Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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POLYPEPTIDE
The present invention relates to polynucleotides, herein referred to as
CASB612
polynucleotides, polypeptides encoded thereby (referred to herein as CASB612
polypeptides), recombinant materials and methods for their production. In
another aspect,
the invention relates to methods for using such polypeptides and
polynucleotides, including
the treatment of cancer and autoimmune diseases and other related conditions.
In a further
aspect, the invention relates to methods for identifying agonists and
antagonists/inhibitors
using the materials provided by the invention, and treating conditions
associated with
1o CASB612 polypeptide imbalance with the identified compounds. In a still
further aspect,
the invention relates to diagnostic assays for detecting diseases associated
with inappropriate
CASB612 polypeptide activity or levels.
Polypeptides and polynucleotides of the present invention are believed to be
important
immunogens for specific prophylactic or therapeutic immunization against
tumours, because
they are specifically expressed or highly over-expressed in tumours compared
to normal
cells and can thus be targeted by antigen-specific immune mechanisms leading
to the
destruction of the tumour cell. They can also be used to diagnose the
occurrence of tumour
cells. Furthermore, their inappropriate expression in certain circumstances
can cause an
2o induction of autoimmune, inappropriate immune responses, which could be
corrected
through appropriate vaccination using the same polypeptides or
polynucleotides. In this
respect the most important biological activities to our purpose are the
antigenic and
immunogenic activities of the polypeptide of the present invention. A
polypeptide of the
present invention may also exhibit at least one other biological activity of a
CASB612
polypeptide, which could qualify it as a target for therapeutic or
prophylactic intervention
different from that linked to the immune response.
Functional genomics relies heavily on high-throughput DNA sequencing
technologies and
the various tools of bioinformatics to identify gene sequences of potential
interest from the
3o many molecular biology databases now available. cDNA libraries enriched for
genes of
relevance to a particular tissue or physiological situation can be constructed
using recently
developed subtractive cloning strategies. Furthermore, cDNAs found in
libraries of certain
tissues and not others can be identified using appropriate electronic
screening methods.
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High throughput genome- or gene-based biology allows new approaches to the
identification
and cloning of target genes for usefiil immune responses for the prevention
and vaccine
therapy of diseases such as cancer and autoimmunity.
In a first aspect, the present invention relates to CASB612 polypeptides. Such
peptides
include isolated polypeptides comprising an amino acid sequence which has at
least 70%
identity, preferably at least 80% identity, more preferably at least 90%
identity, yet more
preferably at least 95% identity, most preferably at least 97-99% identity, to
that of SEQ
1o ID N0:2 over the entire length of SEQ ID N0:2. Such polypeptides include
those
comprising the amino acid of SEQ ID N0:2.
Further peptides of the present invention include isolated polypeptides in
which the
amino acid sequence has at least 70% identity, preferably at least 80%
identity, more
15 preferably at least 90% identity, yet more preferably at least 95%
identity, most
preferably at least 97-99% identity, to the amino acid sequence of SEQ ID N0:2
over the
entire length of SEQ ID N0:2. Such polypeptides include the polypeptide of SEQ
ID
N0:2.
2o Further peptides of the present invention include isolated polypeptides
encoded by a
polynucleotide comprising the sequence contained in SEQ ID NO:1.
The invention also provides an immunogenic fragment of a CASB612 polypeptide,
that is a
contiguous portion of the CASB612 polypeptide which has the same or similar
25 immunogenic properties to the polypeptide comprising the amino acid
seqeunce of SEQ >D
N0:2. That is to say, the fragment (if necessary when coupled to a carrier) is
capable of
raising an immune response which recognises the CASB612 polypeptide. Such an
immunogenic fragment may include, for example, the CASB612 polypeptide lacking
an N-
terminal leader sequence, a transmembrane domain or a C-terminal anchor
domain. In a
3o preferred aspect the immunogenic fragment of CASB612 according to the
invention
comprises substantially all of the extracellular domain of a polypeptide which
has at least
70% identity, preferably at least 80% identity, more preferably at least 90%
identity, yet
2
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more preferably at least 95% identity, most preferably at least 97-99%
identity, to that of
SEQ ID N0:2 over the entire length of SEQ ID N0:2
The polypeptides or immunogenic fragment of the invention may be in the form
of the
"mature" protein or may be a part of a larger protein such as a precursor or a
fusion
protein. It is often advantageous to include an additional amino acid sequence
which
contains secretory or leader sequences, pro-sequences, sequences which aid in
purification such as multiple histidine residues, or an additional sequence
for stability
during recombinant production. Furthermore, addition of exogenous polypeptide
or lipid
to tail or polynucleotide sequences to increase the immunogenic potential of
the final
molecule is also considered.
In one aspect, the invention relates to genetically engineered soluble fusion
proteins
comprising a polypeptide of the present invention, or a fragment thereof, and
various
portions of the constant regions of heavy or light chains of immunoglobulins
of various
subclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is the
constant part of
the heavy chain of human IgG, particularly IgGI, where fusion takes place at
the hinge
region. In a particular embodiment, the Fc part can be removed simply by
incorporation
of a cleavage sequence which can be cleaved with blood clotting factor Xa.
Furthermore,
this invention relates to processes for the preparation of these fusion
proteins by genetic
engineering, and to the use thereof for drug screening, diagnosis and therapy.
A further
aspect of the invention also relates to polynucleotides encoding such fusion
proteins.
Examples of fusion protein technology can be found in International Patent
Application
Nos. W094/29458 and W094/22914.
The proteins may be chemically conjugated, or expressed as recombinant fusion
proteins
allowing increased levels to be produced in an expression system as compared
to non-
fused protein. The fusion partner may assist in providing T helper epitopes
(immunological fusion partner), preferably T helper epitopes recognised by
humans, or
assist in expressing the protein (expression enhancer) at higher yields than
the native
recombinant protein. Preferably the fusion partner will be both an
immunological fusion
partner and expression enhancing partner.
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Fusion partners include protein D from Haemophilus influenza B and the non-
structural
protein from influenzae virus, NS 1 (hemagglutinin). Another immunological
fusion
partner is the protein known as LYTA. Preferably the C terminal portion of the
molecule
is used. Lyta is derived from Streptococcus pneumoniae which synthesize an N-
acetyl-L-
alanine amidase, amidase LYTA, (coded by the lytA gene {Gene, 43 (1986) page
265-
272} an autolysin that specifically degrades certain bonds in the
peptidoglycan backbone.
The C-terminal domain of the LYTA protein is responsible for the affinity to
the choline
or to some choline analogues such as DEAE. This property has been exploited
for the
development of E.coli C-LYTA expressing plasmids useful for expression of
fusion
to proteins. Purification of hybrid proteins containing the C-LYTA fragment at
its amino
terminus has been described {Biotechnology: 10, ( 1992) page 795-798 } . It is
possible to
use the repeat portion of the Lyta molecule found in the C terminal end
starting at residue
178, for example residues 188 - 305.
The present invention also includes variants of the aforementioned
polypeptides, that is
polypeptides that vary from the referents by conservative amino acid
substitutions, whereby
a residue is substituted by another with like characteristics. 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; and among the basic residues Lys and Arg; or aromatic
residues Phe
2o and Tyr. Particularly preferred are variants in which several, 5-10, 1-5, 1-
3, 1-2 or 1 amino
acids are substituted, deleted, or added in any combination.
Polypeptides of the present invention can be prepared in any suitable manner.
Such
polypeptides include isolated naturally occurring polypeptides, recombinantly
produced
polypeptides, synthetically produced polypeptides, or polypeptides produced by
a
combination of these methods. Means for preparing such polypeptides are well
understood
in the art.
In a further aspect, the present invention relates to CASB612 polynucleotides.
Such
3o polynucleotides include isolated polynucleotides comprising a nucleotide
sequence encoding
a polypeptide which has at least 70% identity, preferably at least 80%
identity, more
preferably at least 90% identity, yet more preferably at least 95% identity,
to the amino
acid sequence of SEQ ID N0:2, over the entire length of SEQ ID N0:2. In this
regard,
4
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polypeptides which have at least 97% identity are highly preferred, whilst
those with at least
98-99% identity are more highly preferred, and those with at least 99%
identity are most
highly preferred. Such polynucleotides include a polynucleotide comprising the
nucleotide
sequence contained in SEQ m NO:1 encoding the polypeptide of SEQ >D N0:2.
Further polynucleotides of the present invention include isolated
polynucleotides comprising
a nucleotide sequence that has at least 70% identity, preferably at least 80%
identity, more
preferably at least 90% identity, yet more preferably at least 95% identity,
to a nucleotide
sequence encoding a polypeptide of SEQ ll~ N0:2, over the entire coding
region. In this
to regard, polynucleotides which have at least 97% identity are highly
preferred, whilst those
with at least 98-99% identity are more highly preferred, and those with at
least 99% identity
are most highly preferred.
Further polynucleotides of the present invention include isolated
polynucleotides
15 comprising a nucleotide sequence which has at least 70% identity,
preferably at least 80%
identity, more preferably at least 90% identity, yet more preferably at least
95% identity,
to SEQ ID NO:1 over the entire length of SEQ >D NO:1. In this regard,
polynucleotides
which have at least 97% identity are highly preferred, whilst those with at
least 98-99%
identiy are more highly preferred, and those with at least 99% identity are
most highly
2o preferred. Such polynucleotides include a polynucleotide comprising the
polynucleotide of
SEQ m NO:1 as well as the polynucleotide of SEQ )D NO:1. Said polynucleotide
can be
inserted in a suitable plasmid or recombinant microrganism vector and used for
immunization ( see for example Wolffet. al., Science 247:1465-1468 (1990);
Corr et. al., J.
Exp. Med. 184:1555-1560 (1996); Doe et. al., Proc. Natl. Acad. Sci. 93:8578-
8583 (1996)).
25 The invention also provides polynucleotides which are complementary to all
the above
described polynucleotides.
The invention also provides a fragment of a CASB612 polynucleotide which when
administered to a subject has the same immunogenic properties as the
polynucleotide of
3o SEQ ID NO:l.
The invention also provides a polynucleotide encoding an immunological
fragment of a
CASB612 polypeptide as hereinbefore defined.
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The nucleotide sequence of SEQ ID NO:1 shows no homology with any lrnown gene.
The
nucleotide sequence of SEQ ID NO:1 is a cDNA sequence and comprises a
polypeptide
encoding sequence (nucleotide 133 to 1242) encoding a polypeptide of 369 amino
acids, the
polypeptide of SEQ ID N0:2. The nucleotide sequence encoding the polypeptide
of SEQ
ID N0:2 may be identical to the polypeptide encoding sequence contained in SEQ
ID
NO:1 or it may be a sequence other than the one contained in SEQ ID NO:l,
which, as a
result of the redundancy (degeneracy) of the genetic code, also encodes the
polypeptide
of SEQ ID N0:2. The polypeptide of the SEQ ID N0:2 is not related to other
lrnown
proteins.
Preferred polypeptides and polynucleotides of the present invention are
expected to have,
inter alia, similar biological fimctions/properties to their homologous
polypeptides and
polynucleotides. Furthermore, preferred polypeptides, immunological fragments
and
polynucleotides of the present invention have at least one activity of either
SEQ m NO:1 or
SEQ 117 N0:2, as appropriate.
The present invention also relates to partial or other incomplete
polynucleotide and
polypeptide sequences which were first identified prior to the determination
of the
2o corresponding full length sequences of SEQ ID NO:1 and SEQ 117 N0:2.
Accordingly, in a fiuther aspect, the present invention provides for an
isolated
polynucleotide which:
(a) comprises a nucleotide sequence which has at least 70% identity,
preferably at least
80% identity, more preferably at least 90% identity, yet more preferably at
least 95%
identity, even more preferably at least 97-99% identity to SEQ ID N0:3 over
the entire
length of SEQ ID N0:3;
(b) has a nucleotide sequence which has at least 70% identity, preferably at
least 80%
identity, more preferably at least 90% identity, yet more preferably at least
95% identity,
3o even more preferably at least 97-99% identity, to SEQ ID NO:l over the
entire length of
SEQ ID N0:3;
(c) the polynucleotide of SEQ ID N0:3; or
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(d) a nucleotide sequence encoding a polypeptide which has at least 70%
identity,
preferably at least 80% identity, more preferably at least 90% identity, yet
more
preferably at least 95% identity, even more preferably at least 97-99%
identity, to the
amino acid sequence of SEQ 1D N0:4, over the entire length of SEQ ID N0:4;
as well as the polynucleotide of SEQ ID N0:3.
The present invention further provides for a polypeptide which:
(a) comprises an amino acid sequence which has at least 70% identity,
preferably at least
80% identity, more preferably at least 90% identity, yet more preferably at
least 95%
1o identity, most preferably at least 97-99% identity, to that of SEQ ID N0:2
over the entire
length of SEQ ID N0:4;
(b) has an amino acid sequence which is at least 70% identity, preferably at
least 80%
identity, more preferably at least 90% identity, yet more preferably at least
95% identity,
most preferably at least 97-99% identity, to the amino acid sequence of SEQ ID
N0:2
15 over the entire length of SEQ D7 N0:4;
(c) comprises the amino acid of SEQ ID N0:4; and
(d) is the polypeptide of SEQ ID N0:4;
as well as polypeptides encoded by a polynucleotide comprising the sequence
contained
in SEQ ID N0:3.
The nucleotide sequence of SEQ ID N0:3 and the peptide sequence encoded
thereby are
derived from EST (Expressed Sequence Tag) sequences. It is recognised by those
skilled
in the art that there will inevitably be some nucleotide sequence reading
errors in EST
sequences (see Adams, M.D. et al, Nature 377 (supp) 3, 1995). Accordingly, the
nucleotide sequence of SEQ ID N0:3 and the peptide sequence encoded therefrom
are
therefore subject to the same inherent limitations in sequence accuracy.
Furthermore, the
peptide sequence encoded by SEQ 1D N0:3 comprises a region of identity or
close
homology and/or close structural similarity (for example a conservative amino
acid
difference) with the closest homologous or structurally similar protein.
Polynucleotides of the present invention may be obtained, using standard
cloning and
screening techniques, from a cDNA library derived from mRNA in cells of human
colon
cancer, lung cancer, uterine cancer, and fetal tissues (for example Sambrook
et al.,
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Molecular Cloning: A Laboratory Manual, 2"d Ed., Cold Spring harbor Laboratory
Press,
Cold Spring harbor, N.Y. (1989)). Polynucleotides of the invention can also be
obtained
from natural sources such as genomic DNA libraries or can be synthesized using
well
known and commercially available techniques.
When polynucleotides of the present invention are used for the recombinant
production of
polypeptides of the present invention, the polynucleotide may include the
coding sequence
for the mature polypeptide, by itself; or the coding sequence for the mature
polypeptide in
reading frame with other coding sequences, such as those encoding a leader or
secretory
1o sequence, a pre-, or pro- or prepro- protein sequence, or other fusion
peptide portions. For
example, a marker sequence which facilitates purification of the fused
polypeptide can be
encoded. In certain preferred embodiments of this aspect of the invention, the
marker
sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen,
Inc.) and
described in Gentz et al., Proc Natl Acad Sci USA (1989) 86:821-824, or is an
HA tag. The
15 polynucleotide may also contain non-coding 5' and 3' sequences, such as
transcribed, non
translated sequences, splicing and polyadenylation signals, ribosome binding
sites and
sequences that stabilize mRNA.
Further embodiments of the present invention include polynucleotides encoding
polypeptide
2o variants which comprise the amino acid sequence of SEQ 1D N0:2 and in which
several, for
instance from S to 10, 1 to 5, 1 to 3, 1 to 2 or l, amino acid residues are
substituted, deleted
or added, in any combination.
Polynucleotides which are identical or sufficiently identical to a nucleotide
sequence
25 contained in SEQ >D NO:1, may be used as hybridization probes for cDNA and
genomic
DNA or as primers for a nucleic acid amplification (PCR) reaction, to isolate
full-length
cDNAs and genomic clones encoding polypeptides of the present invention and to
isolate
cDNA and genomic clones of other genes (including genes encoding paralogs from
human
sources and orthologs and paralogs from species other than human) that have a
high
30 sequence similarity to SEQ ID NO:1. Typically these nucleotide sequences
are 70%
identical, preferably 80% identical, more preferably 90% identical, most
preferably 95%
identical to that of the referent. The probes or primers will generally
comprise at least 15
nucleotides, preferably, at least 30 nucleotides and may have at least SO
nucleotides.
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Particularly preferred probes will have between 30 and 50 nucleotides.
Particularly
preferred primers will have between 20 and 25 nucleotides. In particular,
polypeptides or
polynucleotides derived from sequences from homologous animal origin could be
used as
immunogens to obtain a cross-reactive immune response to the human gene.
A polynucleotide encoding a polypeptide of the present invention, including
homologs from
species other than human, may be obtained by a process which comprises the
steps of
screening an appropriate library under stringent hybridization conditions with
a labeled
probe having the sequence of SEQ ID NO: 1 or a fragment thereof; and isolating
full-length
1 o cDNA and genomic clones containing said polynucleotide sequence. Such
hybridization
techniques are well known to the skilled artisan. Preferred stringent
hybridization conditions
include overnight incubation at 42oC in a solution comprising: 50% formamide,
SxSSC
(150mM NaCI, lSmM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x
Denhardt's
solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon
sperm
15 DNA; followed by washing the filters in O.lx SSC at about 65oC. Thus the
present
invention also includes polynucleotides obtainable by screening an appropriate
library
under stingent hybridization conditions with a labeled probe having the
sequence of SEQ ID
NO: l or a fragment thereof.
2o The skilled artisan will appreciate that, in many cases, an isolated cDNA
sequence will be
incomplete, in that the region coding for the polypeptide is short at the 5'
end of the
cDNA.
There are several methods available and well known to those skilled in the art
to obtain
25 full-length cDNAs, or extend short cDNAs, for example those 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 the technique, exemplified
by the
MarathonTM technology (Clontech Laboratories Inc.) for example, have
significantly
simplified the search for longer cDNAs. In the MarathonTM technology, cDNAs
have
3o been prepared from mRNA extracted from a chosen tissue and an'adaptor'
sequence
ligated onto each end. Nucleic acid amplification (PCR) is then carried out to
amplify the
'missing' S' end of the cDNA using a combination of gene specific and adaptor
specific
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oligonucleotide primers. The PCR reaction is then repeated using 'nested'
primers, that is,
primers designed to anneal within the amplified product (typically an adaptor
specific
primer that anneals further 3' in the adaptor sequence and a gene specific
primer that
anneals further 5' in the known gene sequence). The products of this reaction
can then be
analysed by DNA sequencing and a full-length cDNA constructed either by
joining the
product directly to the existing cDNA to give a complete sequence, or carrying
out a
separate full-length PCR using the new sequence information for the design of
the S'
primer.
1 o Recombinant polypeptides of the present invention may be prepared by
processes well
known in the art from genetically engineered host cells comprising expression
systems.
Accordingly, in a further aspect, the present invention relates to an
expression system which
comprises a polynucleotide of the present invention, to host cells which are
genetically
engineered with such expression sytems and to the production of polypeptides
of the
15 invention by recombinant techniques. Cell-free translation systems can also
be employed to
produce such proteins using RNAs derived from the DNA constructs of the
present
invention.
For recombinant production, host cells can be genetically engineered to
incorporate
2o expression systems or portions thereof for polynucleotides of the present
invention.
Introduction of polynucleotides into host cells can be effected by methods
described in many
standard laboratory manuals, such as Davis et al., Basic Methods in Molecular
Biology
(1986) and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.,
Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). Preferred
such methods
25 include, for instance, calcium phosphate transfection, DEAF-dextran
mediated transfection,
transvection, microinjection, cationic lipid-mediated transfection,
electroporation,
transduction, scrape loading, ballistic introduction or infection.
Preferably the proteins of the invention are coexpressed with thioredoxin in
trans (TIT).
30 Coexpression of thioredoxin in trans versus in cis is preferred to keep
antigen free of
thioredoxin without the need for protease. Thioredoxin coexpression eases the
solubilisation of the proteins of the invention. Thioredoxin coexpression has
also a
significant impact on protein purification yield, on purified-protein
solubility and quality.
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Representative examples of appropriate hosts include bacterial cells, such as
Streptococci,
Staphylococci, E. coli, Streptomyces and Bacillus subtilis cells; fungal
cells, such as yeast
cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera
Sf~ cells;
animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes
melanoma
cells; and plant cells.
A great variety of expression systems can be used, for instance, chromosomal,
episomal
and virus-derived systems, e.g., vectors derived from bacterial plasmids, from
to bacteriophage, from transposons, from yeast episomes, from insertion
elements, from
yeast chromosomal elements, from viruses such as baculoviruses, papova
viruses, such as
SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses
and
retroviruses, and vectors derived from combinations thereof, such as those
derived from
plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The
15 expression systems may contain control regions that regulate as well as
engender
expression. Generally, any system or vector which is able to maintain,
propagate or
express a polynucleotide to produce a polypeptide in a 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 set forth in
Sambrook et
2o al., Molecular Cloning, A Laboratory Manual (supra). Appropriate secretion
signals may
be incorporated into the desired polypeptide to allow secretion of the
translated protein
into the lumen of the endoplasmic reticulum, the periplasmic space or the
extracellular
environment. These signals may be endogenous to the polypeptide or they may be
heterologous signals.
The expression system may also be a recombinant live microorganism, such as a
virus or
bacterium. The gene of interest can be inserted into the genome of a live
recombinant
virus or bacterium. Inoculation and in vivo infection with this live vector
will lead to in
vivo expression of the antigen and induction of immune responses. Viruses and
bacteria
3o used for this purpose are for instance: poxviruses (e.g; vaccinia, fowlpox,
canarypox),
alphaviruses (Sindbis virus, Semliki Forest Virus, Venezuelian Equine
Encephalitis
Virus), adenoviruses, adeno-associated virus, picornaviruses (poliovirus,
rhinovirus),
herpesviruses (varicella zoster virus, etc), Listeria, Salmonella , Shigella,
BCG. These
11
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viruses and bacteria can be virulent, or attenuated in various ways in order
to obtain live
vaccines. Such live vaccines also form part of the invention.
Polypeptides of the present invention can be recovered and purified from
recombinant cell
cultures by well-known methods including ammonium sulfate or ethanol
precipitation, acid
extraction, anion or cation exchange chromatography, phosphocellulose
chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite
chromatography and lectin chromatography. Most preferably, ion metal affinity
chromatography (1MAC) is employed for purification. Well known techniques for
1o refolding proteins may be employed to regenerate active conformation when
the polypeptide
is denatured during intracellular synthesis, isolation and or purification.
Another important aspect of the invention relates to a method for inducing ,
re-enforcing
or modulating an immunological response in a mammal which comprises
inoculating the
mammal with a fragment or the entire polypeptide or polynucleotide of the
invention,
adequate to produce antibody and/or T cell immune response for prophylaxis or
for
therapeutic treatment of cancer and autoimmune disease and related conditions.
Yet
another aspect of the invention relates to a method of inducing, re-enforcing
or
modulating immunological response in a mammal which comprises, delivering a
polypeptide of the present invention via a vector or cell directing expression
of the
polynucleotide and coding for the polypeptide in vivo in order to induce such
an
immunological response to produce immune responses for prophylaxis or
treatment of
said mammal from diseases.
A fizrther aspect of the invention relates to an immunological/vaccine
formulation
(composition) which, when introduced into a mammalian host, induces, re-
enforces or
modulates an immunological response in that mammal to a polypeptide of the
present
invention wherein the composition comprises a polypeptide or polynucleotide of
the
invention or an immunological fragment thereof as herein before defined.The
vaccine
3o formulation, may fizrther comprise a suitable Garner. Since a polypeptide
may be broken
down in the stomach, it is preferably administered parenterally (for instance,
subcutaneous, intramuscular, intravenous, or intradermal injection).
Formulations
suitable for parenteral administration include aqueous and non-aqueous sterile
injection
12
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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
formulations may be presented in unit-dose or multi-dose containers, for
example, sealed
ampoules and vials and may be stored in a freeze-dried condition requiring
only the
addition of the sterile liquid Garner immediately prior to use.
A further aspect of the invention relates to the in vitro induction of immune
responses to a
fragment or the entire polypeptide or polynucleotide of the present invention
or a
1o molecule comprising the polypeptide or polynucleotide of the present
invention, using
cells from the immune system of a mammal, and reinfusing these activated
immune cells
of the mammal for the treatment of disease. Activation of the cells from the
immune
system is achieved by in vitro incubation with the entire polypeptide or
polynucleotide of
the present invention or a molecule comprising the polypeptide or
polynucleotide of the
15 present invention in the presence or absence of various immunomodulator
molecules.
A further aspect of the invention relates to the immunization of a mammal by
administration of antigen presenting cells modified by in vitro loading with
part or the
entire polypeptide of the present invention or a molecule comprising the
polypeptide of
the present invention and administered in vivo in an immunogenic way.
Alternatively,
20 antigen presenting cells can be transfected in vitro with a vector
containing a fragment or
the entire polynucleotide of the present invention or a molecule comprising
the
polynucleotide of the present invention, such as to express the corresponding
polypeptide,
and administered in vivo in an immunogenic way.
25 The vaccine formulation of the invention may also include adjuvant systems
for
enhancing the immunogenicity of the formulation. Preferably the adjuvant
system raises
preferentially a TH1 type of response.
An immune response may be broadly distinguished into two extreme catagories,
being a
3o humoral or cell mediated immune responses (traditionally characterised by
antibody and
cellular effector mechanisms of protection respectively). These categories of
response
have been termed TH1-type responses (cell-mediated response), and TH2-type
immune
responses (humoral response).
13
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Extreme TH1-type immune responses may be characterised by the generation of
antigen
specific, haplotype restricted cytotoxic T lymphocytes, and natural killer
cell responses.
In mice TH1-type responses are often characterised by the generation of
antibodies of the
IgG2a subtype, whilst in the human these correspond to IgGl type antibodies.
TH2-type
immune responses are characterised by the generation of a broad range of
immunoglobulin isotypes including in mice IgGl, IgA, and IgM.
It can be considered that the driving force behind the development of these
two types of
1o immune responses are cytokines. High levels of THl-type cytokines tend to
favour the
induction of cell mediated immune responses to the given antigen, whilst high
levels of
TH2-type cytokines tend to favour the induction of humoral immune responses to
the
antigen.
15 The distinction of TH1 and TH2-type immune responses is not absolute. In
reality an
individual will support an immune response which is described as being
predominantly
TH1 or predominantly TH2. However, it is often convenient to consider the
families of
cytokines in terms of that described in murine CD4 +ve T cell clones by
Mosmann and
Coffinan (Mosmann, T.R. and Coffman, R.L. (1989) THI and TH2 cells: different
20 patterns of lymphokine secretion lead to different functional properties.
Annual Review of
Immunology, 7, p145-173). Traditionally, THl-type responses are associated
with the
production of the INF-y and IL-2 cytokines by T-lymphocytes. Other cytokines
often
directly associated with the induction of THl-type immune responses are not
produced by
T-cells, such as IL-12. In contrast, TH2- type responses are associated with
the secretion
25 of IL-4, IL-5, IL-6 and IL-13.
It is known that certain vaccine adjuvants are particularly suited to the
stimulation of
either TH1 or TH2 - type cytokine responses. Traditionally the best indicators
of the
TH1:TH2 balance of the immune response after a vaccination or infection
includes direct
3o measurement of the production of TH1 or TH2 cytokines by T lymphocytes in
vitro after
restimulation with antigen, and/or the measurement of the IgGI :IgG2a ratio of
antigen
specific antibody responses.
14
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Thus, a TH1-type adjuvant is ore which preferentially stimulates isolated T-
cell
populations to produce high levels of THl-type cytokines when re-stimulated
with
antigen in vitro, and promotes development of both CD8+ cytotoxic T
lymphocytes and
antigen specific immunoglobulin responses associated with TH1-type isotype.
Adjuvants which are capable of preferential stimulation of the TH1 cell
response are
described in International Patent Application No. WO 94/00153 and WO 95/17209.
3 De-O-acylated monophosphoryl lipid A (3D-MPL) is one such adjuvant. This is
known
1o from GB 2220211 (Ribi). Chemically it is a mixture of 3 De-O-acylated
monophosphoryl lipid A with 4, 5 or 6 acylated chains and is manufactured by
Ribi
Immunochem, Montana. A preferred form of 3 De-O-acylated monophosphoryl lipid
A
is disclosed in European Patent 0 689 454 B1 (SmithKline Beecham Biologicals
SA).
15 Preferably, the particles of 3D-MPL are small enough to be sterile filtered
through a
0.22micron membrane (European Patent number 0 689 454).
3D-MPL will be present in the range of 10~g - 100~,g preferably 25-SOpg per
dose
wherein the antigen will typically be present in a range 2-SO~g per dose.
2o Another preferred adjuvant comprises QS21, an Hplc purified non-toxic
fraction derived
from the bark of Quillaja Saponaria Molina. Optionally this may be admixed
with 3 De-
O-acylated monophosphoryl lipid A (3D-MPL), optionally together with an
carrier.
The method of production of QS21 is disclosed in US patent No. 5,057,540.
Non-reactogenic adjuvant formulations containing QS21 have been described
previously
(WO 96/33739). Such formulations comprising QS21 and cholesterol have been
shown
to be successful TH1 stimulating adjuvants when formulated together with an
antigen.
3o Further adjuvants which are preferential stimulators of THl cell response
include
immunomodulatory oligonucleotides, for example unmethylated CpG sequences as
disclosed in WO 96/02555.
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WO 00/43511 PCT/EP00/00347
Combinations of different TH1 stimulating adjuvants, such as those mentioned
hereinabove, are also contemplated as providing an adjuvant which is a
preferential
stimulator of TH1 cell response. For example, QS21 can be formulated together
with 3D-
MPL. The ratio of QS21 : 3D-MPL will typically be in the order of 1 : 10 to 10
: l;
preferably 1:5 to S : 1 and often substantially 1 : 1. The preferred range for
optimal
synergy is 2.5 : 1 to 1 : 1 3D-MPL: QS21.
Preferably a carrier is also present in the vaccine composition according to
the invention.
The Garner may be an oil in water emulsion, or an aluminium salt, such as
aluminium
to phosphate or aluminium hydroxide.
A preferred oil-in-water emulsion comprises a metabolisible oil, such as
squalene, alpha
tocopherol and Tween 80. In a particularly preferred aspect the antigens in
the vaccine
composition according to the invention are combined with QS21 and 3D-MPL in
such an
emulsion. Additionally the oil in water emulsion may contain span 85 and/or
lecithin
and/or tricaprylin.
Typically for human administration QS21 and 3D-MPL will be present in a
vaccine in the
range of lp,g - 200p,g, such as 10-100p,g, preferably 10~g - SOpg per dose.
Typically the
oil in water will comprise from 2 to 10% squalene, from 2 to 10% alpha
tocopherol and
from 0.3 to 3% tween 80. Preferably the ratio of squalene: alpha tocopherol is
equal to
or less than 1 as this provides a more stable emulsion. Span 85 may also be
present at a
level of 1%. In some cases it may be advantageous that the vaccines of the
present
invention will further contain a stabiliser.
Non-toxic oil in water emulsions preferably contain a non-toxic oil, e.g.
squalane or
squalene, an emulsifier, e.g. Tween 80, in an aqueous carrier. The aqueous
Garner may
be, for example, phosphate buffered saline.
3o A particularly potent adjuvant formulation involving QS21, 3D-MPL and
tocopherol in
an oil in water emulsion is described in WO 95/17210.
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The present invention also provides a polyvalent vaccine composition
comprising a vaccine
formulation of the invention in combination with other antigens, in particular
antigens useful
for treating cancers, autoimmune diseases and related conditions. Such a
polyvalent vaccine
composition may include a TH-1 inducing adjuvant as hereinbefore described.
This invention also relates to the use of polynucleotides, in the form of
primers derived from
the polynucleotides of the present invention, and of polypeptides, in the form
of antibodies
or reagents specific for the polypeptide of the present invention, as
diagnostic reagents.
1o The identification of genetic or biochemical markers in blood or tissues
that will enable the
detection of very early changes along the carcinogenesis pathway will help in
determining
the best treatment for the patient. Surrogate tumour markers, such as
polynucleotide
expression, can be used to diagnose different forms and states of cancer. The
identification
of expression levels of the polynucleotides of the invention will be useful in
both the
15 staging of the cancerous disorder and grading the nature of the cancerous
tissue. The staging
process monitors the advancement of the cancer and is determined on the
presence or
absence of malignant tissue in the areas biopsied. The polynucleotides of the
invention can
help to perfect the staging process by identifying markers for the aggresivity
of a cancer, for
example the presence in different areas of the body. The grading of the cancer
describes
2o how closely a tumour resembles normal tissue of its same type and is
assessed by its cell
morphology and other markers of differentiation. The polynucleotides of the
invention can
be useful in determining the tumour grade as they can help in the
determination of the
differentiation status of the cells of a tumour.
25 The diagnostic assays offer a process for diagnosing or determining a
susceptibility to
cancers, autoimmune disease and related conditions through diagnosis by
methods
comprising determining from a sample derived from a subject an abnormally
decreased or
increased level of polypeptide or mRNA. This method of diagnosis is known as
differential expression. The expression of a particular gene is compared
between a
3o diseased tissue and a normal tissue. A difference between the
polynucleotide-related
gene, mRNA, or protein in the two tissues is compared, for example in
molecular weight,
amino acid or nucleotide sequence, or relative abundance, indicates a change
in the gene,
17
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WO 00/43511 PCT/EP00/00347
or a gene which regulates it, in the tissue of the human that was suspected of
being
diseased.
Decreased or increased expression can be measured at the RNA level. PolyA RNA
is
first isolated from the two tissues and the detection of mRNA encoded by a
gene
corresponding to a differentially expressed polynucleotide of the invention
can be
detected by, for example, in situ hybridization in tissue sections, reverse
trascriptase-
PCR, using Northern blots containing poly A+ mRNA, or any other direct or
inderect
RNA detection method. An increased or decreased expression of a given RNA in a
1o diseased tissue compared to a normal tissue suggests that the transcript
and/or the expressed
protein has a role in the disease. Thus detection of a higher or lower level
of mRNA
corresponding to SEQ ID NO 1 or 3 relative to normal level is indicative of
the presence
of cancer in the patient.
mRNA expression levels in a sample can be determined by generation of a
library of
expressed sequence tags (ESTs) from the sample. The relative representation of
ESTs in
the library can be used to assess the relative representation of the gene
transcript in the
starting sample. The EST analysis of the test can then be compared to the EST
analysis
of a reference sample to determine the relative expression levels of the
polynucleotide of
interest.
Other mRNA analyses can be carried out using serial analysis of gene
expression (SAGE)
methodology (Velculescu et. Al. Science (1995) 270:484) , differential display
methodology (For example, US 5,776,683) or hybridization analysis which relies
on the
specificity of nucleotide interactions.
Alternatively, the comparison could be made at the protein level. The protein
sizes in the
two tissues may be compared using antibodies to detect polypeptides in Western
blots of
protein extracts from the two tissues. Expression levels and subcellular
localization may
3o also be detected immunologically using antibodies to the corresponding
protein. Further
assay techniques that can be used to determine levels of a protein, such as a
polypeptide of
the present invention, in a sample derived from a host are well-known to those
of skill in the
art. A raised or decreased level of polypeptide expression in the diseased
tissue compared
18
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WO 00/43511 PCT/EP00/00347
with the same protein expression level in the normal tissue indicates that the
expressed
protein may be involved in the disease.
In the assays of the present invention, the diagnosis can be determined by
detection of gene
product expression levels encoded by at least one sequence set forth in SEQ ID
NOS: 1 or 3.
A comparison of the mRNA or protein levels in a diseased versus normal tissue
may also be
used to follow the progression or remission of a disease.
A large number of polynucleotide sequences in a sample can be assayed using
1o polynucleotide arrays. These can be used to examine differential expression
of genes and to
determine gene function. For example, arrays of the polynucleotide sequences
SEQ ID NO:
1 or 3 can be used to determine if any of the polynucleotides are
differentially expressed
between a normal and cancer cell. In one embodiment of the invention, an array
of
oligonucleotides probes comprising the SEQ ID NO: 1 or 3 nucleotide sequence
or
15 fragments thereof can be constructed to conduct efficient screening of
e.g., genetic
mutations. 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,
Vol 274, pp 610-613 (1996)).
"Diagnosis" as used herein includes determination of a subject's
susceptibility to a
disease, determination as to whether a subject presently has the disease, and
also the
prognosis of a subject affected by the disease.
The present invention, further relates to a diagnostic kit for performing a
diagnostic assay
which comprises:
(a) a polynucleotide of the present invention, preferably the nucleotide
sequence of SEQ
ID NO: 1 or 3, or a fragment thereof ;
(b) a nucleotide sequence complementary to that of (a);
(c) a polypeptide of the present invention, preferably the polypeptide of SEQ
ID NO: 2 or
4, or a fragment thereof; or
(d) an antibody to a polypeptide of the present invention, preferably to the
polypeptide of
SEQ ID N0:2 or 4.
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The nucleotide sequences of the present invention are also valuable for
chromosomal
localisation. The sequence is specifically targeted to, and can hybridize
with, a particular
location on an individual human chromosome. The mapping of relevant sequences
to
chromosomes according to the present invention is an important first step in
correlating
those sequences with gene associated disease. Once a sequence has been mapped
to a
precise chromosomal location; the physical position of the sequence on the
chromosome can
be correlated with genetic map data. Such data are found in, for example, V.
McKusick,
Mendelian Inheritance in Man (available on-line through Johns Hopkins
University Welch
1o Medical Library). The relationship between genes and diseases that have
been mapped to
the same chromosomal region are then identified through linkage analysis
(coinheritance of
physically adjacent genes).The differences in the cDNA or genomic sequence
between
affected and unaffected individuals can also be determined.
15 The polypeptides of the invention or their fragments or analogs thereof, or
cells expressing
them, can also be used as immunogens to produce antibodies immunospecific for
polypeptides of the present invention. 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.
In a further aspect the invention provides an antibody immunospecific for a
polypeptide
according to the invention or an immunological fragment thereof as
hereinbefore defined.
Preferably the antibody is a monoclonal antibody
Antibodies generated against polypeptides of the present invention may be
obtained by
administering the polypeptides or epitope-bearing fragments, analogs or cells
to an animal,
preferably a non-human animal, using routine protocols. For preparation of
monoclonal
antibodies, any technique which provides antibodies produced by continuous
cell line
cultures can be used. Examples include the hybridoma technique (Kohler, G. and
Milstein,
C., Nature (1975) 256:495-497), the trioma technique, the human B-cell
hybridoma
technique (Kozbor et al., Immunology Today (1983) 4:72) and the EBV-hybridoma
technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, 77-96, Alan
R. Liss,
Inc., 1985).
CA 02358899 2001-07-19
WO 00/43511 PCT/EP00/00347
Techniques for the production of single chain antibodies, such as those
described in U.S.
Patent No. 4,946,778, can also be adapted to produce single chain antibodies
to polypeptides
of this invention. Also, transgenic mice, or other organisms, including other
mammals, may
be used to express humanized antibodies.
The above-described antibodies may be employed to isolate or to identify
clones expressing
the polypeptide or to purify the polypeptides by affinity chromatography.
The antibody of the invention may also be employed to prevent or treat cancer,
particularly
ovarian and colon cancer, autoimmune disease and related conditions.
Another aspect of the invention relates to a method for inducing or modulating
an
immunological response in a mammal which comprises inoculating the mammal with
a
polypeptide of the present invention, adequate to produce antibody and/or T
cell immune
response to protect or ameliorate the symptoms or progression of the disease.
Yet
another aspect of the invention relates to a method of inducing or modulating
immunological response in a mammal which comprises, delivering a polypeptide
of the
present invention via a vector directing expression of the polynucleotide and
coding for
the polypeptide in vivo in order to induce such an immunological response to
produce
2o antibody to protect said animal from diseases.
It will be appreciated that the present invention therefore provides a method
of treating
abnormal conditions such as, for instance, cancer and autoimmune diseases, in
particular,
ovarian and colon cancer, related to either a presence of, an excess of, or an
under-
expression of, CASB612 polypeptide activity.
The present invention further provides for a method of screening compounds to
identify
those which stimulate or which inhibit the function of the CASB612
polypeptide. In
general, agonists or antagonists may be employed for therapeutic and
prophylactic purposes
3o for such diseases as hereinbefore mentioned. Compounds may be identified
from a variety
of sources, for example, cells, cell-free preparations, chemical libraries,
and natural product
mixtures. Such agonists, antagonists or inhibitors so-identified may be
natural or modified
substrates, ligands, receptors, enzymes, etc., as the case may be, of the
polypeptide; or may
21
CA 02358899 2001-07-19
WO 00/43511 PCT/EP00/00347
be structural or functional mimetics thereof (see Coligan et al., Current
Protocols in
Immunology 1(2):Chapter S (1991)). Screening methods will be known to those
skilled in
the art. Further screening methods may be found in for example D. Bennett et
al., J Mol
Recognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem, 270(16):9459-
9471
(1995) and references therein.
Thus the invention provides a method for screening to identify compounds which
stimulate
or which inhibit the function of the polypeptide of the invention which
comprises a method
selected from the group consisting of
l0 (a) measuring the binding of a candidate compound to the polypeptide (or to
the cells or
membranes bearing the polypeptide) or a fusion protein thereof by means of a
label
directly or indirectly associated with the candidate compound;
(b) measuring the binding of a candidate compound to the polypeptide (or to
the cells or
membranes bearing the polypeptide) or a fusion protein thereof in the presense
of a
15 labeled competitior;
(c) testing whether the candidate compound results in a signal generated by
activation or
inhibition of the polypeptide, using detection systems appropriate to the
cells or cell
membranes bearing the polypeptide;
(d) mixing a candidate compound with a solution containing a polypeptide of
claim 1, to
20 form a mixture, measuring activity of the polypeptide in the mixture, and
comparing the
activity of the mixture to a standard; or
(e) detecting the effect of a candidate compound on the production of mRNA
encoding
said polypeptide and said polypeptide in cells, using for instance, an ELISA
assay.
The polypeptide of the invention may be used to identify membrane bound or
soluble
receptors, if any, through standard receptor binding techniques known in the
art. Well
known screening methods may also be used to identify agonists and antagonists
of the
polypeptide of the invention which compete with the binding of the polypeptide
of the
invention to its receptors, if any.
Thus, in another aspect, the present invention relates to a screening kit for
identifying
agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for
polypeptides of the
22
CA 02358899 2001-07-19
WO 00/43511 PCT/EP00/00347
present invention; or compounds which decrease or enhance the production of
such
polypeptides, which comprises:
(a) a polypeptide of the present invention;
(b) a recombinant cell expressing a polypeptide of the present invention;
(c) a cell membrane expressing a polypeptide of the present invention; or
(d) antibody to a polypeptide of the present invention;
which polypeptide is preferably that of SEQ ID N0:2.
It will be readily appreciated by the skilled artisan that a polypeptide of
the present
1o invention may also be used in a method for the structure-based design of an
agonist,
antagonist or inhibitor of the polypeptide, by:
(a) determining in the first instance the three-dimensional structure of the
polypeptide;
(b) deducing the three-dimensional structure for the likely reactive or
binding sites) of
an agonist, antagonist or inhibitor;
15 (c) synthesing candidate compounds that are predicted to bind to or react
with the
deduced binding or reactive site; and
(d) testing whether the candidate compounds axe indeed agonists, antagonists
or
inhibitors.
2o Gene therapy may also be employed to effect the endogenous production of
CASB612
polypeptide by the relevant cells in the subject. For an overview of gene
therapy, see
Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic
Approaches,
(and references cited therein) in Human Molecular Genetics, T Strachan and A P
Read,
BIOS Scientific Publishers Ltd (1996).
Vaccine preparation is generally described in Pharmaceutical Biotechnology,
Vo1.61
Vaccine Design - the subunit and adjuvant approach, edited by Powell and
Newman,
Plenurn Press, 1995. New Trends and Developments in Vaccines, edited by Voller
et al.,
University Park Press, Baltimore, Maryland, U.S.A. 1978. Encapsulation within
3o liposomes is described, for example, by Fullerton, U.S. Patent 4,235,877.
Conjugation of
proteins to macromolecules is disclosed, for example, by Likhite, U.S. Patent
4,372,945
and by Armor et al., U.S. Patent 4,474,757.
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WO 00/43511 PCT/EP00/00347
The amount of protein in each vaccine dose is selected as an amount which
induces an
immunoprotective response without significant, adverse side effects in typical
vaccinees.
Such amount will vary depending upon which specific immunogen is employed.
Generally, it is expected that each dose will comprise 1-1000p,g of protein,
preferably
2-100p,g, most preferably 4-40pg. An optimal amount for a particular vaccine
can be
ascertained by standard studies involving observation of antibody titres and
other
responses in subjects. Following an initial vaccination, subjects may receive
a boost in
about 4 weeks.
"Isolated" means altered "by the hand of man" from the natural state. If an
"isolated"
composition or substance occurs in nature, it has been changed or removed from
its
original environment, or both. For example, a polynucleotide or a polypeptide
naturally
present in a living animal is not "isolated," but the same polynucleotide or
polypeptide
separated from the coexisting materials of its natural state is "isolated", as
the term is
employed herein.
"Polynucleotide" generally refers to any polyribonucleotide or
polydeoxribonucleotide,
which may be unmodified RNA or DNA or modified RNA or DNA including single and
double stranded regions.
"Variant" refers to a polynucleotide or polypeptide that differs from a
reference
polynucleotide or polypeptide, but retains essential properties. A typical
variant of a
polynucleotide differs in nucleotide sequence from another, reference
polynucleotide.
Changes in the nucleotide sequence of the variant may or may not alter the
amino acid
sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide
changes
may result in amino acid substitutions, additions, deletions, fusions and
truncations in the
polypeptide encoded by the reference sequence, as discussed below. A typical
variant of
a polypeptide differs in amino acid sequence from another, reference
polypeptide.
Generally, differences are limited so that the sequences of the reference
polypeptide and
3o the variant are closely similar overall and, in many regions, identical. A
variant and
reference polypeptide may differ in amino acid sequence by one or more
substitutions,
additions, deletions in any combination. A substituted or inserted amino acid
residue
may or may not be one encoded by the genetic code. A variant of a
polynucleotide or
24
CA 02358899 2001-07-19
WO 00/43511 PCT/EP00/00347
polypeptide may be a naturally occurnng such as an allelic variant, or it may
be a variant
that is not known to occur naturally. Non-naturally occurnng variants of
polynucleotides
and polypeptides may be made by mutagenesis techniques or by direct synthesis.
"Identity," as known in the art, is a relationship between two or more
polypeptide sequences
or two or more polynucleotide sequences, as determined by comparing the
sequences. In the
art, "identity" also means the degree of sequence relatedness between
polypeptide or
polynucleotide sequences, as the case may be, as determined by the match
between
strings of such sequences. "Identity" and "similarity" can be readily
calculated by known
1o methods, including but not limited to those described in (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 I, 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; and Carillo, H., and Lipman, D.,
SIAM J.
Applied Math., 48: 1073 (1988). Preferred methods to determine identity are
designed to
give the largest match between the sequences tested. Methods to determine
identity and
similarity are codified in publicly available computer programs. Preferred
computer
2o program methods to determine identity and similarity between two sequences
include, but
are not limited to, the GCG program package (Devereux, J., et al., Nucleic
Acids
Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al.,
J.
Molec. Biol. 21 S: 403-410 (1990). The BLAST X program is publicly available
from
NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH
Bethesda,
MD 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). The well
known Smith
Waterman algorithm may also be used to determine identity.
The preferred algorithm used is FASTA. The preferred parameters for
polypeptide or
polynuleotide sequence comparison using this algorithm include the following:
3o Gap Penalty: l2
Gap extension penalty: 4
Word size: 2, max 6
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WO 00/43511 PCT/EP00/00347
Preferred parameters for polypeptide sequence comparison with other methods
include
the following:
1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)
Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad.
Sci.
USA. 89:10915-10919 (1992)
Gap Penalty: 12
Gap Length Penalty: 4
A program useful with these parameters is publicly available as the "gap"
program from
1o Genetics Computer Group, Madison WI. The aforementioned parameters are the
default
parameters for polypeptide comparisons (along with no penalty for end gaps).
Preferred parameters for polynucleotide comparison include the following:
1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)
Comparison matrix: matches = +10, mismatch = 0
Gap Penalty: 50
Gap Length Penalty: 3
A program useful with these parameters is publicly available as the "gap"
program from
2o Genetics Computer Group, Madison WI. The aforementioned parameters are the
default
parameters for polynucleotide comparisons.
By way of example, a polynucleotide sequence of the present invention may be
identical
to the reference sequence of SEQ ID NO:l, that is be 100% identical, or it may
include
up to a certain integer number of nucleotide alterations as compared to the
reference
sequence. Such alterations are selected from the group consisting of at least
one
nucleotide deletion, substitution, including transition and transversion, or
insertion, and
wherein said alterations may occur at the 5' or 3' terminal positions of the
reference
nucleotide sequence or anywhere between those terminal positions, interspersed
either
3o individually among the nucleotides in the reference sequence or in one or
'more
contiguous groups within the reference sequence. The number of nucleotide
alterations is
determined by multiplying the total number of nucleotides in SEQ ID NO:1 by
the
26
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WO 00/43511 PCT/EP00/00347
numerical percent of the respective percent identity(divided by 100) and
subtracting that
product from said total number of nucleotides in SEQ ID NO:1, or:
nn ~ xn - (xn' Y)
wherein nn is the number of nucleotide alterations, xn is the total number of
nucleotides
in SEQ ID NO:1, and y is, for instance, 0.70 for 70%, 0.80 for 80%, 0.85 for
85%, 0.90
for 90%, 0.95 for 95%,etc., and wherein any non-integer product of xn and y is
rounded
down to the nearest integer prior to subtracting it from xn. Alterations of a
polynucleotide sequence encoding the polypeptide of SEQ ID N0:2 may create
nonsense,
missense or frameshift mutations in this coding sequence and thereby alter the
1o polypeptide encoded by the polynucleotide following such alterations.
Similarly, a polypeptide sequence of the present invention may be identical to
the
reference sequence of SEQ ID N0:2, that is be 100% identical, or it may
include up to a
certain integer number of amino acid alterations as compared to the reference
sequence
such that the % identity is less than 100%. Such alterations are selected from
the group
consisting of at least one amino acid deletion, substitution, including
conservative and
non-conservative substitution, or insertion, and wherein said alterations may
occur at the
amino- or carboxy-terminal positions of the reference polypeptide sequence or
anywhere
between those terminal positions, interspersed either individually among the
amino acids
2o in the reference sequence or in one or more contiguous groups within the
reference
sequence. The number of amino acid alterations for a given % identity is
determined by
multiplying the total number of amino acids in SEQ ID N0:2 by the numerical
percent of
the respective percent identity(divided by 100) and then subtracting that
product from
said total number of amino acids in SEQ ID N0:2, or:
naSxa - (xa ~ Y)
wherein na is the number of amino acid alterations, xa is the total number of
amino acids
in SEQ ID N0:2, and y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for
85% etc.,
and wherein any non-integer product of xa and y is rounded down to the nearest
integer
prior to subtracting it from xa.
27
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"Homolog" is a generic term used in the art to indicate a polynucleotide or
polypeptide
sequence possessing a high degree of sequence relatedness to a subject
sequence. Such
relatedness may be quntified by determining the degree of identity and/or
similarity between
the sequences being compared as hereinbefore described. Falling within this
generic term
are the terms "ortholog", meaning a polynucleotide or polypeptide that is the
functional
equivalent of a polynucleotide or polypeptide in another species and "paralog"
meaning a
functionally similar sequence when considered within the same species.
28
CA 02358899 2001-07-19
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EXAMPLES
Example 1
Real-time RT-PCR analysis
Real-time RT-PCR (LT. Gibson. 1996. Genome Research: 6,996) is used to compare
mRNA transcript abundance of the candidate antigen in tumour and normal colon
tissues
from multiple patients. In addition, mRNA levels of the candidate gene are re-
evaluated
by this approach in a panel of normal tissues.
Total RNA is extracted from snap frozen colon tissue biopsies using TriPure
reagent
(Boehringer). Total RNA from normal tissues is from InVitrogen as above. Poly-
A+
to mRNA is purified from total RNA after DNAase treatment using oligo-dT
magnetic
beads (Dynal). Quantification of the mRNA is performed by spectrofluorimetry
(BioRad)
using SybrII dye (Molecular Probes). Primers for amplification are designed
with the
Perkin-Elmer Primer Express software using default options for TaqMan
amplification
conditions.
15 Real-time reactions are assembled according to standard PCR protocols using
2 ng of
purified mRNA for each reaction. SybrI dye (Molecular Probes) is added at a
final
dilution of 1/75000 for real-time detection. Amplification (40 cycles) and
real-time
detection is performed in a PE 7700 system. Ct values are calculated using the
7700
Sequence Detector software for the tumour (CtT) and normal (CtN) samples of
each
2o patient. The difference between Ct values (CtN-CtT) is a direct measure of
the difference
in transcript levels between the tumour and normal tissues. As Ct values are
log-linearly
related to copy number and that the efficiency of PCR amplification under the
prevailing
experimental conditions is close to the theoretical amplification efficiency,
2 ~~tN-ccT> is an
estimate of the relative transcript levels in the two tissues (i.e. fold mRNA
over-
25 expression in tumor).The percentage of over-expressing patients and the
average level of
mRNA over-expression in the tumours of these patients is calculated from the
data set of
6 patients (duplicate measures).
TABLE 3:
Patients over-expressing CASB612Average level of over-expression
in colon tumours in colon tumours
(%) (fold)
5/6 13
29
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Table 4: Real-time RT-PCR Ct values for CASB612 in 6 paired colon samples
NIT NIT NIT NIT NIT NIT
experiment 1 37/32.531.5/29.530/24.527/29.533.5%2727.5/25
Ct
CtN-CtT 4.5 2 5.5 -2.5 6.5 2.5
experiment 2 32/30 30/27.527.5/23.525.5/2730.5/2626.5/23.5
Ct
CtN-CtT 2 2.5 4 -1.5 4.5 3
N: normal colon
T: colon tumor
Example 2
Identification of the full length cDNA sequence
1 o Colon tumour cDNA libraries are constructed using the Lambda Zap system
(Stratagene)
from 2 pg of polyA+ mRNA as described in the supplied protocol. 1.5 x106
independent
phage are plated for each screening of the library. Phage plaques are
transferred onto nylon
filters, hybridised using a cDNA probe labelled with AlkPhos Direct (Amersham
Pharmacia) and positive phage are detected by chemiluminescence. The positive
phage are
15 excised from the agar plat, eluted in SOOp.I SM buffer and confirmed by
gene-specific PCR.
Eluted phage are converted to single strand M13 bacteriophage by in vivo
excision. The
bacteriophage is then converted to double strand plasmid DNA by infection of
E. coli.
Infected bacteria are plated and submitted to a second round of screening with
the cDNA
probe. Plasmid DNA is purified from positive bacterial clones and submitted to
Southern
2o blot analysis to estimated the size of the cDNA inserts. CDNA inserts from
multiple
independent clones are sequenced on both strands.
When the full length gene cannot be obtained directly from the cDNA library,
missing
sequence is isolated using RACE technology (Marathon Kit, ClonTech.). This
approach
25 relies on reverse transcribing mRNA into double strand cDNA, ligating
linkers onto the ends
of the cDNA and amplifying the desired extremity of the cDNA using a gene-
specific
primer and one of the linker oligonucleotides. Marathon PCR products are
cloned into a
plasmid and sequenced.
3o Example 3:
3.1 Expression and purification of tumour-specific antigens
CA 02358899 2001-07-19
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Expression in microbial hosts is used to produce the antigen of the invention
for vaccine
purposes and to produce protein fragments or whole protein for rapid
purification and
generation of antibodies needed for characterization of the naturally
expressed protein by
immunohistochemistry or for follow-up of purification.
Recombinant proteins may be expressed in two microbial hosts, E. coli and in
yeast
(such as Saccharomyces cerevisiae or Pichia pastoris). This allows the
selection of the
expression system with the best features for this particular antigen
production. In
general, the recombinant antigen will be expressed in E. coli and the reagent
protein
expressed in yeast.
1o The expression strategy first involves the design of the primary structure
of the
recombinant antigen. In general an expression fusion partner (EFP) is placed
at the N
terminal extremity to improve levels of expression that could also include a
region useful
for modulating the immunogenic properties of the antigen, an immune fusion
partner
(IFP). In addition, an affinity fusion partner (AFP ) useful for facilitating
further
15 purification is included at the C-terminal end.
When the recombinant strains are available, the recombinant product is
characterized by
the evaluation of the level of expression and the prediction of further
solubility of the
protein by analysis of the behavior in the crude extract.
After growth on appropriate culture medium and induction of the recombinant
protein
20 expression, total extracts are analyzed by SDS-PAGE. The recombinant
proteins are
visualized in stained gels and identified by Western blot analysis using
specific
antibodies.
A comparative evaluation of the different versions of the expressed antigen
will allow the
selection of the most promising candidate that is to be used for further
purification and
25 immunological evaluation.
The purification work follows a classical approach based on the presence of an
His
affinity tail in the recombinant protein. In a typical experiment the
disrupted cells are
filtered and the acellular extracts loaded onto an Ion Metal Affinity
Chromatography
(IMAC; Ni~NTA from Qiagen) that will specifically retain the recombinant
protein.
3o The retained proteins are eluted by 0-500 mM Imidazole gradient (possibly
in presence of
a detergent) in a phosphate buffer. This step is optimally followed by an
Anion Exchange
resin step and a Size Exclusion chromatography step depending on the success
of the
Imac step and the nature of the contaminants.
31
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3.2 Antibody production and immunohistochemistry
Small amounts of relatively purified protein can be used to generate
immunological
tools in order to
a) detect the expression by immunohistochemistry in normal or cancer tissue
sections;
b) detect the expression, and to follow the protein during the purification
process
(ELISA/ Western Blot); or
c) characterise/ quantify the purified protein (ELISA).
to 3.2.1 Polyclonal antibodies:
Immuni~atinn
2- 3 Rabbits are immunized , intramuscularly (LM.) , 3 times at 3 weeks
intervals with
100ug of protein, formulated in the adjuvant 3D-MPL/QS21 . 3 weeks after each
immunisation a blood sample is taken and the antibody titer estimated in the
serum by
t5 ELISA using the protein as coating antigen following a standard protocol.
ELISA
96 well microplates (maxisorb Nunc) are coated with Sp,g of protein overnight
at 4°C.
After lhour saturation at 37°C with PBS NCS 1%, serial dilution of the
rabbit sera is
2o added for 1H 30 at 37°C (starting at 1/10). After 3 washings in PBS
Tween, anti rabbit
biotinylated anti serum (Amersham ) is added (1/5000). Plates are washed and
peroxydase coupled streptavidin (1/5000) is added for 30 min at 37°C.
After washing,
501 TMB (BioRad) is added for 7 min and the reaction then stopped with HzS04
0.2M.
The OD can be measured at 450 nm and midpoint dilutions calculated by
SoftmaxPro.
3.2.2 Monoclonal antibodies:
Immuni~atinn
5 BALB/c mice are immunized 3 times at 3 week intervals with S ~.g of purified
protein.
Bleedings are performed 14 days post II and 1 week post 3. The sera is tested
by Elisa
on purified protein used as coated antigen. Based on these results (midpoint
dilution >
10000 ) one mouse is selected for fusion
Fu.cinnl H,4 Tcnlnrtinn
32
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Spleen cells are fused with the SP2/0 myeloma according to a standard protocol
using
PEG 40% and DMSO 5%. Cells are then seeded in 96 well plates 2.5 x104 - 105
cells/well and resistant clones will be selected in HAT medium. The
supernatant of these
hybridomas will be tested for their content in specific antibodies and when
positive,
will be submitted to 2 cycles of limited dilution . After 2 rounds of
screening, 3
hybridomas will be chosen for ascitis production.
3.2.3 Immunohistochemistry
When antibodies are available, immuno staining is performed on normal or
cancer
1 o tissue sections, in order to determine
~ the level of expression of the protein antigen of the invention in cancer
relative to
normal tissue or
~ the proportion of cancers of a certain type expressing the antigen
~ if other cancer types also express the antigen
~ the proportion of cells expressing the antigen in a cancer tissue
~ the cellular localisation of the antigen
Tissue sample preparation
After dissection, the tissue sample is mounted on a cork disk in OCT compound
and
rapidly frozen in isopentane previously super cooled in liquid nitrogen (-
160°C). The
block will then be conserved at -70°C until use. 7-10~,m sections will
be realized in a
cryostat chamber (-20, -30°C).
Staining
Tissue sections are dried for 5 min at room Temperature (RT), fixed in acetone
for
l Omin at RT,dried again, and saturated with PBS 0.5% BSA 5% serum. After 30
min at
RT either a direct or indirect staining is performed using antigen specific
antibodies. A
direct staining leads to a better specificity but a less intense staining
whilst an indirect
staining leads to a more intense but less specific staining.
3.3 Analysis of human cellular immune responses to the antigen of the
invention
The immunological relevance of the antigen of the invention can be assessed by
in vitro
33
CA 02358899 2001-07-19
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priming of human T cells. All T cell lymphocyte lines and dendritic cells are
derived
from PBMCs (peripheral blood mononuclear cells) of healthy donors (preferred
HLA-A2
subtype). An HLA-A2.1lKb transgenic mice is also used for screening of HLA-
A2.1
peptides.
Newly discovered antigen-specific CD8+ T cell lines are raised and maintained
by
weekly in vitro stimulation. The lytic activity and the y-IFN production of
the CD8 lines
in response to the antigen or antigen derived-peptides is tested using
standard procedures.
to Two strategies to raise the CD8+ T cell lines are used: a peptide-based
approach and a
whole gene-based approach. Both approaches require the full-length cDNA of the
newly
discovered antigen in the correct reading frame to be either cloned in an
appropriate
delivery system or to be used to predict the sequence of HLA binding peptides.
15 Peptide-based approach
The HLA-A2 binding peptide sequences are predicted by the Parker's algorithm.
Peptides
are then screened in the HLA-A2.1/Kb transgenic mice model (Vitiello et al.).
Briefly,
transgenic mice are immunized with adjuvanted HLA-A2 peptides, those unable to
induce a CD8 response (as defined by an efficient lysis of peptide-pulsed
autologous
2o spleen cells) will be further analyzed in the human system.
Human dendritic cells (cultured according to Romani et al.) will be pulsed
with peptides
and used to stimulated CD8-sorted T cells (by Facs). After several weekly
stimulations,
the CD8 lines will be first tested on peptide-pulsed autologous BLCL (EBV-B
transformed cell lines). To verify the proper in vivo processing of the
peptide, the CD8
25 lines will be tested on cDNA-transfected tumour cells (HLA-A2 transfected
LnCaP,
Skov3 or CAMA tumour cells).
Whole gene-based approach
CD8+ T cell lines will be primed and stimulated with either gene-gun
transfected
3o dendritic cells, retrovirally transduced B7.1-transfected fibroblastes,
recombinant pox
virus (Kim et al.) or adenovirus (Butterfield et al.) infected dentridic
cells. Virus infected
cells are very efficient to present antigenic peptides since the antigen is
expressed at high
level but can only be used once to avoid the over-growth of viral T cells
lines.
34
CA 02358899 2001-07-19
WO 00/43511 PCT/EP00/00347
After alternated stimulations, the CD8 lines are tested on cDNA-transfected
tumour cells
as indicated above. Peptide specificity and identity is determined to confirm
the
immunological validation.
References
Vitiello et al. (L. Sherman), J. Exp. Med., J. Exp. Med, 1991, 173:1007-1015.
Romani et al., J. Exp. Med., 1994, 180:83-93.
Kim et al., J. Immunother., 1997, 20:276-286.
1o Butterfield et al., J. Immunol., 1998, 161:5607-5613.
All publications, including but not limited to patents and patent
applications, cited in this
specification are herein incorporated by reference as if each individual
publication were
specifically and individually indicated to be incorporated by reference herein
as though
~ s fully set forth.
CA 02358899 2001-07-19
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SEQUENCE INFORMATION
SEQ ID NO:1
GAACCCCGCCGCGCGCGCTTTGAATTTCCCAGCCGCTAAGCGCCTCTCCTGGTCGGTGTCCCAGGCAGAAAGGTGCAGA
G
ACCCATCTGACGCAGGACTCCAGGTAGACAAGCTCCAGCAGAGAGTGGCCAGatgtggtgcctggagcgactccgcttg
g
gtcctgagtgccttcggcggagcggagactggcttctcccgggtcgggcccgcggagccaagtctcgcaccaccgccgc
g
tgcgcaaatgtgctcactccggaccgcatccctgagttctgcatcccgccacggctcatgccccgcctggccttggctg
c
gctccggaattcttgggtcgaagaagcagggatggacgagggcgccggccgcacagactgggacccgcgctcgcaggcc
g
cgctgtcactgccgcacctgccccgtgtgcgcaccgcctacggcttctgcgcgctgctcgagagcccgcacacgcgccg
c
aaggagtcgctcctgctcgggggcccgcccgcgccccggccccgggcccacacctacggcggcggcggcggcccggacg
c
ccycctggggaccctgygcgkcccgcgaggtccgggcccggccacccccgcggcccccggcggtccccgcctgccccag
g
acgcgctcgctgcggggccccgccgctgccgcctcctgcgcgtccccgacgggctgctgagtcgcgcgctgcgggctgg
a
aggagtcgccgcctggcccgcgtccgctccgtctccagcgggaacgaggacgaggagcgccgcgcgggatccgagtccc
c
ggcccgggccccctcctcgagcccgctgtcatccagggccccgcttcctgagcgcctggaggccaagggcaccgtggct
c
tgggccgcgccggcgacgccctgcgcctggctgctgagtactgtccgggaacccggcgtctccgcctccggctgctccg
c
gcggagagcctggtcggaggcgcccccgggccccgcgccgtccgctgccgcctcagcctcgtcctgcggccgccgggca
c
ygcgcktyggcaatgcagcrctgtggtggggcgcagccgcaaggcctcctttgaccaggacttttgcttcgacggcctc
t
cggaagacgaggtgcgccgcctggccgttcgcgtcaaggcccgggatgagggtcgcggccgggatcggggccgcctgct
g
ggccagggtgagctgtccctgggcgccctcctgctgctctgaGGGCCCAGCCCTCCCCGGGGCGCTCTGCCCTGGAGAC
T
CCGGACACTGACAGCCGCGTGGTACAGAATAAACGTTATTTATTTTTTTATTTATGATACTGCTTTATTGACGTTTTAC
T
TCCTCACCATCCACATTTGTCATCGTCTRTTGGTAAAGAAGAAAAAGATAATGACTCCCTGTTCTGTTCACAGGACCCC
C
ATATCTCTTTCCAGACCATTTTTGCATTCCAAGGAAAAATGGGTTGGCTTGGGACTGGGAGAGAAAGGAAGTACACCCA
T
CTGCATTGTTT
SEQ ID N0:2
MWCLERLRLGPECLRRSGDWLLPGRARGAKSRTTAACANVLTPDRIPEFCIPPRLMPRLALAALRNSWVEEAGMDEGAG
R
3O
TDWDPRSQAALSLPHLPRVRTAYGFCALLESPHTRRKESLLLGGPPAPRPRAHTYGGGGGPDAXLGTLXXPRGPGPATP
A
APGGPRLPQDALAAGPRRCRLLRVPDGLLSRALRAGRSRRLARVRSVSSGNEDEERRAGSESPARAPSSSPLSSRAPLP
E
RLEAKGTVALGRAGDALRLAAEYCPGTRRLRLRLLRAESLVGGAPGPRAVRCRLSLVLRPPGTAXXQCSXVVGRSRKAS
F
SEQ ID N0:3
GCGGGGCCCCGCCGCTGCCGCATCCTGCGCGTCCCCGACGGGCTGCTGAGTCGCGCGCTGCGGGCTGGAAGGAGTCGCC
G
CCTGGCCCGCGTCCGCTCCGTCTCCAGCGGGAACGAGGACGAGGAGCGCCGCGCGGGATCCGAGTCCCCGGCCCGGGCC
C
CCTCCTCGAGCCCGCTGTCATCCAGGGCCCCGCTTCCTGAGCGCCTGGAGGCCAAGGGCACCGTGGCTCTGGGCCGCGC
C
GGCGACGCCCTGCGCCTGGCTGCTGAGTACTGTCCGGGAACCCGGCGTCTCCGCCTCCGGCTGCTCCGCGCGGAGAGCC
T
GGTCGGAGGCGCCCCCGGGCCCCGCGCAGTCCGCTGCCGCCTCA
SEQ ID N0:4
AGPRRCRILRVPDGLLSRALRAGRSRRLARVRSVSSGNEDEERRAGSESPARAPSSSPLSSRAPLPERLEAKGTVALGR
A
GDALRLAAEYCPGTRRLRLRLLRAESLVGGAPGPRAVRCRL
36
CA 02358899 2001-07-19
WO 00/43511 PCT/EP00/00347
SEQUENCE LISTING
<110> SmithKline Beecham Biologicals S.A.
<120> Novel Compounds
<130> BC45216
<160> 4
<170> FastSEQ for Windows Version 3.0
<210> 1
<211> 1531
<212> DNA
<213> homo sapiens
<400>
1
gaaccccgccgcgcgcgctttgaatttcccagccgctaagcgcctctcctggtcggtgtc60
ccaggcagaaaggtgcagagacccatctgacgcaggactccaggtagacaagctccagca120
gagagtggccagatgtggtgcctggagcgactccgcttgggtcctgagtgccttcggcgg180
agcggagactggcttctcccgggtcgggcccgcggagccaagtctcgcaccaccgccgcg240
tgcgcaaatgtgctcactccggaccgcatccctgagttctgcatcccgccacggctcatg300
ccccgcctggccttggctgcgctccggaattcttgggtcgaagaagcagggatggacgag360
ggcgccggccgcacagactgggacccgcgctcgcaggccgcgctgtcactgccgcacctg420
ccccgtgtgcgcaccgcctacggcttctgcgcgctgctcgagagcccgcacacgcgccgc480
aaggagtcgctcctgctcgggggcccgcccgcgccccggccccgggcccacacctacggc540
ggcggcggcggcccggacgcccycctggggaccctgygcgkcccgcgaggtccgggcccg600
gccacccccgcggcccccggcggtccccgcctgccccaggacgcgctcgctgcggggccc660
cgccgctgccgcctcctgcgcgtccccgacgggctgctgagtcgcgcgctgcgggctgga720
aggagtcgccgcctggcccgcgtccgctccgtctccagcgggaacgaggacgaggagcgc780
cgcgcgggatccgagtccccggcccgggccccctcctcgagcccgctgtcatccagggcc840
ccgcttcctgagcgcctggaggccaagggcaccgtggctctgggccgcgccggcgacgcc900
ctgcgcctggctgctgagtactgtccgggaacccggcgtctccgcctccggctgctccgc960
gcggagagcctggtcggaggcgcccccgggccccgcgccgtccgctgccgcctcagcctc1020
gtcctgcggccgccgggcacygcgcktyggcaatgcagcrctgtggtggggcgcagccgc1080
aaggcctcctttgaccaggacttttgcttcgacggcctctcggaagacgaggtgcgccgc1140
ctggccgttcgcgtcaaggcccgggatgagggtcgcggccgggatcggggccgcctgctg1200
ggccagggtgagctgtccctgggcgccctcctgctgctctgagggcccagccctccccgg1260
ggcgctctgccctggagactccggacactgacagccgcgtggtacagaataaacgttatt1320
tatttttttatttatgatactgctttattgacgttttacttcctcaccatccacatttgt1380
catcgtctrttggtaaagaagaaaaagataatgactccctgttctgttcacaggaccccc1440
atatctctttccagaccatttttgcattccaaggaaaaatgggttggcttgggactggga1500
gagaaaggaagtacacccatctgcattgttt 1531
<210> 2
<211> 320
<212> PRT
<213> homo sapiens
<400> 2
Met Trp Cys Leu Glu Arg Leu Arg Leu Gly Pro Glu Cys Leu Arg Arg
$5 1 5 10 15
Ser Gly Asp Trp Leu Leu Pro Gly Arg Ala Arg Gly Ala Lys Ser Arg
20 25 30
Thr Thr Ala Ala Cys Ala Asn Val Leu Thr Pro Asp Arg Ile Pro Glu
35 40 45
Phe Cys Ile Pro Pro Arg Leu Met Pro Arg Leu Ala Leu Ala Ala Leu
50 55 60
Arg Asn Ser Trp Val Glu Glu Ala Gly Met Asp Glu Gly Ala Gly Arg
CA 02358899 2001-07-19
WO 00/43511 PCT/EP00/00347
65 70 75 80
Thr Asp Asp ArgSerGln AlaAla LeuSerLeuProHisLeu
Trp Pro
85 90 95
Pro Arg Arg AlaTyrGly PheCys AlaLeuLeuGluSerPro
Val Thr
100 105 110
His Thr Arg GluSerLeu LeuLeu GlyGlyProProAlaPro
Arg Lys
115 120 125
Arg Pro Ala ThrTyrGly GlyGly GlyGlyProAspAlaXaa
Arg His
130 135 140
Leu Gly Leu XaaProArg GlyPro GlyProAlaThrProAla
Thr Xaa
145 150 155 160
Ala Pro Gly ArgLeuPro GlnAsp AlaLeuAlaAlaGlyPro
Gly Pro
165 170 175
Arg Arg Arg LeuArgVal ProAsp GlyLeuLeuSerArgAla
Cys Leu
180 185 190
Leu Arg Gly SerArgArg LeuAla ArgValArgSerValSer
Ala Arg
195 200 205
Ser Gly Glu GluGluArg ArgAla GlySerGluSerProAla
Asn Asp
210 215 220
Arg Ala Ser SerProLeu SerSer ArgAlaProLeuProGlu
Pro Ser
225 230 235 240
Arg Leu Ala GlyThrVal AlaLeu GlyArgAlaGlyAspAla
Glu Lys
245 250 255
Leu Arg Ala GluTyrCys ProGly ThrArgArgLeuArgLeu
Leu Ala
260 265 270
Arg Leu Arg GluSerLeu ValGly GlyAlaProGlyProArg
Leu Ala
275 280 285
Ala Val Cys LeuSerLeu ValLeu ArgProProGlyThrAla
Arg Arg
290 295 300
Xaa Xaa Cys XaaValVal GlyArg SerArgLysAlaSerPhe
Gln Ser
305 310 315 320
<210> 3
<211> 364
<212> DNA
<213> homo
sapiens
<400> 3
gcggggcccc gtccccgacg ggctgctgag 60
gccgctgccg tcgcgcgctg
catcctgcgc
cgggctggaa gtccgctccg tctccagcgg 120
ggagtcgccg gaacgaggac
cctggcccgc
gaggagcgcc gcccgggccc cctcctcgag 180
gcgcgggatc cccgctgtca
cgagtccccg
tccagggccc gccaagggca ccgtggctct 240
cgcttcctga gggccgcgcc
gcgcctggag
ggcgacgccc tgtccgggaa cccggcgtct 300
tgcgcctggc ccgcctccgg
tgctgagtac
ctgctccgcg gcccccgggc cccgcgcagt 360
cggagagcct ccgctgccgc
ggtcggaggc
ctca 364
<210> 4
<211> 121
<212> PRT
<213> homo
sapiens
<400> 4
Ala Gly Arg CysArgIle LeuArg ValProAspGlyLeuLeu
Pro Arg
1 5 10 15
Ser Arg Leu AlaGlyArg SerArg ArgLeuAlaArgValArg
Ala Arg
20 25 30
Ser Val Ser AsnGluAsp Glu ArgArgAlaGlySerGlu
Ser Gly Glu
35 40 45
Ser Pro Arg ProSerSer Pro LeuSerSerArgAlaPro
Ala Ala Ser
50 55 60
Leu Pro Arg GluAlaLys Thr ValAlaLeuGlyArgAla
Glu Leu Gly
70 75 80
2
