Note: Descriptions are shown in the official language in which they were submitted.
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1
INTERFERON GAMMA-LIKE PROTEIN
This invention relates to a protein, termed INSP037, herein identified as an
interferon
gamma-like secreted protein of the four helical bundle cytokine fold, and to
the use of
this protein and nucleic acid sequences from the encoding gene in the
diagnosis,
prevention and treatment of disease.
All publications, patents and patent applications cited herein are
incorporated in full
by reference.
BACKGROUND
The process of drug discovery is presently undergoing a fundamental revolution
as the
era of functional genomics comes of age. The teen "functional genomics"
applies to
an 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.
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.
Various institutions and commercial organisations 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 to Secreted Proteins
The ability of 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 fusion of a
secretory
vesicle with the plasma 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.
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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.
Introduction to Cytokines
Cytokines are a family of growth factors primarily secreted from leukocytes,
and are
messenger proteins that act as potent regulators capable of effecting cellular
processes
at sub-nanomolar concentrations. Interleukins, neurotrophins, growth factors,
interferons and chemokines all define cytokine families that work in
conjunction with
cellular receptors to regulate cell proliferation and differentiation. Their
size allows
cytokines to be quickly transported around the body and degraded when
required.
Their role in controlling a wide range of cellular functions, especially the
immune
response and cell growth has been revealed by extensive resear ch over the
last twenty
years (Boppana, S.B (1996) Indian. J. Pediatr. 63(4):447-52). Cytokines, as
for other
growth factors, are differentiated from classical hormones by the fact that
they are
produced by a number of different cell types rather than just one specific
tissue or
gland, and also effect a broad range of cells via interaction with specific
high affinity
receptors located on target cells.
All cytokine communication systems show both pleiotropy (one messenger
producing
multiple effects) and redundancy (each effect is produced by more than one
messenger (Tringali, G. et al (2000) Therapie. 55(1):171-5; Tessarollo, L.
(1998)
Cytolcine Growth Factor Rev. 9(2):125-137). An individual cytokine's effects
on a cell
can also be dependent on its concentration, the concentration of other
cytokines, the
temporal sequence of cytokines, and the internal state of the cell (for
example, it may
be affected by the cell cycle, presence of neighbouring cells, cancerous).
Although cytolcines are typically small proteins (under 200 amino acids) they
are
often formed from larger precursors which are post-translationally spliced.
This, in
addition to mRNA alternative splicing pathways, give a wide spectrum of
variants of
each cytokine each of which may differ substantially in biological effect.
Membrane
and extracellular matrix associated forms of many cytokines have also been
isolated
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(Okada-Ban, M. et al (2000) Int. J. Biochem. Cell Biol. 32(3):263-267; Atamas,
S.P.
(1997) Life Sci. 61(12):1105-1112).
Cytokines can be grouped into families, though most are unrelated.
Categorisation is
usually based on secondary structure composition, as sequence similarity is
often very
low. The families are named after the archetypal member e.g. IFN-like, IL2-
like, IL1-
like, Il-6 like and TNF-like (Zlotnik, A. et al., (2000) Immunity. 12(2):121-
127).
Studies have shown cytokines are involved in many important reactions in multi-
cellular organisms such as immune response regulation (Nishihira, J. (1998)
Int. J.
Mol. Med. 2(1):17-28), inflammation (Kim, P.K. et al., (2000) Surg. Clin.
North. Am.
80(3):885-894), wound healing (Clark, R.A. (1991) J. Cell Biochem. 46(1):1-2),
embryogenesis and development, and apoptosis (Flad, H.D. et al., (1999)
Pathobiology. 67(5-6):291-293). Pathogenic organisms (both viral and
bacterial) such
as HIV and Kaposi's sarcoma-associated virus encode anti-cytokine factors as
well as
cytolcine analogues, which allow them to interact with cytokine receptors and
control
the body's immune response (Sozzani, S. et al., (2000) Ph arm. Acta. Helv.
74(2-
3):305-312; Aoki, Y. et al., (2000) J. Hematother. Stem Cell Res. 9(2):137-
145).
Virally encoded cytokines, virokines, have been shown to be required for
pathogenicity of viruses due to their ability to mimic and subvert the host
immune
system.
Cytokines may be useful for the treatment, prevention and/or diagnosis of a
wide
variety of medical conditions and diseases, including immune disorders, such
as
autoimmune disease, rheumatoid arthritis, osteoarthritis, psoriasis, systemic
lupus
erythematosus, and multiple sclerosis, inflammatory disorders, such as
allergy,
rhinitis, conjunctivitis, glomerulonephritis, uveitis, Crohn's disease,
ulcerative colitis,
inflammatory bowel disease, pancreatitis, digestive system inflammation,
sepsis,
endotoxic shock, septic shock, cachexia, myalgia, anlcylosing spondylitis,
myasthenia
gravis, post-viral fatigue syndrome, pulmonary disease, respiratory distress
syndrome,
asthma, chronic-obstructive pulmonary disease, airway inflammation, wound
healing,
endometriosis, dermatological disease, Behcet's disease, neoplastic disorders,
such as
melanoma, sarcoma, renal tumour, colon tumour, haematological disease,
myeloproliferative disorder, Hodglcin's disease, osteoporosis, obesity,
diabetes, gout,
cardiovascular disorders, reperfusion injury, atherosclerosis, ischaemic heart
disease,
cardiac failure, stroke, liver disease, AIDS, AIDS related complex,
neurological
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disorders, male infertility, ageing and infections, including plasmodium
infection,
bacterial infection and viral infection.
Clinical use of cytokines has focused on their role as regulators of the
immune system
(Rodriguez, F.H. et al., (2000) Curr. Pharm. Des. 6(6):665-680) for instance
in
promoting a response against thyroid cancer (Schmutzler, C. et al., (2000)
143(1):15-
24). Their control of cell growth and differentiation has also made cytokines
anti-
cancer targets (Lazax-Molnar, E. et al., (2000) Cytokine. 12(6):547-554; Gado,
K.
(2000) 24(4):195-209). Novel mutations in cytokines and cytokine receptors
have
been shown to confer disease resistance in some cases (van Deventer, S.J. et
al.,
(2000) Intensive Care Med. 26 (Suppl 1):S98:S102). The creation of synthetic
cytokines (muteins) in order to modulate activity and remove potential side
effects has
also been an important avenue of research (Shanafelt, A.B. et al., (1998)
95(16):9454-
9458).
Thus, cytokine molecules have been shown to play a role in diverse
physiological
functions, many of which can play a role in disease processes. Alteration of
their
activity is a means to alter the disease phenotype and as such identification
of novel
cytolcine molecules is highly relevant as they may play a role in or be useful
in the
development of treatments for the diseases identified above, as well as other
disease
states.
Introduction to Interferons
Interferons are members of the four-helical bundle family of cytokines. They
are
classified as Type I or Type II depending on their structure and their
stability in acid
medium. Type I interferons are classified into five groups on the basis of
their
sequence: interferon-alpha (IFN-a), interferon-beta (IFN-(3), interferon-omega
(IFN-
0) and interferon-tau (IFN-i). The only Type II interferon so far identified
is
interferon-gamma (IFN-y) which is produced by activated T cells and NK cells.
The genes for Type I interferons are clustered on human chromosome 9. In
humans, it
is estimated that there are at least 14 IFN-oc non-allelic genes and the
number of
naturally-occurring IFN-a proteins is increased further by allelic forms of
IFN-a
genes (Jussain et al, 1996, J. Interferon Cytokine Res 16: 853-9).
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Interferons exert their cellular activities by binding to specific membrane
receptors on
the cell surface, so initiating a complex sequence of intracellular events.
Type I
interferons induce a wide variety of biological responses which include
antiviral,
immunomodulatory and anti-proliferative effects and, as a result of these
effects, they
5 have proved to be effective in the treatment of diverse diseases and
conditions.
Interferons are potent antiviral agents and alpha-interferons, in particular,
have been
found to be useful in the treatment of a variety of viral infections including
human
papillomavirus infection, Hepatitis B and Hepatitis C infections (Jaeckel et
al, 2001,
345(2): 1452-7). Type I interferons also inhibit cellular proliferation and
alpha-
interferons have been used clinically for many years in the treatment of a
variety of
malignancies including hairy cell leukaemia, multiple myeloma, chronic
lymphocytic
leukaemia, low-grade lymphoma, Kaposi's sarcoma, chronic myelogenous
leukaemia,
renal-cell carcinoma, and ovarian cancer. In addition, type I interferons are
useful in
treating autoimmune diseases, with interferon-beta having been approved for
the
treatment of multiple sclerosis.
Interferon-tau was initially identified in conceptus homogenates in ruminants
although it has since been identified in humans (see W096/35789). Although
interferon-tau displays many similar activities to other Type-I interferons,
it also
displays some different effects. In particular, it has an anti-luteolyic
effect which
promotes the establishment and maintenance of pregnancy (Martal et al, Reprod.
Fertil Dev., 1997, 9(3): 355-80). In addition, whilst viral induction of
interferon-alpha
and interferon-beta is transient, lasting a few hours, viral induction of
interferon-tau
expression can last several days and has been found to have antiretroviral
effects
against HIV-1 (Dereuddre-Bosquet et al, J. Acquir. Immune Defic Syndr. Hum.
Retrovirol, 1996, 11(3): 241-6).
Type II interferons (including interferon gamma) may be useful for the
treatment,
prevention and/or diagnosis of medical conditions and diseases which include
immune disorders, such as autoimmune disease, rheumatoid arthritis,
osteoarthritis,
psoriasis, systemic lupus erythematosus, and multiple sclerosis, myastenia
gravis,
Guillain-Barre syndrome, Graves disease, autoimmune alopecia, scleroderma,
psoriasis (Kimball et al., Arch Dermatol 2002 Oct:138(10):1341-6) and graft-
versus-
host disease (Miura Y., et al., Blood 2002 Oct 1:100(7):2650-8), monocyte and
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neutrophil dysfunction, attenuated B cell function, inflammatory disorders,
such as
acute inflammation, septic shock, asthma, anaphylaxis, eczema, dermatitis,
allergy,
rhinitis, conjunctivitis, glomerulonephritis, uveitis, Sjogren's disease
(Anaya et al., J
Rheumatol 2002 Sep; 29(9):1874-6), Crohn's disease (Schmit A. et al., Eur
Cytokine
Netw 2002 Jul-Sep:13(3):298-305), ulcerative colitis, inflammatory bowel
disease,
pancreatitis, digestive system inflammation, ulcerative colitis, sepsis,
endotoxic
shock, septic shock, cachexia, myalgia, ankylosing spondylitis, myasthenia
gravis,
post-viral fatigue syndrome, pulmonary disease, respiratory distress syndrome,
asthma, chronic-obstructive pulmonary disease, airway inflammation, wound
healing,
type I and type II diabetes, endometriosis, demnatological disease, Behcet's
disease,
immuno-deficiency disorders, chronic lung disease (Oei J et al., Acta Paediatr
2002:91(11):1194-9), aggressive and chronic periodontitis (Gonzales JR, et
al., J clin
Periodontol 2002 Sep:29(9):816-22), cancers including carcinomas, sarcomas,
lymphomas, renal tumour, colon tumour, Hodgkin's disease, melanomas, such as
metastatic melanomas (Vaishampayan U, Clin Cancer Res 2002 Dec:B(12):3696-
701), mesotheliomas, Burkitt's lymphoma, neuroblastoma, haematological
disease,
nasopharyngeal carcinomas, leukemias, myelomas, myeloproliferative disorder
and
other neoplastic diseases, osteoporosis, obesity, diabetes, gout,
cardiovascular
disorders, reperfusion injury, atherosclerosis, ischaemic heart disease,
cardiac failure,
stroke, liver disease such as chronic hepatitis (Semin Liver Dis 2002:22 Suppl
1:7),
AIDS (Dereuddre-Bosquet N., et al., J Acquir Immune Defic Syndr Hum Retroviol
1996 Mar l: 11(3):241-6), AIDS related complex, neurological disorders,
fibrotic
diseases, male infertility, ageing and infections, including plasmodium
infection,
bacterial infection, fungal diseases, such as ringworm, histoplasmosis,
blastomycosis,
aspergillosis, cryptococcosis, sporotrichosis, coccidioidocomycosis,
paracoccidiomycosis and candidiasis, diseases associated with antimicrobial
immunity (Bogdan, Current Opinion in Immunology 2000, 12:419-424), Peyronie's
disease (Lacy et al., Int J Impot Res 2002 Oct:l4(5):336-9), tuberculosis
(Dieli et al.,
J Infect Dis 2002 Dec 15;186(12):1835-9), and viral infection (Pfeffer LM,
Semin
Oncol 1997 Jun 24:59-63-69).
In summary, secreted proteins that are members of the four helical bundle
cytokine
family have been shown to play a role in diverse physiological functions, many
of which
can play a role in disease processes. W particular, inteuerons have been found
to play an
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important role in a variety of physiological processes and as a result, have
proved to be
useful in the treatment of a wide range of diseases. However, there remains a
need for
the identification of novel interferons to enable new drugs to be developed
for the
treatment and prevention of disease, including those diseases mentioned above.
THE INVENTION
The invention is based on the discovery that the INSP037 protein is an
interferon
gamma-like secreted protein of the four helical bundle cytokine fold.
In one embodiment of the first aspect of the invention, there is provided a
polypeptide, which polypeptide:
(i) comprises the amino acid sequence as recited in SEQ ID N0:2;
(ii) is a fragment thereof that is an interferon gamma-like secreted protein
of the
four helical bundle cytokine fold, or having an antigenic determinant in
common with the polypeptides of (i); or
(iii) is a functional equivalent of (i) or (ii).
According to a second embodiment of this first aspect of the invention, there
is
provided a polypeptide which:
(i) consists of the amino acid sequence as recited in SEQ ID N0:2;
(ii) is a fragment thereof that is an interferon gamma-like secreted protein
of the
four helical bundle cytokine fold, or having an antigenic determinant in
common with the polypeptides of (i); or
(iii) is a functional equivalent of (i) or (ii).
The polypeptide having the sequence recited in SEQ ID NO:2 is referred to
hereafter
as "the INSP037 polypeptide". INSP037 is also referred to herein as
IPAAA44548.
Preferably, the INSP037 polypeptides according to the first aspect of the
invention
function as an interferon gamma-lilce secreted protein of the four helical
bundle
cytokine fold. The term "interferon gamma-lilce secreted protein of the four
helical
bundle cytokine fold" will be understood by the skilled person, who will
readily be
able to ascertain whether a polypeptide functions as a member of this class
using one
of a variety of assays known in the art. The presence of a four helical bundle
cytokine
fold may be identified by an analysis of protein sequence and secondary
structure.
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Interferon activity is often measured as an anti-viral activity or
antiproliferative
activity on cancer cells. Examples of assays may be found in Schiller J.H., J
Interferon Res 1986; 6(6):615-25, Gibson, U.E. et al., J Immunol Methods
(1989) 20;
125(1-2):105-13 and Chang et al., J. Biol. Chem. (2002) 277(9):7118-7126.
In a second aspect, the invention provides a purified nucleic acid molecule
which
encodes a polypeptide of the first aspect of the invention.
Preferably, the purified nucleic acid molecule comprises the nucleic acid
sequence as
recited in SEQ ID N0:1 (encoding the INSP037 polypeptide). Preferably, the
purified nucleic acid molecule consists of the nucleic acid sequence as
recited in SEQ
ID NO:1 (encoding the INSP037 polypeptide) or is a redundant equivalent or
fragment of this sequence.
In a third aspect, the invention provides a purified nucleic acid molecule
which
hybridizes under high stringency conditions with a nucleic acid molecule of
the
second aspect of the invention.
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.
Examples of such vectors include pDESTl4-IPAAA44548-6HIS (see Figure 10),
PCRII-TOPO-IPAAA44548 (see Figure 11), pDESTl4-IPAAA44548-6HIS (see
Figure 12) and pEAKI2D-IPAAA44548-6HIS (see Figure 13).
In a fifth aspect, the invention provides a host cell transformed with a
vector of the
fourth aspect of the invention.
In a sixth aspect, the invention provides a ligand which binds specifically
to, and
which preferably inhibits the secreted protein activity, more preferably
inhibits the
interferon gamma-like activity of a polypeptide of the first aspect of the
invention.
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.
A compound of the seventh aspect of the invention may either increase
(agonise) or
decrease (antagonise) the level of expression of the gene or the activity of
the
polypeptide.
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Importantly, the identification of the function of the INSP037 polypeptide
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. Using these
methods,
it will now be possible to identify inhibitors or antagonists of INSP037, such
as, for
example, monoclonal antibodies, which may be of use in modulating INSP037
activity in vivo in clinical applications. Such compounds are likely to be
useful in
counteracting the IFNy-like activity of the INSP037 polypeptides.
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 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, for use in therapy or diagnosis of diseases
in which
interferons are implicated, particularly IFN-y-like polypeptides. Such
diseases
include, but are not limited to, immune disorders, such as autoimmune disease,
rheumatoid arthritis, osteoaxthritis, psoriasis, systemic lupus erythematosus,
and
multiple sclerosis, myastenia gravis, Guillain-Barre syndrome, Graves disease,
autoimmune alopecia, sclerodenna, psoriasis (Kimball et al., Arch Dermatol
2002
Oct:138(10):1341-6) and graft-versus-host disease (Miura Y., et al., Blood
2002 Oct
1:100(7):2650-8), monocyte and neutrophil dysfunction, attenuated B cell
function,
inflammatory disorders, such as acute inflammation, septic shock, asthma,
anaphylaxis, eczema, dermatitis, allergy, rhinitis, conjunctivitis,
glomerulonephritis,
uveitis, Sjogren's disease (Anaya et al., J Rheumatol 2002 Sep; 29(9):1874-6),
Crohn's disease (Schmit A. et al., Eur Cytokine Netw 2002 Jul-Sep:l3(3):298-
305),
ulcerative colitis, inflammatory bowel disease, pancreatitis, digestive system
inflammation, ulcerative colitis, sepsis, endotoxic shock, septic shoclc,
cachexia,
myalgia, ankylosing spondylitis, myasthenia gravis, post-viral fatigue
syndrome,
pulmonary disease, respiratory distress syndrome, asthma, chronic-obstructive
pulmonary disease, airway inflammation, wound healing, type I and type II
diabetes,
endometriosis, dermatological disease, Behcet's disease, immuno-deficiency
disorders, chronic lung disease (Oei J et al., Acta Paediatr 2002:91(11):1194-
9),
aggressive and chronic periodontitis (Gonzales JR, et al., J clin Periodontol
2002
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Sep:29(9):816-22), cancers including carcinomas, sarcomas, lymphomas, renal
tumour, colon tumour, Hodgkin's disease, melanomas, such as metastatic
melanomas
(Vaishampayan U, Clin Cancer Res 2002 Dec:B(12):3696-701), mesotheliomas,
Burkitt's lymphoma, neuroblastoma, haematological disease, nasopharyngeal
5 carcinomas, leukemias, myelomas, myeloproliferative disorder and other
neoplastic
diseases, osteoporosis, obesity, diabetes, gout, cardiovascular disorders,
reperfusion
injury, atherosclerosis, ischaemic heart disease, cardiac failure, stroke,
liver disease
such as chronic hepatitis (Semin Liver Dis 2002:22 Suppl 1:7), AIDS (Dereuddre-
Bosquet N., et al., J Acquir Immune Defic Syndr Hum Retroviol 1996 Mar l:
10 11(3):241-6), AIDS related complex, neurological disorders, fibrotic
diseases, male
infertility, ageing and infections, including plasmodium infection, bacterial
infection,
fungal diseases, such as ringworm, histoplasmosis, blastomycosis,
aspergillosis,
cryptococcosis, sporotrichosis, coccidioidocomycosis, paracoccidiomycosis and
candidiasis, diseases associated with antimicrobial immunity (Bogdan, Current
Opinion in Immunology 2000, 12:419-424), Peyronie's disease (Lacy et al., Int
J
Impot Res 2002 Oct:l4(5):336-9), tuberculosis (Dieli et al., J Infect Dis 2002
Dec
15;186(12):1835-9), and viral infection (Pfeffer LM, Semin Oncol 1997 Jun
24:59-
63-69).
These moieties of the first, second, third, fourth, fifth, sixth or seventh
aspect of the
invention may also be used in the manufacture of a medicament for the
treatment of
such diseases.
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 catTied out ih 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.
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
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11
of the invention with a biological sample under conditions suitable for the
formation
of a ligand-polypeptide complex; and (b) detecting said complex.
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.
Preferably, the disease diagnosed by a method of the ninth aspect of the
invention is a
disease in which interferons are implicated, as described above.
In a tenth aspect, the invention provides for the use of the polypeptide of
the first
aspect of the invention as an interferon gamma-like secreted protein of the
four helical
bundle cytokine fold. One suitable use of INSP037 is use as an adjuvant in
bacterial,
fungal or viral infections, in conjunction with well-established treatments.
Other
potential uses include use of INSP037 to activate macrophages, and to increase
expression of MHC molecules and antigen processing components. Experimental
results included herein confirm the predicted IFNy-like activity of INSP037.
This
discovery opens a series of interesting therapeutic applications for the
protein per' se,
in that the polypeptides of the invention can be tested for suitability for
use in lcnown
applications of IFNy, such as in anti-cancer applications (see, for example,
Vaishampayan U, Clin Cancer Res 2002 Dec:B(12):3696-701). It will also now be
possible to identify inhibitors or antagonists of INSP037, such as, for
example,
monoclonal antibodies, which may be of use in further studies of INSP037
activity i~
vivo or in clinical applications.
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.
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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
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 the manufacture of a
medicament for
the diagnosis or treatment of a disease in which interferons are implicated.
Such
diseases include those described above in connection with the eighth aspect of
the
invention.
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.
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,
vector, host cell, 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,
vector, host
cell, 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.
Preferably, the disease is a disease in which interferons are implicated, as
described
above.
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
regimes for
the identification of compounds that are effective in the treatment or
diagnosis of such
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13
a disease.
Preferably, the disease is a disease in which interferons are implicated, as
described
above.
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.
Standard abbreviations for nucleotides and amino acids are used in this
specification.
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.
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 c~ 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).
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).
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14
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.
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).
Polypeptides may contain amino acids other than 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-linking, cyclization, disulphide
bond
formation, demethylation, formation of covalent cross-linlcs, formation of
cysteine,
formation of pyroglutamate, formylation, 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.
Modifications can occur anywhere in a polypeptide, including the peptide
backbone,
the amino acid side-chains and the amino or carboxyl termini. 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.
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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
5 present in the amino acid sequence of the polypeptide in question. For
instance,
glycosylation patterns vary between different types of host cell.
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
10 proteins), synthetically-produced polypeptides or polypeptides that are
produced by a
combination of these methods.
The functionally-equivalent polypeptides of the first aspect of the invention
may be
polypeptides that are homologous to the INSP037 polypeptides. Two polypeptides
are
said to be "homologous", as the term is used herein, if the sequence of one of
the
15 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., and 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 ). Preferably, percentage identity, as referred to herein, is
as
determined using BLAST version 2.1.3 using the default parameters specified by
the
NCBI (the National Center for Biotechnology Information;
http://www.ncbi.nlm.nih.gov/) [Blosum 62 matrix; gap open penalty=11 and gap
extension penalty=1].
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
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16
substitutions, insertions or deletions) of the INSP037 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, l 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 not 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.
Typically, greater than 80% 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
INSP037 polypeptide, or with active fragments thereof, of greater than 80%.
More
preferred polypeptides have degrees of identity of greater than 90%, 95%, 98%
or
99%, respectively.
The functionally-equivalent polypeptides of the first aspect of the invention
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 search database
may
be used (see PCT application published as WO 01/69507) to identify
polypeptides of
presently-unknown function which, while having low sequence identity as
compared
to the INSP037 polypeptides, are predicted to be interferon gamma-lilce
secreted
proteins of the four helical bundle cytokine fold by virtue of sharing
significant
structural homology with the 1NSP037 polypeptide sequences. 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.
The polypeptides of the first aspect of the invention also include fragments
of the
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17
INSP037 polypeptides and fragments of the functional equivalents of these
polypeptides, provided that those fragments retain interferon gamma-like
activity, or
have an antigenic determinant in common with these polypeptides.
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
INSP037
polypeptides or one of its 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
more).
Small fragments may form an antigenic determinant.
Such fragments may be "free-standing", i.e. not part of or fused to other
amino acids
or polypeptides, or they may be comprised within a lar ger 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.
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 purify 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.
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 antibodies
thus bind
to the polypeptides of the first aspect of the invention.
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
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18
cell-surface receptors.
Preferably, the affinity is at least 1.5-fold, 2-fold, 5-fold 10-fold, 100-
fold, 103-fold,
104-fold, 105-fold, 106-fold or greater for a polypeptide of the invention
than for
known IFNy-like polypeptides.
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 immunise the animal can 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, thyroglobulin and
keyhole
limpet haemocyanin. The coupled polypeptide is then used to immunise the
animal.
Serum from the immunised animal is collected and treated according to known
procedures, for example by immunoaffinity chromatography.
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).
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.
Chimeric antibodies, in which non-human variable regions axe 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.
The antibody may be modified to make it less immunogenic in an individual, for
example by humanisation (see Jones et al., Nature, 321, 522 (1986); Verhoeyen
et al.,
Science, 239, 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.
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19
USA, 88, 34181 (1991); and Hodgson et al., Bio/Technology, 9, 421 (1991)). The
term "humanised 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 humanised antibody thus
closely
resembles a human antibody but has the binding ability of the donor antibody.
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.
Phage display technology may be utilised to select genes which encode
antibodies
with 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, 624-628).
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.
Preferred nucleic acid molecules of the second and third aspects of the
invention are
those which encode a polypeptide sequences as recited in SEQ ID N0:2 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).
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).
Nucleic acid molecules of the present invention may be in the form of RNA,
such as
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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
5 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 ih vivo transcription of DNA sequences.
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
10 the non-coding strand, also referred to as the anti-sense strand.
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
15 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).
20 A nucleic acid molecule which encodes the polypeptide of SEQ ID N0:2 may be
identical to the coding sequence of the nucleic acid molecule shown in SEQ ID
NO: l .
These molecules also may have a different sequence which, as a result of the
degeneracy of the genetic code, encodes a polypeptide of SEQ ID N0:2. 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.
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21
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-
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.
Among variants in this regard are vaxiants 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.
The nucleic acid molecules of the invention can also be engineered, using
methods
generally known in the art, for a variety of reasons, 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.
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
ilivention and
the sequence of a heterologous protein so that the polypeptide may be cleaved
and
purified away from the heterologous protein.
The nucleic acid molecules of the invention also include antisense molecules
that are
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22
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 invention, as will be knomn 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 al., Science 241, 456 (1988);
Dervan et
al., Science 251, 1360 (1991).
The term "hybridization" as used here refers to the association of two nucleic
acid
molecules with one another by hydrogen bonding. Typically, one molecule will
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]).
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).
"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 axe defined as overnight incubation at
42°C in a
solution comprising 50% formamide, SXSSC (150mM NaCI, lSmM trisodium
citrate), SOmM sodium phosphate (pH7.6), Sx Denhardts solution, 10% dextran
sulphate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by
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23
washing the filters in O.1X SSC at approximately 65°C. Low stringency
conditions
involve the hybridisation reaction being carried out at 35°C (see
Sambrook et al.
[supra]). Preferably, the conditions used for hybridization are those of high
stringency.
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 INSP037 polypeptide (SEQ ID N0:2) 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 the nucleic acid molecules
having the
sequence produced by SEQ ID NO:l or 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% or 99% 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 INSP037 polypeptides.
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.
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 INSP037 polypeptides and to
isolate
cDNA and genomic clones of homologous or onthologous genes that have a high
sequence similarity to the gene encoding this polypeptide.
In this regard, the following techniques, among others known in the art, may
be
utilised and are discussed below for purposes of illustration. Methods for 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 Coip, Cleveland, OH), Taq polymerase
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24
(Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, IL), 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, NV), the Peltier Thermal Cycler
(PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377
DNA Sequencers (Perkin Elmer).
One method for isolating a nucleic acid molecule encoding a polypeptide with
an
equivalent function to that of the INSP037 polypeptides 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 Yorlc, 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) are particularly useful probes. 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.
In many cases, isolated cDNA sequences will be incomplete, in that the region
encoding the polypeptide will 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 MarathonTM technology (Clontech Laboratories Inc.), for
example, have significantly simplified the search for longer cDNAs. A slightly
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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)
5 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., l, 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-
10 3060). Additionally, one may use PCR, nested primers, and PromoterFinderTM
libraries to walk genomic DNA (Clontech, Palo Alto, CA). This process avoids
the
need to screen libraries and is useful in fording intron/exon junctions.
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,
15 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.
In one embodiment of the invention, the nucleic acid molecules of the present
20 invention may be used for chromosome localisation. In this technique, a
nucleic acid
molecule is specifically tar geted 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
25 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
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26
sequences mapping to that area may represent associated or regulatory genes
for
further 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.
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. Such
inappropriate
expression may be of a temporal, spatial or quantitative nature.
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, SM 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 synthesised in vita°o 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.
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.
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 eulcaryotic.
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 lcnown to those of slcill 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,
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27
San Diego, London, Boston, New York, Sydney, Tokyo, Toronto).
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 Saznbrook 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.
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
chromosomes (HACs) may also be employed to deliver larger fragments of DNA
than
can be contained and expressed in a plasmid.
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.
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
(196)
and Sambroolc et al.,[supra]. Particularly suitable methods include calcium
phosphate
transfection, DEAE-dextran mediated transfection, transvection,
microinjection,
cationic lipid-mediated transfection, electroporation, transduction, scrape
loading,
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28
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.
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 extracellulax 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.
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 cause 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 cellulax proteins to carry out transcription and translation. Such
regulatory
sequences may waxy 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, CA) or pSportlTM 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 fiom 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.
An expression vector is constructed so that the particular nucleic acid coding
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29
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.
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.
For long-teen, 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.
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 (BHK), monlcey kidney (COS), C127, 3T3, BHK, HEK
293, Bowes melanoma and human hepatocellular carcinoma (for example Hep G2)
cells and a number of other cell lines.
In the baculovirus system, the materials for baculovirus/insect cell
expression systems
are commercially available in lcit form from, inter alia, Invitrogen, San
Diego CA (the
"MaxBac" kit). These techniques are generally lalown to those slcilled in the
art and
are described fully in Surmners and Smith, Texas Agricultural Experiment
Station
Bulletin No. 1555 (197). Particularly suitable host cells for use in this
system include
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insect cells such as Drosophila S2 and Spodoptera S~ cells.
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 US 5,693,506; US 5,659,122; and US 5,608,143. Additional examples
of
5 genetic expression in plant cell culture has been described by Zenk,
Phytochemistry
30, 3861-3863 (1991).
In particular, all plants from which protoplasts can be isolated and cultured
to give
whole regener ated plants can be utilised, so that whole plants are recovered
which
contain the transferred gene. Practically all plants can be regenerated from
cultured
10 cells or tissues, including but not limited to all major species of sugar
cane, sugar
beet, cotton, fruit and other trees, legumes and vegetables.
Examples of particularly preferred bacterial host cells include Streptococci,
Staphylococci, E. coli, St~eptofnyces and Bacillus subtilis cells.
Examples of particularly suitable host cells for fungal expression include
yeast cells
15 (for example, S. cef°evisiae) and Aspergillus cells.
Any number of selection systems are known in the art that may be used to
recover
transformed cell lines. Examples include the herpes simplex virus thymidine
lcinase
(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- or
aprt~
20 cells, respectively.
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 G-418 (Colbere-Garapin,
F.
25 et al (1981) J. Mol. Biol. 150:1-14) and als or pat, which confer
resistance to
chlorsulfiuon and phosphinotricin acetyltransferase, respectively. Additional
selectable genes have been described, examples of which will be clear to those
of skill
in the art.
Although the presence or absence of marker gene expression suggests that the
gene of
30 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
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31
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 the marker gene in response to induction or
selection
usually indicates expression of the tandem gene as well.
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, MN) and Maddox,
D.E. et al. (1983) J. Exp. Med, 158, 1211-1216).
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 vitf~o 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, MI);
Promega (Madison WI); and U.S. Biochemical Corp., Cleveland, OH)).
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.
Nucleic acid molecules according to the present invention may also be used to
create
transgenic animals, particularly rodent animals. Such transgenic animals fomn
a
further aspect of the present invention. This may be done locally by
modification of
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32
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.
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
chromatography,
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.
Specialised vector constructions may also be used to facilitate purification
of proteins,
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 extensionlaffinity
purification system (Immunex Corp., Seattle, WA). The inclusion of cleavable
linker
sequences such as those specific for Factor XA or enterokinase (Invitrogen,
San
Diego, CA) 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 enterolcinase cleavage site provides a means 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).
If the polypeptide is to be expressed for use in screening assays, generally
it is
preferred that it be secreted into the culture medium of the host cell in
which it is
expressed. In this event, the polypeptides of the invention may be purified
from the
culture medium may be harvested prior to use in the screening assay, for
example
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33
using standard protein purification techniques such as gel exclusion
chromatography,
ion-exchange chromatography or affinity chromatography. Examples of suitable
methods of protein purification are provided in the Examples herein. If
polypeptide is
produced intracellularly, the cells must first be lysed before the polypeptide
is
recovered.
Alternatively, it may be preferred that the polypeptides of the invention be
expressed
as cell-surface fusion proteins. 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.
The polypeptide of the invention can be used to screen libraries of compounds
in any
of a variety of drug screening techniques. Such compounds may activate
(agonise) or
inhibit (antagonise) 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.
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).
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.
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
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34
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.
A preferred method for identifying a ligand for the IFN~y-like polypeptides of
the
present invention comprises:
(a) contacting a cell expressing on the surface thereof a putative binding
partner
for a IFN-like polypeptide of the invention, the putative binding partner
being
capable of providing a detectable signal in response to the binding of a
polypeptide of the present invention, (or associated with a second component
capable of providing a detectable signal in response to the binding of a
polypeptide of the present invention), to the putative binding partner, with a
polypeptide of the present invention to be screened under conditions to permit
binding to the putative binding partner; and
(b) determining whether the polypeptide of the present invention binds to and
activates or inhibits the putative binding partner by measuring the level of a
signal generated from the interaction of the polypeptide of the present
invention with the putative binding partner.
A further preferred method for identifying a ligand for the IFNy-like
polypeptides of
the present invention comprises:
(a) contacting a cell expressing on the surface thereof a putative binding
partner
for a IFNy-like polypeptide of the invention, the putative binding partner
being capable of providing a detectable signal in response to the binding of a
polypeptide of the present invention, (or associated with a second component
capable of providing a detectable signal in response to the binding of a
polypeptide of the present invention), to the putative binding partner, with a
polypeptide of the present invention to permit binding to the putative binding
partner; and
(b) determining whether the polypeptide of the present invention binds to and
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activates or inhibits the putative binding partner by comparing the level of a
signal generated from the interaction of the polypeptide of the present
invention with the putative binding partner with the level of a signal in the
absence of the polypeptide of the present invention.
5 . 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 INSP037 polypeptides.
In another embodiment of the method for identifying an agonist or antagonist
of a
polypeptide of the present invention comprises:
10 determining the inhibition of binding of a polypeptide of the present
invention to cells
which have a ligand expressed at the surface thereof, or to cell membranes
containing
such a ligand, in the presence of a candidate compound under conditions to
permit
polypeptide binding to the ligand, and determining the amount of polypeptide
bound
to the ligand. A compound capable of causing reduction of binding of a
polypeptide
15 of the present invention is considered to be an agonist or antagonist.
Preferably the
polypeptide of the invention is labelled.
More particularly, a method of screening for an antagonist or agonist compound
comprises the steps of:
(a) incubating a labelled polypeptide of the present invention with a whole
cell
20 expressing a ligand according to the invention on the cell surface, or a
cell
membrane containing a ligand of the invention,
(b) measuring the amount of labelled polypeptide bound to the whole cell or
the
cell membrane;
(c) adding a candidate compound to a mixture of labelled polypeptide and the
25 whole cell or the cell membrane of step (a) and allowing the mixture to
attain
equilibrium;
(d) measuring the amount of labelled polypeptide bound to the whole cell or
the
cell membrane after step (c); and
(e) comparing the difference in the labelled polypeptide bound in step (b) and
(d),
30 such that the compound which causes the reduction in binding in step (d) is
considered to be an agonist or antagonist.
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36
The polypeptides may be found to modulate a variety of physiological and
pathological processes in a dose-dependent manner in the above-described
assays.
Thus, the "functional equivalents" of the polypeptides of the invention
include
polypeptides that exhibit any of the same modulatory 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 polypeptides of the invention, preferably
the
"functional equivalents" will exhibit substantially similar dose-dependence in
a given
activity assay compared to the polypeptides of the invention.
Alternatively, simple binding assays may be used, in which the adherence of a
test
compound to a surface 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 neutralising 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.
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.
Assay methods that are also included within the terms of the present invention
are
those that involve the use of the genes and polypeptides of the invention in
overexpression or ablation assays. Such assays involve the manipulation of
levels of
these genes/polypeptides in cells and assessment of the impact of this
manipulation
event on the physiology of the manipulated cells. For example, such
experiments
reveal details of signalling and metabolic pathways in which the particular
genes/polypeptides are implicated, generate information regarding the
identities of
polypeptides with which the studied polypeptides interact and provide clues as
to
methods by which related genes and proteins are regulated.
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37
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 W084/03564). 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-neutralising
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.
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 higand binding and crosslinking assays in which the pohypeptide 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 phasmon resonance (supplied by Biacore
AB,
Uppsala, Sweden) and spectroscopy. Binding assays may be used for the
purification
and cloning of the r eceptor, but 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.
The invention also includes a screening kit useful in the methods for
identifying
agonists, antagonists, ligands, receptors, substrates, enzymes, that are
described
above.
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.
The invention also provides pharmaceutical compositions comprising a
polypeptide,
nucleic acid, higand 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.
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38
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.
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.
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 0.01 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.
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.
Pharmaceutically acceptable salts can be used therein, for example, mineral
acid salts
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39
such as hydrochlorides, hydrobromides, phosphates, sulphates, and the lilce;
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).
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.
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.
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 W098/20734), 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.
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.
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,
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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 humanised to minimise their immunogenicity, as
described previously.
5 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.
In an alternative approach, expression of the gene encoding the polypeptide
can be
10 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)
15 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.
20 (1994) In: Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches,
Futura
Publishing Co., Mt. Kisco, NY). The complementary sequence or 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 ivy situ from expression ih vivo.
25 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
crl., 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
30 mRNAs into functional polypeptide. Ribozymes may be synthesised with a
natural
ribose phosphate baclcbone 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
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41
may contain modified bases.
RNA molecules may be modified to increase intracellular stability and half
life.
Possible modifications include, but are not limited to, the addition of
flanlcing
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
recognised
by endogenous endonucleases.
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.
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
pemnanently the inappropriate production of the polypeptide by replacing a
defective
gene with a corrected therapeutic gene.
Gene therapy of the present invention can occur ih 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, i~ vivo gene therapy does not require isolation and purification
of a
patient's cells.
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 CuiT. Top.
Microbiol.
Immunol., 158, 39-66 (1992) or adeno-associated virus (AAV) vectors as
described
by Muzyczlca, N., in Curr. Top. Microbiol. Immunol., 158, 97-129 (1992) and
U.S.
Patent No. 5,252,479. For example, a nucleic acid molecule encoding a
polypeptide of
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42
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 Strachan and A
P
Read, BIOS Scientific Publishers Ltd).
Another approach is the administration of "naked DNA" in which the therapeutic
gene
is directly injected into the bloodstream or muscle tissue.
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.
Vaccines according to the invention may either be prophylactic (ie. to prevent
infection) or therapeutic (ie. to treat disease after infection). Such
vaccines comprise
immunising antigen(s), immunogen(s), polypeptide(s), proteins) 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.
Since polypeptides may be broken down in the stomach, vaccines comprising
polypeptides are preferably administered parenterally (for instance,
subcutaneous,
intramuscular, intravenous, or intradernal 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 non-
aqueous
sterile suspensions which may include suspending agents or thickening agents.
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
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43
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.
Genetic delivery of antibodies that bind to polypeptides according to the
invention
may also be effected, for example, as described in International patent
application
W098/55607.
The technology referred to as jet injection (see, for example,
www.powderject.com)
may also be useful in the formulation of vaccine compositions.
A number of suitable methods for vaccination and vaccine delivery systems are
described in International patent application WO00/29428.
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.
Nucleic acid molecules for diagnosis may be obtained from a subject's cells,
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), strand 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); Birkemneyer et al., J.
Virol.
Meth., 35, 117-126 (1991); Van Brunt, J., Bio/Technology, 8, 291-294 (1990))
prior
to analysis.
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
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44
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.
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.
To aid the detection of nucleic acid molecules in the above-described methods,
an
amplification step, for example using PCR, may be included.
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 unhybridised 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 unhybridised 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.
Such diagnostics are particularly useful for prenatal and even neonatal
testing.
Point mutations and other sequence differences between the reference gene and
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"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
5 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
10 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.
DNA sequence differences may also be detected by alterations in the
electrophoretic
mobility of DNA fragments in gels, with or without denaturing agents, or by
direct
15 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 S 1 protection or the chemical cleavage method (see Cotton et
al., Proc.
Natl. Acad. Sci. USA (1985) 85: 4397-4401).
In addition to conventional gel electrophoresis and DNA sequencing, mutations
such
20 as microdeletions, aneuploidies, translocations, inversions, can also be
detected by in
situ analysis (see, for example, Keller et al., DNA Probes, 2nd Ed., Stoclcton
Press,
New Yorlc, 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
25 applied method and numerous reviews of FISH have appeared (see, for
example,
Trachuclc et al., Science, 250, 559-562 (1990), and Traslc et al., Trends,
Genet., 7,
149-154 (1991)).
In another embodiment of the invention, an array of oligonucleotide probes
comprising a nucleic acid molecule according to the invention can be
constructed to
30 conduct efficient screening of genetic variants, mutations 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
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46
(1996), Vol 274, pp 610-613).
In one embodiment, the array is prepared and used according to the methods
described in PCT application W095/11995 (Chee et al); Lockhart, D. J. et al.
(1996)
Nat. Biotech. 14: 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
in PCT
application W095/251116 (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 blot 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.
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.
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.
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47
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 talcen 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.
Antibodies which specifically bind to a polypeptide of the invention may be
used for
the diagnosis of conditions or diseases characterised 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.
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.
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.
In one aspect of the invention, a diagnostic lcit may comprise a first
container
containing a nucleic acid probe that hybridises under stringent conditions
with a
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48
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
further
comprise a third container holding an agent for digesting unhybridised RNA.
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.
To detect polypeptides according to the invention, a diagnostic kit may
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.
Such kits will be of use in diagnosing a disease or susceptibility to disease,
particularly immune disorders, such as autoimmune disease, rheumatoid
arthritis,
osteoarthritis, psoriasis, systemic lupus erythematosus, and multiple
sclerosis,
myastenia gravis, Guillain-BaiTe syndrome, Graves disease, autoimmune
alopecia,
sclerodemna, psoriasis (Kimball et al., Arch Dermatol 2002 Oct:138(10):1341-6)
and
graft-versus-host disease (Miura Y., et al., Blood 2002 Oct 1:100(7):2650-8),
monocyte and neutrophil dysfunction, attenuated B cell function, inflammatory
disorders, such as acute inflammation, septic shock, asthma, anaphylaxis,
eczema,
dermatitis, allergy, rhinitis, conjunctivitis, glomerulonephritis, uveitis,
Sjogren's
disease (Anaya et al., J Rheumatol 2002 Sep; 29(9):1874-6), Crohn's disease
(Schmit
A. et al., Eur Cytokine Netw 2002 Jul-Sep:l3(3):298-305), ulcerative colitis,
inflammatory bowel disease, pancreatitis, digestive system inflammation,
ulcerative
colitis, sepsis, endotoxic shock, septic shock, cachexia, myalgia, ankylosing
spondylitis, myasthenia gravis, post-viral fatigue syndrome, pulmonary
disease,
respiratory distress syndrome, asthma, chronic-obstructive pulmonary disease,
airway
inflammation, wound healing, type I and type II diabetes, endometriosis,
dermatological disease, Behcet's disease, immuno-deficiency disorders, chronic
lung
disease (Oei J et al., Acta Paediatr 2002:91(11):1194-9), aggressive and
chronic
periodontitis (Gonzales JR, et al., J clin Periodontol 2002 Sep:29(9):816-22),
cancers
including carcinomas, sarcomas, lymphomas, renal tumour, colon tumour,
Hodgkin's
disease, melanomas, such as metastatic melanomas (Vaishampayan U, Clin Cancer
CA 02527091 2005-11-24
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49
Res 2002 Dec:B(12):3696-701), mesotheliomas, Burkitt's lymphoma,
neuroblastoma,
haematological disease, nasopharyngeal carcinomas, leulcemias, myelomas,
myeloproliferative disorder and other neoplastic diseases, osteoporosis,
obesity,
diabetes, gout, cardiovascular disorders, reperfusion injury, atherosclerosis,
ischaemic
heart disease, cardiac failure, stroke, liver disease such as chronic
hepatitis (Semin
Liver Dis 2002:22 Suppl 1:7), AIDS (Dereuddre-Bosquet N., et al., J Acquir
Immune
Defic Syndr Hum Retroviol 1996 Mar 1: 11(3):241-6), AIDS related complex,
neurological disorders, fibrotic diseases, male infertility, ageing and
infections,
including plasmodium infection, bacterial infection, fungal diseases, such as
ringworm, histoplasmosis, blastomycosis, aspergillosis, cryptococcosis,
sporotrichosis, coccidioidocomycosis, paracoccidiomycosis and candidiasis,
diseases
associated with antimicrobial immututy (Bogdan, Current Opinion in Immunology
2000, 12:419-424), Peyronie's disease (Lacy et al., Int J Impot Res 2002
Oct:l4(5):336-9), tuberculosis (Dieli et al., J Infect Dis 2002 Dec
15;186(12):1835-9),
and viral infection (Pfeffer LM, Semin Oncol 1997 Jun 24:59-63-69).
Various aspects and embodiments of the present invention will now be described
in
more detail by way of example, with particular reference to INSP037
polypeptides.
It will be appreciated that modification of detail may be made without
departing from
the scope of the invention.
Brief description of the Figures
Figure 1: Results from Inpharmatica Genome Threader query using SEQ ID N0:2.
Figure 2: Alignment generated by Inpharmatica Genome Threader between SEQ ID
N0:2 and closest related structure.
Figure 3: INSP037 predicted nucleotide sequence (comprising SEQ ID NO:1) with
translation (SEQ ID N0:2).
Figure 4: 1NSP037 cloned nucleotide sequence (comprising SEQ ID NO:l) with
translation (SEQ ID N0:2), demonstrating that the predicted and cloned
sequence for
INSP037 are identical.
Figure 5: Map of PCRII-TOPO-IPAAA44548.
Figure 6: Map of expression vector pEAKl2d.
Figure 7: Map of plasmid pDONR201.
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Figure 8: Map of expression vector pEAKl2d-IPAAA44548-6HIS.
Figure 9: Map of E. coli expression vector pDESTI4.
Figure 10: Map of plasmid pDESTI4-IPAAA44548-6HIS.
Figure 11: Nucleotide sequence of PCRII-TOPO-IPAAA44548.
5 Figure 12: Nucleotide sequence of pDESTI4-IPAAA44548-6HIS.
Figure 13: Nucleotide sequence of pEAKI2D-IPAAA44548-6HIS.
Figure 14: The NCBI-NR results for INSP037 (SEQ ID N0:2) showing no 100%
match, thus demonstrating INSP037 to be novel.
Figure 15: The NCBI-month-as results for INSP037 (SEQ ID N0:2) showing no 100%
10 match, thus demonstrating INSP037 to be novel.
Figure 16A: The translated nucleotide database NCBI-month-nt results for
INSP037
(SEQ ID N0:2) showing no 100% match, thus demonstrating INSP037 to be novel.
Figure 16B: The NCBI-nt results for INSP037 (SEQ ID N0:2) showing no 100%
match, thus demonstrating INSP037 to be novel.
15 Figure 17: Results of an investigation of INSP037 activity in a marine
model of ConA-
induced fulminant hepatitis.
Figure 18: Positive control showing effects of IL-6 upon a marine model of
ConA-
induced fulminant hepatitis.
Examples
20 Example 1: Identification of INSP037
The polypeptide sequence derived from SEQ ID NO:2 which represents the
translation of exons from INSP037 was used as a query in the Inpharmatica
Genome
Threader tool against protein structures present in the PDB database. The top
match is
the structure of a four helical bundle cytokine family member. The top match
aligns to
25 the query sequence with a Genome Threader confidence of 84% (Figure 1).
Figure 2
shows the alignment of the INSP037 query sequence to the sequence of Bovine
interferon-gamma (PDB-1 d9g) a member of the four helical bundle cytokine
family
(Randal et al Acta Crystallogr D Biol Crystallogr. 2000 Jan;56 (Pt 1):14-24).
Note
that the INSP037 polypeptide sequence is referred to as "IPAAA445" in Figure
2.
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Members of the four helical bundle cytokine family of proteins are of
therapeutic
importance.
Figure 16B shows that INSP037 can be found on Homo sapiens chromosome 3. As
described above, all Type I interferons are clustered on chromosome 9.
Therefore, the
location of the INSP037 gene on chromosome 3 (3q25.33, chr3:157121275-
157121511 (on hgl5/build 33)) is in accordance with its aimotation herein as
an IFN-
gamma like interferon, and thus as a Type II interferon.
Example 2: Cloning of INSP037 (IPAAA445481 from cDNA libraries
cDNA libraries
Human cDNA libraries (in bacteriophage lambda (~,) vectors) were purchased
from
Stratagene or Clontech or prepared at the Serono Pharmaceutical Research
Institute in
7~ ZAP or 7~ GT10 vectors according to the manufacturer's protocol
(Stratagene).
Bacteriophage ~, DNA was prepared from small scale cultures of infected E.coli
host
strain using the Wizard Lambda Preps DNA purification system according to the
manufacturer's instructions (Promega, Corporation, Madison WL) The list of
libraries and host strains used is shown in Table 1.
Table 1: Human cDNA libraries
Library Tissuelcell source Vector Host strainSupplierCat.
no.
human fetal brain Zap II XL1-Blue Stratagene936206
MRF'
2 human ovary GT10 LE392 ClontechHL1098a
3 human pituitary GT10 LE392 ClontechHL1097a
4 human placenta GT11 LE392 ClontechHL1075b
5 human testis GT11 LE392 ClontechHL1010b
human sustanta nigra GT10 LE392 in house
human fetal brain GT10 LE392 in house
human cortex brain GT10 LE392 in house
human colon GT10 LE392 ClontechHL1034a
10 human fetal brain GT10 LE392 ClontechHL1065a
11 human fetal lung GT10 LE392 ClontechHL1072a
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12 human fetal kidney GT10 LE392 ClontechHL1071a
13 human fetal liver GT10 LE392 ClontechHL1064a
14 human bone marrow GT10 LE392 ClontechHL1058a
15 human peripheral blood GT10 LE392 ClontechHL1050a
monocytes
16 human placenta GT10 LE392 in house
17 human SHSYSY GT10 LE392 in house
18 human 0373 cell line GT10 LE392 in house
19 human CFPoc-1 cell lineUni Zap XL1-Blue Stratagene936206
MRF'
2o human retina GT10 LE392 ClontechHL1132a
21 human urinary bladder GT10 LE392 in house
22 human platelets Uni Zap XL1-Blue in house
MRF'
23 human neuroblastoma GT10 LE392 in house
Kan + TS
24 human bronchial smooth GT10 LE392 in house
muscle
25 human bronchial smooth GT10 LE392 in house
muscle
26 human Thymus GT10 LE392 ClontechHL1127a
27 human spleen 5' stretchGT11 LE392 ClontechHL1134b
28 human peripherical bloodGT10 LE392 ClontechHL1050a
monocytes
29 human testis GT10 LE392 ClontechHL1065a
30 human fetal brain GT10 LE392 ClontechHL1065a
31 human substancia Nigra GT10 LE392 ClontechHL1093a
32 human placenta#11 GT11 LE392 ClontechHL1075b
33 human Fetal brain GT10 LE392 Clontechcustom
34 human placenta #59 GT10 LE392 ClontechHL5014a
35 human pituirary GT10 LE392 ClontechHL1097a
36 human pancreas #63 Uni Zap XL1-Blue Stratagene937208
XR MRF'
37 human placenta #19 GT11 LE392 ClontechHL1008
38 human liver 5'strech GT11 LE392 ClontechHL1115b
39 human uterus Zap-CMV XL1-Blue Stratagene980207
XR MRF'
4o human kidney large-insertTripIEx2XL1-Blue ClontechHL5507u
cDNA library
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Gene specific cloning primers for PCR
Pairs of PCR primers having a length of between 18 and 25 bases were designed
for
amplifying the full length sequence of the virtual cDNA using Primer Designer
Software, as shown in Table 2 below (Scientific & Educational Software, PO Box
72045, Durham, NC 27722-2045, USA). PCR primers were optimized to have a Tm
close to 55 + 10 °C and a GC content of 40-60%. Primers were selected
which had
high selectivity for the target sequence IPAAA44548 (little or no none
specific
priming).
Table II: INSP037 Cloning primers
PrimerName Sequence (5'-3) Position Tm oGC
C
CP1 2C5 GCA TCA ACA ACA TCC 28 58 40
AGT AA
Forward primer
CP2 2C6 CAT TCT AAA GTG TGC 291C 57 40
CAT CT
Reverse Primer
PCR of virtual cDNAs from phage library DNA
Full-length virtual cDNA encoding IPAAA44548 (Figure 3) was obtained as a PCR
amplification product of 264 by (Figure 4) using gene specific cloning primers
(CP 1
and CP2, Figure 3 and Table 2). The PCR was performed in a final volume of
SOyI
containing 1X AmpliTaqTM buffer, 200 ~,M dNTPs, 50 pmoles each of cloning
primers primers, 2.5 units of AmpliTaqTM (Perkin Elmer) and 100 ng of each
phage
library DNA using an MJ Research DNA Engine, programmed as follows: 94
°C, 1
min; 40 cycles of 94 °C, 1 min, x °C, and y min and 72
°C, (where x is the lowest Tm
- 5 °C and y = 1 min per kb of product); followed by 1 cycle at 72
°C for 7 min and a
holding cycle at 4 °C.
The amplification products were visualized on 0.8 % agarose gels in 1 X TAE
buffer
(Life Technologies) and PCR products migrating at the predicted molecular mass
were purified from the gel using the Wizard PCR Preps DNA Purification System
(Promega). PCR products eluted in 50 y1 of sterile water were either sub-
cloned
directly or stored at -20 °C.
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Subcloning, of PCR Products
PCR products were subcloned into the topoisomerase I modified cloning vector
(pCR
II TOPO) using the TOPO TA cloning lcit purchased from the Invitrogen
Corporation
(cat. No. K4600-01 and K4575-O1 respectively) using the conditions specified
by the
manufacturer. Briefly, 4 ~l of gel purified PCR product from the human
pituitary
library (library number 3) amplification was incubated for 15 min at room
temperature with 1 ~1 of TOPO vector and 1 ~,1 salt solution. The reaction
mixture
was then transformed into E. coli strain TOP10 (Invitrogen) as follows: a 50
~,1
aliquot of One Shot TOP10 cells was thawed on ice and 2 ~l of TOPO reaction
was
added. The mixture was incubated for 15 min on ice and then heat shoclced by
incubation at 42 °C for exactly 30 s. Samples were returned to ice and
250 ~,1 of warm
SOC media (room temperature) was added. Samples were incubated with shaking
(220 rpm) for 1 h at 37 °C. The transformation mixture was then plated
on L-broth
(LB) plates containing ampicillin (100 yg/ml) and incubated overnight at 37
°C.
Ampicillin resistant colonies containing cDNA inserts were identified by
colony PCR.
Colony PCR
Colonies were inoculated into 50 ~1 sterile water using a sterile toothpick. A
10 ~.~1
aliquot of the inoculum was then subjected to PCR in a total reaction volume
of 20 ~1
as described above, except the primers pairs used were SP6 (5') and T7. The
cycling
conditions were as follows: 94 °C, 2 min; 30 cycles of 94 °C, 30
sec, 47 °C, 30 sec
and 72 °C for 1 min); 1 cycle, 72 °C, 7 min. Samples were then
maintained at 4 °C
(holding cycle) before further analysis.
PCR reaction products were analyzed on 1 % agarose gels in 1 X TAE buffer.
Colonies which gave the expected PCR product size (264 by cDNA + 1 ~7 by due
to
the multiple cloning site or MCS) were grown up overnight at 37 °C in 5
ml L-Broth
(LB) containing ampicillin (50 ~,g /ml), with shaking at 220 rpm at 37
°C.
Plasmid DNA preparation and Sequencing
Miniprep plasmid DNA was prepared from 5 ml cultures using a Qiaprep Turbo
9600
robotic system (Qiagen) or Wizard Plus SV Minipreps kit (Promega cat. no.
1460)
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according to the manufacturer's instructions. Plasmid DNA was eluted in 100
~,1 of
sterile water. The DNA concentration was measured using an Eppendorf BO
photometer. Plasmid DNA (200-500 ng) was subjected to DNA sequencing with T7
primer and SP6 primer using the BigDyeTerminator system (Applied Biosystems
cat.
5 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.
Example 3: Construction of plasmids for expression of INSP037~IPAAA44548) in
10 HEK293/EBNA cells
A pCRII-TOPO clone containing the full coding sequence (ORF) of IPAAA44548
identified by DNA sequencing (Figure 5) was then used to subclone the insert
into the
mammalian cell expression vector pEAKl2d (Figure 6) using the GatewayTM
cloning
methodology (Invitrogen). The cloned sequence contains a single nucleotide
15 substitution A134G (Figure 4).
Generation of Gatewa~patible IPAAA44548 ORF fused to an in frame 6HIS tag
sequence.
The first stage of the Gateway cloning process involves a two step PCR
reaction
which generates the ORF of IPAAA44548 flanked at the 5' end by an attB 1
20 recombination site and Kozak sequence, and flanked at the 3' end by a
sequence
encoding an in frame 6 histidine (6HIS) tag, a stop codon and the attB2
recombination site (Gateway compatible cDNA). The first PCR reaction (in a
final
volume of 50 ~,~1) contains: 25 ng of pCR II TOPO-IPAAA44548 (plasmid 13124
and
Figure 5), 2 ~1 dNTPs (SmM), 5~,1 of lOX Pfx polymerase buffer, 0.5 ~.l each
of gene
25 specific primer (100 ~,M) (EX1 forward and EX1 reverse) and 0.5 ~l Platinum
Pfx
DNA polymerase (Invitrogen). The PCR reaction was performed using an initial
denaturing step of 95°C for 2 min, followed by 12 cycles of 94
°C, 15 sec and 68°C
for 30 sec. PCR products were purified directly from the reaction mixture
using the
Wizard PCR prep DNA purification system (Promega) according to the
30 manufacturer's instructions. The second PCR reaction (in a final volume of
50 ~,1)
contained 10 ~1 purified PCR product, 2 ~,1 dNTPs (5 mM), 5 ~.l of lOX Pfx
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polymerase buffer, 0.5 E~l of each Gateway conversion primer (100 ~,M) (GCP
forward and GCP reverse) and 0.5 ~,1 of Platinum Pfx DNA polymerase. The
conditions for the 2nd PCR reaction were: 95 °C for 1 min; 4 cycles of
94 °C, 15 sec;
45 °C, 30 sec and 68 °C for 3.5 min; 25 cycles of 94 °C,
15 sec; 55 °C , 30 sec and 68
°C, 3.5 min. PCR products were purified as described above.
Alternatively for expression of IPAAA44548 in E.coli, an ORF was generated
which
contained a Shine Dalgarno sequence upstream of the initiating methionine
codon
using gene specific primers (EX3 - forward and EX2 - reverse) in the first
PCR, and
primers GCPF and GCPR using the same conditions as described above. The
resultant
PCR product was called SD-IPAAA44548.
Subclonin~ of Gatewa~patible IPAAA44548 ORF into Gateway entry vector
pDONR201 and expression vector pEAKl2d
The second stage of the Gateway cloning process involves subcloning of the
Gateway modified PCR product into the Gateway entry vector pDONR201
(Invitrogen, Figure 7) as follows: 5 ~l of purified PCR product is incubated
with 1.5
~1 pDONR201 vector (0.1 ~,g/yl), 2 ~l BP buffer and 1.5 ~l of BP clonase
enzyme
mix (Invitrogen) at RT for 1 h. The reaction was stopped by addition of
proteinase K
(2 ~,g) and incubated at 37°C for a further 10 min. An aliquot of this
reaction (2 ~,l)
was transformed into E. coli DH10B cells by electroporation using a Biorad
Gene
Pulser. Transformants were plated on LB-kanamycin plates. Plasmid mini-prep
DNA
was prepared from 1-4 of the resultant colonies using Wizaxd Plus SV Minipreps
kit
(Promega), and 1.5 ~.1 of the plasmid eluate was then used in a recombination
reaction
containing 1.5 y1 pEAKl2d vector (Figure 6) (0.1 ~.g / ~,l), 2 ~.l LR buffer
and 1.5 ~1
of LR clonase (Invitrogen) in a final volume of 10 ~1. The mixture was
incubated at
RT for 1 h, stopped by addition of proteinase K (2 fig) and incubated at
37°C for a
further 10 min. An aliquot of this reaction (1 ~.1) was used to transform E.
coli DH10B
cells by electroporation.
Clones containing the correct insert were identified by performing colony PCR
as
described above except that pEAKl2d primers (pEAKl2d F and pEAKl2d R) were
used for the PCR. Plasmid mini prep DNA was isolated from clones containing
the
correct insert using a Qiaprep Turbo 9600 robotic system (Qiagen) or manually
using
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a Wizard Plus SV minipreps kit (Promega) and sequence verified using the
pEAKl2d
F and pEAKl2d R primers.
CsCI gradient purified maxi-prep DNA of plasmid pEAKl2d-IPAAA44548-6HIS
(plasmid ID number 11775, Figure 8) was prepared from a 500 ml culture of
sequence
verified clones (Sambrook J. et al., in Molecular Cloning, a Laboratory
Manual, 2°d
edition, 1989, Cold Spring Harbor Laboratory Press), resuspended at a
concentration
of 1 ~g/~,1 in sterile water and stored at -20 C.
Construction of exuression vector pEAKl2d
The vector pEAKl2d is a Gateway Cloning System compatible version of the
mammalian cell expression vector pEAKl2 (purchased from Edge Biosystems) in
which the cDNA of interest is expressed under the control of the human EFla,
promoter. pEAKl2d was generated as described below:
pEAKl2 was digested with restriction enzymes HindIII and NotI, made blunt
ended
with Klenow (New England Biolabs) and dephosphorylated using calf intestinal
alkaline phosphatase (Roche). After dephosphorylation, the vector was ligated
to the
blunt ended Gateway reading frame cassette C (Gateway vector conversion
system,
Invitrogen cat no. 11828-019) which contains AttR recombination sites flanking
the
ccdB gene and chloramphenicol resistance, and transformed into E.coli DB3.1
cells
(which allow propagation of vectors containing the ccdB gene). Mini prep DNA
was
isolated from several of the resultant colonies using a Wizard Plus SV
Minipreps kit
(Promega) and digested with AseI / EcoRI to identify clones yielding a 670 by
fragment, indicating that the cassette had been inserted in the correct
orientation. The
resultant plasmid was called pEAKl2d (Figure 6).
Subcloning of Gatewa.~patible SD - IPAAA44548 ORF into Gateway
vector pDONR201 and E coli expression vector pDESTI4.
Gateway compatible SD-IPAAA44548 ORF containing an in frame 3' 6HIS tag
coding sequence and a 5' upstream Shine Dalgarno sequence was subcloned into
pDONR201 using BP clonase. The resultant plasmid was then used in a
recombination reaction with the E.coli expression vector pDESTl4 (purchased
from
Invitrogen, Figure 9) using LR clonase as described above. The resultant
expression
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plasmid (pDESTI4-IPAAA44548-6HIS) (Figure 10, plasmid ID 12896) was
sequence verified as described above. For expression in E coli, CsCI purified
maxi-
prep DNA was re-transformed into E.coli host strain BL21. The expression of
the
inserted cDNA is under the control of a T7 promoter.
Table 3: Primers for IPAAA44548 subcloning and sequencing
Primer Name Sequence(5'-3')
GCP I-C1 attB1-K G GGG TACAAAAAA GCA GGC
Forward ACA TTC
AGT
TTG
GCC
ACC
GCP 22A3 attB2-stop-His6-GGG CACTTTGTACAA
Reverse R GAC ATGGTGATGGAA
ATG AGC
GTG TGG
GTT
TCA
GTG
GCP-SD III-A1 attB1- G GGG TACAAAAAA GCA GGC
shineDalgarno-p ACA TTC
Forward AGT
TTG
G~.
~G~1
G2
EXl 32A5 attBlp- GCA TTCGCCACCATGACTTCA CCA AAC
Forward TPAAA44548-1F GGC GAA
CTA
A
EX2 32A8 IPAAA44548- GTG GTGATGGTG TGTGCC ATC TGC
Reverse H6p-2348 ATG AAG ATT
TCT
EX3 II-I8 AAA GGCTTC,C-AA,GGA GATATA CAT ATG
forward 44548ShineDalgarno-1FGCA AACGAACT ACT
TCA
CCA
pEAKl2-F 32D1 GCC TTGGCACTTGATGT
AGC
pEAKl2-R 32D2 GAT GGTGGACGTGTCAG
GGA
SP6 ATT GTGACACTATAG
TAG
T7 TAA GACTCACTATAGGG
TAC
pDESTI4-R TGG CAGCCAACTCAGCTT
CAG
Underlined sequence = Kozak sequence
Bold = Stop codon
Italic sequence = His tag
Shaded sequence = Shine Dalgarno sequence (Ribosome binding site)
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Example 4: Identification of cDNA libraries containing IPAAA44548
PCR products obtained with CP l and CP2 and migrating at the correct size (264
bp)
were identified in libraries 3, 8 and 12 (pituitary, brain cortex and fetal
lcidney
respectively).
Example 5: Expression in mammalian cells of the cloned, IPAAA44548-S-6HIS
(plasmid number 12118)
Cell culture
Human Embryonic Kidney 293 cells expressing the Epstein-Barr virus Nuclear
Antigen (HEK293-EBNA, Invitrogen) were 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 were seeded in 2x T225 flasks (50
ml per
flask in DMEM / F12 (1:l) containing 2% FBS seeding medium (JRH) at a density
of
2x105 cells/ ml). The next day (transfection day0) the transfection took place
by using
the JetPEITM reagent (2~.1/~g of plasmid DNA, PolyPlus-transfection). For each
flask,
113 ~g of plasmid (No. 12118) was co-transfected with 2.3 ~,g of GFP
(fluorescent
reporter gene). The transfection mix was then added to the 2xT225 flasks and
incubated at 37°C (5%C02) for 6 days.
Confirmation of positive transfection was done by qualitative fluorescence
examination at day l and day 6 (Axiovert 10 Zeiss).
On day 6 (harvest day), supernatants (100m1) from the two flasks were pooled
and
centrifuged (4°C, 400g) and placed into a pot bearing a unique
identifier.
One aliquot (SOOuI) was kept for QC of the 6His-tagged protein (internal
bioprocessing QC).
Scale-up batches were produced following the protocol called "PEI transfection
of
suspension cells" referenced BP / PEI/ HH/02/04 with PolyEthyleneImine from
Polysciences as transfection agent.
This protocol was based on the following proportions:
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For 400 ml spinner: 1E6 hek293EBNA cells / ml in 200m1 FEME 1% FBS
400 yg (plasmid No. 12118) diluted into 1 Oml FEME 1 % and 800ug PEI added
90 minutes post-transfection, FEME 1% medium added to reach 400-ml total
volume.
Spinner left in culture for 6 days until harvest.
5 Purification process
The culture medium sample (100 or 400 ml) containing the recombinant protein
with
a C-terminal 6His tag was diluted with one volume cold buffer A (50 mM
NaH2P04;
600 mM NaCI; 8.7 % (w/v) glycerol, pH 7.5) to a final volume of 200 and 800
ml,
respectively. The sample was filtered through a 0.22 um sterile filter
(Millipore, 500
10 ml filter unit) and kept at 4°C in a sterile square media bottle
(Nalgene).
The purification was performed at 4°C on the VISION workstation
(Applied
Biosystems) connected to an automatic sample loader (Labomatic). The
purification
procedure was composed of two sequential steps, metal affinity chromatography
on a
Poros 20 MC (Applied Biosystems) column charged with Ni ions (4.6 x 50 mm,
0.83
15 ml), followed by gel filtration on a Sephadex G-25 medium (Amersham
Phannacia)
column (1,0 x 10 cm).
For the first chromatography step the metal affinity column was regenerated
with 30
column volumes of EDTA solution (100 mM EDTA; 1 M NaCI; pH 8.0), recharged
with Ni ions through washing with 15 column volumes of a 100 mM NiSOø
solution,
20 washed with 10 column volumes of buffer A, followed by 7 column volumes of
buffer B (50 mM NaHZP04; 600 mM NaCI; 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 was transferred, by the Labomatic
sample
loader, into a 200 ml sample loop and subsequently charged onto the Ni metal
affinity
25 column at a flow rate of 10 mlhnin. In case of the 400 ml scale up samples
the
transfer and charging procedure was repeated 4 times. The column was
subsequently
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 were elution of the column. The recombinant
His-
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tagged protein was finally eluted with 10 column volumes of buffer B at a flow
rate of
2 ml/min, and the eluted protein was collected in a 1.6 ml fraction.
For the second chromatography step, the Sephadex G-25 gel-filtration column
was
regenerated with 2 ml of buffer D (1.137 M NaCI; 2.7 mM KCI; 1.5 mM KH2PO4; 8
mM Na~HP04; pH 7.2), and subsequently equilibrated with 4 column volumes of
buffer C (137 mM NaCI; 2.7 mM KCI; 1.5 mM KH2P04; 8 mM Na2HP0~; 20
(w/v) glycerol; pH 7.4). The peak fraction eluted from the Ni-column was
automatically through the integrated sample loader on the VISION loaded onto
the
Sephadex G-25 column and the protein was eluted with buffer C at a flow rate
of 2
ml/min. The desalted sample was recovered in a 2.2 ml fraction. The fraction
was
filtered through a 0.22 um sterile centrifugation filter (Millipore), frozen
and stored at
-80C. An aliquot of the sample was analyzed on SDS-PAGE (4-12 % NuPAGE gel;
Novex) by coomassie staining and Western blot with anti-His antibodies.
Coomassie staining. The NuPAGE gel was stained in a 0.1 % coomassie blue 8250
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 was
clear and the protein bands clearly visible.
Western blot. Following the electrophoresis the proteins were
electrotransferred from
the gel to a nitrocellulose membrane at 290 mA for 1 hour at 4°C. The
membrane was
bloclced with 5 % milk powder in buffer E (137 mM NaCI; 2.7 mM KCI; 1.5 mM
KH2PO4; 8 mM Na2HP04; 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, 0.2ug/ml each; Santa Cruz) in 2.5 % milk powder in buffer E
overnight
at 4°C. After further 1 hour incubation at room temperature, the
membrane was
washed with buffer E (3 x 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 hours at room temperature. After washing
with
buffer E (3 x 10 minutes), the membrane was developed with the ECL kit
(Amersham
Pharmacia) for 1 min. The membrane was subsequently exposed to a Hyperfilm
(Amersham Pharmacia), the film developed and the western blot image visually
analysed.
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Protein assay. The protein concentration was determined using the BCA protein
assay
kit (Pierce) with bovine serum albumin as standard in samples that showed
detectable
protein bands by coomassie staining.
Expression of IPAAA44548-SEC- 6HIS in bacterial cells (vplasmid No. 128961
The method below describes the use of E. Coli BL-21 DE3 bacterial strain for
producing the protein. "BL21 DE3" are part of T7 RNA polymerase-based
expression
systems widely used for over-expressing recombinant proteins.
Transformation of bacterial strain BL21 (DE3):
We used the procedure of TSS method, the protocol has been taken from: Chung,
C.T
et al., Proc. Natl. Acad. Sci. USA (1989) 86:2172-2175.
10-100 ng DNA (2 ~l) of the recombinant plasmid No. 12896 were added to
competent BL21 for TSS method and placed 20 minutes on ice. SOC mediwn (0.8
ml) were added and the tube was incubated at 37°C, 200 rpm for 1 hour.
From this
culture 20 ~.l and 200 ~,1 were sampled and plated on LB plates containing
Ampicillin
(40 ~.g/ml final concentration) and left overnight at 37°C.
The next day, 3 colonies were isolated and used for preparation of the
glycerol stocks,
tested for expression in shake flaslcs experiments before transferring
production into a
fennenter (one out of the three was chosen for large scale, as they were all
performing
the same in shalce flasks).
Preparation of a seed stock for long term storage of the recombinant E. Coli
strain:
A 5 ml tube containing LB medium with Ampicillin 40 ~,g/ml (final
concentration)
was inoculated with a single colony from a fresh agar plate. Bacteria were
grown
overnight at 37 C, 200 rpm. The next morning, 50 ~.l of the overnight culture
was
sampled in order to inoculate a fresh 5 ml LB tube (+ antibiotics) and
incubated 2-3
hours at 37°C, 200 rpm in order to bring bacteria to the exponential
growth phase.
5 ml glycerol at 20 % was then added to the culture and mixed. 1.5 ml were
dispensed
in each of 5 cryogenic vials which constitute a seed stock stored at -
80°C (internal
Glycerol stock).
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Expression at the 5-litre scale:
The recombinant strain was propagated in a 5-litres Biolafitte stirred tank
reactor
(working containing 5-litres of ECPM 1 medium (having a composition as
reported in
Table 4) with appropriate antibiotic (40 ~.g/ml final concentration) and 0.5 %
Glucose
in order to avoid pre-induction of the T7 promoter. Only The research-grade
run 2464
was prepared and sent to purification.
The inoculum was prepared in a 500-ml LB (+ antibiotics, 0.5 % Glucose) shake
flask starting from one loop of frozen bacteria (scraped from one of the
glycerol seed
stock vial) and grown for 9 hours before automatic inoculation. When cells
reached
OD 10, (usually after 7 to 9 hours growth), the protein production was induced
with
IPTG: 1 mM final concentration. Induction lasted 3 hours.
Fermenter setting conditions throughout growth and induction were set at: 50
dissolved oxygen concentration, 300 to 700 rpm depending on p02, pH7Ø The
P02
was maintained by air sparging +/- O2 at 25 ml/min. A 5-ml sample was talcen
every
hour and optical density was measured at 600 nm.
The cells were harvested and centrifuged at 4 000 rpm (in Sorvall RC 3B). The
pellet
was kept frozen at-20 °C until further processing.
Presence of the protein in the cells extract was assessed by Coomassie
staining of a
SDS-PAGE.
Table 4 : ECPMl composition
Component Source Comment Conc.Unit Steril.Type
CaC12.2H20STOCK SOL. stock sol.=1.32 10 m1/1 HT MAIN
g/1
CAS.AA Sigma Enzymatic Hydrolysate20 g/1 HT MAIN
~
GLYCEROL 0.87 ~ or anhydrous 46 g/1 HT MAIN
glycerol
__..._.._......._.....___..._.___.__.__....__.._.
._._..__......__.._...._._._...._..._.______..._..__..
.._______.__...__.._..______.-.._______._.____._ _ _.
___.._._._.._.__.._...
K2HP04 STOCK-SOL. ~_..__._ __~.._._.........._.____
_...._.___._..._._._MAIN
stock sol. = 400 10 _..._.___..HT
g/1 m1/1
K2S04 STOCK SOL Stock Sol=104 22.7 m1/1 HT MAIN
g/1 ~
KH2P04 STOCK SOL. stock sol.= 100 10 m1/1 HT MAIN
g/1
i I _
MgC12.6H20stock sol Stock Sol= 1M 2 m1/1 FI ADD
~
NH4C1 ~ ~ STOCK ~ stock sol.= 10 ~ml/1HT MAIN-
4~~ SOL.~ ~ 100 g/1 ~~~~~~~~~~~~ -
Z ~
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64
TRACE ~ STOCK SOL. ~ stock sol composition in 10 m1/1 HT MAIN
Y.E ~ Difco ( 3 g/1 HT MAIN
A few drops of Antifoam PPG P2000 are added.
Table 5 : Trace Elements
Component Comment Conc Unit Steril.Type
Amonium molb adjust pH 7-8 as 0.01 g/1 HT MAIN
necessary
Co(N03)26H20 0.01 g/1 HT MAIN
CuCl2 2H20 ~--~~-~~ 0.01 g/1 HT MAIN
m~
EDTA ~ dissolved in approx.800m15 g/1 HT MAIN
FeCl3 6H20 0.5 g/1 ' FI MAIN
Zn0 ~ ~ ~ ~HT-~ MAIN(
0.05 g/1
Each element was separately dissolved in HCl
Purification process
67 g of the frozen bacteria paste was suspended in 270 ml of buffer A (50 mM
NaH2PO4; 600 mM NaCI; 1 mM PMSF; 1 mM benzamidine; 8.7 % (w/v) glycerol,
pH 7.5) supplemented with 1 tablet of complete EDTA-free protease inbitors
(Roche)
/50 ml. The bacteria were disrupted by two passages through the Z-plus cell
disrupter
(Constant Cell Disruption Systems) at 1300 bar.
The sample was subsequently centrifuged at 36,000 x g for 30 min. The
supernatant
(300 ml) was loaded, at a flow rate of 4 ml/min, onto a Ni-NTA-Agarose column
(2.5
x 3.0 cm) equilibrated in buffer A.
The column was washed with 100 ml buffer A followed by 85 ml 20 mM imidazole
in
buffer A. Proteins were eluted at a flow rate of 3 ml/min by a 300 ml linear
gradient
of 20 to 250 mM imidazole in buffer A and fractions of 7.5 ml were collected.
A
sample of every second fraction was diluted 1/6 in reducing SDS-sample buffer,
15 u1
loaded /well on a 4-12 % NuPage gel (Novex) and after electrophoresis the gel
was
stained with coomassie blue.
Fractions with the highest IPAAA44548 concentration (fractions 36-42) were
pooled,
total volume was 53 ml (Pool N1). Fractions on both sides of pool N1 with a
lower
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purity and concentration (fractions 32-35 + 43-44) were pooled into pool N2
with a
volume of 44 ml.
The pools from the Ni-column were further purified on a Q-Sepharose Fast flow
column (1.5 x 12 cm) equilibrated in buffer B (50 mM Tris-HCI, 1 mM
benzamidine,
5 pH 7.5). 52 ml of pool N1 was diluted with 300 ml buffer B and 648 ml H20 to
a final
volume of 1000 ml. The sample was loaded onto the column at a flow rate of 5
ml/min, the column washed with 150 ml buffer B and proteins were eluted with a
160
ml linear gradient of 0 to 400 mM NaCI in buffer B. Fractions of 2 ml were
collected
and analyzed by coomassie stained SDS-PAGE as described above. Fractions 28-30
10 (Pool Ql) contained one protein band at the expected molecular weight of
9.6 kDa.
Fractions 31-33 (Pool Q2) in addition contained a protein band at
approximately 20
kDa, indicating dimer formation.
43 ml of pool N2 from the Ni-column was diluted with 300 ml buffer B and 657
ml
H20 to 1000 ml. The sample was loaded onto the Q-Sepharose column, the protein
15 was eluted and fractions analyzed as described for pool Nl. Fractions 28-30
(Pool Q3)
contained one protein band at the expected molecular weight of 9.6 kDa. Each Q-
pool
had a vohune of 5.5 ml.
The pools from the Q-Sepharose column were passed over a Superdex G75 gel
filtration column (HiLoad 16/60, Pharmacia). The column was washed with 0.5 M
20 NaOH and equilibrated in PBS. The column was run at a flow rate of 1 ml/min
and 5
ml of the pools was loaded onto the column. Fractions of 2 ml were collected
and
analyzed by coomassie stained SDS-PAGE as described above.
IPAAA44548 from pool Q1 eluted in fractions 31-35 (9.5 ml) (S1), from pool Q2
the
protein eluted in two peaks, in fraction 31-34 (7.5 ml) (S2) and in fractions
26-28 (5.8
25 ml) (S3), and the protein in pool Q3 eluted in fractions 32-35 (7.5 ml)
(S4). When
analyzed on non-reducing SDS-PAGE pool S3 showed to contain over 80 % of the
protein as dimers, whereas the other pools contained only traces of dimers.
The pools
S 1 and S2 had comparable purity and concentration and were pooled into one
pool
Slb (9.5+7.5=17 ml).
30 Protein concentrations were determined by measuing absorption at 280 nm,
using the
calculated molar extinction coefficient of 7,090 and molecular weight of
9,625. The
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66
molecular mass of the protein, determined by mass spectrometry, was found to
be
9,624.6 in pools Slb and S4. The molecular mass in pool S3 was determined to
be
19,252.2, confimning disulphide bridged dimers in this pool. The pools were
assayed
for LPS and contained between 1.1 and 3.4 U/mg.
Summary of the purified pools:
Pool Concentration Total amount Lot number
Pool Slb 2.1 mg/ml 35.7 mg 2
Pool S3 1.7 mg/ml 9.8 mg 3
Pool S4 0.95 mg/ml 7.1 mg 6
A total of 52 mg pure protein was recovered, or 0.77 mg/g bacteria paste. All
three
pools were over 97 % pure on RP-HPLC.
Example 6: Ih vivo characterisation of IPAA44548 (INSP037)
The IPAA44548 (INSP037) protein (IPAAA44548-6-HIS and IPAAA44548-ATT-
6HIS) was shown in vit~~o to induce IFNy secretion by Concanavalin A (ConA)
and
Phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells
(hPBMC) (preliminary data, not shown). On the basis of those data, it was
decided to
test the activity of IPAAA44548 (INSP037) in an in vivo ConA model by
electrotransfer, as described below.
Concanavalin A (ConA)-induced liver hepatitis
Toxic liver disease represents a worldwide health problem in humans for which
pharmacological treatments have yet to be discovered. For example, active
chronic
hepatitis leading to liver cirrhosis is a disease state, in which liver
parenchyma) cells
are progressively destroyed by activated T cells. ConA-induced liver toxicity
is one of
three experimental models of T-cell dependent apoptotic and necrotic liver
injury
described in mice. Gal N ( D-Galactosamine) sensitized mice challenged with
either
activating anti-CD3 monoclonal AB or with superantigen SEB develop severe
apoptotic and secondary necrotic liver injury (Dusters S, Gastroenterology.
1996
Aug;lll(2):462-71). Injection of the T-cell mitogenic plant lectin ConA to non-
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67
sensitized mice results also in hepatic apoptosis that preceeds necrosis. ConA
induces
the release of systemic TNFa and IFNy and various other cytokines. Both TNFa
and
IFNy are critical mediators of liver injury. Transaminase release 8 hours
after the
insult indicates severe liver destruction.
Several cell types have been shown to be involved in liver damage, including
CD4 T
cells, macrophages and natural killer cells (Kaneko J Exp Med 2000, 191, 105-
114).
Anti-CD4 antibodies block activation of T cells and consequently liver damage
(Tiegs
et al. 1992, J Clin Invest 90, 196-203). Pre-treatment of mice with monoclonal
antibodies against CD8 failed to protect, whereas deletion of macrophages
prevented
the induction of hepatitis.
A study was undertaken to investigate the role of IPAA44548, a IFNy like
protein, in
ConA-induced liver hepatitis. Several cytokines have been shown either to be
critical
in inducing or in conferring protection from ConA-induced liver damage. TNFa
for
example is one of the first cytokines produced after ConA injection and anti-
TNFa
antibodies confer protection against disease (Seino et al. 2001, Annals of
surgery 234,
681). IFNy appears also to be a critical mediator of liver injury, since anti-
IFNy
antisenun significantly protect mice, as measured by decreased levels of
transaminases in the blood of ConA-treated animals (see Kusters et al.,
above). In
liver injury, increased production of IFNy was observed in patients with
autoimmune
or viral hepatitis. In addition transgenic mice expressing IFNy in the liver
develop
liver injury resembling chronic active hepatitis (Toyonaga et al. 1994, PNAS
91, 614-
618). IFNy may also be cytotoxic to hepatocytes, since in vitf~o IFNy induces
cell
death in mouse hepatocytes that was accelerated by TNF (Morita et al. 1995,
Hepatology 21, 1585-1593).
Other molecules have been described to be protective in the ConA model. A
single
aclininistration of rhIL-6 completely inhibited the release of transaminases
(Mizuhara
et al. 1994, J. Exp. Med. 179, 1529-1537).
cDNA electrotransfer into muscle fibers in order to achieve systemic
expression of a
protein of interest
Among the non-viral techniques for gene transfer in vivo, the direct injection
of
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68
plasmid DNA into the muscle and subsequent electroporation is simple,
inexpensive
and safe. The post-mitotic nature and longevity of myofibers permits stable
expression of transfected genes, although the transfected DNA does not usually
undergo chromosomal integration (Somiari et al. 2000, Molecular Therapy
2,178).
Several reports have demonstrated that secretion of muscle-produced proteins
into the
blood stream can be achieved after electroporation of corresponding cDNAs
(Rizzuto
et al. PNAS, 1996, 6417; Aihara H et al., 1998, Nature Biotech 16, 867). In
addition,
in vivo efficacy of muscle expressed Epo and IL-18BP in disease models has
been
shown (Rizzuto, 2000, Human Gene Therapy 41, 1891; Mallat, 2001, Circulation
research 89, 41).
The following material and methods were employed in this Example:
Animals
In all the studies male C57/BL6 male (8 weeks old) were used. In general, 7
animals
per experimental group are used. Mice were maintained in standard conditions
under
a 12-hour light-dark cycle, provided irradiated food and water ad libituf~z.
Muscle Electrotransfer
Choice of vecto~°
His or StrepII tagged hIL-6 or IPAAA44548 genes were cloned in the Gateway
compatible pDESTl2.2 containing the CMV promoter.
Elects°opo~ati~h Protocol
Mice were anaesthetised with gas (isofluran, Baxter, Ref: ZDG9623). Hindlimbs
were shaved and an echo graphic gel was applied. Hyaluronidase was injected in
the
posterior tibialis mucle with (20U in 50' ~,l sterile NaCI 0.9%, Sigma, Ref.
H3631).
After 10 min, 100 ~,g of plasmid (50 ~,g per leg in 25 ~,l of sterile NaCI
0.9%) was
injected in the same muscle. The DNA was prepared in the Buffer PBS-L-
Glutamate
(6 mg/ml; L-Glutamate, Sigma, P4761) before intra-muscular injection. For
electrotransfer, the electric field was applied for each leg with the
ElectroSquarePorator (BTX, ref ECM830) at 75 Volts during 20 ms for each
pulse, 10
pulses with an interval of 1 second in a unipolar way with 2 round electrodes
(size 0.5
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69
mm diameter) (Mir LM et al, Proc Natl Acad Sci U S A. 1999 Apr 13;96(8):4262-7
and Haas K et al., Neuron. 2001 Mar;29(3):583-91.).
Readouts
Blood Sampling
100 ~.l of blood was sampled from the eye at various 1.30h, 6h and 8h time-
points. At
the time of sacrifice, blood was taken from the heart.
Detection of cytokines and tf°ansaminases in blood sarfzples
IL-2, IL-5, IL-4, TNFa and IFNy cytokine levels were measured using the
THl/TH2
CBA assay (BD 551287). ASpartate AminoTransferase (ASAT), ALanine Amino
Transferase ALAT and urea blood parameters were determined using the COBAS
instrument (Hitachi).
ConA induction
Mice female C57/B16 (from IFFA CREDO), 8 weeks old animals; ConA (purchased
from Sigma, ref.C7275). ConA was injected at different doses at time 0 i.v and
blood
samples were taken at 1.30, 6 or 8 hours post-injection. Cytokine and ASAT
ALAT
measurements were performed like described above.
IL-6 p~etr°eatynent i~t the ConA model
CHO cell produced hIL-6 was injected 1 hour before ConA injection.
IPAAA44548 ahd IL-6 elects°otf°ansfe~
At day 0 electrotransfer of IPAAA44548 or hIL-6 vectors as well as the empty
vector
(negative control) was performed (according to the above protocol). At day 5
after
electrotransfer, ConA (20mg/kg) was injected iv and blood sampled at 3 time-
points
(1.30, 6, 24 hours). Cytolcines, ASAT and ALAT measurements were performed
like
described above.
Results
In vivo, in this marine model of ConA-induced fulminant hepatitis, treatment
using
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cDNA electrotransfer with IPAAA44548 showed an increase in circulating levels
of
TNF-a, IL-2, and IFN-'y (see Figure 17 A-C). In addition ASAT and ALAT levels
were increased with respect to the control (Figure 17, D and E).
Results in Figure 18 A-F represent the positive control of the experiment
(rhIL-6
5 known to block pro-inflammatory response induced by ConA). We used either
the
pDEST12.2hII,-6-STREPII or the pDESTl2.2 STREPII electrotransfer vectors in
order to
express hIL-6 in the blood and thus show subsequent protection from ConA
induced liver
toxicity.
Our experiments show that expression of IPAAA44548 protein in serum using
10 electrotransfer increases the level of pro-inflammatory cytokines at a
systemic level
after ConA challenge and exacerbates liver disease as measured by increased
transaminase levels.
These results confirm the predicted IFNy-like activity of IPAAA44548 and open
a
series of interesting therapeutic applications for the protein per se. For
example,
15 known applications of IFNy may now be investigated for suitability to
IPAAA44548
(e.g. anti-cancer activity). It will also now be possible to identify
inhibitors or
antagonists of IPAAA44548, such as for example monoclonal antibodies, which
may
be of use in fiu-ther studies of IPAAA44548 activity irT vivo or in clinical
applications.
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71
INSP037 Sequence Information:
SEQ ID NO: 1 (Nucleotide sequence of INSP037)
1 ATGACTTCAC CAAACGAACT AAATAAGCTG CCATGGACCA ATCCTGGAGA
S 51 AACAGAGATA TGTGACCTTT CAGACACAGA ATTCAAAATA TCTGTGTTGA
101 AGAACCTCAA AGAAATTCAA GATAACACAG AGAAGGAATC CAGAATTCTA
151 TCAGACAAAT ATAAGAAACA GATTGAAATA ATTAAAGGGA ATCAAGCAGA
201 AATTCTGGAG TTGAGAAATG CAGATGGCAC ACTTTAG
SEQ ID NO: 2 (Protein sequence of INSP037)
1 MTSPNELNKL PWTNPGETEI CDLSDTEFKT SVLKNLKEIQ DNTEKESRIL
51 SDKYKKQIEI IKGNQAEILE LRNADGTL
