San Diego,
Calif.) between the purification domain and the polypeptide of the
invention may be used to facilitate purification. One such
expression vector provides for expression of a fusion protein
containing the polypeptide of the invention fused to several
histidine residues preceding a thioredoxin or an enterokinase
cleavage site. The histidine residues facilitate purification by
IMAC (immobilised metal ion affinity chromatography as described in
Porath, J. et al (1992), Prot. Exp. Purif. 3: 263-281) while the
thioredoxin or enterokinase cleavage site provides a mean-s for
purifying the polypeptide from the fusion protein. A discussion of
vectors which contain fusion proteins is provided in Kroll, D. J.
et al. (1993; DNA Cell Biol. 12:441-453).
[0153] If the polypeptide is to be expressed for use in screening
assays, generally it is preferred that it be produced at the
surface of the host cell in which it is expressed. In this event,
the host cells may be harvested prior to use in the screening
assay, for example using techniques such as fluorescence activated
cell sorting (FACS) or immunoaffinity techniques. If the
polypeptide is secreted into the medium, the medium can be
recovered in order to recover and purify the expressed polypeptide.
If polypeptide is produced intracellularly, the cells must first be
lysed before the polypeptide is recovered.
[0154] The polypeptide of the invention cam be used to screen
libraries of compounds in any of a variety of drug screening
techniques. Such compounds may activate (agonise) or inhibit
(antagonize) the level of expression of the gene or the activity of
the polypeptide of the invention and form a further aspect of the
present invention Preferred compounds are effective to alter the
expression of a natural gene which encodes a polypeptide of the
first aspect of the invention or to regulate the activity of a
polypeptide of the first aspect of the invention.
[0155] Agonist or antagonist compounds may be isolated from, for
example, cells, cell-free preparations, chemical libraries or
natural product mixtures. These agonists or antagonists may be
natural or modified substrates, ligands, enzymes, receptors or
structural or functional mimetics. For a suitable review of such
screening techniques, see Coligan et al., Current Protocols in
Immunology 1 (2): Chapter 5 (1991).
[0156] Compounds that are most likely to be good antagonists are
molecules that bind to the polypeptide of the invention without
inducing the biological effects of the polypeptide upon binding to
it. Potential antagonists include small organic molecules,
peptides, polypeptides and antibodies that bind to the polypeptide
of the invention and thereby inhibit or extinguish its activity. In
this fashion, binding of the polypeptide to normal cellular binding
molecules may be inhibited, such that the normal biological
activity of the polypeptide is prevented.
[0157] The polypeptide of the invention that is employed in such a
screening technique may be free in solution, affixed to a solid
support, borne on a cell surface or located intracellularly. In
general, such screening procedures may involve using appropriate
cells or cell membranes that express the polypeptide that are
contacted with a test compound to observe binding, or stimulation
or inhibition of a functional response. The functional response of
the cells contacted with the test compound is then compared with
control cells that were not contacted with the test compound. Such
an assay may assess whether the test compound results in a signal
generated by activation of the polypeptide, using an appropriate
detection system. Inhibitors of activation are generally assayed in
the presence of a known agonist and the effect on activation by the
agonist in the presence of the test compound is observed.
[0158] A preferred method for identifying an agonist or antagonist
compound of a poly peptide of the present invention comprises:
(a) contacting a cell expressing on the surface thereof the
polypeptide according to the first aspect of the invention, the
polypeptide being associated with a second component capable of
providing a detectable signal in response to the binding of a
compound to the polypeptide, with a compound to be screened under
conditions to permit binding to the polypeptide; and (b)
determining whether the compound binds to and activates or inhibits
the polypeptide by measuring the level of a signal generated from
the interaction of the compound with the polypeptide.
[0159] A further preferred method for identifying an agonist or
antagonist of a polypeptide of the invention comprises:
(a) contacting a cell expressing on the surface thereof the
polypeptide, the polypeptide being associated with a second
component capable of providing a detectable signal in response to
the binding of a compound to the polypeptide, with a compound to be
screened under conditions to permit binding to the polypeptide; and
(b) determining whether the compound binds to and activates or
inhibits the polypeptide by comparing the level of a signal
generated from the interaction of the compound with the polypeptide
with the level of a signal in the absence of the compound.
[0160] In further preferred embodiments, the general methods that
are described above may further comprise conducting the
identification of agonist or antagonist in the presence of labelled
or unlabelled ligand for the polypeptide.
[0161] In another embodiment of the method for identifying an
agonist or antagonist of a polypeptide of the present invention
comprises:
determining the inhibition of binding of a ligand to cells which
have a polypeptide of the invention on the surface thereof, or to
cell membranes containing such a polypeptide, in the presence of a
candidate compound under conditions to permit Winding to the
polypeptide, and determining the amount of ligand bound to the
polypeptide. A compound capable of causing reduction of binding of
a ligand is considered to be an agonist or antagonist. Preferably
the ligand is labelled.
[0162] More particularly, a method of screening for a polypeptide
antagonist or agonist compound comprises the steps of:
(a) incubating a labelled ligand with a whole cell expressing a
polypeptide according to the invention on the cell surface, or a
cell membrane containing a polypeptide of the invention, (b)
measuring the amount of labelled ligand bound to the whole cell or
the cell membrane; (c) adding a candidate compound to a mixture of
labelled ligand and the whole cell or the cell membrane of step (a)
and allowing the mixture to attain equilibrium; (d) measuring the
amount of labelled ligand bound to the whole cell or the cell
membrane after step (c); and (e) comparing the difference in the
labelled ligand bound in step (b) and (d), such that the compound
which causes the reduction in binding in step (d) is considered to
be an agonist or antagonist.
[0163] The INSP085 polypeptides may also be found to modulate
immune and/or nervous system cell proliferation and differentiation
in a dose-dependent manner in the above-described assays. Thus, the
"functional equivalents" of the INSP085 polypeptides include
polypeptides that exhibit any of the same growth and
differentiation regulating activities in the above-described assays
in a dose-dependent manner. Although the degree of dose-dependent
activity need not be identical to that of the INSP085 polypeptides,
preferably the "functional equivalents" will exhibit substantially
similar dose-dependence in a given activity assay compared to the
INSP085 polypeptides.
[0164] In certain of the embodiments described above, simple
binding assays may be used, in which the adherence of a test
compound to a si face bearing the polypeptide is detected by means
of a label directly or indirectly associated with the test compound
or in an assay involving competition with a labelled competitor. In
another embodiment, competitive drug screening assays may be used,
in which neutralizing antibodies that are capable of binding the
polypeptide specifically compete with a test compound for binding.
In this manner, the antibodies can be used to detect the presence
of any test compound that possesses specific binding affinity for
the polypeptide.
[0165] Assays may also be designed to detect the effect of added
test compounds on the production of mRNA encoding the polypeptide
in cells. For example, an ELISA may be constructed that measures
secreted or cell-associated levels of polypeptide using monoclonal
or polyclonal antibodies by standard methods known in the art, and
this can be used to search for compounds that may inhibit or
enhance the production of the polypeptide from suitably manipulated
cells or tissues. The formation of binding complexes between the
polypeptide and the compound being tested may then be measured.
[0166] Another technique for drug screening which may be used
provides for high throughput screening of compounds having suitable
binding affinity to the polypeptide of interest (see International
patent application WO84/03564). In this method, large numbers of
different small test compounds are synthesised on a solid
substrate, which may then be reacted with the polypeptide of the
invention and washed. One way of immobilising the polypeptide is to
use non-neutralizing antibodies. Bound polypeptide may then be
detected using methods that are well known in the art. Purified
polypeptide can also be coated directly onto plates for use in the
aforementioned drug screening techniques.
[0167] The polypeptide of the invention may be used to identify
membrane-bound or soluble receptors, through standard receptor
binding techniques that are known in the art, such as ligand
binding and crosslinking assays in which the polypeptide is
labelled with a radioactive isotope, is chemically modified, or is
fused to a peptide sequence that facilitates its detection or
purification, and incubated with a source of the putative receptor
(for example, a composition of cells, cell membranes, cell
supernatants, tissue extracts, or bodily fluids). The efficacy of
binding may be measured using biophysical techniques such as
surface plasmon resonance and spectroscopy. Binding assays may be
used for the purification and cloning of the receptor, bat may also
identify agonists and antagonists of the polypeptide, that compete
with the binding of the polypeptide to its receptor. Standard
methods for conducting screening assays are well understood in the
art.
[0168] The invention also includes a screening kit useful in the
methods fox identifying agonists, antagonists, ligands, receptors,
substrates, enzymes, that are described above.
[0169] The invention includes the agonists, antagonists, ligands,
receptors, substrates and enzymes, and other compounds which
modulate the activity or antigenicity of the polypeptide of the
invention discovered by the methods that are described above.
[0170] The invention also provides pharmaceutical compositions
comprising a polypeptide, nucleic acid, ligand or compound of the
invention in combination with a suitable pharmaceutical carrier.
These compositions may be suitable as therapeutic or diagnostic
reagents, as vaccines, or as other immunogenic compositions, as
outlined in detail below.
[0171] According to the terminology used herein, a composition
containing a polypeptide, nucleic acid, ligand or compound [X] is
"substantially free of" impurities [herein, Y] when at least 85% by
weight of the total X+Y in the composition is X. Preferably, X
comprises at least about 90% by weight of the total of X+Y in the
composition, more preferably at least about 95%, 98% or even 99% by
weight.
[0172] The pharmaceutical compositions should preferably comprise a
therapeutically effective amount of the polypeptide, nucleic acid
molecule, ligand, or compound of the invention. The term
"therapeutically effective amount" as used herein refers to an
amount of a therapeutic agent needed to treat, ameliorate, or
prevent a targeted disease or condition, or to exhibit a detectable
therapeutic or preventative effect. For any compound, the
therapeutically effective dose can be estimated initially either in
cell culture assays, for example, of neoplastic cells, or in animal
models, usually mice, rabbits, dogs, or pigs. The animal model may
also be used to determine the appropriate concentration range and
route of administration. Such information can then be used to
determine useful doses and routes for administration in humans.
[0173] The precise effective amount for a human subject will depend
upon the severity of the disease state, general health of the
subject, age, weight, and gender of the subject, diet, time and
frequency of administration, drug combination(s), reaction
sensitivities, and tolerance/response to therapy. This amount can
be determined by routine experimentation and is within the
judgement of the clinician. Generally, an effective dose will be
from 0.sub.--01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10
mg/kg Compositions may be administered individually to a patient or
may be administered in combination with other agents, drugs or
hormones.
[0174] A pharmaceutical composition may also contain a
pharmaceutically acceptable carrier, for administration of a
therapeutic agent. Such carriers include antibodies and other
polypeptides, genes and other therapeutic agents such as liposomes,
provided that the carrier does not itself induce the production of
antibodies harmful to the individual receiving the composition, and
which may be administered without undue toxicity. Suitable carriers
may be large, slowly metabolised macromolecules such as proteins,
polysaccharides, polylactic acids, polyglycolic acids, polymeric
amino acids, amino acid copolymers and inactive virus
particles.
[0175] Pharmaceutically acceptable salts can be used therein, for
example, mineral acid salts such as hydrochlorides, hydrobromaides,
phosphates, sulphates, and be like; and the salts of organic acids
such as acetates, propionates, malonates, benzoates, and the like.
A thorough discussion of pharmaceutically acceptable carriers is
available in Remington's Pharmaceutical Sciences (Mack Pub. Co.,
N.J. 1991).
[0176] Pharmaceutically acceptable carriers in therapeutic
compositions may additionally contain liquids such as water,
saline, glycerol and ethanol. Additionally, auxiliary substances,
such as wetting or emulsifying agents, pH buffering substances, and
the like, may be present in such compositions. Such carriers enable
the pharmaceutical compositions to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions,
and the like, for ingestion by the patient.
[0177] Once formulated, the compositions of the invention can be
administered directly to the subject. The subjects to be treated
can be animals; in particular, human subjects can be treated.
[0178] The pharmaceutical compositions utilised in this invention
may be administered by any number of routes including, but not
limited to, oral, intravenous, intramuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal or
transcutaneous applications (for example, see WO98/20734),
subcutaneous, intraperitoneal, intranasal, enteral, topical,
sublingual, intravaginal or rectal means. Gene guns or hyposprays
may also be used to administer the pharmaceutical compositions of
the invention. Typically, the therapeutic compositions may be
prepared as injectables, either as liquid solutions or suspensions;
solid forms suitable for solution in, or suspension in, liquid
vehicles prior to injection may also be prepared.
[0179] Direct delivery of the compositions will generally be
accomplished by injection, subcutaneously, intraperitoneally,
intravenously or intramuscularly, or delivered to the interstitial
space of a tissue. The compositions can also be administered into a
lesion. Dosage treatment may be a single dose schedule or a
multiple dose schedule.
[0180] If the activity of the polypeptide of the invention is in
excess in a particular disease state, several approaches are
available. One approach comprises administering to a subject an
inhibitor compound (antagonist) as described above, along with a
pharmaceutically acceptable carrier in an amount effective to
inhibit the function of the polypeptide, such as by blocking the
binding of ligands, substrates, enzymes, receptors, or by
inhibiting a second signal, and thereby alleviating the abnormal
condition. Preferably, such antagonists are antibodies. Most
preferably, such antibodies are chimeric and/or humanized to
minimise their immunogenicity, as described previously.
[0181] In another approach, soluble forms of the polypeptide that
retain binding affinity for the ligand, substrate, enzyme,
receptor, in question, may be administered. Typically, the
polypeptide may be administered in the form of fragments that
retain the relevant portions.
[0182] In an alternative approach, expression of the gene encoding
the polypeptide can be inhibited using expression blocking
techniques, such as the use of antisense nucleic acid molecules (as
described above), either internally generated or separately
administered Modifications of gene expression can be obtained by
designing complementary sequences or antisense molecules (DNA, RNA,
or PNA) to the control, 5' or regulatory regions (signal sequence,
promoters, enhancers and introns) of the gene encoding the
polypeptide. Similarly, inhibition can be achieved using "triple
helix" base-pairing methodology Triple helix pairing is useful
because it causes inhibition of the ability of the double helix to
open sufficiently for the binding of polymerases, transcription
factors, or regulatory molecules. Recent therapeutic advances using
triplex DNA have been described in the literature (Gee, J. E. et
al. (1994) In: Huber, B. E. and B. I. Carr, Molecular and
Immunologic Approaches, Futura Publishing Co., Mt Kisco, N.Y.). The
complementary sequence on antisense molecule may also be designed
to block translation of mRNA by preventing the transcript from
binding to ribosomes. Such oligonucleotides may be administered or
may be generated in situ from expression in vivo.
[0183] In addition, expression of the polypeptide of the invention
may be prevented by using ribozymes specific to its encoding mRNA
sequence. Ribozymes are catalytically active RNAs that can be
natural or synthetic (see for example Usman, N, et al., Curr. Opin.
Struct. Biol (1996) 6(4), 527-33). Synthetic ribozymes can be
designed to specifically cleave mRNAs at selected positions thereby
preventing translation of the mRNAs into functional polypeptide.
Ribozymes may be synthesised with a natural ribose phosphate
backbone and natural bases, as normally found in RNA molecules.
Alternatively the ribozymes may be synthesised with non-natural
backbones, for example, 2'-O-methyl RNA, to provide protection from
ribonuclease degradation and may contain modified bases.
[0184] RNA molecules may be modified to increase intracellular
stability and half-life. Possible modifications include, but are
not limited to, the addition of flanking sequences at the 5' and/or
3' ends of the molecule or the use of phosphorothioate or 2'
O-methyl rather than phosphodiesterase linkages within the backbone
of the molecule. This concept is inherent in the production of PNAs
and can be extended in all of these molecules by the inclusion of
non-traditional bases such as inosine, queosine and butosine, as
well as acetyl-, methyl-, thio- and similarly modified forms of
adenine, cytidine, guanine, thymine and uridine which are not as
easily recognized by endogenous endonucleases.
[0185] For treating abnormal conditions related to an
under-expression of the polypeptide of the invention and its
activity, several approaches are also available. One approach
comprises administering to a subject a therapeutically effective
amount of a compound that activates the polypeptide, i.e., an
agonist as described above, to alleviate the abnormal condition.
Alternatively, a therapeutic amount of the polypeptide in
combination with a suitable pharmaceutical carrier may be
administered to restore the relevant physiological balance of
polypeptide.
[0186] Gene therapy may be employed to effect the endogenous
production of the polypeptide by the relevant cells in the subject
Gene therapy is used to treat permanently the inappropriate
production of the polypeptide by replacing a defective gene with a
corrected therapeutic gene.
[0187] Gene therapy of the present invention can occur in vivo or
ex vivo. Ex vivo gene therapy requires the isolation and
purification of patient cells, the introduction of a therapeutic
gene and introduction of the genetically altered cells back into
the patient. In contrast, in vivo gene therapy does not require
isolation and purification of a patients cells.
[0188] The therapeutic gene is typically "packaged" for
administration to a patient. Gene delivery vehicles may be
non-viral, such as liposomes, or replication-deficient viruses,
such as adenovirus as described by Berkner, K. L., in Curr. Top.
Microbiol. Immunol., 158, 39-66 (1992) or adeno-associated virus
(AAV) vectors as described by Muzyczka, N., in Curr. Top.
Microbiol. Immunol., 158, 97-129 (1992) and U.S. Pat. No.
5,252,479. For example, a nucleic acid Molecule encoding a
polypeptide of the invention may be engineered for expression in a
replication-defective retroviral vector. This expression construct
may then be isolated and introduced into a packaging cell
transduced with a retroviral plasmid vector containing RNA encoding
the polypeptide, such that the packaging cell now produces
infectious viral particles containing the gene of interest These
producer cells may be administered to a subject for engineering
cells in vivo and expression of the polypeptide in vivo (see
Chapter 20, Gene Therapy and other Molecular Genetic-based
Therapeutic Approaches, (and references cited therein) in Human
Molecular Genetics (1996), T Stracham and A P Read, BIOS Scientific
Publishers Ltd).
[0189] Another approach is the administration of "naked DNA" in
which the therapeutic gene is directly injected into the
bloodstream or muscle tissue.
[0190] In situations in which the polypeptides or nucleic acid
molecules of the invention are disease-causing agents, the
invention provides that they can be used in vaccines to raise
antibodies against the disease causing agent.
[0191] Vaccines according to the invention may either be
prophylactic (i.e. to prevent infection) or therapeutic (i.e. to
treat disease after infection). Such vaccines comprise immunizing
antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic
acid, usually in combination with pharmaceutically-acceptable
carriers as described above, which include any carrier that does
not itself induce the production of antibodies harmful to the
individual receiving the composition. Additionally, these carriers
may function as immunostimulating agents ("adjuvants").
Furthermore, the antigen or immunogen may be conjugated to a
bacterial toxoid, such as a toxoid from diphtheria, tetanus,
cholera, H. pylori, and other pathogens.
[0192] Since polypeptides may be broken down in the stomach,
vaccines comprising polypeptides are preferably administered
parenterally (for instance, subcutaneous, intramuscular,
intravenous, or intradermal injection). Formulations suitable for
parenteral administration include aqueous and non-aqueous sterile
injection solutions which may contain anti-oxidants, buffers,
bacteriostats and solutes which render the formulation isotonic
with the blood of the recipient, and aqueous and nor-aqueous
sterile suspensions which may include suspending agents or
thickening agents.
[0193] The vaccine formulations of the invention may be presented
in unit-dose or multi-dose containers. For example, sealed ampoules
and vials and may be stored in a freeze-dried condition requiring
only the addition of the sterile liquid carrier immediately prior
to use. The dosage will depend on the specific activity of the
vaccine and can be readily determined by routine
experimentation.
[0194] Genetic delivery of antibodies that bind to polypeptides
according to the invention may also be effected, for example, as
described in International patent application WO98/55607.
[0195] The technology referred to as jet injection (see, for
example, www.powderject.com) may also be useful in the formulation
of vaccine compositions.
[0196] A number of suitable methods for vaccination and vaccine
delivery systems are described in International patent application
WO00/29428.
[0197] This invention also relates to the use of nucleic acid
molecules according to the present invention as diagnostic
reagents. Detection of a mutated form of the gene characterised by
the nucleic acid molecules of the invention which is associated
with a dysfunction will provide a diagnostic tool that can add to,
or define, a diagnosis of a disease, or susceptibility to a
disease, which results from under-expression, over-expression or
altered spatial or temporal expression of the gene. Individuals
carrying mutations in the gene may be detected at the DNA level by
a variety of techniques.
[0198] Nucleic acid molecules for diagnosis may be obtained from a
subjects sells, such as from blood, urine, saliva, tissue biopsy or
autopsy material. The genomic DNA may be used directly for
detection or may be amplified enzymatically by using PCR, ligase
chain reaction (LCR), stand displacement amplification (SDA), or
other amplification techniques (see Saiki et al., Nature, 324,
163-166 (1986); Bej, et al., Crit. Rev. Biochem. Molec. Biol., 26,
301-334 (1991); Birkenmeyer et al., J. Virol. Meth., 35, 117-126
(1991); Van Brunt, J., Bio/Technology, 8, 291-294 (1990)) prior to
analysis.
[0199] In one embodiment, this aspect of the invention provides a
method of diagnosing a disease in a patient, comprising assessing
the level of expression of a natural gene encoding a polypeptide
according to the invention and comparing said level of expression
to a control level, wherein a level that is different to said
control level is indicative of disease. The method may comprise the
steps of:
a) contacting a sample of tissue from the patient with a nucleic
acid probe under stringent conditions that allow the formation of a
hybrid complex between a nucleic acid molecule of the invention and
the probe; b) contacting a control sample with said probe under the
same conditions used in step a); c) and detecting the presence of
hybrid complexes in said samples; wherein detection of levels of
the hybrid complex in the patient sample that differ from levels of
the hybrid complex in the control sample is indicative of
disease.
[0200] A further aspect of the invention comprises a diagnostic
method comprising the steps of:
a) obtaining a tissue sample from a patient being tested for
disease, b) isolating a nucleic acid molecule according to the
invention from said tissue sample; and c) diagnosing the patient
for disease by detecting the presence of a mutation in the nucleic
acid molecule which is associated with disease.
[0201] To aid the detection of nucleic acid molecules in the
above-described methods, an amplification step, for example using
PCR, may be included.
[0202] Deletions and insertions can be detected by a change in the
size of the amplified product in comparison to the normal genotype.
Point mutations can be identified by hybridizing amplified DNA to
labelled RNA of the invention or alternatively, labelled antisense
DNA sequences of the invention. Perfectly-matched sequences can be
distinguished from mismatched duplexes by RNase digestion or by
assessing differences in melting temperatures. The presence or
absence of the mutation in the patient may be detected by
contacting DNA with a nucleic acid probe that hybridises to the DNA
under stringent conditions to form a hybrid double-stranded
molecule, the hybrid double-stranded molecule having an
unhybridized portion of the nucleic acid probe strand at any
portion corresponding to a mutation associated with disease; and
detecting the presence or absence of an unhybridized portion of the
probe strand as an indication of the presence or absence of a
disease-associated mutation in the corresponding portion of the DNA
strand.
[0203] Such diagnostics are particularly useful for prenatal and
even neonatal testing.
[0204] Point mutations and other sequence differences between the
reference gene and "mutant" genes can be identified by other
well-known techniques, such as direct DNA sequencing or
single-strand conformational polymorphism, (see Orita et al.,
Genomics, 5, 874-879 (1989)). For example, a sequencing primer may
be used with double-stranded PCR product or a single-stranded
template molecule generated by a modified PCR The sequence
determination is performed by conventional procedures with
radiolabelled nucleotides or by automatic sequencing procedures
with fluorescent-tags. Cloned DNA segments may also be used as
probes to detect specific DNA segments. The sensitivity of this
method is greatly enhanced when combined with PCR. Further, point
mutations and other sequence variations, such as polymorphisms, can
be detected as described above, for example, through the use of
allele-specific oligonucleotides for PCR amplification of sequences
that differ by single nucleotides.
[0205] DNA sequence differences may also be detected by alterations
in the electrophoretic mobility of DNA fragments in gels, with car
without denaturing agents, or by direct DNA sequencing (for
example, Myers et al., Science (1985) 230:1242) Sequence changes at
specific locations may also be revealed by nuclease protection
assays, such as RNase and SI protection or the chemical cleavage
method (see Cotton et at., Proc. Natl. Acad Sci USA (1985) 85:
43974401).
[0206] In addition to conventional gel electrophoresis and DNA
sequencing, mutations such as microdeletions, aneuploidies,
translocations, inversions, can also be detected by in site
analysis (see, for example, Keller et al., DNA Probes, 2nd Ed.,
Stockton Press, New York, N.Y., USA (1993)), that is, DNA or RNA
sequences in cells can be analysed for mutations without need for
their isolation and/or immobilisation onto a membrane. Fluorescence
in situ hybridization (FISH) is presently the most commonly applied
method and numerous reviews of FISH have appeared (see, for
example, Trachuck et al., Science, 250, 559-562 (1990), and Trask
et al., Trends, Genet. 7, 149-154 (1991)).
[0207] In another embodiment of the invention, an array of
oligonucleotide probes comprising a nucleic acid molecule according
to the invention can be constructed to conduct efficient screening
of genetic variants, mutation and polymorphisms. Array technology
methods are well known and have general applicability and can be
used to address a variety of questions in molecular genetics
including gene expression, genetic linkage, and genetic variability
(see for example: M. Chee et al., Science (1996), Vol 274, pp
610-613).
[0208] In one embodiment, the array is prepared and used according
to the methods described in PCT application WO95/11995 (Chee et
al); Lockhart, D. J. et al. (1996) Nat. Biotech. L4: 1675-1680);
and Schena, M. et al. (1996) Proc. Natl. Acad. Sci. 93:
10614-10619). Oligonucleotide pairs may range from two to over one
million. The oligomers are synthesized at designated areas on a
substrate using a light-directed chemical process. The substrate
may be paper, nylon or other type of membrane, filter, chip, glass
slide or any other suitable solid support. In another aspect, an
oligonucleotide may be synthesized on the surface of the substrate
by using a chemical coupling procedure and an ink jet application
apparatus, as described is PCT application WO95/25116
(Baldeschweiler et al.). In another aspect, a "gridded" array
analogous to a dot (or slot) blot may be used to arrange and link
cDNA fragments or oligonucleotides to the surface of a substrate
using a vacuum system, thermal, UV, mechanical or chemical bonding
procedures. An array, such as those described above, may be
produced by hand or by using available devices (slot b-lot or dot
blot apparatus), materials (any suitable solid support), and
machines (including robotic instruments), and may contain 8, 24,
96, 384, 1536 or 6144 oligonucleotides, or any other number between
two and over one million which lends itself to the efficient use of
commercially-available instrumentation.
[0209] In addition to the methods discussed above, diseases may be
diagnosed by methods comprising determining, from a sample derived
from a subject, an abnormally decreased or increased level of
polypeptide or mRNA. Decreased or increased expression can be
measured at the RNA level using any of the methods well known in
the art for the quantitation of polynucleotides, such as, for
example, nucleic acid amplification, for instance PCR, RT-PCR,
RNase protection, Northern blotting and other hybridization
methods.
[0210] Assay techniques that can be used to determine levels of a
polypeptide of the present invention in a sample derived from a
host are well-known to those of skill in the art and are discussed
in some detail above (including radioimmunoassays,
competitive-binding assays, Western Blot analysis and ELISA
assays). This aspect of the invention provides a diagnostic method
which comprises the steps of: (a) contacting a ligand as described
above with a biological sample under conditions suitable for the
formation of a ligand-polypeptide complex; and (b) detecting said
complex.
[0211] Protocols such as ELISA, RIA, and FACS for measuring
polypeptide levels may additionally provide a basis for diagnosing
altered or abnormal levels of polypeptide expression. Normal or
standard values for polypeptide expression are established by
combining body fluids or cell extracts taken from normal mammalian
subjects, preferably humans, with antibody to the polypeptide under
conditions suitable for complex formation The amount of standard
complex formation may be quantified by various methods, such as by
photometric means.
[0212] Antibodies which specifically bind to a polypeptide of the
invention may be used for the diagnosis of conditions or diseases
characterized by expression of the polypeptide, or in assays to
monitor patients being treated with the polypeptides, nucleic acid
molecules, ligands and other compounds of the invention. Antibodies
useful for diagnostic purposes may be prepared in the same manner
as those described above for therapeutics. Diagnostic assays for
the polypeptide include methods that utilise the antibody and a
label to detect the polypeptide in human body fluids or extracts of
cells or tissues. The antibodies may be used with or without
modification, and may be labelled by joining them, either
covalently or non-covalently, with a reporter molecule. A wide
variety of reporter molecules known in the art may be used, several
of which are described above.
[0213] Quantities of polypeptide expressed in subject, control and
disease samples from biopsied tissues are compared with the
standard values. Deviation between standard and subject values
establishes the parameters for diagnosing disease. Diagnostic
assays may be used to distinguish between absence, presence, and
excess expression of polypeptide and to monitor regulation of
polypeptide levels during therapeutic intervention. Such assays may
also be used to evaluate the efficacy of a particular therapeutic
treatment regimen in animal studies, in clinical trials or in
monitoring the treatment of an individual patient.
[0214] A diagnostic kit of the present invention may comprise:
(a) a nucleic acid molecule of the present invention; (b) a
polypeptide of the present invention; or (c) a ligand of the
present invention.
[0215] In one aspect of the invention, a diagnostic kit may
comprise a first container containing a nucleic acid probe that
hybridises under stringent conditions with a nucleic acid molecule
according to the invention; a second container containing primers
useful for amplifying the nucleic acid molecule; and instructions
for using the probe and primers for facilitating the diagnosis of
disease. The kit may fiber comprise a third container holding an
agent for digesting unhybridized RNA.
[0216] In an alternative aspect of the invention, a diagnostic kit
may comprise an array of nucleic acid molecules, at least one of
which may be a nucleic acid molecule according to the
invention.
[0217] To detect polypeptide according to the invention, a
diagnostic kit ray comprise one or more antibodies that bind to a
polypeptide according to the invention; and a reagent useful for
the detection of a binding reaction between the antibody and the
polypeptide.
[0218] Such kits will be of use in diagnosing a disease or
susceptibility to disease in which members of the IL-8 like
chemokine family are implicated. Such diseases may include cell
proliferative disorders, including neoplasm, melanoma, lung,
colorectal, breast, pancreas, head and neck and other solid
tumours; myeloproliferative disorders, such as leukemia,
non-Hodgkin lymphoma, leukopenia, thrombocytopenia, angiogenesis
disorder, Kaposis' sarcoma; autoimmune/inflammatory disorders,
including allergy, inflammatory bowel disease, arthritis, psoriasis
and respiratory tract inflammation, asthma, and organ transplant
rejection; cardiovascular disorders, including hypertension,
oedema, angina, atherosclerosis, thrombosis, sepsis, shock,
reperfusion injury, and ischemia; neurological disorders including
central nervous system disease, Alzheimers disease, brain injury,
amyotrophic lateral sclerosis, and pain; developmental disorders;
metabolic disorders including diabetes mellitus, osteoporosis, and
obesity, AIDS and renal disease; infections including viral
infection, bacterial infection, fungal infection and parasitic
infection and other pathological conditions. Preferably, the
diseases are those in which members of the IL-8 like chemokine
family are implicated such as arthritis, rheumatoid arthritis (RA),
psoriatic arthritis, osteoarthritis, systemic lupus erythematosus
(SLE), systemic sclerosis, scleroderma, polymyositis,
glomerulonephritis, fibrosis, lung fibrosis and inflammation,
allergic or hypersensitivity diseases, dermatitis, asthma, chronic
obstructive pulmonary disease (COPD), inflammatory bowel disease
(IBD), Crohn's diseases, ulcerative colitis, multiple sclerosis,
septic shock, HIV infection, transplant rejection, wound healing,
metastasis, endometriosis, hepatitis, liver fibrosis, cancer,
analgesia, and vascular inflammation related to atherosclerosis.
Such kits may also be used for the detection of reproductive
disorders including infertility.
[0219] Various aspects and embodiments of the present invention
will now be described in more detail by way of example, with
particular reference to the INSP085 polypeptides.
[0220] It will be appreciated that modification of detail may be
made without departing from the scope of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0221] FIG. 1: Top ten results from BLAST against NCBI
non-redundant database using SEQ ID NO:8 (INSP085 full protein
sequence).
[0222] FIG. 2: Alignment generated by BLAST between SEQ ID NO:8
(INSP085 full protein sequence) and the top five hits, small
inducible cytokine subfamily B member 15 (Mus musculus), K60
protein (Gallus gallus), sheep IL-8 precursor (Ovis aries), pig
IL-8 precursor (Sus scrofa) and bovine IL-8 precursor (Bos
taurus).
[0223] FIG. 3: Sig P cleavage site prediction for INSP085.
[0224] FIG. 4: Sig P peptide prediction for INSIP085.
[0225] FIG. 5: INSP085 coding exon organization in genomic DNA and
position of PCR primers.
[0226] FIG. 6: Nucleotide sequence and translation of cloned
INSP085 ORF
[0227] FIG. 7: Map of pENTR-INSP085-6HIS
[0228] FIG. 8: Map of pEAK12d-INSP085-6HIS
EXAMPLES
Example 1
INSP085 Protein BLAST Results
[0229] The INSP085 polypeptide sequence, shown in SEQ ID NO:8, was
used as a BLAST query against the NCBI non-redundant sequence
database. As can be seen in FIG. 1, the top hit is for an inducible
cytokine (chemokines were considered to be part of the cytokine
family for a long time). The third to fifth hits are all for IL-8
precursors, thus providing flirter evidence that INSP085 is a
member of the IL-8 like protein family.
Example 2
INSP085 Signal Sequence
[0230] FIGS. 3 and 4 show that INSP085 is predicted to possess a
signal peptide at the start of the protein. As the data in FIG. 3
clearly shows, the signal peptide cleavage site is thought to be
between residues 16 and 17 of the INSP085 full protein sequence.
FIG. 4 shows that this sequence is not an anchor peptide, so it is
likely that INSP085 is a secreted protein (0.818 probability)
(Nielsen, H. et al 1997, Protein Engineering, 10, 1-6; Nielsen, H.,
and Krogh, A.: Prediction of signal peptides and signal anchors by
a hidden Markov model. In Proceedings of the Sixth International
Conference on Intelligent Systems for Molecular Biology (ISMB 6),
AAAI Press, Menlo Park, Calif., pp. 122-130 (1998)).
Example 3
Cloning of INSP085 by Exon Assemble
[0231] 1. PCR Amplification of Exons Encoding INSP085 from Genomic
DNA.
[0232] PCR primers were designed to amplify exons 1, 2 and 3 of
INSP085 (table 1). The reverse primer for exon 1 (INSP085-exon1R)
has an overlap of 18 bases with exon 2 of INSP085 at its 5' end.
The forward primer for exon 2 (INSP085-exon2F) has an 18 bp overlap
with exon 1 of INSP085 at its 5' end. The reverse primer for exon 2
(INSP085-exon2R) has an overlap of 18 bases with exon 3 at its 5'
end. The forward primer for exon 3 (NSP085-exon3F) contains a 18 bp
overlap with exon 2 at its 5' end.
[0233] To generate exon 1 of INSP085, the PCR reaction was
performed in a final volume of 100 .mu.l and contained 1.5 .mu.l of
genomic DNA (0.1 .mu.g/.mu.l Clontech cat no. 65550-1), 2 .mu.l of
10 mM dNTPs (Amersham Pharmacia Biotech), 6 .mu.l of INSP085-exon1F
(10 .mu.M), 6 .mu.l of INSP085-exon1R (10 .mu.M), 5 .mu.l of
10.times. Pfu buffer and 1 .mu.l of Pfu polymerase (3 U/.mu.l)
(Promega cat. no. M774B). The PCR conditions were 94.degree. C. for
2 min; 35 cycles of 9-4.degree. C. for 30s, 60.degree. C. for 30s
and 72.degree. C. for 1 min; an additional elongation cycle of
72.degree. C. for S main; and a holding cycle of 4.degree. C.
Reaction products were loaded onto a 1.5% agarose gel (1.times.
TAE) and PCR products of the correct size (158 bp) were
gel-purified using a Qiaquick Gel Extraction Kit (Qiagen cat. no.
28704) and eluted in 50 .mu.l of elution buffer (Qiag en). Exon 1
was subcloned into pCR4Blunt TOPO vector (Invitrogen) by incubating
4 .mu.l of get purified PCR product, with 1 .mu.l of salt solution
and 1 .mu.l of topoisomerase modified pCR4 Blunt-TOPO vector.
[0234] The reaction mixture was incubated at RT for 30 mm. An
aliquot of this reaction (2 .mu.l) was used to transform 50 .mu.l
of E. coli Top 10 multishot cells (Invitrogen) by heat shock as
follows: cells and DNA were mixed in a 12 ml polypropylene tube.
The mixture was stored on ice for 15 min then heat shocked at
42.degree. C. for exactly 30 sec. Samples were then stored on ice
for a further 2 min, then diluted by addition of 250 .mu.l of room
temperature SOC medium and incubated for 1 h at 37.degree. C. with
shaking. Transformants (300 .mu.l) were plated on LB plates
containing 100 .mu.g/ml of ampicillin and incubated over night at
37.degree. C. Mini prep DNA was prepared from 5 ml cultures
prepared from 30 of the resultant colonies using a Qiaprep Turbo
9600 robotic system (Qiagen). Mini-prep DNA was eluted in 50 .mu.l
of elution buffer. Plasmid mini prep DNA (200-500 ng) was then
subjected to DNA sequencing with T7 and T3 sequencing primers using
the BigDyeTerminator system (Applied Biosystems cat. no. 4390246)
according to the manufacturer's instructions. Sequencing reactions
were purified using Dye-Ex columns (Qiagen) or Montage SEQ 96
cleanup plates (Millipore cat. no. LSKS09624) then analyzed on an
Applied Biosystems 3700 sequencer. One of the clones containing the
correct sequence of exon 1 was then used as template for further
amplification of exon 1 in a 50 .mu.l PCR reaction containing 0.3
.mu.l of miniprep DNA, 2 .mu.l of 5 mM dNTPs (Amersham Pharmacia
Biotech), 6 .mu.l of INSP085-exon1F (10 .mu.M), 6 .mu.l of
INSP085-exon1R (10 .mu.M), 5 .mu.l of 10.times. Pfu buffer and 0.5
.mu.l of Pfu polymerase (3 U/.mu.l) (Promega cat no. M774B). The
PCR conditions were 94.degree. C. for 2 min; 30 cycles of
94.degree. C. for 30s, 60.degree. C. for 30 s and 72.degree. C. for
1 min; an additional elongation cycle of 72.degree. C. for 3 min;
and a holding cycle oaf 4.degree. C. Reaction products were loaded
onto a 1.5% agarose gel (1.times.TAE) and PCR products of the
correct size (168 bp) were gel-purified using a Qiaquick Gel
Extraction Kit (Qiagen cat. no. 28704) and eluted in 30 .mu.l of
elution buffer (Qiagen).
[0235] To generate exon 2 of INSP085, the PCR reaction was
performed in a final volume of 100 .mu.l and contained 1.5 .mu.l of
human genomic DNA (0.1 .mu.g/.mu.l, Novagen cat. No. 69237), 2
.mu.l of 10 mM dNTs (Amersham Pharmacia Biotech), 6 .mu.l of
INSP085-exon2F (10 .mu.M), 6 .mu.l of INSP085-exon2R (10 .mu.M), 5
.mu.l of 10.times. Pwo buffer and 1 .mu.l of Pwo polymerase (5
U/.XI.l) (Roche, cat. No. 1 644 955). The PCR conditions were
94.degree. C. for 2 min; 35 cycles of 94.degree. C. for 30s,
60.degree. C. for 30s and 72.degree. C. for 1 min; an additional
elongation cycle of 72.degree. C. for 5 rain; and a holding cycle
of 4.degree. C. Reaction products were loaded onto a 1.5% agarose
gel (1.times.TAE) and PCR products of the correct size (143 bp)
were gel-purified using a Qiaquick Gel Extraction Kit (Qiagen cat.
no. 28704) and eluted in 30 .mu.l of elution buffer (Qiagen).
[0236] To generate exon 3 of INSP085, the PCR reaction was
performed in a final volume of 100 .mu.l and contained 1.5 .mu.l of
human genomic DNA (0.1 .mu.g/.mu.l, Novagen cat. No 69237), 2 .mu.l
of 10 mM dNTPs (Amersham Pharmacia Biotech), 6 .mu.l of
INSP085-exon3F (10 .mu.M), 6 .mu.l of INSP085-exon3R (10 .mu.M), 5
.mu.l of 10.times. Pwo buffer and 1 .mu.l of Pwo polymerase (5
U/.mu.l) (Roche, cat. No. 1 644 955). The PCR conditions were
94.degree. C. for 2 min; 35 cycles of 94.degree. C. for 30s,
60.degree. C. for 30s and 72.degree. C. for 1 min; an additional
elongation cycle of 72.degree. C. for 5 min; and a holding cycle of
4.degree. C. Reaction products were loaded onto a 1.5% agarose gel
(1.times.TAE) and PCR products of the correct size (131 bp) were
gel-purified using a Qiaquick Gel Extraction Kit (Qiagen cat. no.
28704) and eluted in 30 .mu.l of elution buffer (Qiagen).
2. Assembly of Exons 1-3 to Generate the INSP085 ORF
[0237] Exons 1, 2 and 3 were assembled in a 50 .mu.l PCR reaction
containing 2 .mu.l of gel purified exon 1, 5 .mu.l of gel purified
exon 2, 5 .mu.l of gel purified exon 3, 2 .mu.l of 5 mM dNTPs, 64
.mu.l of INSP085-EX1 (10 .mu.M), 641 of INSP085-EX2 (10 .mu.M), 5
.mu.l of 10.times. Pfu buffer, and 0.5 .mu.l of Pfu polymerase (3
U/.mu.l) (Promega). The INSP085-EX1 primer contains a partial attB1
site and Kozak sequence at the 5' end. The INSP085-EX2 primer
contains a 5 HIS sequence at its 5' end. The reaction conditions
were: 94.degree. C., 4 min; 10 cycles of 94.degree. C. for 30s,
48.degree. C. for 30s and 70.degree. C. for 2 min; 25 cycles of
94.degree. C. for 30s, 52.degree. C., for 30s and 70.degree. C. for
2 min; an additional elongation step of 70.degree. C. for 10 min;
and a holding cycle at 4.degree. C. Reaction products were analysed
on a 1.5% agarose gel (1.times.TAE). PCR products of the correct
size (387 bp) were gel purified using a Qiaquick Gel Extraction Kit
(Qiagen cat. no. 28704) and eluted in 50 .mu.l of elution buffer
(Qiagen). The resultant PCR product contains the ORF of
INSP085.
3. Subcloning of the INSP085 ORF into pDONR201
[0238] The INSP085 ORF was subcloned into pDONR201 using the
Gateway.TM. cloning system (Invitrogen). A partial attB1
recombination site was added to the 5' end of INSP085 ORF and a 6
HIS tag sequence, stop codon and attB2 recombination site was added
to the 3' end of the INSP085 ORF in a 50 .mu.l PCR reaction
containing 2 .mu.l of gel purified INSP085-ORF PCR product, 2 .mu.l
of 5 mM dNTPs (Amersham Pharmacia Biotech), 6 .mu.l of GCP-F (10
.mu.M), 6 .mu.l of GCP-R (10 .mu.M), 5 .mu.l of 10.times. Pfu
buffer and 0.5 .mu.l of Pfu polymerase (5 U/.mu.l) in a final
volume of 50 .mu.l. The PCR conditions wee 94.degree. C. for 2 min;
30 cycles of 94.degree. C. for 30s; 55.degree. C. for 30s and
72.degree. C. for 1 min; an additional elongation step of
12.degree. C. for 3 mm and a holding cycle of 4.degree. C. Reaction
products were analysed on a 1.5% agarose gel (1.times.TAE) and PCR
products of the correct size (445 bp, corresponding to
Gateway-modified NSP085 ORF) were gel purified using a Qiaquick Gel
Extraction Kit (Qiagen cat. no. 28704) and eluted in 50 .mu.l of
elution buffer (Qiagen). Gateway-modified INSP085 ORF was then
transferred to pDONR201 using BP clonase as follows: 5 .mu.l of
Gateway-modified INSP085 ORF was incubated with 1.5 .mu.l pDONR201
(0.1 .mu.g/.mu.l), 2 .mu.l BP buffer and 1.5 .mu.l of BP clonase
enzyme mix (Invitrogen) at RT for Oh. The reaction was stopped by
addition of 1 .mu.l proteinase K (2 .mu.g) and incubated at
37.degree. C. for a further 10 min. An aliquot of this reaction (1
.mu.l) was used to transform 20 .mu.l of E. coli DH10B cells
(Invitrogen) (diluted 1/5 in sterile water) by electroporation
using a Biorad Gene Pulser according to the manufacturer's
recommendations. Electroporated cells were transferred to 12 ml
polypropylene tubes, diluted by addition of 900 .mu.l of room
temperature LB medium and incubated for 1 h at 37.degree. C. with
shaking. Transformants (100 .mu.l) were plated on LB plates
containing 40 .mu.g/ml of kanamycin and incubated cover night at
37.degree. C. Mini prep DNA was prepared from 5 ml overnight
cultures from 8 of the resultant colonies using a Qiaprep Turbo
9600 robotic system (Qiagen). Mini-prep D>NA was eluted in 50
.mu.l off elution buffer. Plasmid mini prep DNA (200-500 ng) was
then subjected to DNA sequencing with pENTR-F and pENTR-R
sequencing primers using the BigDyeTerminator system (Applied
Biosystems cat. no. 4390246) according to the manufacturer's
instructions. Sequencing reactions were purified using Dye-Es
columns (Qiagen) or Montage SEQ 96 cleanup plates (Millipore cat.
no. LSKS09624) then analyzed on an Applied Biosystems 3700
sequencer.
4. Subcloning of the INSP085 ORF to Expression Vector pEAK12d
[0239] Plasmid eluate (1.5 .mu.l) from a pDONR201 clot containing
the correct sequence of the INSP085 ORF (pENTR-INSP085-6HIS,
plasmid ID no. 13414, FIG. 4) was then used in a recombination
reaction containing 1.5 .mu.l pEAK1.2d vector (0.1 .mu.g/.mu.l), 20
.mu.l LR buffer and 1.5 .mu.l of LR clonase (Invitrogen) in a final
volume of 10 .mu.l. The mixture was incubated at RT for 1 h,
stopped by addition of 1 .mu.l proteinase K (2 .mu.g) and incubated
at 37.degree. C. for a further 10 min. An aliquot of this reaction
(1 .mu.l) was used to transform 20 .mu.l of E. coli DH10B cells
(Invitrogen) (diluted 1/5 in sterile water) by electroporation
using a Biorad Gene Pulser according to the manufacturer's
recommendations. Electroporated cells were transferred to 12 ml
polypropylene tubes, diluted by addition of 900 .mu.l of room
temperature SOC medium and incubated for 1 h at 37.degree. C. with
shaking. Transformants (100 .mu.l) were plated on LB plates
containing 100 .mu.g/ml of ampicillin and incubated at 37.degree.
C. overnight. Mini prep DNA was prepared from 5 ml overnight
cultures from 4 of the resultant colonies using a Qiaprep Turbo
9600 robotic system (Qiagen) as described above. Mini-prep DNA was
eluted in 100 .mu.l of elution buffer. Plasmid mini prep DNA
(200-500 ng) was then subjected to DNA sequencing with pEAK12-F and
pEAK12-R sequencing primers using the BigDyeTerminator system
(Applied Biosystems cat no. 4390246) according to the
manufacturer's instructions. Sequencing reactions were purified
using Dye-Ex columns (Qiagen) or Montage SEQ 96 cleanup plates
(Millipore cat. no. LSKS09624) then analyzed on an Applied
Biosystems 3700 sequencer.
[0240] CsCl gradient purified maxi-prep DNA was prepared from a 500
ml culture of a sequence verified clone, pEAK12d-INSP085-6HIS
(plasmid ID no. 13413, FIG. 5) (Sambrook J. et al., in Molecular
Cloning, a Laboratory Manual, 2.sup.nd edition, 1989, Cold Spring
Harbor Laboratory Press), resuspended at a concentration of 1
.mu.g/.mu.l in sterile water and stored at -20.degree. C.
TABLE-US-00001 TABLE 1 Primers for INSP085 cloning and sequencing
Primer Sequence (5'-3') GCP Forward G GGG ACA AGT TTG TAC AAA AAA
GCA GGC TTC GCC ACC GCP Reverse GGG GAC CAC TTT GTA CAA GAA AGC TGG
GTT TCA ATG GTG ATG GTG ATG GTG INSP085-exon1F ATG AGC TTG GCT ATC
CTC ATA TGG T INSP085-exon1R ##STR00001## INSP085-exon2F
##STR00002## INSP085-exon2R ##STR00003## INSP085-exon3F
##STR00004## INSP085-exon3R TTA GCG TTT GTT ACC TGG GTT ATG AC
INSP085-EX1 GCA GGC TTC GCC ACC ATG AGC TTG GCT ATC CTC AT
INSP085-EX2 GTG ATG GTG ATG GTG GCG TTT GTT ACC TGG GTT AT pEAK12-F
GCC AGC TTG GCA CTT GAT GT pEAK12-R GAT GGA GGT GGA CGT GTC AG
pENTR-F TCG CGT TAA CGC TAG CAT GGA TCT C pENTR-R GTA ACA TCA GAG
ATT TTG AGA CAC T7 TAA TAC GAC TCA CTA TAG GG T3 CTC CCT TTA GTG
AGG GTA ATT Underlined sequence = Kozak sequence Bold = Stop codon
Italic sequence = His tag Highlighted sequence = overlap with
adjacent exon
Example 4
Expression and Purification of INSP085
[0241] Further experiments may now be performed to determine the
tissue distribution and expression levels of the INSP085
polypeptides in vivo, on the basis of the nucleotide and amino acid
sequence disclosed herein.
[0242] The presence of the transcripts for INSP085 may be
investigated by PCR of cDNA from different human tissues. The
INSP085 transcripts may be present at very low levels in the
samples tested. Therefore, extreme care is needed in the design of
experiments to establish the presence of a transcript in various
human tissues as a small amount of genomic contamination in the RNA
preparation will provide a false positive result. Thus, all RNA
should be treated with DNAse prior to use for reverse
transcription. In addition, for each tissue a control reaction may
be set up in which reverse transcription was not undertaken (a-ve
RT control).
[0243] For example, 1 .mu.g of total RNA from each tissue may be
used to generate cDNA using Multiscript reverse transcriptase (ABI)
and random hexamer primers. For each tissue, a control reaction is
set up in which all the constituents are added except the reverse
transcriptase (-ve RT control). PCR reactions are set up for each
tissue on the reverse transcribed RNA samples and the minus RT
controls. INSP085-specific primers may readily be designed on the
basis of the sequence information provided herein. The presence of
a product of the correct molecular weight in the reverse
transcribed sample together with the absence of a product in the
minus RT control may be taken as evidence for the presence of a
transcript in that tissue. Any suitable cDNA libraries may be used
to screen for the INSP085 transcripts, not only those generated as
described above.
[0244] The tissue distribution pattern of the INSP085 polypeptides
will provide further useful information in relation to the function
of those polypeptides.
[0245] In addition, further experiments may now be performed using
the pEAK12d-INSP085-6HIS expression vectors. Transfection of
mammalian cell lines with these vectors may enable the high level
expression of the INSP085 proteins and thus enable the continued
investigation of the functional characteristics of the INSP085
polypeptides. The following material and methods are an example of
those suitable in such experiments:
Cell Culture
[0246] Human Embryonic Kidney 293 cells expressing the Epstein-Barr
virus Nuclear Anti gen (WK293-EBNA, Invitrogen) are maintained in
suspension in Ex-cell VPRO serum-free medium (seed stock,
maintenance medium, JRH). Sixteen to 20 hours prior to transfection
(Day-1), cells are seeded in 2.times. T225 flasks (50 ml per flask
in DMEM/F12 (1:1) containing 2% FBS seeding medium (JRH) at a
density of 2.times.10.sup.5 cells/ml). The next day (transfection
day 0) transfection takes place using the JetPEI.TM. reagent (2
.mu.l/.mu.g of plasmid DNA, PolyPlus-transfection). For each flask,
plasmid. DNA is co-transfected with GFP (fluorescent reporter gene)
DNA. The transfection mix is then added to the 2.times.1225 flasks
and incubated at 37.degree. C. (5% CO.sub.2) for 6 days.
Confirmation of positive transfection may be carried out by
qualitative fluorescence examination at day 1 and day 6 (Axio-vert
10 Zeiss).
[0247] On day 6 (harvest day), supernatants from the two flasks are
pooled and centrifuged e.g. 4.degree. C., 400 g) and placed into a
pot bearing a unique identifier. One aliquot (500 .mu.l) is kept
for QC of the 6H is-tagged protein (internal bioprocessing QC).
[0248] Scale-up batches may be produced by following the protocol
called "PEI transfection of suspension cells", referenced
BP/PEI/HH/02/04, with Polyethyleneimine from Polysciences as
transfection agent
Purification Process
[0249] The culture medium sample containing the recombinant protein
with a C-terminal 6H is tag is diluted with cold buffer A (50 mM
NaH.sub.2PO.sub.4; 600 ml % NaCl; 8.7% (w/v) glycerol, pH 7.5). The
sample is filtered then through a sterile filter (Millipore) and
kept at 4.degree. C. in a sterile square media bottle
(Nalgene).
[0250] The purification is performed at 4.degree. C. on the VISION
workstation (Applied Biosystems) connected to an automatic sample
loader (Labomatic). The purification procedure is composed of two
sequential steps, metal affinity chromatography on a Poros 20 MC
(Applied Biosystems) column charged with Ni ions (4.6.times.50 mm,
0.83 ml), followed by gel filtration on a Sephadex G-25 medium
(Amersham Pharmacia) column (1.0.times.10 cm).
[0251] For the first chromatography step the metal affinity column
is regenerated with 3 column volumes of EDTA solution (100M EDTA;
1M NaCl; pH 8.0), recharged with Ni ions through washing with 15
column volumes of a 100 mM NiSO.sub.4 solution, washed with 10
column volumes of buffer A, followed by 7 column volumes of buffer
B (50 mM NaH.sub.2PO.sub.4; 600 mM NaCl; 8.7% (w/v) glycerol, 400
mM; imidazole, pH 7.5), and finally equilibrated with 15 column
volumes of buffer A containing 15 mM imidazole. The sample is
transferred, by the Labomatic sample loader, into a 200 ml sample
loop and subsequently charged onto the Ni metal affinity column at
a flow rate of 10 ml/min. The column is washed with 12 column
volumes of buffer A, followed by 28 column volumes of buffer A
containing 20 mM imidazole. During the 20 mM imidazole wash loosely
attached contaminating proteins are eluted from the column. The
recombinant His-tagged protein is finally eluted with 10 column
volumes of buffer B at a flow rate of 2 ml/min, and the eluted
protein is collected.
[0252] For the second chromatography step, the Sephadex G-25
gel-filtration column is regenerated with 2 ml of buffer D (1.137M
NaCl; 2.7 nm t KCl; 1.5 mM KH.sub.2PO.sub.4; 8 mM
Na.sub.2HPO.sub.4; pH 7.2), and subsequently equilibrated with 4
column volumes of buffer C (137 mM NaCl; 2.7 mM KCl; 1.5 mM
KH.sub.2PO.sub.4; 8 mM Na.sub.2HPO.sub.4; 20% (w/v) glycerol; pH
7.4). The peak fraction eluted from the Ni-column is automatically
loaded onto the Sephadex G-25 column through the integrated sample
loader on the VISION and the protein is eluted with buffer C at a
flow rate of 2 ml/min. The fraction was filtered through a sterile
centrifugation filter (Millipore), frozen and stored at -80.degree.
C. An aliquot of the sample is analyzed on SDS-PAGE (4-12% NuPAGE
gel; Novex) Western blot with anti-His antibodies. The NuPAGE gel
may be stained in a 0.1% Coomassie blue R250 staining solution (30%
methanol, 10% acetic acid) at room temperature for 1 h and
subsequently destained in 20% methanol, 7.5% acetic acid until the
background is clear and the protein bands clearly visible.
[0253] Foil owing the electrophoresis the proteins are
electrotransferred from the gel to a nitrocellulose membrane. The
membrane is blocked with 5% milk powder in buffer E (137 mM NaCl;
2.7 mM KCl; 1.5 mM KH.sub.2PO.sub.4; 8 mM Na.sub.2HPO.sub.4; 0.1%
Tween 20, pH 7.4) for 1 h at room temperature, and subsequently
incubated with a mixture of 2 rabbit polyclonal anti-His antibodies
(G-18 and H-15, 0.2 .mu.g/ml each, Santa Cruz) in 2.5% milk powder
in buffer E overnight at 4.degree. C. After a further 1 hour
incubation at room temperature, the membrane is washed with buffer
E (3.times.10 min), and then incubated with a secondary
HRP-conjugated anti-rabbit antibody (DAKO, HRP 0399) diluted 1/3000
in buffer E containing 2.5% milk powder for 2 hoes at room
temperature. After washing with buffer E (3.times.10 minutes), the
membrane is developed with the ECL kit (Amersham Pharmacia) for 1
min. The membrane is subsequently exposed to a Hyperfilm (Amersham
Pharmacia), the film developed and the western blot image visually
analysed.
[0254] For samples that showed detectable protein bands by
Coomassie staining, the protein concentration may be determined
using the BCA protein assay kit (Pierce) with bovine serum albumin
as standard.
[0255] Furthermore, overexpression or knock-down of the expression
of the polypeptides in cell lines may be used to determine the
effect on transcriptional activation of the host cell genome.
Dimerisation partners, co-activators and co-repressors of the
INSP085 polypeptide may be identified by immunoprecipitation
combined with Western blotting and immunoprecipitation combined
with mass spectroscopy.
Sequence CWU 1
1
281140DNAHomo sapiens 1atgagcttgg ctatcctcat atggtggcag ctgcatttta
acagagcagg ggtagaaact 60cctaggcttc ttaccttgtt ggcctggaac atctacatca
tcacttttaa cacttcctat 120tggccaaagc aagtcatgag 140247PRTHomo
sapiens 2Met Ser Leu Ala Ile Leu Ile Trp Trp Gln Leu His Phe Asn
Arg Ala1 5 10 15Gly Val Glu Thr Pro Arg Leu Leu Thr Leu Leu Ala Trp
Asn Ile Tyr 20 25 30Ile Ile Thr Phe Asn Thr Ser Tyr Trp Pro Lys Gln
Val Met Arg 35 40 453107DNAHomo sapiens 3agttggatgt cagtgtattc
agacacattc tgactttatt cctcaccaat tcattaaaaa 60tgatcagttg atacataagg
atcctttctg cagaagaaaa gaagtca 107436PRTHomo sapiens 4Val Gly Cys
Gln Cys Ile Gln Thr His Ser Asp Phe Ile Pro His Gln1 5 10 15Phe Ile
Lys Asn Asp Gln Leu Ile His Lys Asp Pro Phe Cys Arg Arg 20 25 30Lys
Glu Val Lys 355113DNAHomo sapiens 5aacataaggt atgtgttgcc ttaattgctt
gtatagttga ggtaaaaatt gttggccaga 60aaatagtctg gaatcatttt agaagttgtc
ataacccagg taacaaacgc taa 113636PRTHomo sapiens 6His Lys Val Cys
Val Ala Leu Ile Ala Cys Ile Val Glu Val Lys Ile1 5 10 15Val Gly Gln
Lys Ile Val Trp Asn His Phe Arg Ser Cys His Asn Pro 20 25 30Gly Asn
Lys Arg 357360DNAHomo sapiens 7atgagcttgg ctatcctcat atggtggcag
ctgcatttta acagagcagg ggtagaaact 60cctaggcttc ttaccttgtt ggcctggaac
atctacatca tcacttttaa cacttcctat 120tggccaaagc aagtcatgag
agttggatgt cagtgtattc agacacattc tgactttatt 180cctcaccaat
tcattaaaaa tgatcagttg atacataagg atcctttctg cagaagaaaa
240gaagtcaaac ataaggtatg tgttgcctta attgcttgta tagttgaggt
aaaaattgtt 300ggccagaaaa tagtctggaa tcattttaga agttgtcata
acccaggtaa caaacgctaa 3608119PRTHomo sapiens 8Met Ser Leu Ala Ile
Leu Ile Trp Trp Gln Leu His Phe Asn Arg Ala1 5 10 15Gly Val Glu Thr
Pro Arg Leu Leu Thr Leu Leu Ala Trp Asn Ile Tyr 20 25 30Ile Ile Thr
Phe Asn Thr Ser Tyr Trp Pro Lys Gln Val Met Arg Val 35 40 45Gly Cys
Gln Cys Ile Gln Thr His Ser Asp Phe Ile Pro His Gln Phe 50 55 60Ile
Lys Asn Asp Gln Leu Ile His Lys Asp Pro Phe Cys Arg Arg Lys65 70 75
80Glu Val Lys His Lys Val Cys Val Ala Leu Ile Ala Cys Ile Val Glu
85 90 95Val Lys Ile Val Gly Gln Lys Ile Val Trp Asn His Phe Arg Ser
Cys 100 105 110His Asn Pro Gly Asn Lys Arg 115992DNAHomo sapiens
9ggggtagaaa ctcctaggct tcttaccttg ttggcctgga acatctacat catcactttt
60aacacttcct attggccaaa gcaagtcatg ag 921031PRTHomo sapiens 10Gly
Val Glu Thr Pro Arg Leu Leu Thr Leu Leu Ala Trp Asn Ile Tyr1 5 10
15Ile Ile Thr Phe Asn Thr Ser Tyr Trp Pro Lys Gln Val Met Arg 20 25
3011312DNAHomo sapiens 11ggggtagaaa ctcctaggct tcttaccttg
ttggcctgga acatctacat catcactttt 60aacacttcct attggccaaa gcaagtcatg
agagttggat gtcagtgtat tcagacacat 120tctgacttta ttcctcacca
attcattaaa aatgatcagt tgatacataa ggatcctttc 180tgcagaagaa
aagaagtcaa acataaggta tgtgttgcct taattgcttg tatagttgag
240gtaaaaattg ttggccagaa aatagtctgg aatcatttta gaagttgtca
taacccaggt 300aacaaacgct aa 31212103PRTHomo sapiens 12Gly Val Glu
Thr Pro Arg Leu Leu Thr Leu Leu Ala Trp Asn Ile Tyr1 5 10 15Ile Ile
Thr Phe Asn Thr Ser Tyr Trp Pro Lys Gln Val Met Arg Val 20 25 30Gly
Cys Gln Cys Ile Gln Thr His Ser Asp Phe Ile Pro His Gln Phe 35 40
45Ile Lys Asn Asp Gln Leu Ile His Lys Asp Pro Phe Cys Arg Arg Lys
50 55 60Glu Val Lys His Lys Val Cys Val Ala Leu Ile Ala Cys Ile Val
Glu65 70 75 80Val Lys Ile Val Gly Gln Lys Ile Val Trp Asn His Phe
Arg Ser Cys 85 90 95His Asn Pro Gly Asn Lys Arg
1001337DNAArtificial SequencePrimer GCP Forward 13ggggacaagt
ttgtacaaaa aagcaggctt cgccacc 371451DNAArtificial SequencePrimer
GCP reverse 14ggggaccact ttgtacaaga aagctgggtt tcaatggtga
tggtgatggt g 511525DNAArtificial SequencePrimer INSP085-exon1F
15atgagcttgg ctatcctcat atggt 251641DNAArtificial SequencePrimer
INSP085-exon1R 16atacactgac atccaactct catgacttgc tttggccaat a
411738DNAArtificial SequencePrimer INSP085-exon2F 17gccaaagcaa
gtcatgagag ttggatgtca gtgtattc 381841DNAArtificial SequencePrimer
INSP085-exon2R 18caacacatac cttatgtttg acttcttttc ttctgcagaa a
411941DNAArtificial SequencePrimer INSP085-exon3F 19gcagaagaaa
agaagtcaaa cataaggtat gtgttgcctt a 412026DNAArtificial
SequencePrimer INSP085-exon3R 20ttagcgtttg ttacctgggt tatgac
262135DNAArtificial SequencePrimer INSP085-EX1 21gcaggcttcg
ccaccatgag cttggctatc ctcat 352235DNAArtificial SequencePrimer
INSP085-EX2 22gtgatggtga tggtggcgtt tgttacctgg gttat
352320DNAArtificial SequencePrimer pEAK12-F 23gccagcttgg cacttgatgt
202420DNAArtificial SequencePrimer pEAK12-R 24gatggaggtg gacgtgtcag
202525DNAArtificial SequencePrimer pENTR-F 25tcgcgttaac gctagcatgg
atctc 252624DNAArtificial SequencePrimer pENTR-R 26gtaacatcag
agattttgag acac 242720DNAArtificial SequencePrimer T7 27taatacgact
cactataggg 202821DNAArtificial SequencePrimer T3 28ctccctttag
tgagggtaat t 21
* * * * *
References