Note: Descriptions are shown in the official language in which they were submitted.
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579 -
1
TITLE
"MUCINS"
f=IELD OF THE INVENTION
THIS INVENTION relates generally to nucleic acids
corresponding to mammalian Mucin genes, and to polypeptides encoded
thereby. More particularly, the present invention provides isolated nucleic
acids which correspond to Mucin regulatory genes that are predominantly
expressed in the colon. These Mucin genes are associated with disease
conditions including colorectal cancer, breast cancer, cystic fibrosis,
respiratory diseases, inflammatory bowel disease, ulcerative colitis and
Crohn's disease and/or any other conditions associated with aberrant Mucin
expression, altered properties of mucus or epithelial inflammatory processes
involving Mucins. In particular, the present invention provides methods for
the diagnosis and therapy of the abovementioned disease conditions.
BACN;GROUND OF THE INVENTION
The increasing sophistication of recombinant DNA technology
is greatly facilitating research and development in the medical and allied
health fields. This is particularly the case in cancer research. However,
despite the effectiveness of this powerful technology, progress has been
slow in developing effective recombinant DNA-derived therapeutic or
diagnostic agents for cancers. One difficulty has been a lack of
understanding of many cancers and other disease conditions. Regulatory
genes are an imporlrant component of these complex regulatory
mechanisms.
Cancer suppressor genes, for example, are regulators of cell
growth and differentiation (Weinberg et al., 1995, Ann. NY Acad. Sci. 758
331 ). The paradigm for their role in cancer is that they are traps-acting and
recessive at the cellular level; loss of one homologue has no effect on cell
function and homozygous inactivation is required for carcinogenesis
(Cavenee et al., 1983, Nature 305 779).
Colorectal cancers contribute to a major proportion of the
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
2
mortality and morbidity associated with cancer development. There is a
particular need, therefore, to understand the complex regulatory
mechanisms associated with colorectal cancers as well as cancers in
anatomically adjacent regions.
The epithelial mucins are a family of secreted and cell surface
glycoproteins expressed by epithelial tissues. They are characterised by a
central polymorphic tandem repeat structure, which comprises most of the
protein backbone, and .a large number of O-linked carbohydrate side chains
(Gum et al., 1995, Biochem. Soc. Traps. 23 795). The complex structure and
large size of these molecules makes it difficult to characterise them using
classical biochemical techniques. The genes are also difficult to clone
because of their large size and the presence of GC-rich tandem repeats.
Ten mucin genes havE: been identified; MUC3, MUC4, MUCSAC, MUCSB,
MUC6 and MUC8 have been partially cloned and full-length cDNA clones
are available for MUC1, MUC2, MUC7 and MUC9.
Mucins .are known to contribute to pathology in a number of
epithelial diseases including cystic fibrosis (CF), inflammatory bowel disease
(IBD) and adenocarcinomas. Gastrointestinal mucins which have been
described to date include: the transmembrane mucins MUC1 and MUC4; the
gel-forming mucins MUC2, MUCSAC and MUC6; and MUC3 which has an
unclear structure and function.
As used herein, Mucin genes or isolated nucleic acids
corresponding thereto will be expressed in italicized form as MUC. Mucin
polypeptides will be expressed as MUC.
Immunohistochemical staining and Western blotting analysis
with mature MUC1-specific antibodies revealed that MUC1 became
ectopically expressed in colorectal tumours and levels were significantly
higher in primary tumours of patients with metastases. Experimentally
increased expression of gel-forming mucins resulted in increased metastasis
in colon cancer cells in xenograft metastasis models (Ho et al., 1995, Int. J.
Oncol. 7 913). Northern blot analysis has been employed to investigate
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
3
expression of MUC9, MUC2, MUC3 and MUC4 in paired normal and colonic
tumour tissues and in nine colorectal cancer (CRC) cell lines (Ogata et al.,
1992, Cancer Res. 52 5971 ). MUCH and MUC4 were present in colonic
mucosa with similar expression levels in carcinomas, but occasionally
elevated levels of MUC4 were apparent. Levels of MUC2 and MUC3 were
decreased by varying degrees in the tumours of most patients. There was
no apparent correlation between the expression of any mucin gene and the
site, stage or histological type of tumour. All four mucin genes were
expressed at low levels or not at all in the nine CRC cell lines under
investigation; MUCH transcripts were detected in COL0205, MUC2 and
MUC4 probes hybridised weakly to all nine cell lines, and MUC3 expression
was observed in five of the lines. Using a combination of in situ
hybridisation
and immunohistochemistry, Chang et a! (Chang et al., 1994,
Gastroenterology 107 ~'.8) also found MUC2 and MUC3 were downregulated
in CRC. A more recent in situ hybridisation study found expression of MUC2
and MUC3 mRNA was markedly reduced in poorly, moderately and well-
differentiated colorectal tumours but preserved in mucinous carcinomas
(Weiss et ai., 1996, ,J. Histochem. Cytochem. 44 1161 ). It is noted that
MUC3 is located on human chromosome 7q22, or an equivalent location on
other mammalian chromosomes, and is primarily expressed under normal
conditions in the small intestine (Shekels et al., 1998, Biochem J. 330 1301
).
OBJECT OF THE INVENTION
The present inventors have realized that the Mucins constitute
an incomplete family of genes and gene products implicated in a variety of
disease conditions. Surprisingly, the present inventors have identified novel
Mucin genes located on human chromosome 7q22, and isolated novel
nucleic acids corresponding thereto. Furthermore, the present inventors
have found that these; novel Mucin genes are predominantly expressed in
the colon, and may be involved in cancer of the large bowel, cystic fibrosis,
breast cancer, inflammatory bowel disease, ulcerative colitis respiratory
diseases and Crohn's disease and/or any ather conditions associated with
CA 02333853 2001-O1-16
WO 00/04142 PCT/AlJ99/00579
4
aberrant Mucin expression, altered properties of mucus or epithelial
inflammatory processes involving Mucins.
It is therefore an object of the invention to provide novel Mucin
genes and isolated nucleic acids corresponding thereto.
SUMMARY OF THE INVENTION
The present invention is broadly directed to an isolated MUC
nucleic acid which corresponds to a MUC gene located on mammalian
chromosome 7q22, or on a mammalian chromosome structurally or
functionally equivalent thereto, which MUC gene is normally predominantly
expressed in the colon.
In a first aspect, the MUC gene of the present invention is
MUC11.. Accordingly, "a MUC11 nucleic acid" means an isolated nucleic
acid of the invention which corresponds to the MUC11 gene.
PreferabVy, the isolated MUC11 nucleic acid comprises a
nucleotide sequence Encoding an amino acid sequence which comprises
SGLSEESTTSHSSPGSTHTTLSPASTTT (SEQ ID NO: 1 ).
More preferably, the isolated MUC11 nucleic acid comprises
a nucleotide sequence encoding the amino acid sequence according to SEQ
ID N0:3.
Even more preferably, the isolated MUC11 nucleic acid
comprises a nucleotide sequence according to SEQ ID NO: 2.
In a second aspect, the MUC gene of the present invention is
MUC12. Accordingly, ".a MUC12 nucleic acid" means an isolated nucleic acid
of the invention which corresponds to the MUC12 gene.
Preferat~ly, the isolated MUC12 nucleic acid comprises a
nucleotide sequence encoding an amino acid sequence which comprises
SGLSQESTTFHSSPGSTETTLAPASTTT (SEQ ID N0: 4).
More preferably, the isolated MUC12 nucleic acid comprises
a nucleotide sequence encoding the amino acid sequence according to SEQ
ID N0:6.
Even more preferably, the isolated MUC12 nucleic acid
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
comprises a nucleotide sequence according to SEQ ID NO: 5.
In a third aspect, the present invention resides in an isolated
MUC polypeptide.
In one embodiment, the isolated MUC polypeptide has an
5 amino acid sequence according to SEQ 1D NO: 3, hereinafter referred to as
a "MUC11 polypeptide".
In another embodiment, the isolated MUC polypeptide has an
amino acid sequence according to SEQ ID N0:6, hereinafter referred to as
a "MUC12 polypeptide".
In a fourtlh aspect, the present invention resides in an antibody
specific for a MUC poiypeptide (hereinafter referred to as an anti-MUC
antibody).
Preferably, the anti-MUC antibody is selected from the group
consisting of:-
(i) an anti-MUC11 IgM monoclonal antibody hereinafter
referred to as M11.9; and
(ii) an anti-MUC12 IgM monoclonal antibody hereinafter
referred to as M12.15.
In a fifth aspect, the present invention resides in methods of
detecting a MUC gene, a MUC gene transcript or a MUC polypeptide. The
fifth aspect extends to methods for detecting a polymorphism, deletion,
mutation, truncation or expansion in a MUC gene, a MUC gene transcript or
a MUC polypeptide, or detecting a level of expression thereof. One
embodiment of the fifth aspect is directed to use of an isolated MUC nucleic
acid to determine whether a mammal has a disease condition, or a
predisposition thereto. Another embodiment is directed to use of an isolated
MUC polypeptide to determine whether a mammal has a disease condition,
or a predisposition thE:reto.
In a sixth aspect, the present invention provides a method of
gene therapy of a disease condition in a mammal, said method including
administering to said mammal a gene therapy construct which includes an
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
6
isolated MUC nucleic acid as hereinbefore defined, to thereby alleviate one
or more symptoms of ;>aid disease condition in said mammal.
fn a seventh aspect, the present invention provides a method
of treating a disease condition in a mammal, said method comprising the
step of administering to said mammal a pharmaceutically effective amount
of a MUC polypeptide or an anti-MUC antibody.
In an eigth aspect, the present invention resides in a
pharmaceutical composition comprising a MUC polypeptide or anti-MUC
antibody, together with a pharmaceutically acceptable carrier andlor diluent.
Preferably, the mammal is a human.
As used herein, the "disease condition" is associated with
aberrant Mucin expression, altered properties of mucus or epithelial
inflammatory processes involving Mucins.
Preferably, the disease condition is selected from the group
consisting of colorectal cancer (CRC), cystic fibrosis (CF), inflammatory
bowel disease (IBD), breast cancer (BC), Crohn's disease, ulcerative colitis,
asthma and chronic bronchitis.
More preferably, the disease condition is selected from the
group consisting of colorectal cancer (CRC), cystic fibrosis (CF),
inflammatory bowel disease (IBD) and breast cancer (BC).
As used herein, unless the context requires otherwise, the
word "comprise", or variations such as "comprises" or "comprising", will be
understood to imply the inclusion of a stated element or integer or group of
elements or integers but not the exclusion of any other element or integer or
group of elements or integers.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1(A): Autoradiograph of a differential display gel showing amplified
products from RNA isolated from matched normal colon (N)
and primary colorectal tumor (P) tissues. Differentially
expressed bands dd29 (MUC12) and dd34 (MUC11) are
arrowedl.
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
7
FIG. 1(B): Northern blot analysis of total RNA from patient 101 hybridized
with the dd29 probe to detect a MUC12 gene transcript
(mRNA). Signal corresponding to 18S ribosomal RNA is shown
as a loading control.
FIG. 1{C): Northern blot analysis of RNA from patient 112 hybridized with
the dd34 probe to detect a MUC11 gene transcript (mRNA) .
Signal corresponding to 18S ribosomal RNA is shown as a
loading control.
FIG. 1(D): Multiplex semi-quantitative RT-PCR showing amplification of
MUC12 mRNA transcripts from matched normal colonic
mucosa and primary tumor # 40, normal mucosa from patient
# 81 and six colorectal cancer cell lines. Amplification of ~2
microglok~ulin ((i2 MG) is included as a measure of total RNA.
FIG. 1(E): Multiplex semi-quantitative RT-PCR showing amplification of
MUC11 nnRNA transcripts in matched normal colonic mucosa
and primary tumors of patients # 40, 164, and 97 and six
colorectal cancer cell lines. Amplification of (i2-microglobulin
((3z-MG) its included as a measure of total RNA.
FIG. 1 (F): Multiplex: semi-quantitative RT-PCR showing amplification of
MUC12 mRNA transcripts from matched normal colonic
mucosa and primary tumors # 346, 84, 128, 97 and 316 and
from five unpaired Dukes' stage D tumors (M) # 93, 361, 107,
357 andl 367. Amplification of (32-microglobulin (~ -~VIG) is
included as a measure of total RNA.
FIG. 1(G): Multiplex semi-quantitative RT-PCR showing MUC11 mRNA
transcripts in matched normal colonic mucosa and primary
tumors of patients # 110, 346, 84, 128, and 348 and from five
unpaired Dukes' stage D tumors (M) # 93, 107, 361, 367 and
357. Amplification of pz-microglobulin (biz MG) is included as
a measure of total RNA. Ma denotes molecular size markers
in FIG 1 D-G.
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
FIG. 2: Predicted amino acid sequence of MUC92. Numbering of
amino acids is given on the right. The consensus sequence of
the degenerate tandem repeat structure is shown at the top.
The two cysteine-rich EGF-like domains are double
underlined, a potential coiled-coil domain is in bold, the
hydrophobic domain singly underlined and potential N-
glycosylation sites shaded. The stop codon is denoted by an
asterisk.
F1G. 3: Amino acid sequence alignment of the carboxyl termini of
MUC12, hMUC3 (amino acids 1-366), mMuc3 (Shekels et al.,
1998, supra; amino acids 637-1015), rMuc3 (Gum et al., 1991,
supra; K.hatri et al., 1997, Biochem. Biophys. Acta 1326 7;
amino acids 356-447 and 1-379 respectively), hMUC4
(Moniaux et al., 1998, Biochem. J. 338 1998; amino acids 861-
1156) and rMuc4 (Sheng et al., 1992, J. Biol. Chem. 267
16341; amino acids 451-744). Light shading demonstrates
identity with MUC12 and dark shading highlights all cysteine
residues. Hyphens indicate gaps inserted to optimize the
alignment.
FlG.4: Predicted amino acid sequence of MUC11 showing the
degenerate tandem repeat structure. The consensus
sequenG~ is shown at the top and amino acids not consistent
with this aequence are shown in bold. Hyphens indicate gaps
placed in order to optimize the amino acid alignment. A
potential N-glycosylation site is shaded.
FIG. 5: mRNA tissue distribution of the 7q22 mucin gene family. Only
those tissues showing a positive signal by Northern blot
analysis are represented in the histogram. Sixteen tissues of
neural origin, heart, aorta, skeletal muscle, bladder, stomach,
testis, ovary, spleen, pituitary gland, adrenal gland, thyroid
gland, salivary gland and mammary gland were negative for
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
9
mucin nnRNA expression. Expression was quantified by
densitometry and is shown as a proportion of the tissue showing
highest expression.
FIG.6: Domain organization of the C-termini of human MUC12,
hMUC3, the rodent Muc3 mucins and the rat and human MUC4
mucins. l'he relative size of domains is accurate except that the
N-glycasylated domain adjacent to the mucin domain in MUC4
is shown at approximately one fifth of its actual size. Only the
beginning of the large mucin domains are shown.
FIG. 7: Alignment of the first extracellular EGF-like domain of MUC12
with human EGF-like growth factors. Dark shading highlights
identical amino acids and light shading indicates conservative
amino acid substitutions.
FIG. 8: Schematic representation of MUC 11 cloning (A) and MUC 12
cloning (B).
FIG. 9: Normal colonic expression patterns of MUC11 (A, B) and
MUC12 (C) poiypeptides as determined by anti-MUC mAb
M11.9 and M12.15 immunostaining, respectively. (D) shows
MUC 11 gene transcript (mRNA) expression detected by in sifu
hybridization in normal colonic epithelium and loss of
expression in CRC (top right).
FIG. 10: Expression of MUC11 and MUC12 mRNA in normal colon as
detectedl by RT-PCR. Cytokeratin 20, (CIC20) a colonic
epithelia) marker, was employed as a loading control. 'RC'
denotes right colon, 'TC' the transverse colon, 'LC' the left
colon, ';iC' sigmoid colon; 'CA' refers to the caecum and 'R'
denotes the rectum.
FIG.11: Expression of MUC11 and MUC12 mRNA in CRC cell lines as
detected by RT-PCR. The loading control is (iZ microglobulin
(B2MG) and 'M' denotes the molecular weight marker.
FIG. 12: Expression of MUC11 and MUC12 mRNA in IBD as detected
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
by RT-PC;R. Cytokeratin 20 (CK20) a colonic epithelial marker,
was empNoyed as a loading control. 'N' denotes tissues which
appear macroscopically normal and 'D' refers to tissues
reported t.o have IBD. 'CA' refers to the caecum, 'CO' the colon,
5 'LC' the 4eft colon, 'TC' the transverse colon, 'RS' the recto-
sigmoid colon, 'Sf the small intestine, 'IL'denotes the ileum and
'IP' an ileal pouch.
F1G. 13: Expression of MUC11 and MUC12 mRNA in BC as detected by
RT-PCR. The loading control is (i2 microglobulin denoted by
10 B2MG and the molecular weight marker is denoted by 'M'. The
positive control was normal colonic cDNA from patient 164.
FIG 14: Northern blot analysis of MUC11 expression in normal colon
(N) and primary CRC (P) of six patients, assessed using a
probe corresponding to dd34. The position of ribosomal RNAs
are indicated, and signal from 18S ribosomal RNA was used as
a loading control.
DETAILED DESCF:IPTION OF THE PREFERRED EMBODIMENTS
The present invention is predicated in part on the identification
of novel MUC11 and MUC12 genes which are normally predominantly
expressed in the colon. The isolated MUC nucleic acids and MUC genes of
the invention may be useful in treatment and diagnosis of disease conditions
associated with aberrant Mucin expression, altered properties of mucus or
epithelial inflammatory processes involving Mucins. Such disease conditions
include but are not limited to cancer of the large bowel (CRC), cystic
fibrosis
{CF), inflammatory bowel disease (IBD), respiratory diseases such as asthma
and chronic bronchitis>, breast cancer (BC), ulcerative colitis and Crohn's
disease.
The present invention is particularly directed to cancers of the
large bowel, which includes the colon, rectum and anal canal, such as CRC,
although it extends t.o biochemically, physiologically andlor genetically
related cancers in other parts of the gastrointestinal tract.
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
11
The MUC; genes are, for example, down-regulated in CRC.
By "predominantly expressed" is meant that a MUC gene
transcript or MUC polypeptide encoded by said MUC gene is expressed in
the colon at a level greater than in any other organ.
By "associated with" is meant that the disease condition
displays symptoms consistent with aberrant Mucin expression, altered
properties of mucus or Epithelial inflammation involving Mucins. The disease
association may be merely correlative or may reflect a causative role of
Mucins in the disease condition.
The term "cancer" is used in its broadest sense to include
malignant tumours, carcinomas and sarcomas.
In light of the foregoing, it will be appreciated that a MUC
nucleic acid "corresponds to" a MUC gene by being an isolated nucleic acid
derived from said MUC gene, or a portion thereof. Thus it will be understood
that said gene has components including amino acid coding sequences and
non-coding sequences. Non-coding sequences include, for example, introns
and regulatory sequences which include a promoter, translation initiation and
termination sequences and a polyadenylation sequence, for example. The
isolated MUC nucleic acid may therefore correspond to some or all of the
aforementioned components of the corresponding MUC gene.
It should be noted that MUC terminology has recently
undergone revision. In particular, MUC12 was formerly known as dd 29 or
MUC10. Also, MUC11 was formerly known as dd 34. Therefore, with this in
mind, should the term "MUC10" or "dd29" be encountered herein, it should
in all cases be taken to mean MUC12.
It will also be understood that a MUC polypeptide is encoded
by an isolated MUC nucleic acid or by a MUC gene as hereinbefore defined.
Isolated MUC nucleic acids of the invention may be in DNA
(e.g. cDNA or genomic DNA), RNA {e.g. mRNA) or hybrid DNA:RNA form,
eithre in double-stranded or single-stranded form. For example, single-
stranded MUC nucleic acids include nucleic acids having sequences
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
12
complementary to the nucleotide sequences of SEQ ID N0:2 and SEQ ID
N0:5.
In one embodiment, the isolated MUC nucleic acid of the
invention comprises a nucleotide sequence having at least 60% identity to
the nucleotide sequence according to SEQ 1D N0:2, or a nucleotide
sequence capable of hybridizing thereto under at least low stringency
conditions.
In another embodiment, the isolated MUC nucleic acid of the
invention comprises a nucleotide sequence having at least 60% identity to
the nucleotide sequence according to SEQ ID N0:5, or a nucleotide
sequence capable of hybridizing thereto under at least low stringency
conditions.
According to these embodiments, it is preferable that the
nucleotide sequence has at least 75% identity.
More preferably, the nucleotide sequence has at least 90%
sequence identity.
The terra "identity" is used herein in its broadest sense to
include the number of exact nucleotide or amino acid matches having regard
to an appropriate alignment using a standard algorithm, such as but not
limited to the Geneworl~s program (Intelligenetics). For this purpose, BLAST
family programs may also be useful (Altschul et al., 1997, Nucl. Acids Res.
3389, which is herein incorporated by reference). A detailed discussion
of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS
IN MOLECULAR BIOLOGY, Eds Ausubel et al., (John Wiley & Sons), which
25 is herein incorporated by reference.
According to these embodiments, it is preferable that the
nucleotide sequence is capable of hybridizing under medium stringency
conditions.
More preferably, the nucleotide sequence is capable of
hybridizing under high stringency conditions
Reference herein to low stringency conditions includes and
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
13
encompasses from at least about 1 % v/v to at least about 15% vlv formamide
and from at least about 1 M to at least about 2 M salt for hybridisation at
42°C, and at least about 1 M to at least about 2 M salt for washing at
42°C.
Low stringency conditions also include 1 % Bovine Serum
Albumin (BSA), 1 mM EDTA, 0.5 M NaHP04 (pH 7.2), 7% SDS for
hybridization at 65°C, and (i) 2xSSC, 0.1 % SDS; or (ii) 0.5% BSA, 1 mM
EDTA, 40 mM NaHP04 (pH 7.2), 5% SDS for washing at room temperature.
Medium stringency conditions include and encompass from at
least about 16% v/v to at least about 30% v/v formamide and from at least
about 0.5 M to at least about 0.9 M salt for hybridisation at 42°C, and
at least
about 0.5 M to at least about 0.9 M salt for washing at 42°C.
Medium stringency conditions also include 1 % Bovine Serum
Albumin (BSA), 1 mM EDTA, 0.5 M NaHP04 (pH 7.2), 7% SDS for
hybridization at 65°C, and (i) 2 x SSC, 0.1 % SDS; or (ii) 0.5% BSA, 1
mM
EDTA, 40 mM NaHP0,4 (pH 7.2), 5% SDS for washing at 42°C.
High stringency includes and encompasses from at least about
31 % vlv to at least about 50% v/v formamide and from at least about 0.01 M
to at least about 0.15 M salt for hybridisation at 42°C, and at least
about 0.01
M to at least about 0.15 M salt for washing at 42°C.
High stringency also includes 1 % BSA, 1 mM EDTA, 0.5 M
NaHP04 (pH 7.2), 7% SDS for hybridization at 65°C, and (i) 0.2 x
SSC, 0.1
SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHP04 (pH 7.2), 1 % SDS for
washing at a temperature in excess of 65°C.
In general, washing is carried out at Tm = 69.3 + 0.41 (G + C)
% _ -12°C. However, 'the Tm of a duplex DNA decreases by 1 °C
with every
increase of 1 % in the number of mismatched based pairs.
Although the MUC genes and isolated MUC nucleic acids of
the present invention are exemplified in relation to the human mammalian
species, the present invention extends to orthologs in non-human mammals
such as in primates, laboratory test animals (e.g. mice, rates, rabbits,
guinea
pigs, hamsters), companion animals (e.g. dogs, cats), livestock animals (e.g.
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
14
sheep, pigs, horses, donkeys, cows) and captive wild animals (e.g. deer, fox).
In light o~f the foregoing, the term "MUC homologsr is used to
encompass MUC orthologs, isolated nucleic acids which hybridize to MUC
nucleic acids of the invention and isolated nucleic acids which display at
least 60% sequence identity to isolated MUC nucleic acids.
It will also be appreciated that MUC homologs encompass
single or multiple nuclE:otide substitutions, deletions andlor additions to
the
isolated MUC nucleic acids of the invention, inclusive of mutants, fragments,
parts, portions and segments of the nucleotide sequences of the invention.
The isolated MUC nucleic acids of the present invention and
homologs thereof therefore include oligonucleotides, primers (such as for
PCR), antisense sequences, molecules suitable for use in co-suppression
and fusion nucleic acid molecules. Ribozymes are also contemplated by the
present invention. It will be understood that probes, primers and antisense
sequences correspond to distinct portions of isolated MUC nucleic acids of
the invention, in that they contain nucleotide sequences based on said
distinct portions of an isolated MUC nucleic acid sequence. Such probe and
primer sequences may be based on a MUC sequence of the invention by
being identical thereto, or by being degenerate with respect thereto.
As used herein, "oligonucleotides" are nucleic acids which
comprise a contiguous sequence of no more than seventy (70) nucleotides,
whereas "polynucleotides" are nucleic acids which comprise a contiguous
sequence of more than seventy (70) nucleotides. A "probe" may be an
oligonucleotide or a polynucleotide, either double-stranded or single-
stranded, for use in hybridization techniques such as Northern blotting,
Southern blotting or in situ hybridization. The skilled person will realize
that
in situ hybridization also includes Fluorescence In Situ Hybridization (FISH),
which is used for determining chromosomal localization. In situ hybridization
techniques applicable to the present invention will be described in detail
hereinafter.
A "primer" is a nucleic acid (usually an oligonucleotide) capable
CA 02333853 2001-O1-16
WO 00/04142 PCT/ALl99/00579
of annealing to a nucleic acid template under appropriate conditions of ionic
strength and temperai:ure, which annealed primer can be extended in a
template-dependent fashion by a suitable nucleic acid polymerase (for
example Taq polymerase or SequenaseT""~. It will therefore be understood
5 that primers of the invention may be useful for PCR, sequencing, RACE,
primer extension and the like.
In use, isolated MUC nucleic acids, probes and primers may be
modified such as by end-labeling with ~zP-ATP and T4 polynucleotide kinase
or by random primed labeling with 32P-dCTP and DNA polymerase.
70 Biotinylation is also contemplated, as is modification with
phosphorothiorates,
fluorochromes, digoxigenin, enzymes and peptides, for example.
It is contemplated that diagnostic methods may be employed
which utilize isolated M~JC nucleic acids of the present invention, or
portions
thereof such as probes and PCR primers. Also, diagnostic methods
15 employing MUC polypeptides will be discussed in more detail hereinafter.
Diagnostic methods may include detection of MUC genes,
transcripts and/or polypeptides in samples such as fecal specimens and/or
in colonic biopsies, analysis of serum MUC levels in patients with epithelial
diseases including cancers, breast tissue biopsy samples or in respiratory
mucus samples from patients suffering from CF, asthma or chronic bronchitis.
The diagnostic methods of the present invention may therefore
be applicable to determining whether an individual has a disease condition
associated with aberrant Mucin expression, altered properties of mucus or
epithelial inflammatory processes involving Mucins, or a predisposition to
said disease. It will be appreciated that "predispositionn as used herein
refers
to an increased probability that an individual will contract the disease.
However, it will also be appreciated that the diagnostic methods may also
indicate whether an individual actually suffers from the disease, assist in
assessing the severity of disease, a prognosis of the likely course of disease
and appropriate treatments for the disease. Thus, the diagnostic methods of
the invention may be useful whether or not the individual suffers from one or
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
1fi
more symptoms of the disease.
The present invention therefore contemplates methods of
detecting MUC genes and MUC gene transcripts (e.g. mRNA), such as
involving hybridization techniques (for example, by Northern or Southern
blotting or in situ hybridization) or polynucleotide sequence amplification
techniques (for example RT-PCR). Such methods may detect:-
(i) a polymorphism, deletion, mutation, expansion, and/or
truncation in a MUC gene or MUC gene transcript; and
(ii) a relative level of expression of a MUC gene transcript
(an mRNA transcript derived from a MUC gene).
Such methods of detection facilitate determination of whether
said MUC gene is aberrantly-expressed as an indication of a disease
condition or a predisposition thereto. Also, MUC gene poiymorphisms,
deletions, mutations, truncations or deletions may be detected which indicate
a disease condition or a predisposition thereto.
It will be appreciated, for example, that measurement of a
relative level of expression of a MUC gene transcript facilitates diagnostic
assessment of whether MUC gene expression is downregulated and thereby
indicative of CRC.
Although PCR is the preferred nucleic acid sequence
amplification technique, It will be appreciated that there are a variety of
polynucleotide sequence amplification techniques other than PCR, which
include rolling circlE; amplification {RCA) and strand displacement
amplification (SDA). With regard to RCA, reference is made to W097/19193
which is herein incorporated by reference. With regard to SDA, reference is
made to U.S. Patent No. 5455166, which is herein incorporated by reference.
Detailed PCR methods are provided hereinafter, although the
skilled person is also rE:ferred to Chapter 15 of CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, Eds Ausubel et al., (John Wiley & Sons), which is
herein incorporated by reference, for a detailed discussion and examples of
PCR methods.
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579 _
17
It will also be understood that PCR includes within its scope
RT-PCR and multiplex PCR as will be described in detail hereinafter. Such
methods may be used for qualitative or semi-quantitative analysis. PCR-
based Restriction Fragment Length Palymorphism (PCR-RFLP) methods are
also contemplated, which methods are useful when a polymorphism, deletion
mutation, truncation and/or expansion either introduces or removes one or
more restriction endonuclease sites in a MUC gene.
The skilled person will appreciate that Northern, Southern and
in situ hybridization methods involve formation of a hybrid nucleic acid
comprising a MUC gene or mRNA transcript and a corresponding isolated
MUC nucleic acid or portion thereof.
RNA isolation and Northern hybridization methods are
described in detail herein, although the skilled person is also referred to
Chapter 4 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Eds
Ausubei et ai., (John Wiley & Sons), which is herein incorporated by
reference.
Furthermore, Southern hybridization methods are described in
detail herein, although the skilled person is also referred to sections 2.9A-B
and 2.10 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Eds
Ausubel et al., (John Wiley & Sons), which is herein incorporated by
reference.
Also, determining whether a MUC gene or MUC gene transcript
includes a polymorphism, mutation, deletion, truncation andlor expansion can
be performed using rr~ethods such as PCR-RFLP analysis, Single Strand
Conformational Polymorhpism (SSCP) analysis and Denaturing Gradient Gel
Electrophoresis (DGGE). These techniques have become well known in the
art of mutation detection. A non-limiting example of DGGE is provided in
Folde & Loskoot, 1994, Hum. Mut. 3 83, which is herein incorporated by
reference. A non-limiting example of specific allele detection by PCR-RFLP
and SSCP is provided in Lappalainen ef al., 1995, Genomics 27 274, which
is herein incorporated by reference.
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
18
It is proposed that mutations in MUC11 or MUC12 genes are
associated with bowel cancers (CRC), CF, BC, IBD, chronic bronchitis,
asthma, ulcerative colitis and/or Crohn's disease. These are examples of
disease conditions associated with aberrant Mucin expression, altered
properties of mucus or epithelial inflammatory processes involving Mucins.
The isolated MUC nucleic acids now provide a means for genetic screening
of the abovementioned disease conditions in human and other mammalian
species. Genetic screening may be conducted by determining full expression
or full-length transcript production by Northern blot, cloning and sequencing
~ of the MUC genes or identifying mutations by oligonucleotide hybridisation
or by direct sequencing of PCR amplification products of the MUC genes. In
addition, the present invention extends to nucleic acid molecules having
translation-terminating mutations leading to truncation mutants. The detection
of truncation mutants imay be important for genetic analysis of people with,
for example, cancer of the large bowel or with a propensity to develop large
bowel cancer, determined on, for example, hereditary grounds.
Truncated MUC polypeptides may also be useful in developing
therapeutic agents such as antagonists or for developing antibodies.
Truncational mutants may be readily detected by a direct protein truncation
test. In essence, DNA fragments including PCR amplification products or
corresponding mRNA molecules are subjected to in vitro translation and
optionally also transcription and the translation products assayed by, for
example, SDS-PAGE or by differential antibody binding assays. This assay
may also be employed to screen for agents capable of inducing truncation
mutations or for agents acting as antagonists for truncation mutant-inducing
agents.
Alternatively, MUC polypetides may be assayed by, for
example, by antibody screening such as in an ELISA.
Thus, it will be appreciated that the present invention
contemplates isolated MUC polypeptides, and also:-
(i) polypeptides which comprise an amino acid sequence
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
19
having at least 60% identity to a MUC polypeptide
amino acid sequence, preferably at least 75% identity
thereto, or more preferably at least 90% identity thereto;
and
(ii) polypeptides encoded by MUC homologs.
Such polypeptides are hereinafter referred to as "MUC
homologs".
The MUt; polypeptide homologs of the invention include amino
acid substitution(s), deletions) and/or additions) to a MUC polypeptide
sequence. Particular examples include antigenic fragments and analogues
useful in immunoassays and as therapeutic agents as well as other
fragments carrying B cell andlor T cell linear or conformational epitopes.
Additions to the amino acid sequence include fusion partners in the form of
peptides or polypeptides, which create a MUC fusion polypeptide.
Fusion polypeptides include the MUC polypeptide(s) together
with fusion partners such as HIS6, glutathione-s-transferase (GST),
thioredoxin (TR) and maltose binding protein (MBP). Fusion partners greatly
assist recombinant synthetic polypeptide purification by virtue of each fusion
partner affording affiniity purification by a specific affinity matrix.
Preferably,
the fusion polypeptide also includes a protease-specific cleavage site, so
that
the fusion partner may be cleaved and removed following purification to leave
a substantially unmodified MUC polypeptide.
The usE: of fusion partners for purification of recombinant
expressed polypeptides is well known in the art. Indeed, there are a variety
of commercial sources. applicable to fusion partners and purification systems
such as the QlAexpressT"" (HIS)s system, the Pharmacia GST purification
system and the New (England Biolabs MBP system.
Also within the scope of fusion partners are "epitope tags".
Such tags are well known in the art and include c-myc, influenza
hemagglutinin and FLAG tags.
Furthermore, Green Fluorescent Protein (GFP) is a well known
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
z0
fusion partner applicable to MUC polypeptides of the invention. A particularly
useful application of GFP fusion partners is in the visible identification of
cells or tissues which express a GFP-MUC fusion polypeptide of the
invention. Identification may be performed by flow cytometry or fluorescence
microscopy, as are well known in the art.
The MUC polypeptides and MUC homologs of the invention
may be in recombinant form of may be chemically synthesized, as is well
known in the art. Chemical synthesis is preferably suited to production of
MUC peptides. As used herein, "peptides" have no more than fifty (50)
contiguous amino acids.
Preferably, MUC polypeptides are in recombinant form.
In order to produce recombinant MUC polypeptides, isolated
MUC nucleic acids of the present invention may be ligated into an expression
vector to form an expression construct capable of directing expression of said
MUC nucleic acid in a prokaryotic cell (for example, E. col~~ or in a
eukaryotic
cell (for example, yeast cells, fungal cells, insect cells, mammalian cells or
plant cells).
Suitably, the expression vector comprises one or more
regulatory elements which direct expression of the nucleic acid ligated in
said
expression construct. Such regulatory sequences include promoters,
enhancers, splice donor/acceptor sites, polyadenylation sequences,
translation initiation (Kozak sequences) and translation termination signals.
Suitable promoters may be constitutive (for example, CMV- or SV40-derived
promoters) or inducible (for example, Zn responsive metallothionein
promoters) or repressible (tet-repressible promoters).
Exemplary methods useful for recombinant protein expression
and purification, including fusion polypeptides, can be found in Chapters 16
of CURRENT PROTOCOLS fN MOLECULAR BIOLOGY (Eds. Ausubel et al.;
John Wiley & Sons Inc., 1997 Edition) and Chapters 5 and 6 of CURRENT
PROTOCOLS IN PROTEIN SCIENCE (Eds. Coligan et al.; John Wiley &
Sons Inc., 1997 Edition) which are herein incorporated by reference.
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
21
"Analogues" of the MUC polypeptides of the invention
contemplated herein include, but are not limited to, modification to side
chains, incorporating of unnatural amino acids and/or their derivatives during
peptide, polypeptide or protein synthesis and the use of crosslinkers and
other methods which impose conformational constraints on the proteinaceous
molecule or their analogues. Such chemical analogues may be useful in
providing stable means for diagnostic purposes or for producing agonists or
antagonists or for producing stable molecules for use in natural product
screening.
Examples of side chain modifications contemplated by the
present invention include modifications of amino groups such as by reductive
alkylation by reaction with an aldehyde followed by reduction with NaBH4;
amidination with methylacetimidate; acylation with acetic anhydride;
carbamoylation of amiino groups with cyanate; trinitrobenzylation of amino
groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino
groups with succinic anhydride and tetrahydrophthalic anhydride; and
pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with
NaBH4.
The guanidine group of arginine residues may be modified by
the formation of heterocyclic condensation products with reagents such as
2,3-butanedione, phenylglyoxal and glyoxal.
The carboxyl group may be modified by carbodiimide activation
via O-acylisourea formation followed by subsequent derivitisation, for
example, to a corresponding amide.
Sulphydryl groups may be modified by methods such as
carboxymethylation with iodoacetic acid or iodoacetamide; performic acid
oxidation to cysteic acid; formation of a mixed disulphides with other thiol
compounds; reaction with maleimide, malefic anhydride or other substituted
maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate,
4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-
chloromercuri-4-nitrophenol and other mercurials; carbamoylation with
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
22
cyanate at alkaline pl-~.
Tryptophan residues may be modified by, for example,
oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-
hydroxy-5-nitrobenzyl bromide or sulphenyi halides. Tyrosine residues on
the other hand, may be altered by nitration with tetranitromethane to form a
3-nitrotyrosine derivative.
Modification of the imidazole ring of a histidine residue may be
accomplished by alkylation with iodoacetic acid derivatives or N-
carbethoxylation with diethylpyrocarbonate.
Examples of incorporating unnatural amino acids and
derivatives during peptide synthesis include, but are not limited to, use of
norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid,
6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine,
sarcosine, 4-amino-3-hydroxy-6-methyiheptanoic acid, 2-thienyl alanine
andlor D-isomers of amino acids. A list of unnatural amino acids,
contemplated herein us shown in Table 1. Crosslinkers can be used, for
example, to stabilise tertiary conformation, using homo-bifunctional
crosslinkers such as the bifunctional imido esters having (CHZ)~ spacer
groups with n=1 to n=6, glutaraldehyde, N-hydroxysuccinimide esters and
hetero-bifunctional reagents which usually contain an amino-reactive moiety
such as N-hydroxysuccinimide and another group specific-reactive moiety
such as maleimido or dithio moiety (SH) or carbodiimide (COON). In
addition, peptides can be conformationally constrained by, for example,
incorporation of Ca and Na-methylamino acids, introduction of double bonds
between Ca and Cs atoms of amino acids and the formation of cyclic peptides
or analogues by introducing covalent bonds such as forming an amide bond
between the N and C termini, between two side chains or between a side
chain and the N or C i:erminus.
The present invention further contemplates chemical analogues
of the polypeptides of the invention capable of acting as antagonists or
agonists thereof, or which can act as functional analogues thereof. Chemical
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
23
analogues may not necessarily be derived from the polypeptides of the
invention, but may share certain conformational similarities. Alternatively,
chemical analogues may be specifically designed to mimic certain
physiochemical properties of MUC poypeptides. Chemical analogues may
be chemically synthesised or may be detecked following, for example, natural
product screening. Useful sources for screening for natural products include
coral, reefs, sea beds, river beds, plants, microorganisms and aqua and
antarctic environments.
Still anather aspect of the present invention is directed to
antibodies specific for MUC polypeptides andlor homologs thereof.
In one embodiment, the anti-MUC antibody is M11.9.
In another embodiment, the anti-MUC antibody is M12.15.
A detailed method of anti-MUC antibody preparation is provided
hereinafter.
In this regard, it will be understood that anti-MUC polypeptide
antibodies may be produced by immunization with MUC polypeptides or MUC
peptides.
In particular, it is also likely that naturally-occurring anti-MUC
antibodies may well have naturally arisen against MUC polypeptides.
In light of the foregoing, it will be appreciated that "anti-MUC
antibody" as used herein is an antibody specific for, or at least binds to, a
MUC polypeptide, irrespective of how the anti-MUC antibody was produced.
The anti-MUC antibodies of the present invention may be
useful as therapeutic or diagnostic agents.
For example, a MUC polypeptide or homolog can be used to
screen for naturally occurring anti-MUC antibodies. These may occur, for
example in some autoimmune diseases. Alternatively, anti-MUC antibodies
can be used to screen for MUC polypeptides. Techniques for such assays
are well known in the art and include, for example, sandwich assays and
ELISA. Knowledge of endogenous MUC polypeptide levels may be important
for diagnosis of large bowel cancer or a predisposition to large bowel cancers
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
24
or for monitoring certain therapeutic protocols. This knowledge may also be
important in other epithelial cancers such as cancer of the breast.
Anti-MUC antibodies of the present invention may be
monoclonal or polyclonal. Alternatively, fragments of antibodies may be
used such as Fab fragments. Furthermore, the present invention extends to
recombinant and synthetic antibodies and to antibody hybrids. A "synthetic
antibody" is considered herein to include fragments and hybrids of
antibodies. The antibodies of this aspect of the present invention are
particularly useful for immunotherapy and may also be used as a diagnostic
tool for assessing cancer development or cancer cell apoptosis or monitoring
the program of a therapeutic regimum.
For example, anti-MUC antibodies can be used to screen for
endogenous MUC polypeptides. The latter would be important, for example,
as a means for screening for levels of the MUC polypeptide in a cell extract
or other biological fluid or purifying the MUC polypeptide made by
recombinant means from culture supernatant fluid. Techniques for the
assays contemplated herein are known in the art and include, for example,
sandwich assays and ELISA.
It is within the scope of this invention to include any second
antibodies (monoclonal, polyclonaf or fragments of antibodies or synthetic
antibodies) directed to~ the first mentioned antibodies discussed above. Both
the first and second antibodies may be used in detection assays or a first
antibody may be used with a commercially available anti-immunoglobulin
antibody. An antibody as contemplated herein includes any antibody specific
to any region of the MUC polypeptide.
Both polyclonal and monoclonal antibodies are obtainable by
immunization with the enzyme or protein and either type is utilizable for
immunoassays. The rnethods of obtaining both types of sera are well known
in the art. Polyclonal sera are less preferred but are relatively easily
prepared by infection of a suitable laboratory animal with an effective amount
of a MUC polypeptid~, or antigenic parts thereof, collecting serum from the
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
animal, and isolating specific sera by any of the known immunoadsorbent
techniques. Although antibodies produced by this method are utilizable in
virtually any type of immunoassay, they are generally less favoured because
of the potential heterogeneity of the product.
5 The use of monoclonal antibodies in an immunoassay is
particularly preferred because of the ability to produce them in large
quantities and the horniogeneity of the product. The preparation of hybridoma
cell lines for monoclonal antibody production derived by fusing an immortal
cell line and lymphocytes sensitized against the immunogenic preparation
10 can be done by techniques which are well known to those who are skilled in
the art.
The present invention contemplates a method for detecting a
MUC polypeptide in a protein extract obtained from a mammal, said method
including the step of farming a complex between an anti-MUC antibody and
15 a MUC polypeptide, and then detecting said complex.
The presence of a MUC polypeptide may be determined in a
number of ways such as by Western blotting and ELISA procedures. A wide
range of immunoassay techniques are available as can be seen by reference
to U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653. These include both
20 single-site and two-site or "sandwich" assays of the non-competitive types,
as well as in the traditional competitive binding assays. These assays also
include direct binding of a labelled antibody to a target.
Sandwich assays are among the most useful and commonly
used assays and are favoured for use in the present invention. A number of
25 variations of the sandwich assay technique exist and all are intended to be
encompassed by the present invention. Briefly, in a typical forward assay,
an unlabelled antibody is immobilized to a solid substrate and the sample to
be tested brought into contact with the bound molecule. After a suitable
period of incubation, for a period of time sufficient to allow formation of an
antibody-antigen complex, a second antibody specific to the antigen, labelled
with a reporter molecule capable of producing a detectable signal is then
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
26
added and incubated, allowing time sufficient for the formation of another
complex of antibody-antigen-labelled antibody. Any unreacted material is
washed away and the presence of the antigen is determined by measurement
of a signal produced by the reporter molecule. The results may either be
qualitative, by simple observation of the visible signal, or may be
quantitated
by comparing with a control sample containing known amounts of hapten.
Variations on the forward assay include a simultaneous assay, in which both
sample and labelled antibody are added simultaneously to the bound
antibody. These techniques are well known to those skilled in the art,
including any minor variations as will be readily apparent. In accordance with
the present invention the protein extract might be a cell extract, tissue
biopsy
or possibly serum, saliva, mucosal secretions, lymph, tissue fluid and
gastrointestinal fluid. T'he extract is, therefore, generally a biological
sample.
In the typical forward sandwich assay, a first antibody having
specificity for MUC or antigenic parts thereof, is either covalently or
passively
bound to a solid surface. The solid surtace is typically glass or a polymer,
the most commonly used polymers being cellulose, polyacrylamide, nylon,
polystyrene, polyvinyl chloride or polypropylene. The solid supports may be
in the form of tubes, beads, discs of microplates, or any other surface
suitable for conducting an immunoassay. The binding processes are well-
known in the art and generally consist of cross-finking covalently binding or
physically adsorbing, the polymer-antibody complex is washed in preparation
for the test sample. An aliquot of the sample to be tested is then added to
the solid phase complex and incubated for a period of time sufficient {e.g. 2-
40 minutes or overnight if more convenient) and under suitable conditions
(e.g. from 4°C to 3T°C) to allow binding of any subunit present
in the
antibody. Following the incubation period, the solid phase complex is
washed and dried and incubated with a second antibody which is specific for
a portion of the antigen (i.e. MUC). The second antibody is linked to a
reporter molecule which is used to indicate the binding of the second
antibody to MUC.
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
27
An alternative method involves immobilizing the target
molecules in the biological sample and then exposing the immobilized target
to specific antibody which may or may not be labelled with a reporter
molecule. Depending on the amount of target and the strength of the
reporter molecule signal, a bound target may be detectable by direct labelling
with the antibody. Alternatively, a second labelled antibody, specific to the
first antibody is exposed to the target-first antibody complex to form a
target-
first antibody-second antibody tertiary complex. The complex is detected by
the signal emitted by the reporter molecule.
By "reporter molecule" as used in the present specification, is
meant a molecule which, by its chemical nature, provides an analytically
identifiable signal which allows the detection of antigen-bound antibody.
Detection may be either qualitative or quantitative. The most commonly used
reporter molecules in this type of assay are either enzymes, fluorochromes
or radionuclide containing molecules (i.e. radioisotopes) and
chemiluminescent molecules.
In the case of an enzyme immunoassay, an enzyme is
conjugated to the second antibody, such as via glutaraldehyde or periodate
amongst other means. As will be readily recognized, however, a wide variety
of different conjugation techniques exist, which are readily available to the
skilled artisan. Commonly used enzymes include horseradish peroxidase,
glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst
others. The substrates to be used with the specific enzymes are generally
chosen for the production, upon hydrolysis by the corresponding enzyme, of
a detectable colour change. It is also possible to employ fluorogenic
substrates, which yield a fluorescent product rather than the chromogenic
substrates noted above. In all cases, the enzyme-labelled antibody is added
to the first antibody-antigen complex, allowed to bind, and then the excess
reagent is washed away. A solution containing the appropriate substrate is
then added to the complex of antibody-antigen-antibody. The substrate will
react with the enzyme linked to the second antibody, giving a qualitative
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
28
visual signal, which may be further quantitated, usually
spectrophotometrically, to give an indication of the amount of antigen which
was present in the sample. The term "reporter molecule" also extends to use
of cell agglutination or inhibition of agglutination such as red blood cells
on
latex beads, and the like.
Also, fluorescent compounds, such as fluorescein and
rhodamine, may be chemically coupled to antibodies without altering their
binding capacity. When activated by illumination with light of a particular
wavelength, the fluorochrome-labelled antibody adsorbs the light energy,
inducing a state of excitability in the molecule, followed by eneossion of the
light at a characteristic colour visually detectable with a light microscope.
As
in the EIA, the fluorescent labelled antibody is allowed to bind to the first
antibody-hapten complex. After washing off the unbound reagent, the
remaining tertiary complex is then exposed to light of the appropriate
wavelength and the fluorescence observed indicates the presence of the
antigen of interest. Immunofluorescene and EIA techniques are both very
well established in the art. Other reporter molecules, such as radioisotope,
chemiluminescent or bioluminescent molecules, may also be employed.
The MUC; genes of the present invention are likely to function
in cell adhesion, signal transduction, growth regulation, epithelial cell
protection andlor immunological reactions. The classical gel-forming mucins
function in protecting and lubricating epithelial tissues (particularly those
of
the respiratory and gastrointestinal tracts) by forming a layer of
viscoelastic
gel. These new mucins, MUC11 and MUC12, show structural similarity to
MUC1. MUC1 can be secreted, but unlike the classical mucins, it is primarily
a type I transmembrane protein that interacts and complexes with other
adhesion molecules, and is involved in signal transduction. MUC12 has an
EGF growth factor-like domain, is likely to be a transmembrane protein and
has a putative tyrosine phosphorylation site that may participate in
intracellular signalling. It is hypothesised that loss of MUC12 may be
associated with poor prognosis in CRC.
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
29
The isolated MUC nucleic acids of the present invention are,
therefore, considered in one embodiment, to correspond to cancer
suppressor genes. Suppression may mean total inhibition of any
development of large bowel cancer or a limitation of the severity of or an
amelioration of the condition resulting from a large bowel cancer. The MUC
nucleic acids of the present invention are also considered in another
embodiment to be capable of modulating disease conditions such as CRC,
BC, IBD, CF, asthma, chronic bronchitis, ulcerative colitis and/or Crohn's
disease
Cystic fibrosis (CF) is an inherited disease of epithelial cell
chloride ion transport that affects multiple organ systems. It is the most
common cause of severe, progressive lung disease and exocrine pancreatic
insufficiency in childhood. The cystic fibrosis transmembrane conductance
regulator (CFTR) gene located on chromosome 7q22 encodes a large single
chain protein that forms a chloride channel. Virtually all of the morbidity
and
mortality associated with mutations in the CFTR gene causing cystic fibrosis
arise from respiratory disease due to chronic infection and mucus
obstruction. The precise mechanism of mucus accumulation in cystic fibrosis
is controversial. Data suggest that CFTR malfunction may trigger mucin
secretion and alter mucus properties, andlor bacterial infection triggers the
hypersecretion of mucin in CF patients. The gene of the present invention
is expressed in the colon, pancreas, small intestine, and lung, all tissues
where mucus obstruction occurs. Accordingly, aberrant expression of the
genes may contribute to cystic fibrosis.
Aberrant mucin expression is also a recognised component of
IBD. Inflammatory bowel disease is characterised by considerable alterations
in glycosylation, sialyation and sulphation of glycoproteins. It is unclear
whether the changes in mucus production are a cause or response to the
disease. Susceptibility genes for inflammatory bowel disease have been
localised to chromosomes 3, 12 and 7q22. Accordingly, the MUC genes of
the present invention are considered candidates for susceptibility genes for
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
IBD. Up or down regulation, or altered secretion of one of these mucins may
influence the quality of colonic mucus and therefore the pathology of these
diseases. Certain inherited forms of these genes may indicate a
predisposition to IBD.
5 The identification of MUC genes and isolated MUC nucleic
acids permits the generation of a range of therapeutic methods and
compositions. Such therapeutics may modulate MUC gene expression and
the activity of MUC polypeptides. Modulators contemplated by the present
invention includes agonists and antagonists of MUC gene expression.
10 Antagonists of MUC: gene expression include antisense molecules,
ribozymes and co-suppression molecules. Agonists include molecules which
increase promoter activity or interfere with negative mechanisms. Agonists
of MUC include molecules which overcome any negative regulatory
mechanism. Antagonists of MUC poiypeptides include antibodies and
15 inhibitor peptide fragments. Another class of therapeutics may be designed
to mimic or block intracellular signal transduction by MUC polypeptides.
In accordance with the present invention, it is proposed that
MUC functions as a suppressor of cancer development in the large bowel.
Hereditary cancers arise with loss of the wild-type gene. In addition,
20 germline mutations underlying large bowel cancer are inactivated for the
MUC genes and, therefore, hereditary cancers have no functional copy of the
gene. Furthermore, sporadic large bowel cancers arise with somatic loss of
both copies of the gene. The present invention extends to the use of
modulating levels of expression of MUC genes or their translation products
25 in the context of cancers related thereto.
Thus, the present invention contemplates a method of gene
therapy of a mammal. .Such a method utilizes a gene therapy construct which
includes an isolated MUC nucleic acid ligated into a gene therapy vector
which provides one or more regulatory sequences that direct expression of
30 said nucleic acid in said mammal.
Such regulatory sequences may include a promoter, an
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
31
enhancer, a polyadenylation sequence, splice donor/acceptor sequences
and translation termination and intiation sequences.
Typically, gene therapy vectors are derived from viral DNA
sequences such as adenovirus, adeno-associated viruses, herpes-simplex
viruses and retroviruses. Suitable gene therapy vectors currently available
to the skilled person may be found in Robbins et al., 1998, Trends
Biotechnol. 16 35, for example, which is herein incorporated by reference.
If "anti-sense" therapy is contemplated, then one or more
selected portions of a MUC nucleic acid may be oriented 3'-~5' in the gene
therapy vector.
Administration of the gene therapy construct to said mammal,
preferably a human, rnay include delivery via direct oral intake, systemic
injection, or delivery to,elected tissues) or cells, or indirectly via
delivery to
cells isolated from the mammal or a compatible donor. An example of the
latter approach would be stem-cell therapy, wherein isolated stem cells
having potential for growth and differentiation are transfected with the
vector
comprising the MUC nucleic acid. The stem-cells are cultured for a period
and then transferred to the mammal being treated.
Delivery of said gene therapy construct to cells or tissues of
said mammal or said compatible donor may be facilitated by microprojectile
bombardment, liposome mediated transfection (e.g. lipofectin or
lipofectamine), electroporation, calcium phosphate or DEAE-dextran-
mediated transfection, for example. A discussion of suitable delivery methods
may be found in Chapter 9 of CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY (Eds. Ausubel et al.; John Wiley & Sons lnc., 1997 Edition), for
example, which is herein incorporated by reference.
For example, a MUC nucleic acid may be introduced into a cell
to enhance the ability of that cell to survive, conversely, MUC antisense
sequences such as 3'-~ 5' oligonucleotides may be introduced to decrease
the survival capacity of any cell expressing an endogenous MUC gene.
In this regard, increased MUC expression or activity is
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
32
important in conditions of repressing cancer growth and/or development.
Decreased MUC expression or activity may be important, for example, in the
treatment of cystic fibrosis or the treatment of inflammatory bowel disease.
Accordingly, the present invention contemplates a
pharmaceutical composition comprising a MUC polypeptide or a derivative
thereof or a modulatar of MUC gene expression or activity, inclusive of anti
MUC antibodies. These components are referred to herein as the "active
ingredients", and are suitably provided in combination with one or more
pharmaceutically-acceptable carriers andlor diluents.
The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions (where water soluble) and sterile powders for the
extemporaneous preparation of sterile injectable solutions. It must be stable
under the conditions of manufacture and storage and must be preserved
against the contaminating action of microorganisms such as bacteria and
fungi. The carrier can be a solvent or, for example, water, ethanol, polyol
(for
example, glycerol, propylene glycol and liquid polyethylene glycol, and the
like) or suitable mixtures thereof as well as vegetable oils. The preventions
of the action of microorganisms can be brought about by various antibacterial
and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic
acid, thirmersal and the like. In many cases, it will be preferable to include
isotonic agents, for example, sugars or sodium chloride. Prolonged
absorption of the injectable compositions can be brought about by the use in
the compositions of agents delaying absorption, for example, aluminum
monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the
active compounds in the required amount in the appropriate solvent with
various of the other ingredients enumerated above, as required, followed by
filtered sterilization. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation are vacuum
drying and the freeze-drying technique which yield a powder of the active
ingredient plus any additional desired ingredient from previously
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
33
sterile-filtered solution thereof.
When the active ingredients are suitably protected they may be
orally administered, for example, with an inert diluent or with an assimilable
edible carrier, or it may be enclosed in hard or soft shell gelatin capsule,
or
it may be compressed into tablets, or it may be incorporated directly with the
food of the diet. For oral therapeutic administration, the active compound
may be incorporated 'with excipients and used in the form of ingestible
tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups,
wafers, and the like. Such compositions and preparations should contain at
least 1 % by weight of ac,~tive compound. The percentage of the compositions
and preparations may, of course, be varied and may conveniently be
between about 5 to about 80% of the weight of the unit. The amount of
active compound in such therapeutically useful compositions in such that a
suitable dosage will bE: obtained. Preferred compositions or preparations
according to the present invention are prepared so that an oral dosage unit
form contains between about 0.1 Ng and 2000 mg of active ingredient.
The tablets, troches, pills, capsules and the like may also
contain the components as listed hereafter: a binder such as gum, acacia,
corn starch or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent such as corn starch, potato starch, alginic acid and the
like; a lubricant such as magnesium stearate; and a sweetening agent such
a sucrose, lactose or saccharin may be added or a flavouring agent such as
peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit
form is a capsule, it may contain, in addition to materials of the above type,
a liquid carrier. Various other materials may be present as coatings or to
otherwise modify the physical form of the dosage unit. For instance, tablets,
pills, or capsules may be coated with shellac, sugar or both. A syrup or
elixir
may contain the active compound, sucrose as a sweetening agent, methyl
and propylparabens as preservatives, a dye and flavouring such as cherry
or orange flavour. Of course, any material used in preparing any dosage unit
form should be pharmaceutically pure and substantially non-toxic in the
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
34
amounts employed. In addition, the active compounds) may be incorporated
into sustained-release preparations and formulations.
Pharmaceutically acceptable carriers and/or diluents include
any and all solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents and the like. The use of
such media and agents for pharmaceutically active substances is well known
in the art. Except insofar as any conventional media or agent is incompatible
with the active ingredient, use thereof in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be incorporated
into the compositions.
It is especially advantageous to formulate parenteral
compositions in dosagE~ unit form for ease of administration and uniformity of
dosage. Dosage unit firm as used herein refers to physically discrete units
suited as unitary dosages for the mammalian subjects to be treated; each
unit containing a predetermined quantity of active material calculated to
produce the desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the novel dosage unit forms of
the invention are dictated by and directly dependent on (a) the unique
characteristics of the active material and the particular therapeutic effect
to
be achieved, and (b) the limitations inherent in the art of compounding such
an active material for the treatment of disease in living subjects having a
diseased condition in 'which bodily health is impaired.
The principal active ingredient is compounded for convenient
and effective administration in effective amounts with a suitable
pharmaceutically acceptable carrier in dosage unit form as hereinbefore
disclosed. A unit dosage form can, for example, contain the principal active
compound in amounts ranging from 0.5 Ng to about 2000 mg. Expressed in
proportions, the active compound is generally present in from about 0.5 Ng
to about 2000 mg/mt. of carrier. In the case of compositions containing
supplementary active ingredients, the dosages are determined by reference
to the usual dose and manner of administration of the said ingredients. It is
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/005'79
also convenient to represent the effective amounts of active ingredients as
an amount per kg body weight. For example, the present invention
encompasses effective amounts for 0.005 Ng/kg body weight at 2000 mg/kg
body weight.
5 The pharmaceutical composition may also comprise genetic
molecules such as a vector capable of transfecting target cells where the
vector carries a nucleic acid molecule capable of modulating MUC gene
expression or MUC polypeptide activity. The vector may, for example, be a
viral vector.
10 From the foregoing, it is apparant that therapeutic methods and
compositions of the invention are useful in the treatment of disease
conditions associated with aberrant Mucin expression, altered properties of
mucus or epithelial inflammatory processes involving Mucins.
Preferably, the disease condition is selected from the group
15 consisting of colorectal cancer (CRC), cystic fibrosis (CF), inflammatory
bowel disease (IBD), breast cancer (BC), Crohn's disease, ulcerative colitis,
asthma and chronic bronchitis.
More preferably, the disease condition is selected from the
group consisting of colorectal cancer (CRC), cystic fibrosis (CF),
20 inflammatory bowel disease (IBD) and breast cancer (BC). although not
limited thereto. The therapeutic methods of the invention may therefore be
used to alleviate one or more symptoms of diseases or be used as
prophylactic treatments to prevent, or reduce the likelihood of, said symptoms
from occurring.
25 The present invention is further described by the following non-
limiting Examples.
EXAMPLES
EXAMPLE 1: Tissue Specimens
Tissue specimens were collected from patients undergoing
30 surgery (Dukes' A n=5; Dukes' B n=5, Dukes C n=5, Dukes' D n=5). Colonic
specimens were obtained from patients undergoing either colectomy or
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
36
partial hepatectomy far colorectal carcinoma. Samples of normal colonic
mucosa, primary colon cancer, liver metastases (if present) and adjacent
normal liver were rapidly excised from operative specimens, snap-frozen in
liquid nitrogen and stored at -70°C until use. Care was taken to
exclude
normal mucosal tissue from tumour samples. functional tissue specimens
from four tumours of each Dukes' stage were randomly selected for in situ
hybridisation. Tissues were fixed for 24-48 hours in 10% v/v buffered
formalin, dehydrated in ethanol, cleaned in chloroform and embedded in
parraffin wax. Biopsy specimens of normal colonic epithelium from four
10' distinct regions of the colon were collected via colonoscopy from each of
three healthy individuals undergoing routine colonoscopic screening.
Similarly, intestinal biopsies were obtained via colonoscopy from ten patients
with inflammatory bowel disease. Specimens were snap frozen and stored
at -70°C until RNA was extracted as per Example 3 below.
EXAMPLE 2: Cell Lines and Culture
Seven human colonic tumour lines were obtained: LIM1215,
LIM2405, LIM1863, LIM1899 (Ludwig Institute, Melbourne, Australia), HT29
(ATCC HTB38), SW480 (ATCC CCL 228) and SW620 (ATCC CCL 227).
LIM1215 and SW620 are each derived from CRC metastases. Cell lines were
maintained in RPMI 1640 with 10% v/v fetal calf serum, 2 mM glutamate, 25
mM HEPES, 60 mg/ml penicillin G and 100 mglml streptomycin sulfate and
incubated in 5% vlv COZ and 95% v/v air at 37°C. Cultures were passaged
twice weekly using standard techniques. The following breast carcinoma
lines were included in this study: KPL-1 (a gift of Dr Junichi Kurebayashi,
Suzuki, Japan), MA11 (a gift of Dr Philip Rye, Oslo, Norway), BT 20,
DU4475, MCF-7, MDA-MB-453, SK-Br-3, T47D, UACC-893, ZR-75-1 and
ZR-75-30 (ATCC, Rockville, MD), and MDA-MB-435 and MDA-MB-468 (a gift
of Dr. Janet Price, MD Anderson Cancer Center, Houston, TX). All breast
cancer cell cultures were maintained in RPMI-1640 medium supplemented
with 10% fetal calf serum and 0.006% penicillin and 0.01 % streptomycin with
the following exceptions: DU-4475 in RPMI-1640 with 20% FCS, KPL-1 was
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
37
maintained in DMEM with 5% FCS, MA11 in 1:1 Ham's F12:RPM1-1640 with
10% FCS, SK-Br-3 in McCoy's medium with 15% FCS, and UACG-893 in
RPMI-1640 with 15% FCS.
EXAMPLE 3: RNA Extraction
Total RNA was isolated by the method of Chomczynski and
Sacchi (Chomczynski et al., 1987, Anal. Biochem. 162 156). Cells were
resuspended in RNA. extraction buffer (4 M guanidinium isothiocyanate
containing 25 mM sodium citrate, pH7.0, 0.5 % w/v sodium lauroyl sarcosine
(SLS) and 0.1 M 2-mercaptoethanol). Tissue samples were homogenised in
RNA extraction buffer. Extracted RNA was dissolved in RNase free water and
the concentration and purity determined by spectrophotometry at 260 and
280 nm (Sambrook et al., Molecular Cloning, A Laboratory Manual. 2nd Ed.
Cold Spring Harbour Laboratory Press. Cold Spring Harbour, NY, 1989). The
integrity of the RNA was assessed by denaturing agarose gel electrophoresis
and samples transferred to HYBOND N {Amersham, Bucks, England)
membrane by capillary blotting.
EXAMPLE 4: DNA Seauencing
Approximately 500 ng of DNA were employed in a cycle
sequencing reaction with 2.5 pmol of primer and 4 N1 of Dye terminator or
dRhodamine reaction mix (DNA Cycle Sequencing Kits, Perkin Elmer,
Norwalk, CT,) in a total volume of 10 NI. Reaction mixes contained Amplitaq
DNA polymerise, dNTPs and fluorescently labelled dideoxynucleotides (dye
terminators). Cycling reactions were as follows: 25 cycles of denaturation at
96°C (30 s), primer annealing at 50°C (15 s) and extension at
60°C (4 min).
Unincorporated nucleotides were removed by ethanol precipitation. The
reactions were analysed on a Model 373A automated DNA sequencer
(Applied Biosystems) run by technical staff in the core sequencing facility of
the Queensland Institute of Medical Research.
EXAMPLE 5: Identification by Differential Disptay of Two cDNAs
~ncodina Mucins Downreaulated in Colorectal
cancer
CA 02333853 2001-O1-16
WO 00/04142 PC'T/AU99/00579
38
The differential display method was devised from the original
technique described by Liang & Pardee, 1992, Science 257 967. Total RNA
was isolated by the method as described previously. Reverse transcription
was carried out using one of four anchored primers, T,2MG, T,2MC, T,2MA
and T~2MT (Operon Technologies Inc., Alameda, CA) and Superscript RNAse
H- reverse transcriptase (Gibco BRL, Gaithersburg, MD). One arbitrary
10mer primer (Operon Technologies Inc.) was selected at random to be
employed in a PCR with the appropriate anchored primer. Two patients, 101
and 112, were analysed simultaneously and duplicates of two separate
reverse transcription reactions electrophoresed on each gel. Gels were put
down wet and autoradiographed for 1-3 days. DNA was removed from gel
slices by boiling and reamplified by PCR. Bands were then cloned into
pGEM-T {Promega Corporation, Madison, WI) and sequenced. Sequences
were analysed by multiple sequence similarity searches using BLAST
algorithms (Altshcul et al., 1990, supra) accessed through the National
Centre of Biotechnology Information (NCBI; http:llwww.ncbi.nlm.nih.gov).
Differential display was performed on RNA from paired normal
colonic mucosa and primary colorectal cancers. Using a PCR primer
combination of T~2MG and 10mer 5'-ACTTCGCCAC-3' (SEQ ID N0:7),
bands dd29 (MUC12) and dd34 (MUC91) were both amplified from normal
colonic mucosal RNA of two patients and were consistently downregulated
in the tumors from these patients in multiple PCR reactions (FIG. 1A).
Following reamplification PCR, discrete bands of approximately 720 by for
dd29 and 530 by for dd34 were isolated and cloned into pGEM-T. Sequence
analysis showed that both cDNAs were novel, with no match in any database
accessed through the NCBI. Repetitive segments typical of mucin tandem
repeats were observed in dd34.
EXAMPLE 6: Northern Blot Analysis
Northern blot analysis was performed on paired normal and
tumor total RNA extracted from the same patients employed in the differential
display experiment. dd29 (MUC12) and dd34 (MUC11) were random primer-
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
39
labeled using a Megaprime DNA labeling system (Amersham, Aylesbury, UK)
and hybridization performed at 65°C in buffer containing 7% SDS, 0.26 M
Na2HP04, 1 mM EDTA, 1 % BSA.
Northern blot analyses of dd29 (FIG. 1 B) and dd34 (FIG. 1 C)
with colonic total RNA used for the differential display reactions revealed a
polydisperse signal beginning near the top of the gel for RNA isolated from
normal colonic mucosa and no signal in tumor-derived RNA. Probe dd29
showed some cross-hybridization to ribosomal RNA. Polydispersity of signal
is a hallmark of mucin RNA blots due to shearing of very high molecular
weight transcripts.
EXAMPLE 7: Multiplex Semi-guantitative RT PCR
Multiplex semi-quantitative RT-PCR was pertormed on total
RNA isolated from six colorectal cancer cell lines and from paired normal
colonic mucosa and tumor colorectal cancer tissues from 20 patients, eve of
each Dukes' stage. Informed consent was obtained from each subject after
approval by the appropriate hospital Ethics Committee. PCR products were
quantitated relative to a [iz-microglobulin cDNA amplification control using
densitometry. First strand cDNA synthesis was accomplished using 1 pg of
total RNA. PCR amplification of cDNA was pertormed in a total volume of 25
pl containing 1 NI of the first strand cDNA synthesis reaction products, 2.5
pl
10x Taq polymerise buffer (25 mM TAPS (tris-[hydroxymethylJ-methyl-amino-
propane-sulfonic acid, ;>odium salt) pH 9:3, 50 mM KCI), 2 mM dNTPs, 25 mM
MgCl2, 20 pmol each of the forward and reverse primers, and 2.5 U Taq
polymerise. Gene-specific forward and reverse primers for MUC92 and
MUCH were designed to produce PCR products of 510 by and 169 by
respectively. Primers for [32-microglobulin generated a PCR product of 247
by (Gussow et al., 1987, J. Immunol. 139 3132). Primers were:
MUC12F1; 5'-TGAAGGGCGACAATCTTCCTC-3' (SEQ ID N0:8);
MUC12R1; 5'-TACACGAGGCTCTTGGCGATGTTG-3' (SEQ ID N0:9);
MUC11 F1; 5'-CAGGC;GTCAGTCAGGAATCTACAG-3' (SECI ID N0:10);
MUC11 R1; 5'-GAGGCTGTGGTGTTGTCAGGTAAG-3' (SEQ ID N0:11 );
CA 02333853 2001-O1-16
WO 00/04142 PCT1AU99/00579
~i-21 F; 5'-TGAATTGCTATGTGTCTGGGT-3' (SEQ ID N0:12);
~i-21 R; 5'-CCTCCATGATGCTGCTTACAT-3' (SEQ ID N0:13);
MUC12TOTF1;5'-AGCCAACCAGGCTCAGCTCT-3' (SEQ ID N0:14); and
MUC12TOTR1;5'-GC~'CACACAGTGGATGCTACC-3' (SEQ ID N0:15}.
5 After an initial denaturation step of 94°C for 5 minutes, the
amplification conditions were: 21 cycles of denaturation at 94°C (30 s)
for
MUC12, (24 cycles of denaturation at 94°C (30 s) for MUC11);
annealing at
60°C (30 s) and extension at 72°C (30 s). PCR products were
electrophoresed on 1.2% 1x TBE gels and photographed.
10 Due to the polydisperse signals obtained by :Northern analysis,
expression of MUC19 and MUC12 was examined in a range of colorectal
cancer cell lines and tissue mRNAs by multiplex semi-quantitative RT-PCR.
dd29 was not expressE:d in any of six colorectal cancer cell lines examined
(FIG. 1 D). In contrast, dd34 showed a different pattern of expression, with
15 HT29; LIM1215, LIM1899, LIM1863 lines revealing very faint PCR products,
and SW620 and SW480 lines showing relatively high levels of expression
{FIG.1 E). For tumor tissue-derived RNA, downregulation was defined as
amplified band intensity less than 30% of that observed from paired normal
colon tissue. dd29 was found to be downregulated or absent in 6115 {40%)
20 tumors with paired normal samples, and at low levels in 3/5 (60%) Dukes'
stage D samples (where normal colon was not available for comparison)
(FIG.1 F). dd34 was downregulated in the tumors of 12!15 (80%) paired
samples and expressed at low levels in 415 (80%) Dukes' stage D samples.
One of five Dukes' stage D samples showed relatively high levels of
25 expression of dd34 (FIG. 1 G). Significantly, 13115 (87%) colorectal
cancers
showed downregulation of at least one of these mucin genes, with 5115
(33%) showing downregulation of both genes.
EXAMPLE 8: Differential Tissue Distribution of MUC11, MUC12
and MUC3 mRNAs
30 A human RNA "master blot" (Clontech, Palo Alto, CA, catalogue
number 7770-1 ) with RNA from 50 different tissues and controls was used to
CA 02333853 2001-O1-16
WO 00/04142. PCT/AU99/00579
41
examine mucin gene expression. DNA fragments encoding dd29, dd34 and
MUC3 (Genbank Accession No. M55405, a gift from Dr. Sandra Gendler,
Mayo Clinic, Scotsdale, Arizona) were excised from vector and radiolabeled
as described above. Hybridization was performed as per the manufacturer's
instructions. The master blot was reprobed with a radiolabeled ~3-actin cDNA
as a loading control.
Analysis of the tissue distribution of MUC11, MUC12 and MUC3
transcripts in RNA isolated from 50 different normal tissues showed a distinct
pattern of expression for each gene (FiG. 5). MUC12 and MUC11 showed
highest expression in colon but had different patterns in other organs, mainly
restricted to those of epithelial type. MUC11 had a wider epithelial
distribution than MUC12 which was restricted to expression in the colon, and
weakly in the pancreas, prostate and uterus. Consistent with published
findings (Van Klinken et al., 1997, Biochem. Biophys. Res. Comm. 238 143),
MUC3 was found to be predominantly expressed in the small intestine and
at much lower levels in the colon. Interestingly, it was also present in the
thymus.
EXAMPLE 9: Extendin4 the Seyuences of dd29 and dd34
The strategy employed in the cloning of_MUC11 and MUC12 is
shown in FIGS. 8A and 8B respectively.
9.1 Library ~~creenin4.
A hgt11 human fetal brain 5'-STRETCH PLUS cDNA library
(Clontech, Palo Alto, C;A) was screened using radiolabeled dd29 and dd34.
~ DNA was extracted and inserts were excised, cloned into pBSK- and
sequenced.
9.2 PCR to extend the seqruence of dd34 by IinkinQ clones 2 and li5
Screening of the fetal brain library with clone dd34, yielded two
new cDNA clones: clone 2 (1043 bp) and clone li5 (1045 bp). Clone dd34
was a perfect match to the middle of the larger clone 2. cDNA from clone 1i5,
however, was highly homologous but not identical to the cDNA from clone
dd34. To ascertain whether these partial cDNAs arose from a single mRNA
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
42
transcript, RT-PCR was carried out using combinations of forward and
reverse primers specific for each cDNA in an attempt to link them. RT-PCR
was performed on total RNA extracted from normal colon in a stringent
touchdown PCR using high fidelity DyNAzyme DNA polymerase (Finnzymes,
Espoo, Finland). Primer combination MUC11 F1 and IiSR {5'-
GGGAACACTGTGGTTTCAGTTGAG-3'; SEQ ID N0:16) yielded a PCR
product of 2 kb demonstrating that these two cDNAs were derived from a
single transcript. This product was cloned into pGEM-T and sequenced.
9.3 PCR library screening - to extend seguence of dd29
~ Forward and reverse primers for dd29 (dd29F1 and dd29R1 )
were used in combination with a T7 vector-derived primer in a stringent
touchdown PCR to screen an ulcerative colitis (UC) plasmid library (a gift
from Dr. Jonathon Fawcett, Queensland Institute of Medical Research,
Brisbane, Australia). Amplified products were purified, cloned into pGEM-T
and sequenced.
EXAMPLE 10: Seguence Analysis of dd29 (MUC~2)
The sequence of dd29 revealed that it was amplified as a result
of priming of random 'I Omer at both ends of the PCR product and that it did
not contain a 3' untranslated region (3'-UTR) or poly A tail. Screening of an
UC cDNA library with ~dd29-specific primers extended the sequence 840 by
in the 5' direction and 800 by in the 3' direction to the poly A tail (Genbank
Accession Number AF147790). To confirm contiguous cDNA sequence,
primers MUC12TOTF'1 and MUC12TOTR1 were designed to produce an
expected PCR product of 1532 bp; primers corresponded to bases 230-250
and 1742-1762, respectively, in SEQ ID N0:6. In a stringent touchdown PCR
amplification procedure an intense discrete product of the expected size was
identified from normal colonic cDNA and cDNA from the Caco-2 colonic
cancer cell line. This reaction confirmed the reported MUC12 cDNA
sequence.
Conceptual translation of the composite MUC92 cDNA reveals
the presence of serinelthreonine and proline-rich degenerate tandem repeats
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
43
(FIG. 2) consistent with this protein being a member of the epithelial mucin
family. The deduced 28 amino acid tandem repeat structure is shown in FIG.
2. Following the mucin-repeat domain, MUC12 contains two cysteine-rich
EGF-like domains separated by a 150 amino acid non-mucin-like sequence
(amino acids 261-410) containing five N-glycosylation sites and a potential
coiled-coil domain. The second cysteine-rich EGF-like domain is immediately
followed by a putative transmembrane domain containing 26 hydrophobic or
uncharged amino acids, and a cytoplasmic tail of 75 amino acids at the
carboxyl terminus.
Sequence alignment of MUC12, human MUC3 (hMUC3), rat
Muc3 (rMuc3), mouse Muc3 (mMuc3), human MUC4 (hMUC4) and rMu~ is
shown in FIG. 3. When aligned by the transmembrane amino acid
sequences, MUC12 was found to have areas of significant homology to
rMuc3, mMuc3 and hMUC3, including perfect conservation of eight cysteine
residues in the second EGF-like domain. With inclusion of three small gaps,
each of these cysteines also align with those in rat and human MUC4.
Interestingly, all six mucins contain a conserved EGF-like sequence of
Cx(5)GPxCxCx(9)GExC. Furthermore, there is some (4 out of 8) conservation
of the cysteine residues between MUC12 and the human and rodent MUC3
and MUC4 mucins in the first EGF-like domain.
EXAMPLE 11: SeQUence Analysis of dd34 (MUC99)
Clone dd34 (544 bp) was also obtained as a result of priming
of random 10mers at both ends of the PCR product. Screening of a ~gt11
human fetal brain library yielded two positive plaques which hybridized to
dd34, clone li5 (1045 bp) and clone 2 (1043 bp). These two clones
represented opposite ends of a 2.8 kb partial MUC9 9 cDNA sequence
(Genbank Accession Number AF147791 ), the finking of which was
established by PCR (see Methods). Conceptual translation of the MUC91
composite is shown in FIG. 4. The entire 957 amino acid sequence consisted
of serine, threonine and proline-rich tandem repeats of 28 amino acids in
length, consistent with it being derived from a large epithelial mucin. The
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
44
deduced tandem repeat structure and consensus repeat sequence for
MUC11 is shown in FIIG. 4.
EXAMPLE 12: Chromosomal Localization of MUC11 and MUC12
DNA fragments excised from dd29 (720 bp) and dd34 (530 bp}
were nick translated with biotin-14-dATP and hybridized in situ at a final
concentration of 10 ng/N1 to metaphases from two normal males. The
fluorescence in situ hybridization (FISH) method was modified from that
previously described (fallen et al., 1990, Ann. Genet. 33 219) in that
chromosomes were stained before analysis with both propidium iodide as
counterstain and DAPI for chromosome identification. Images of metaphase
preparations were captured by a cooled CCD camera using the CyroVision
Ultra image collection and enhancement system (Applied Imaging Int Ltd,
Sunderland, U.K.).
Twenty metaphases from a normal male were examined for
hybridization to dd29 and dd34 probes. For both genes, all of the
metaphases showed strong signal on one or both chromatids of chromosome
7, at band 7q22 (data not shown). A similar result was obtained using
metaphases from a second normal male.
EXAMPLE 13: Production of monoclonal antibodies reactive with
MUC11 and 12
The following peptides were conjugated to keyhole limpet
haemocyanin (KLH} with the heterobifunctional cross-linking agent m-
maleimidobenzoyl-N-hydroxysuccinimide ester using standard techniques
(Harlow, E. & Lane, D. Antibodies: A .Laboratory Manual, Cold Spring
Harbor, Cold Spring Harbor Laboratory, 1988, which is herein incorporated
by reference):-
MUC11: CFHSRPASTHTTLFTED (SEQ ID NO: 17); corresponding to
part of the degenerate tandem repeat region, specifically amino
acid residues 690-705 deduced from the partial cDNA MUC11
clone, 'with an N-terminal cysteine residue added for
conjugation);
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
MUC12: TYRNF1-EKMNDASSQEC (SEQ ID NO: 18); corresponding to
part of 'the N- glycosylated region, specifically amino acid
residue:. 286-302 deduced from the partial cDNA MUC12
clone, with a C-terminal cysteine residue added for
5 conjugation).
One Balblc mouse was immunised with each KLH-conjugated
peptide as per the following protocol:-
Day 0: KLH-conjugated peptide was diluted to 100 NglmL in
phosphate buffered saline (PBS) and mixed with an equal
10 volume of complete Freund's adjuvant (CFA). Each mouse
was injected intra-peritonealy with 0.5 mL of this mixture.
Day 14: Each mouse was immunised as above but peptide was mixed
with incomplete Freund's adjuvant (IFA).
Day 33: Each mouse was immunised as on day 14.
15 Day 43: Each mouse was bled from the tail to assess antibody
production by ELISA (see below).
Day 53: Each mouse was injected intra-venously with 100 NL of peptide
at 100 Ng/mL in PBS without adjuvant, and with 700 NL mixed
with IFA intra-peritonealy.
20 Day 56: Mice were euthanased, and the spleen removed for fusion with
myeioma cells.
Splenocytes were fused to Ag8 mouse myeloma cells at a ratio
of 5:1 with polyethylene glycol using established methods (Harlow & Lane,
supra).
25 Specific antibody producing clones were screened by a solid
phase antigen antibody capture ELISA with the immunizing peptides bound
to polystyrene assay plates using established methods (Harlow & Lane,
supra). Positive clones were expanded, retested for specific antibody
production and recloned by limiting dilution. Clones were further tested for
30 reactivity with paraffin embedded normal colonic mucosa.
93.1 MUC19 and MUC92 reactive hvbridomas
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
46
Two hybridomas, one reacting with each of MUC11 and MUC12
peptides and with paraffin embedded colonic sections are described in Table
2.
13.2 Immunohistochemical detection of MUC11 and MUC12 in
normal colonic epithelium using antibodies M11.9 and M12.15
Paraffin sections (4 Nm) of normal colonic epithelium were
dewaxed with xylene, hydrated in a graded series of ethanol to water and
treated with 0.1 U/mL neuraminidase (Boehringer, Germany) in 50 mM Na
acetate, 150 mM NaCI, 100 mM CaCl2 buffer, pH 5.5 for 1 hr at room
termperature to remove sialic acid groups. Sections were then treated with
1 % H20z, 0.1 % NaN3; in Tris buffered saline (TBS) for 10 min to quench
endogenous peroxidase activity, and non-specific protein binding blocked
with 4% skim milk in TBS for 15 min. Monoclonal antibodies M11.9 and
M12.15 were semi-purified by PEG precipitation and diluted to 5-50 Ng/mL
in TBS/50% non-immune goat serum and incubated for 2 hours overnight at
room temperature. Sections were washed once with 1 % TX-100 in TBS for
5 min and then twice in TBS for 5 min. Sections were incubated for 30 min
at room temperature with pre-diluted biotinylated goat anti-mouse
immunoglobulins (Zymed, USA) and then washed as above. Sections were
then incubated for 15 min at room temperature with pre-diluted streptavidin-
conjugated horseradish peroxidase (Zymed Laboratories) and then washed
as above. Peroxidase activity was detected using 10 mg/mL 0.05%
diaminobenzidine, 0.03% HZOZ in Tris saline, pH 7.6. Sections were
counterstained with haematoxylin, dehydrated with ethanol, cleared with
xylene and mounted in DePeX.
M11.9 reacts strongly with colonic epithelium, primarily with
columnar cells of the surface epithelium (see FIG. 9A). Both goblet and
columnar cells deep in the crypts are not stained by this antibody (see FIG.
9A). In surface epithelial columnar cells M11.9 reacted with the perinuclear
cytoplasm, lateral cell membranes and most strongly as granular staining in
the subapical cytoplasm (FIG. 9B). This localisation suggests reactivity with
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
47
precursor in the rough endoplasmic reticulum (perinuclear staining),
reactivity
with mature mucin on the lateral membranes at columnar cell junctions with
other cells, and reactivity with processed mature mucin in granules for apical
secretion or incorporation into the apical cell membrane. This pattern of
reactivity is distinct from that seen for other known mucin core proteins.
M12.15 also reacts strongly with colonic epithelium, and like
M11.9 it reacts primarily with columnar cells of the surface epithelium (see
FIG. 9C). However, M12.15 gave a more diffuse cytoplasmic staining pattern
than that seen with M11.9, although, like M11.9, the strongest staining was
in the apical cytoplasm. ,
Imrnunohistochemistry in normal colonic mucosa with these
antibodies demonstrates protein expression of the MUC11 and MUC12 gene,
supporting the mRNA studies. The co-expression of MUC11 and MUC12 in
normal colon is also consistent with the RT-PCR data showing similar levels
of relative expression of these two mucin genes in different regions of the
intestinal tract.
EXAMPLE 14: Expression of MUC11 by in situ hybridization
74.9 Methods
Optimisation of conditions for in situ hybridisation, outlined
below, was based upon published techniques (Rex & Scotting, 1994,
Biochemica 3 24, which is herein incorporated by reference). Riboprobes
were made by in vifro transcription of DNA with SP6 and T7 RNA
polymerases and incorporation of a digoxigenin-labelled uridine triphosphate
(DIG-UTP). The orientation of inserts in pGEM-T was established by
sequencing. Insert in the antisense direction and thus complementary to RNA
template was the hybridisation probe and insert in the sense direction was
used as a negative control. 1 mg of purified linearised plasmid pGEM-T was
labelled in the presence of 1/10 volume 10 x transcription buffer, 1/10 volume
10 x NTP mix (1 mM ATP, CTP, GTP, 0.65 mM UTP, 0.35 mM DIG-UTP),
10U RNase inhibitor and 40U of either SP6 or T7 RNA polymerase. The
reaction was carried oust at 37°C for 2 hours and terminated by
addition of 2
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
48
NI of 0.2M EDTA. Probes were ethanol precipitated with 1 /11 volume 4M LiCI
and placed at -20°C for 2 hours. They were then centrifuged at 12,000 g
for
30 min at 4°C. Pellets were washed with 70% ethanol, air-dried for 10
min
and resuspended in 100m1 of RNase-free water.
Paraffin-embedded functional tissue specimens were sectioned
at 4 Nm onto sterile water and affixed to Vectabond-treated slides (Vector
Laboratories). Sections were dewaxed in xylene, rehydrated and then
incubated for 5 min in 0.2 N HCI. HCI treatment contributes to an
improvement in the signal to noise ratio by extraction of proteins and partial
hydrolysis of target sequences. Slides were washed in sterile water for 5 min,
followed by 5 min in I'BT (PBS and 0.1 % Tween 20). Sections were then
incubated in proteinase K (5 mg/ml) at 37°C for 15 min and washed
briefly in
3 x PBT. They were fixed in 4% paraformaldehyde for exactly 20 min and
prehybridised for 4 hours at 70°C in hybridisation buffer (50%
formamide, 5
x SSC, 1 % SDS, 500 mg/mL tRNA, 50 mg/mL heparin). Denatured probe (0.5
mg/section) was added to hybridation buffer and sections hybridised
overnight at 70°C.
Sections were washed in 2 x wash solution 1 (50% formamide,
5 x SSC, 1% SDS) at E35°C followed by 2 x washes in wash solution 2
(50%
formamide, 2 x SSC) also at 65°C. Sections were then incubated with
anti-
digoxygenin-AP antibody at 1/2000 in PBS overnight at 40°C.
Excess antibody was removed by 3 x 20 minute washes in PBT.
Sections were then washed 2 x 20 minute in NTMT buffer (100 mM Tris, (pH
9.5), 50 mM MgCl2, 100 mM NaCI, 0.1 % Tween 20, 2 mM levamisole).
Hybridisation was viisualised with NBT and BCIP overnight at room
temperature. The reaction was stopped by immersion of slides into 1 x TE
and sections lightly counterstained in eosin. Sections were then dehydrated
through ethanols of iincreasing concentration to xylene and mounted in
DePeX. Slides were photographed within 3 days due to fading of the signal
with time.
14. 2 Detection of MUC 9 9 mRNA b y in situ hybridization
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
49
Intense signal for MUC11 was observed in the columnar cells
of the surface epithelium in all specimens of the normal colon. However, it
was not possible to conclusively identify positive signal in the goblet cells
of
the colonic epithelium. Transcripts for MUC91 were not detected in adjacent
carcinoma of several functional tissue specimens (an example is shown in
FIG. 9D), thus confirming the findings of the differential display and
Northern
blot analyses.
EXAMPLE 15: E~rpression of MUC~1 and MUC92 in normal colon
by RT PCR
The results of RT-PCR experiments to determine the
expression patterns of MUC19 and MUC12 genes in normal colonic
epithelium are shown FIG. 10.
MUC17 and MUC92 are predominantly expressed in the colon,
although the data in FIG. 10 show that in fact their levels of expression vary
within the colon. In this regard, a progressive increase (3-4 fold) in the
expression of both MUC99 and MUC12 was seen from the right colon to the
rectum.
EXAMPLE 16: Expression of MUC1~ and MUC~2 in CRC by RT
PCR and Northern hybridization
The expression patterns of MUC19 and MUC92 in CRC were
investigated by RT-PCR, and the results are shown in FIG. 11. After 40
rounds of amplification, MUC97 expression was observed in all CRC cell
lines under investigation. Similarly, MUC92 expression was observed in all
cell lines, although two cell lines, SW620 and SW116 revealed low levels of
expression.
These observations, together with the downregulation data,
show that although these genes are downregulated in CRCs, they are still
detectable in CRC cell lines. In contrast to the normal gastrointestinal tract
and IBD tissues, the expression of MUC19 and MUC92 in CRCs and in CRC
cell lines show patterns of expression distinct form each other.
Referring to FIG. 14, the results of Northern analysis with a
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
dd34 (MUC19) probe showed that in nucleic acid extracts obtained from
colonic tissue of four (4) of the (6) CRC patients tested, the level of MUC91
mRNA expression was lower relative to normal colonic tissue from the same
patients. Similarly, MUC12 mRNA was downregulated in three (3) of five (5)
5 CRC patients (data not shown).
Such quantitative (e.g. downregulation of these genes and
differential downregulation expression patterns of MUC1 ~ and MUC92) and
also qualitative changes of these genes, e.g. mutations, could be used for
diagnostic and prognostic testing in CRC.
10 EXAMPLE 17: Expression of MUC19 and MUC~2 in !BD by RT PCR
The expression patterns of MUC11 and MUC12 in IBD were
investigated by RT-PCR, and the results are shown in FIG. 12. Cytokeratin
20, (CK20) a colonic epithelial marker, was employed as a loading control
due to the variable epithelial content of IBD tissues. 'N' denotes tissues
15 which appear macroscopically normal and 'D' refers to tissues reported to
have IBD. 'CA' refers to the caecum, 'CO' the colon, 'LC' the left colon, 'TC'
the transverse colon, ''RS' the recto-sigmoid colon, 'SI' the small intestine,
'IL'denotes the ileum and 'IP' an ileal pouch.
Two patients, patient 1 and patient 4, show 3-4 fold upregulated
20 expression of MUC~7 and MUC92 in diseased tissues, compared with the
same intestinal region observed in the 3 normal controls. Patient 6, who has
a history of severe ulcerative colitis in the right colon, also revealed
approximately 3-fold upregulated expression of MUCH 9 and MUC12
compared to the right colon observed in the normal controls.
25 There is coordinate regulation of Mucin expression in the
normal gastrointestinal tract as well as in IBD tissues and upregulation of
both Mucin genes wars observed in 3110 patients. Given the documented
quantitative changes in the expression of MUCH and MUC92, their
expression levels may form the basis of useful diagnostic and prognostic
30 testing for this disease. Qualitative changes in these genes, eg. mutations
may also be useful markers for IBD.
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
51
EXAMPLE 18: Expression of MUC11 and MUC12 in BC by RT PCR
The expression patterns of MUC11 and MUC92 in BC tissue
were investigated by Rl'-PCR, and the results are shown in FIG. 13. After 40
rounds of amplification, MUC99 expression was identified in all breast cancer
cell tines under investigation; at low levels in BT-20, DU4475, MDA-MB-435
and ZR-75-30 cell lines and at higher levels in the remaining nine cell lines.
Eight of the cell lines .showed MUC91 expression higher than the normal
colonic cDNA positive control. MUC19 is clearly highly expressed by most
breast cancers and may impact upon the behaviour of the breast cancer
cells. MUC91 may also be secreted by breast cancers and detection in serum
could form the basis of diagnostic and prognostic testing for breast cancer.
MUC92 expression was only readily identifiable in one breast cancer cell
line, MCF7, although faint bands were observed for BT20, KPL-1 and MA11
cell fines.
EXAMPLE 19: Experimental Summary
Differential display has been used to identify two partial cDNAs,
which encode novel colonic mucin-like proteins. Expression of both cDNAs,
designated MUC11 and MUC12 by the Human Nomenclature Committee,
was commonly downregulated in colorectal cancers.
MUC97 and MUC92 were mapped by FISH to chromosome
band 7q22. The location of another mucin gene, MUC3, at 7q22, suggests
the identification of a new cluster of mucin genes at this locus.
Interestingly,
four genes encoding gel-forming mucins are found in a cluster on
chromosome 11 and these genes appear to have originated from a common
ancestral gene. WhilE; the mucin cDNAs mapped to 7q22 most likely
represent separate genes, it is also possible that they are produced as a
result of alternative mRNA splicing from a single, large mucin gene. Northern
blot analysis for MUC91, MUC12 and MUC3 shows that these encode large
transcripts, estimated to be greater than 12 kb.
Multiple tissue RNA analysis showed no cross-reactivity
between MUC11, MUC92 or MUC3. MUC91 and MUC92 showed
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
52
predominant expression in the colon, while MUC3 was predominantly
expressed in the small intestine and at very low levels in the colon. This
expression pattern constitutes an important point of distinction between
MUC17 and MUC92 genes of the present invention and MUC3. Furthermore,
the sequences of MU'C19 and MUC12 are not homologous with any other
human mucin genes, but show some degree of similarity within their variable
tandem repeat regions to each other (71 % aver 653 bp). However, their clear
differential expression patterns in normal and tumor tissues as well as tumor
cell lines, show that they are distinct from each other, and from MUC3.
While both MUC11 and MUC12 contain variable repeat regions
typical of mucins, MUC12 is putatively a transmembrane mucin with features
suggesting an involvement in growth regulation, a largely unrecognized
function in human mucins. MUC12 is only the fourth human membrane-
anchored epithelial mucin to be described to date, along with MUC1, MUC3
and MUC4. MUC1 has been shown to be involved in cell signaling via
multiple tyrosine phosphorylation sites on its highly conserved cytoplasmic
tail (Zrihan-Licht et al., 1994, FEES Lett. 356 130). At its carboxyl
terminus,
MUC12 possesses a cytoplasmic tail containing a YNNF sequence (amino
acids 557-560 in FIG. 2) which is similar to motifs recognized by SH2
domain-containing proteins (Songyang et al., Mol. Cell. Biol. 14 2777),
suggesting that MUC12, like MUC1, may be involved in signal transduction.
The deduced amino acid sequence of the partial MUC9 9 cDNA
was composed entirely of serinelthreonine-rich tandem repeats. There is a
similarity between the tandem repeat consensus sequences of MUC11 (FIG.
4) and MUC12 (FIG. 2) and these also show limited homology to the MUC3
repeat (ITTETTSHS'T'PSFTSS). These similarities are consistent with
evolution from a common ancestral gene. MUC11 is more widely expressed
than MUC12 and MUC3 however, with RNA detected in gastrointestinal,
respiratory, reproductive and urinary tracts, and unexpectedly in the liver
and
thymus.
The physiological roles of MUC99 and MUC92 in colonic
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579 _
53
epithelium are unknown. MUC11 and MUC12 are commonly downregulated
in colorectal cancer suggesting they may play a role in epithelial cell growth
modulation and/or differentiation. At present, it is not possible to comment
on
whether downregulation of these genes is related to stage of tumor
progression, as only ;?0 patients were analyzed in this study. However,
downregulation appears to be so frequent, that it may be an early event in
tumorigenesis. Given the co-localization of the MUC11 and MUC12 genes
on chromosome 7q22, it is possible that their expression is co-ordinately
regulated and hence they are simultaneously downregulated in a large
proportion of colorectal cancers. The effect of downregulation of these
mucins on normal colonic epithelial cells could be substantial. Mucins are
believed to protect epithelial cells from attack by pathogenic organisms and
from mechanical and chemical damage. Therefore, reduced expression of
these mucins could expose colonic epithelial cells to the harsh environment
of the intestinal lumen. /Furthermore, loss of a transmembrane mucin such as
MUC12 may also contribute to loss of critical cell signaling.
The location of these two novel mucin genes on chromosome
7q22 may have significance for two non-malignant epithelial diseases where
aberrant mucin expre:>sion and/or function is a recognized component of
pathology, namely, inflammatory bowel disease and cystic fibrosis.
Susceptibility genes for inflammatory bowel disease have been located to
chromosomes 3, 12 and 7q22 (Satsangi et al., 1996, Nature Genet. 14 199).
Thus, MUC11 and MUC12 must be considered candidates for involvement
in inflammatory bowel disease given their chromosomal localization,
expression in normal colon, and the documented alterations in mucins in this
disease (Rhodes, 1997, QJM 90 79). Mucins may also play a role in cystic
fibrosis as patients wifh the same CFTR gene mutation do not demonstrate
exactly the same phenotype in terms of mucus obstruction. The existence of
modifier genes has been postulated and mucin genes are obvious
candidates (Harris & Reid, 1997, J. Med. Genet. 35 82). A murine Mucin
gene that shows C-terminal homology with MUC12 has recently been shown
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
54
to be a major constituent of obstructive mucus in the gastrointestinal tract
of
mice with CF (Parmley et al:, 1998, J. Clin. Invest 102 1798).
The CFTR gene lies in the adjacent chromosome band (7q31 )
to the MUC3, MUC99 and MUC92 genes. While the significance of these
findings is not clear, MUC19 and MUC92, which are expressed in many of
the tissues affected by cystic fibrosis, should be considered as candidate
modifier genes involved in the aetiology of this disease.
Mucins are encoded by large genes which have proved difficult
to clone by conventional methods due to the repetitive nature of their tandem
repeat regions. Hereinbefore, the present inventors have unexpectedly
identified by differential display two partial cDNAs which represent novel
mucin genes that are predominantly expressed in colonic epithelium, both of
which are downregulated in colorectal cancer. In this regard, MUCH and
MUC92 differ from the other mucin gene located on chromosome 7q22,
MUC3. These findings together with the sequence homology between the
MUC12 EGF-like domain and EGF receptor-binding growth factors, suggest
MUC11 and MUC12 may function as growth regulators in colonic epithelium.
Downregulation of these two novel mucin genes could be an important and
previously unrecognized step in colorectal carcinogenesis.
Those skilled in the ark will appreciate that the invention
described herein is susceptible to variations and modifications other than
those specifically described. It is to be understood that the invention
includes all such variations and modifications. The invention also includes
all of the steps, features, compositions and compounds referred to or
indicated in this specification, individually or collectively, and any and all
combinations of any two or more of said steps or features.
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
''~viY~:i!Sv:~'..
' f: '
:~~:xa: ~'.<~.
;;;~ i~.xN~y~,l f1
Iv. is
:'!,:?:%vri mjy,'.:.4
' Viis "::Gx
i'i:
;'.',.h''' Lx
k.~ .
:s 'Y,.
:i'.~'
.iv: ~ v.
Viriiifs
-'~i'..'.'SI6~~(n~~~(n
'~.'; ~ E E ~ W r a~
E E E ~E E E E E
: ..~,;:x E E E
'~.v~ZZ;ZZZZZZZZZZZ
. ,~;:
~::x~'~ ~ :;.
w.< ,.:.
~,~, .,~.
::x. }ff
'i,fY k~
:a: Ni:
%; x~t: .:.~:
~f.~Y: .~'i
.,'.%.,ryi~,$xi'..~~. . ::
;:0.~:
~ ....
~~". O U O
~~.."..,.~.~#'~gOOC~NC_c~OCN CCC
.. ~ .~.::.. C C ~p) C V C 'U C N 'C __
4
~c*''f' ::, ~ i: ~p p~ Q 'C ?. ~ E N O _O _~, .-. O O
h f.. '' ~ L_ ~ (B
s. . . ~ tn O- V t O ~, O O C
;~> Yes, ~ u~ p ~ ~n ~, E C
..
:~~ _
~~~.vi ~ '' E a~ '~ ~ a~ °' ~ °' ~ E ~ a ~ E
i:,i'' .~ ~ E ~ ~ E w E E Z Z E E ,
s;~:'..,.~'#'fZZ, ,Z ~, ZZ ~' ZZJJZZ' Z
%r J i Z J Z J ~ ~ ~ J
.a~;.''~ ~;s ,J ' J , J .J 'J
J i . ' ~:~. J J
'r
a,~ > f~,;,:
'# ~:' ~,:
~;;<
':~:,. , ~'r..::
.. . ,.,.
''xs~:
.,xvs:: y..
~fro
it~k~:.<,:
:i;:%Y:'.r. :i;
.~,:~, .
y'y'~.;i f! fj~~.
'~~:s~:?': ~ O ~ X C ~ ~ V) C
'~.''cf:;o'.,v.~i:6.T;','J~r'~:
:~:6.~.~.s:~ a ~O OQ~~~U
:>,x.:::~.:, a o~ v a z ~ U D D O D D
:v U
~<. :..~:
;,~ _. ,...
tiv:: .
~'' ~ :.,,sh::
%nfr~'.y'%.'i~.: a'..
~. ,':
. "r.''.~~.. i. :. .,
:'li~::~..i:'.~~;fEø:~a:
:Gn .~ f,. ';;.:iu:
.~,~: ff;5:; i.fi.
::C4x:;~:Cf F':
::':::< i~:,:;
v
ix, i. i...
:.i4x:f::::
%.;..i~ f-.::;
:..x~;.
~i, i:x.. x~ ~ '':.~i% r
:::':
;~: , ,.i.::::.;~. .O ' mQ
;;~~~ '';s;.x, ~, N O N
:~r;. ~~;;~?. U ~ C ~ ~ C C :D
:; 'f ~~x :, i
<~ :%:
''r' V ?, p ~ '' '' ~ C _(11 N O V O C
. ~' ~ C_ ~ C_
_ ~ C
c: ~ ''~i sj, -C ~ O >~ O ~ (If ~C V
f' . i~w'~:'' '. ''r O O ~ O ~ X 7, ~E ~ N
~s.. a :.. .~ O
':~ :~ ;: ~ ~ V C ~, 7
~.: O ~ >, -n ~ O ~ N O ~ (a fl.~ (~
'CZSUyCQ
: E O C U C U U O ~ ~ N C1
_ E U o
?c' ~c,,. x;:~ ;: ~ C
',;~%s'~:'i~ ~ '~ '~ O V U
.:::: ~ ~E O O
'~:e::::a::
sse' ~,~.~i' '.>: ~LJ
f,
~::~~,. a
:. i.
..' i: ;
w: ~N.:.:.i,:; ~ ~'i:
-'"'.. ,q i':::.
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
56
;::::.::..~.
~::
~:~:~.~:~
. .:
'H;~51f'r
','yiy~:;
%$.;:~;;.,
n;.
.:. f
$ ~fsr:.
' ::
4:
;fi
'i
'
J:
~
>
S
r
t
~
.
v i~ tt
;J a~ ~ a rn rn o m c
V a~ ~: ' ~a o x
r
~
.
.: ;
E
;
;
"
:~
. .c a N . ~ , > ~. .Q a~ c~ ~ ~ '
r:. , a .,... > a~
. ..
;.~ .
,f ~''~'~.'
O
.C
~ Z Z Z c'
3
~Z Z Z Z '. Z
f~~ ~t~
r
Ns
f~ .:
i~ '%::
%..~'
.iJ
fi ~~.
.
s ...
>it. ~
',..<;
tt.;'4
~ r
1
~itS
i. i
kt..
7 Ff:i:
'~':
S
?,':R:,y
:
:
:
: - _ . . _
i';:i:Y:
... ...
'k#
f
Y
f
'
f ~~''
:
f
.r:f...u
i:%~: C
. :.. C C O C ~ ~ C C
C
. ,,. C O O C (a N V ~ >,
~
. .
: _
<y i:!:;.;~ C C C Q ~ C >, ~ O ~ c0
~
C
t'i
...'. (6 " N m .O
i. - ~ 'W.O O ~ O O L ~ >,
.'..
t
.. . .
=
c ~, a~ ~ a ~ ? ~ .~ c c N o
. . .
~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ Z ~ E o
v
~
~ L >, ...
}J. ~
:
. '
~ _ ~ ...,~ ~ '~ N >, ' ~ p ~ ~ V
~'' fl. O
~
., . >' E o ~' E >' ~ cu ~' >' a
4
,..,'.
:yo
~' _..E ~ E , ~ a~ E ~ ~ ~ c ~ o
' J
. t. >. N Z Z ' ~ ~ Z ~ ~ J ~ ~ ~ >,
.: .
'
~
..~'f ~ ' J Z Z Z ~ ' E .-~ , ~ s
Vii: ~ ~
Z
~
r:.t ., , J i i J - Z ~ N O N
J E
~f Z J, ' Z
:::~
'~
.
y, ' .J J '
:
ti i
i J -1 'ZS
. ZS ~ t5
%;~~ ..
'
:
%
'%'%
~
,
.
,
;
i~s: iv;
v ~~
'
~
~
t
::
:...
'
. W:~:,.
.::~ f~:
:h:i.~
~f'
~k
t~
i:
r:i
f! ,N m
f.
,
vb
~
i _
Yi' ~ N O ~ _ ~ O Q. ~ O ~ N
l
~ '
;~~ ~ _ _>, . ...... p >
~ ~
~
'':~''~p p p p p p p p p p p p
''~
.' p
f
r;:;r...
f s;
.
m:rr:
'w~~.'
~ rr:M:
f~~
f'%::
.
.~3~i~r
,c:
~'
s
::~~:<
;;~,
~:,' .
w::
:, f;.y
. ::
'': N
~Y~2
r~
F
:
p~
~
.iYf
/.. .
~
F
'k' . O y C C
f'.~ta. i
;
:
r:
~ O C , C C C
~,..,
:f..
f4 ~ ~ . C ~ ~ C
(~ ~
:: ~ :, ~ ~ ~ L
V C ~ C ~C ' _ N
U Q O
.
_ 'O ~ O = N ~
~' f~%. N ~V = ~ . ~ ~ ~ >
':<.'.t...:s><:
'
'
.
''. ' :.. ~ ~ ~ O ~ ~ O ~ _ ,
s' ~' o _~'"-'E c a ~n ~. a > >
'.. .;
~
~'u~ m
.' L O ' a~ , ' L ~, , 0 u~ o
..r
,r> .~ p c~ E o Q p p ; o E E
: ~.~
:. o p o o p a '
Y~
'
: ~ is
: f ::
.: o o '
;.:. p
~~.~,
.
.
. p
~~f~
,r~ ~f
.
,.
.
~~~
;'.
~r 7 J
t...;
.
; ,~:
~:
.:~
~
.:
:. .
..:...
,.. .
:..t:a:....;<..:.~.
Y.r~::::
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
57
r. v .:
fi. .
~,v
~: >.:.
x ~
:'::~;
>:: ;~'~.
err: ~::
ry.
4. .,st,%.
~~
:i
:r~"
r
.
:
:~.".''i:;'.~
ii$f""'
t i
,'s~'
ii?:
L
. Z ~ E Z
.
~ ~ Z Q Z Z Z Z Z Z Z Z
g
'~'~
~
~ Z
.
t
~"~v~'
r
;
. ,:;
.;rs>
:
~
,' :~
~'
.,.;
';'
;~y::
rF?
.
;
:.<.:
:
~Fy,
:~.v.
.
:
:
,. . ..::
3 ~~~
i
g:.::r
:! .. ~
r' :
~.:
: i.:. wr
J c O C C O (Q C C C C
$ 's..r,,
C
.;sv."
,y C '~~ ~ U (~ 'V y O O O
',:::
iw,r
C
'.'W ' V V
-! i~'Tr: =~' N ~ ~ >' C C C C
' ' _~
'" :. E ~, >' ~ C ~
~
. f~ ~ ~ L ~ ~ ~' >
~ ~
C V ?, >, ~, _ ~. ,
'~,~ ~, >. '~ ~' O ~ L .C >' >' ~ O) p~
~ ~ ~ O O _ N N
. ~
~'~
~,s:" C . ~ ~ ~ ~ Q X U
'r.
r
~
~~l
%W ~ o o ~ E ~ N ~ a ~' E ~ _ ' -'
r
~~
J _ , . a
r :.. - C C Q ~, 0 C
.~''~.5: ~
.
,, ~.~ ~ _ C ~ . O N E n 0 0 0
. . .
. L E ~ cU .C~ .
~ ~ c ~ ~ C ~ ~ -n V j >,
i~ '' f
~
~
.:. ~ N _ , Z ,
f . E ' ~ ~ , U
,... ~t N ~ ~ ,
',>.3~~:_
....
o ~ .....~ c~> E ~ N Z Z Z Z
~ ' . N
< .a;:
r:
'
~
'
, t5 Z Z .; c~f ...
.
%
.~~..v~.
E
.
:;~~: Z Z Z Z Z Z
'ft5
.
sx;>..;
",
.,.;
.
:r:,~
t,<~:
'
.$r <
.' ;.:
f
:
::
.
,
;.
'.~Y''.
'~'
~~ ,,''..
. fi ~:
~:
..
.
.. . _,
<~.'~~.,:'
:;~:
:,::"~:,':,''.~v.'~;
>':'.~:
:
N:~"
. a u~ o u~ o o v~ o C O O
~
;~,~$C
..' w v v~ s- . a~ >. E o Q a ~
.:: r~:>{:m
~
c ~ _
a ~ E E E E
r, E
:
r: '''<c~
. E E E E E E E p
a.;;~~~.,.
E
~t~~r'~,~pD p p C~ p o p p p p p p p p
f:~~'F;.
::,
~<
~
:!' '
v,.'<
,.
'
;:.,.:
;,,~.
av::'~
:,:: ,~~f:~
:r~ 'r.
:;''.'":H'.~','r'::
'~'~ly
y:
'
", N C C
~
i,...
.t:::;.
O
:fir < O
.:. %:
f:.a;.k.,:
~
r
~
..:. ~ C C ~U C N C (a C ' -C C
::y -
.:
3v~
~ ~'
%
~ 'C . E _ O '~jC O ~ ~ C C a N
%% O TJ
N
'. IU N ~ ::. 0 0 ~ ~ O ' ~ ~ O
<..::
'~
, a. t~ ?, ..rC >, N
ta,:
~
:... ,
.,.
'~
; ~ ~ ~ p ~ O ~. C Q _N L ...~,.
.ri.:,. V ~ > O O
;: fl.
~ N m " t~ , ~
'
s.O j, ~' j, '' j L .~ ~' Q ~ ~ ~' j C
3v ' .:.
',
>, L ~ L ~ , ''rN ~' ~ N . L , .
i ~ N
'
't
'
N
s :~ .,.r~ r ~ . ~ E .C ~ y, E ~
'.,:i ~ ~ ~ ..
.C
,'
''
fi
': ' ~...
r .. : L ~ E
' E ~ ~ ~ 47
:::.r.
a~
E E E ~ ~ ~, ~ is , E '
v'~~C ~ ~ ~ ~ ~ a
'
Y ~ ~ R3 ~ t5 p t5
'~~~''"~ E D p t5
~'.
~
.
'fi:"
:
.;. p p p ~ p t5 p '
.~
.
.
.
~
~
'f~;~':4:,~
,::',; p p p p
D
>..~.~,..'
~
.
p
....
''~
' "
"
.....~:.
.
: ...
.........
x
:...
'
..
. .,..
~
'':
:
;;
:
,
f
~
v: ,
,:
,
,
.
..
.
.:
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
58
:~~.k.ir3:~:::
4
..
<:
4
:
~.>.
:4
:)tY
~
-
1',k.
~',~
/
nn~
~u.
:.
w.
.
r
.'i
~:
:
'ihv ' ~ '-' C
~i.';s
'''fir';
:''
v
~~
:
' 0 j O 'O E O ~ ' ' N a .a O N s O
~
~: o c ,c ' ~ a~ ' m E a a a
:,.,: ~ n c~ ~- v, r ~ v~ E U E
y~'..::~y'~ c Z ~ Z . Z Z Z Z Z E c E E c
Z Z Z Z c
:Ir z p z p p
'r:
,
~.:
:M:i,.
.
:>:.
F
rf
%
.
:
'
:
u
<:
~j'
''
:.
%'
k'
%,<:
2;~
t
~r
::?;Y
a;
>,:;~.:.
:~'.''yir
.<~','';..:
I
S<
,
<::::5;. C
,
S.T'r;
~ C C N O C O C N
~ ' ~U ~ C '
N O _ _ _
s: N U 7, U C C (a O C C
%':% V . '
~:
~
'
. _ C
, C C >, ~ V U ~, V >, O ~ O
'~. m
;;:;,;.
V C ' V - ~ >, ~, .",~ f~ C
O)
~: ~, (~ ~, r. ~ p~ p) ~ ~ ~
~?
..
:;:
:: > o
::.:; s , c
:~.: ~, W - ~, ~...o o ~ >, c ,~ c ~ o c a
::~. ~
~ >. ~ ~ ' a ' s . 'r a
:.~.r a " E
;~
: a~ ~ ~, ~ a '- o ~ ~ o ~ s
.<
. ~ ~ ~ a
.' - 0 0 ~ cu c o ~c'o ?''~ ~ a ~
~ o s: _ ~ '- O N ' . U S E
O
,, DL
f. U ~ >' O
'
'
a - ~, ~ V ~~ ~ ~ ~ > : 'c ~ ~ s E
;:~;,.;yy ~ ,
,:
~
.
.
:
:.
'
.~
, >, , , o
, ~ M z ~
;
,
.
s
:x,.:.,xY z Z ~ N~ M ~ ~ E ~ z
~~ '
'
z z Ni ~ ~' ' . '~ ~ p E
. -.-
~.:.,
.
.:::;
o
:;~::. . ri M Z ~ ,
.
.. ... Z Z Z
Z
'':Y W z
:
Y:.%
Ai..
:il
5t~y,
4.1,'
;.
i
,
.:
:;lfb;Y'.$';<:
.f;>f,:
..
r
.
;
<f:''~~'~,'
:::k
,::".'!
h;.
~r
...
'.'
<;I.
Y<:;::
>~~'
n
.
:
;
.
/'
':r
y
J.
.
'
~
!:
, N a ~ C O O
v.
F
:
'~,
'
'
~ ~ ~ l ~. N 7.
~ (~ V , ~
!
s:<;;> ~ ~ E E E E E E ~ E .c E ~
.; E
:.
v.::,.
..<.. E
.:
p D D D a D o o D p D 0 E o
:
; z
.r,,4.,
;
~<i
.
:l
,
v
;
r
:~,~
%<:
..:;:.
'.
fy
v
4:?.
:;:.;
r
<.r.
~;
%%s<,'w
.
G.,:7~
fi
f:
:'::y~.fk..'~~'r'i
Y
<;;;.:.
r..f;. C
:;~'.,<:.
;
:
<:~
.,
<
#;
~s
''
. O
..
;.,
,
;:
r.
rlw"F'~ ~ .a.i~ C ~ >,
~ C C (B C O ~ C
'~; O C C ~ (t1 _ y C O 7
' ' ~ ~ E
.r e t. E E a r C
f
h
i
S
i ~ ' ~ ~
~
1.
S
Fr
}<
_ _
' ' ~ ~ N p V ~ U> >.
<': j, >, j Ifi~ >> _p) _~ _ fi O >' C
Ls::::' ~'
_ , _ ~, >, >~ ~ >, ~ . U ~ ~,
r
>' S. .= L .C .C ~ O .
'i' E ' s u o ~ ~ ~ E E ~ ' a~ cv
...
' E >,
~ ; ~ ~ ~ ~ ~ E E ~ E Z z >, ~ '
h' '
s
= ~ z Z , z z Z ; ~ z ..r
'
~ z z z ,
::f p , z , ~ , p p
. , p ~ p , E p E
fi,:.<:~ p o p p p
.<, o p
,
.
~~~
'
~
: ,
~ .
:~,::.:
,
:~.<.:::
. : z
>:"..:
'',. ::
.c~y,::
,: >
...
x
..:'"'."
::::.':<;,r;::<.:.::
.
..
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
59
;~r::ii~~':.:..,~.-.
:~:
:r~s '' .:Y.
.i~s~r ~,.s' :::;.
k5i,'v..ris5 . .,~y~:
:L i.!y: ~ f'
y::f; ~r'/'r,
ii:::
'~~ ':'i~%" ~ L
:s~~~~fY:; ~ v~7 .L ~ Q> '' v7 ~p C ~ p~ ~ ~ ...,.
'j ~ c Z E E Z Z a. g g ~ ~ ~ '~ Z
w'' D ~ Z Z
vex ~ ' ' .
i
.~;:.
ii
f r'
r;#
z . r.:
%s. : .;r:::
.xø~' . ,
:. : ~r. :
.~;~~ ~.
:';x'~i .': N C ~ C N
:.
C _ ~ U ~ ~ C ~ ~ _~N ~U
k,. , . ~ C V ~ C C ~ C 7, C ~ ~ N
.i.>~<.:.~, ,f:k:: C 'E ~ E Ln '(j C ~ 'V ~, m
>, ..r ... a 'c .c ~ c~ ~ .n ,= o~ °- °
>,.... f' :':r.
.s >. ° ca ° a ~ = s ° ... -- ~,, ° o
Nay.. v r
%ZE~>,~s--..°~o°.Q~°~~w°LStS
E >. ~,
..: ,
,':.~t,~ Q Z ~ ~ E .~ ~' J ZS ~ ~ ~ N E J
.i..~ O ~r~Z Q'Z J ~JJ EN
fr
~ .r i. ~.~. t.;: Z Z ''~i J y..~
v' r.<
Z ~ Z
:: c. ,~.<,s r J
rtv:
"H,.:.:
'~' ..
:.>~'
rff~inri~...
it 'A
47:::.
s: .~~ . S;:r
:{
~'i.~r:~.
',,:,".,.'::
>, ~ ~ m N m ~ ~ C tn
i:;~~~~ - ~ ~ ~ ~ ~ .n ~ Q ca c~ ° vW ~ a~
.,.~.~ . : ''<:Z Z c c_ ~ E- z ~
.,.: i
',.. ~ ~ Cl
r .. y:
...rr~ ..;
~,r ~ <f.
'..''.,~'~::,:
;.:
:iu:.;3~,:. , did:;;
r .;.~' '.;':~i'i.'::
ir:
i:~::
'"
o' : ''~~/~;e,:f~:
i : . ~ :~,'r~.:
rr
'~~'.'"~':~>''.~C~'V~ '~CN,CECy
~ ~ O O _~ U C ~ _c0 ~
V
y>'~'C..'' :>.L. j,'U ~ ~Q..~.7 ~ _N
~r ;:::'i2
:.>' ~ ~~ s s ~ E ~ c .n ~ o E ~ ~ ~ ~ a~
:.. t ,.:
. ~ Z ~ J i
'f.:,w'~..~...,.1' ~ ~ Z , ~ (a -~ _ ~ ~ y5 y3 J
<i~ ~ ~,. J
~ N C~ ~ ?- J J J
rw- .::<~ Z z
ø:.<.~ ~~r:::::,:
:~~:~;:;' r::,r:
,~'~~:~~:
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
~'~ '
~>
Yi ~>~:
:,
.
:. .
. i
Y,i~..
J
; i
:%:YT
.
.
v
'
'
.
~i4
~
t~:
i,
~
:f:. y
. sx.y:
:~~iR:.'
. ri
:r .'s'.,~(
. <f.'-
~~,~s.tiG
':?:
i :ti
:~i .
. . i
i f
t !a'
'
2 i o a .. .-,
*~7
c
, -c c
~ ~ ~
~
:.:h;~ ~ ~ E
,,~.:...t.;'.
'
:',f
r:
v
;
.
:'
i4
i::iJ.
,'.co-~
fr~''r,:
Gf ~':,:.
Y::
mij~
>,~ 3 C
f:
,~:: _~
.:i ~ >
:~ ~~
~f
:} >
hi:. .~~ ~ ~ d C
C ~
. , Q7 C ! to O U a.
. ~
''
~
y C y ~: ~, Q ~, p
<~: ~
U
'
''
i
y. : ~_ V,I
~ O ~
>
f ~
C ~ O L. p C p
:;~; :..tw'
:: t,
N
L L
..: a ~ ~ ~
p o L . a , v
C >, L O . N
C
.:
~ ~ .. ~ .'"'p _ ~
'O
.C N O
:f.
~'~
~~ :n E E a~ ~ .c M >, c
~...
~
v2;.
1 1 ~ 1 ~
..:
<~
~ ~ ~ ~ ~s ~ ~ cu
'~
y4 _~ a~ Z ' >,
; . J
.
. J '.
,: ~ 1
..,.i
J
i':
. F f.~.
f
.
# J
,
._ 4;.
:: f:
3x
: ,x:<:i
:'s,7
*; ,t.
. ."
:. t.w:,
i
s.. t~~':
'r'''k~
;F.:
~3.
..;i:.
~
~.:.~;
fi:
"
;~~ f
>::
. f,:..;_::~
;vs
'~
'
'
~
~f.~
.~
.~~,i
'xi.... w,
~ E U
~3't:Yti~;:> C ~ ~ ~ L .Q
~ '
sf.;tyY, C . Vf 'a''> L
~ Q
y~,~ h
ii
f
~
.
~
::~
:%f,
:S ii'.
k:
'<i.t
/f. ..;;:
:: :G,.
'F,:-.
"
~~'
r
j
fi~f~lf~~,
i' ,~
'
ie:~ti
$ji%IS I
,,
~:,'.Gr%y
:~
. ~ _
:r i.
C,.','s
. .
x:~~.:;.
$~?$
i
?
~
ys., C ~ N ~ N .,..
i.
:w;
:
.
a _ a~ >,
: c
r
. c a c a
: N f6 ~
:p ~ m
"f'f:' ~ > y p ~a c ~ 1
~ a~ ~-
''~~''
.. p . t~ C1 > N >,
';: N C
~
::<. : C ~ L E-.L Q ~ CV
a ~
'
v?;t,, . .
'~ : ~' ; .
;,:
.
'.s
.~7 . a --r:. ~ ~ ~ 1
. . ~,:>,~ u~ . a~
.:a_ ~ ~ ,
..,..,..
'
... .:
~ ~ p ~ X
.
.,:: ..
:
.
,
.;. ~ ~ ~ ~
.,
., ~ ~ ~ L
: ~ ~ J J
1.~'~
~
'
'%'
' , 25
..
.
II
.~%.'',..'...t'':~~:-~ ~ .J 1 ~ U
J
:ry Z c,
:a
. J
::
,~:
:
:;
.y
.:, .
:. ;:,.
>.'>
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
61
TABLE 2
::x'.,-
:::f::f::.'ir'..,..>aa:.::.~:.a.>xii:.:..:.>'....>::.>..>:.>..:rr:.r...>:.a.:-
0:>:.:..>:....r..i:.>::,t.:~:'.:::a:.::.rai::-0.::: t::t::.::::..:.;.:.;...
..;:.~::.::.:..:: ::::::::::::::::::::........ ...............
.:.. a -,::;
..
.. ::v.: i . .i ...,
..>:i.;;
t;irr,::fi$: .. trt'r>::
~:.'~.; 2 ;.; . .,.. tr:.~:.~.:::. :.:. r.. . :w.fi.:. : ,. f .::.
:.::.)w.:;:':.r
>i~,fi:>:i..:..
.rr.4/a~''~'':' : A v. f'ffJ#f''$.t
..t >'i ..x.. ;: '>xJ x '' ; i ~.
. . :':vfi:: ,.yj#>i:Y.<::>ir4: .. ~.:~'iivit.:r rr'~L.'A':~':-0:!
. f::>.. x~:.tr ~~J:~
,,~~, . 9s. .fir:.ri. n
. Mv.. . l f, .:::: i SY.,.\..: ... ~ ..:'i iv. i:::
~:. J.
n, r't ~ r' r . , h
or. 'r~~ .rn~:k': i
v., :: x .>. '.:-0;Fr.
w~. .' . . r
.C i :. s
.,<~%';y'' ~: ::; . ~. ,r., r ..:~':r f.
r . . ..~ /. f.; :.:n ~:x.;,y.;" ,>~> ~,.
y,~ ,G: . , :.:;ixff, .. . . .y:~ir; .. . .Rv>.. .,'.$..'~<$;'.~
f~ ' #:~.'~f:>r u; f
(~!~:~ r:~~.Q' :: ' . >:...! . s~~': Y:
' : ff''' J I f
~ ~ .rxd ~': f
~ : trxl.r ~." ~ . iS i#:T:'~./4 t: . L.
~:: ':' r .... . . ~ ~ r~~:~. ~ . ' :..~ . ..tr.. ... /:$..~
..', :I,,~,,~: .,.... ... r.. ...
'.~~rxf: :~vf> i~c'~w~..r ..;:::,,; . .:.,f..~~,~,.:..~', .. ,
. ..h~Cx . "... ~TF~"~>v 'S'r;::#i: r . ; fi I. . ' ~a:~ .. , n .>..:..; ~~ Y:
~v
:G:i , n.. -0 , ~ .. ntr. rn ~:-0~~ /. .~~~'2~ v. .7 . ~. :. , ;,"'~: '
,: fi: fr~'~:: . v. ~r~'~r:~'~: ~;rr'~.... r-0 x .#;,. y; ..,ø , n tt:.'i~'..
rr : . .c:.. i':fi: ~ . . ~....:....::
. . r. r . ~.. ..~ ; .. . ., n.. . .fi' .n '::Ei f #%,~~~ ~Y ' ...:
'twit:.>;i xi7X':#.:$ : ~'...f'~.:...x . . ; .r~ .t.:3.:~, z'%f:~'~:f: . .:6
;.~~!,"~>y, 3~:~%.'x: tifryfi .~ ;ii.... .: \ns;vv s ~:f'k ::' :: r' 2 ~~~~~~~
:x
,~J'::$:~'... r.:r~:4'i~ftr :$, 11 . .I:: n ..9,:.x .~.r~~,.; s~~~~. ~ >:. ~ :-
0~'.'':f$
r\' f ::'.~f:'s,~~'~S. ~,~4 :,Kyy ~>tt, :: a~l~.i,~r~Hn.. ~ ..~. .ir; ~.i:'
'i: 4tr fitr:~:.h>:, t, ', .. . ' f: r .. . .tr fi. : ~ \ yr
> v.. .i .>. .: ... . :~:,-0~>>:ry .. . .~
.y. t:~ r.r.s. fir:>:ikhv.v~~:~.
~~ 9~:\... .n. : ':.:... .....
.:~: n , r. .;~::: ..~.~kt~'.~.~:~'it:n: ., h.vr.. : vL.~..
: rt . .. \. .:r.. . rf>~ ) . .> iY:>>Y :: r~ :\ :. r
T. .f.x.. ~. ..> . . $'~ v: '>f:~. .r:... ., i~$~ . ~fi '
.i, :.. r : ~ .;,.,,:, v r; . :~'.'., h..4.'.?r ... -0 .. '...: . \ 4
r;:f. .. ....5 ' :.. :.... :::~x; . r'n~~~~:~ ...~~. c':#f!~ . . r'fi ....
,trh: r: ~~::rr,: .'v.:... :'.$ ... ii.~'i:~~~Y:iifiy. ~. ;,:. ....fi
~:i:: ... :'~". :fi.,; .i. . ... f . . Q:f>v: ..,i2 .: i : .. ..~...
r'.~i'% ~ , nvt.tvnv; v. 'i r:.;; :rife "uir . .. ~:. .\.
.''~.::;rr.,r:~..l,ø;~ , , ri:.>.t 4:;:~':' ' k :ay3>.,'t-0$..:rr. r:.y 'f
.:.;.>,:: :o:~~i .
.'..:;~..r r :'. ..:Y : ..rt: . r .~~a. : ::C~:.,~.;:. . : r,.. .:. v>;g~
"':';a#
~,~ .;,:.>r: :':r ,~ , .;.r. '"'r... ~r.. :,..., xt..rr~ .:./ . . ~ir.#:
::.: . n:.~fi'~.: r: t>.;r.: ~;.r.rfx.....tr... i 'fr:> .. .: . :.> . /
...::~!: , n .>. ' s.'~~ :~hf::r :;~.~,.r.$ .,,...,..: :....,,:. :..f'f: ~::
tea':#:f',... :. ,rr'.::,a,Ntr~ ....rr.tr.t..f.~~~.a. /. .
:.. ,,::.,,:. frl "": .2 . . . ~.:., ~ n i"%~vi~>:.... trtr;,.a.: >: r: . . :
:. ~r~C ,.:
':
t.rr..... :~ .::. n r:.". .. :.:,:a;,.. ...r...,>. . 7: >r' ....
....ft r fn ... nsvxvv. . . . .r n >; ih ~.i'fi'~'f. ~ii v::Y::'. . ~ ...
.~.~~':'~:~:!. s.'r;n, ::t ...f. ': J:l. :f... n.$...' r.fi.Y: tn..
.fx... .~,'Lt0 r~.~C : ~. y:: i,;
".::r'tr'~' .:tr:'r::,
~::..
..:'~fi%::Y~.~..>r a: n'v.:. .. . . n~'i-0 '~f~.'~>:)tf r r. ~ . .tG..> >. .
::::.,!: ~-0 ~ :::.,'#2.:; r .. . \.ls>~.. . .
....:fir. h x ':.f... . :.rf. . . ';ifik ~ ."~~4r4a
:n.L:i .Y.r.'.;~r: .trlufi.., r:: :.a> ,v,. .r:n.' ,.'n'.i.;.. ,,,. . , . ::fi
~f ..,. .:a::>:: , . S.
.. ':.. . :.C . r. ::; f . : , rx~:. r :. . . c" r~ ~~
...t..... . . r :~ . ::; ~tr.,.. f: ~, :... . , lrx'"r;
%'::~~ ::, f.: ., r ~'~ . .,t. ...
%::~f f .. . 'r .Y~xafi:...\ .: .r. f.
:.:r. .: n......:.. . ir;~f.~~x.$;':i rr.~ . :.. :.''~i. . : >::.
.f .. !. ' ~t: ' ' #
> :$ ~"f.'~$i:~~ -0. . $x~ v:hv:r ...
:.~,;'rri.:: n.:. ., rr: ., ''" :, t:..,:;'YX"i~~::3:~"~>::i% t. :>: :, ,
tr$.:r.~'.
ra~:ii: . ..f . ..,..k.~.: rfi\ . .. :-0 ...n: ~ :r~t~:'';#:i%:y;: > .y :i: .t
.,.t r::> irJ.',.:.,.~.fir~:.: .. "rxr:..;. .ua.ni: .k..~. ,:. ,~r:.:fi. ,r .
?~:.~:ira.~.x h , h .:,.ii# ...~f$~.
:fi:~ ~s . :nT: a nY ... Yir~ri~.,r,;.~a.~~>.: . 7 . .'i.uf:~ :~i:'
:...~. Y'in.> $ .: tr >$Y...... . .r.~ i . ....~.:... s., y, . /~. : . .'~:~!>
i~ a..~.. .. ~ . , iv:F.
:nf~::::r:.i:~'. ....-0.~:'.:d$:r....rr..s:'.'.' $r :;:~#::i:.....
.>:.:r....rr.~.c:,.. .,ai.: ... ... try: ~vtv:.:
...r.nn...fi.. . . .. \ :n' ,...4 . .. .,.??.. .... : 'rr.'::,. .. x...
f.::.:, ... : ..-0
.......... . ....::::.:..vn.3?~... nw:.:::...;::::::/. . . .....
...:.;...:.:..:/::.x'.'i;#i::.,,...;:::rr .:r..d.;,.,Y.,fi.ii:>i>a f;>;.>;f.;
nrn;:.!,.,.;.:.>:i.,xi.;nfin'~h , r',..,::;xF',t:
M11.9 MUC11 IgM ++++ Reacts with paraffin
embedded tissue,
reactivity enhanced
by pre-treatment of
sections with
neuraminidase
which removes
sialic acid rou s.
M12.15 MUC12 IgM ++++ Reacts with paraffin
embedded tissue,
reactivity enhanced
by pre-treatment of
sections with
neuraminidase
which removes
sialic acid rou s.
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
SEQUENCE LISTING
<110> The Council of the Queensland Institute of Medical
The Corporation of the: Trustees of the order of th
<120> MUCINS
<130> mucinqimr
<140>
<141>
<150> PP4708
<151> 1998-07-16
<160> 18
<170> PatentIn Ver. 2.0
<210> 1
<211> 28
<212> PRT
<213> Homo sapiens
<220>
<221> REPEAT
<222> (1)..(28)
<223> MUC11 consensus tandem repeat sequence
<400> 1
Ser Gly Leu Ser Glu Glu Ser Thr Thr Ser His Ser Ser Pro Gly Ser
1 5 10 15
Thr His Thr Thr Leu Ser Pro ,Ala Ser Thr Thr Thr
20 25
<210> 2
<211> 2872
<212> DNA
<213> Homo Sapiens
<220>
<221> CDS
<222> (1)..(2871)
<400> 2
agg aac agg ccg cac aca aca gca ttc cct ggc agt acc acc atg cca 48
1
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
Arg Asn Arg Pro His Thr Thr Al.a Phe Pro Gly Ser Thr Thr Met Pro
1 5 10 15
ggc gtc agt cag gaa tct aca get tcc cac agc agc cca ggc tcc aca 96
Gly Val Ser Gln Glu Ser Thr Al.a Ser His Ser Ser Pro Gly Ser Thr
20 25 30
gac aca aca ctg tcc cct ggc agt acc aca gca tca tcc ctt ggt cca 144
Asp Thr Thr Leu Ser Pro Gly Ser Thr Thr Ala Ser Ser Leu Gly Pro
35 90 95
gaa tct act acc ttc cac agc ggc cca ggc tcc act gaa aca aca ctc 192
Glu Ser Thr Thr Phe His Ser Gly Pro Gly Ser Thr Glu Thr Thr Leu
50 55 60
tta cct gac aac acc aca gcc i:.cc ggc ctc ctt gaa gca tct acg ccc 240
Leu Pro Asp Asn Thr Thr Ala Ser Gly Leu Leu Glu Ala Ser Thr Pro
65 70 75 80
gtc cac agc agc act gga tcg cca cac aca aca ctg tcc cct gcc ggc 288
Val His Ser Ser Thr Gly Ser ;Pro His Thr Thr Leu Ser Pro Ala Gly
85 90 95
tct aca acc cgt cag gga gaa tct acc acc ttc cag agc tgg cct aac 336
Ser Thr Thr Arg Gln Gly Glu ;5er Thr Thr Phe Gln Ser Trp Pro Asn
100 105 110
tcg aag gac act acc cct gca cct cct act acc aca tca gcc ttt gtt 384
Ser Lys Asp Thr Thr Pro Ala Pro Pro Thr Thr Thr Ser Ala Phe Val
115 120 125
gag cta tct aca acc tcc cac ggc agc ccg agc tca act cca aca acc 432
Glu Leu Ser Thr Thr Ser His Gly Ser Pro Ser Sex Thr Pro Thr Thr
130 135 140
cac ttt tct gcc agc tcc aca acc ttg ggc cgt agt gag gaa tcg aca 480
His Phe Ser Ala Ser ser Thr Thr Leu Gly Arg Ser Glu Glu Ser Thr
145 150 155 160
aca gtc cac agc agc cca gtt gca act gca aca aca ccc tcg cct gcc 528
Thr Val His Ser Ser Pro Val Ala Thr Ala Thr Thr Pro Ser Pro Ala
165 170 175
cgc tcc aca acc tca ggc ctc gtt gaa gaa tct acg acc tac cac agc 576
Arg Ser Thr Thr Ser Gly Leu Val Glu Glu Ser Thr Thr Tyr His Ser
180 185 190
agc ccg ggc tca act caa aca atg cac ttc cct gaa agc gac aca act 624
2
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
Ser Pro Gly Ser Thr Gln Thr M:et His Phe Pro Glu Ser Asp Thr Thr
195 200 205
tca ggc cgt ggt gaa gaa tca aca act tcc cac agc agc aca aca cac 672
Ser Gly Arg Gly Glu Glu Ser Thr Thr Ser His Ser Ser Thr Thr His
210 215 220
aca ata tct tca get cct agc acc aca tct gcc ctt gtt gaa gaa cct 720
Thr Ile Set Ser Ala Pro Ser Thr Thr Ser Ala Leu Val Glu Glu Pro
225 230 235 240
acc agc tac cac agc agc ccg ggc tca act gca aca aca cac ttc cct 768
Thr Ser Tyr His Ser Ser Pro Gly Ser Thr Ala Thr Thr His Phe Pro
245 250 255
gac agc tcc aca acc tca ggc cgt agt gag gaa tca aca gca tcc cac 816
Asp Ser Ser Thr Thr Ser Gly A.rg Ser Glu Glu Ser Thr Ala Ser His
260 265 270
agc aac caa gac gca acg gga aca ata gtc cta cct gcc cgc tcc aca 869
Ser Asn Gln Asp Ala Thr Gly Thr Ile Val Leu Pro Ala Arg Ser Thr
275 280 285
acc tca gtt ctt ctt gga gaa t.ct acg acc tca ccc atc agt tca ggc 912
Thr Ser Val Leu Leu Gly Glu S'~er Thr Thr Ser Pro Ile Ser Ser Gly
290 295 300
tca atg gaa acg aca gcg tta c:cc ggc agt acc aca acg cca ggc ctc 960
Ser Met Glu Thr Thr Ala Leu Pro Gly Ser Thr Thr Thr Pro Gly Leu
305 310 315 320
agt gag aaa tct acc act ttc c:ac agt agc ccc aga tca cca gcc aca 1008
Ser Glu Lys Ser Thr Thr Phe His Ser Ser Pro Arg Ser Pro Ala Thr
325 330 335
aca ctc tca cct gcc agc acg aca agc tca ggc gtc agt gaa gaa tcc 1056
Thr Leu Ser Pro Ala Ser Thr '.Chr Ser Ser Gly Val Ser Glu Glu Ser
340 345 350
acc acc tcc cac agc cga cca ggc tca acg cac aca aca gca ttc cct 1104
Thr Thr Ser His Ser Arg Pro Gly Ser Thr His Thr Thr Ala Phe Pro
355 360 365
gac agc acc acc acg cca ggc ctc agt cgg cat tct aca act tcc cac 1152
Asp Ser Thr Thr Thr Pro Gly Leu Ser Arg His Ser Thr Thr Ser His
370 375 380
agc agc cca ggc tca acg gat aca aca ctg tta cct gcc agc acc acc 1200
3
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
Ser Ser Pro Gly Ser Thr Asp Thr Thr Leu Leu Pro Ala Ser Thr Thr
385 390 395 400
acc tca ggc ccc agt cag gaa tca aca act tcc cac agc agc cca ggt 1248
Thr Ser Gly Pro Ser Gln Glu Ser Thr Thr Ser His Ser Ser Pro Gly
405 410 415
tca act gac aca gca ctg tcc cct ggc agt acc aca gcc tta tcc ttt 1296
Ser Thr Asp Thr Ala Leu Ser Pro Gly Ser Thr Thr Ala Leu Ser Phe
420 425 430
ggt caa gaa tct aca acc ttc cac agc agc cca ggc tcc act cac aca 1344
Gly Gln Glu Ser Thr Thr Phe His Ser Ser Pro Gly Ser Thr His Thr
935 940 445
aca ctc ttc cct gac agc acc aca agc tca ggc atc gtt gaa gca tct 1392
Thr Leu Phe Pro Asp Ser Thr Thr 5er Ser Gly Ile Val Glu Rla Ser
450 455 460
aca cgc gtc cac agc agc act ggc tca cca cgc aca aca ctg tcc cct 1440
Thr Arg Val His Ser Ser Thr Gly Ser Pro Arg Thr Thr Leu Ser Pro
465 470 475 980
gcc agc tcc aca agc cct gga ctt cag gga gaa tct acc gcc ttc cag 1488
Ala Ser Ser Thr Ser Pro Gly Leu Gln Gly Glu Ser Thr Rla Phe Gln
485 490 495
acc cac cca gcc tca act cac acg acg cct tca act cct agc acc gca 1536
Thr His Pro Ala Ser Thr His Thr Thr Pro Ser Thr Pro Ser Thr Ala
500 505 510
aca gcc cct gtt gaa gaa tct aca acc tac cac cgc agc cca agc tcg 1584
Thr Ala Pro Val Glu Glu Ser Thr Thr Tyr His Arg Ser Pro Ser Ser
515 520 525
act cca aca aca cac ttc cct gcc agc tcc aca act tcg ggc cac agt 1632
Thr Pro Thr Thr His Phe Pro Ala Ser 5er Thr Thr Ser Gly His Ser
530 535 540
gag aaa tca aca ata ttc cac agc agc cca gat gca agt gga aca aca 1680
Glu Lys Ser Thr Ile Phe His Ser Ser Pro Asp Ala Ser Gly Thr Thr
545 550 555 560
ccc tca tct gcc cac tcc aca acc tca ggt cgt gga gaa tct aca acc 1728
Pro Ser Ser Ala His Ser Thr Thr Ser Gly Arg Gly Glu Ser Thr Thr
565 570 575
tca cgc atc agt cca ggc tca, act gaa ata aca acg tta cct ggc agt 17'76
4
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
Ser Arg Ile Ser Pro Gly Ser Thr Glu Ile Thr Thr Leu Pro Gly Ser
580 585 590
acc aca aca cca ggc ctc agt gag gca tct acc acc ttc tac agc agc 1824
Thr Thr Thr Pro Gly Leu Ser Glu Ala Ser Thr Thr Phe Tyr Ser Ser
595 600 605
ccc aga tca cca acc aca aca ctc tca cct gcc agc atg aca agc cta 1872
Pro Arg Ser Pro Thr Thr Thr Leu Ser Pro Ala Ser Met Thr Ser Leu
610 615 620
ggc gtc ggt gaa gaa tcc acc acc tcc cgt agc caa cca ggt tct act 1920
Gly Val Gly Glu Glu Ser Thr Thr Ser Arg Ser Gln Pro Gly Ser Thr
625 630 635 640
cac tca aca gtg tca cct gcc agc acc acc acg cca ggc ctc agt gag 1968
His Ser Thr Val Ser Pro Ala Ser Thr Thr Thr Pro Gly Leu Ser Glu
645 650 655
gaa tct acc acc gtc tac agc agc agc cca ggc tca act gaa acc aca 2016
Glu Ser Thr Thr Val Tyr Ser 5er Ser Pro Gly Ser Thr Glu Thr Thr
660 665 670
gtg ttc cct cgc agc acc aca acc tca gtt cgt ggt gaa gag cct aca 2064
Val Phe Pro Arg Ser Thr Thr Thr Ser Val Arg Gly Glu Glu Pro Thr
675 680 685
acc ttc cac agc cgg cca gcc tca act cac aca aca ctg ttc act gag 2112
Thr Phe His Ser Arg Pro Ala 5er Thr His Thr Thr Leu Phe Thr Glu
690 695 700
gac agc acc acc tcg ggc ctc act gaa gaa tct aca gcc ttc ccc ggc 2160
Asp Ser Thr Thr Ser Gly Leu Thr Glu Glu Ser Thr Ala Phe Pro Gly
705 710 715 720
agc cca gcc tcc acc caa aca ggg tta cct gcc aca ctc aca acc gca 2208
Ser Pro Ala Ser Thr Gln Thr Gly Leu Pro Ala Thr Leu Thr Thr Ala
725 730 735
gac ctc ggt gag gaa tca act acc ttt ccc agc agc tca ggc tca act 2256
Asp Leu Gly Glu Glu Ser Thr Thr Phe Pro Ser Ser Ser Gly Ser Thr
740 745 750
gga aca aca ctc tca cct gcc cgc tcc acc acc tct ggc ctc gtt gga 2304
Gly Thr Thr Leu Ser Pro Ala Arg Ser Thr Thr Ser Gly Leu Val Gly
755 760 765
gaa tcc aca ccc tca cgc ctc agt cca agc tca acc gaa aca aca act 2352
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
Glu Ser Thr Pro Ser Arg Leu Ser Pro Ser Ser Thr Glu Thr Thr Thr
770 775 780
tta ccc ggc agt ccc aca aca cca agc ctc agt gag aaa tca acc acc 2400
Leu Pro Gly Ser Pro Thr Thr Pro Ser Leu Ser Glu Lys Ser Thr Thr
785 790 795 800
ttc tac act agc ccc aga tca cca gat gca aca ctc tca cct gca acc 2448
Phe Tyr Thr Ser Pro Arg Ser Pro Asp Rla Thr Leu Ser Pro Ala Thr
805 810 815
aca aca agc tca ggc gtc agc gaa gaa tcc agc aca tcc cac agt caa 2496
Thr Thr Ser Ser Gly Val Ser Glu Glu Ser Ser Thr Ser His Ser Gln
820 825 830
cca ggc tca acg cac aca aca gcg ttc cct gac agc acc acc acc tca 2544
Pro Gly Ser Thr His Thr Thr Ala Phe Pro Asp Ser Thr Thr Thr Ser
835 840 895
ggc ctc agt cag gaa cct aaa act tcc cac agc agc caa ggc tca aca 2592
Gly Leu Ser Gln Glu Pro Lys Thr Ser His Ser Ser Gln Gly Ser Thr
850 855 860
gag gca aca ctg tcc cct ggc agt acc aca gcc tca tcc ctt ggt caa 2640
Glu Ala Thr Leu Ser Pro Gly Ser Thr Thr Ala Ser Ser Leu Gly Gln
865 870 875 880
caa tct aca acc ttc cac agc agc cca ggc gac act gaa acc aca ctc 2688
Gln Ser Thr Thr Phe His Ser Ser Pro Gly Asp Thr Glu Thr Thr Leu
885 890 895
tta cct gat gac acc ata acc tca ggc ctc gtg gag gca tct aca ccc 2736
Leu Pro Asp Asp Thr Ile Thr Ser Gly Leu Val Glu Ala Ser Thr.Pro
900 905 910
acc cac agc agc act ggc tcg cta cac aca aca ctg acc cct gcc agc 2789
Thr His Ser Ser Thr Gly Ser Leu His Thr Thr Leu Thr Pro Ala Ser
915 920 925
tcc aca agc get ggc ctt cag gaa gaa tct act act ttc cag agc tgg 2832
Ser Thr Ser Ala Gly Leu Gln Glu Glu Ser Thr Thr Phe Gln Ser Trp
930 935 940
cca agc tca agt gac aca aca cct tca cct ccc ggc ccg g 2872
Pro Ser Ser Ser Asp Thr Thr Pro Ser Pro Pro Gly Pro
945 950 955
6
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
<210> 3
<211> 957
<212> PRT
<213> Homo Sapiens
<900> 3
Arg Asn Arg Pro His Thr Thr .Ala Phe Pro Gly Ser Thr Thr Met Pro
1 5 10 15
Gly Val Ser Gln Glu Ser Thr.Ala Ser His Ser Ser Pro Gly Ser Thr
20 25 30
Asp Thr Thr Leu Ser Pro Gly Ser Thr Thr Ala Ser Ser Leu Gly Pro
35 40 95
Glu Ser Thr Thr Phe His Ser Gly Pro Gly Ser Thr Glu Thr Thr Leu
50 55 60
Leu Pro Asp Asn Thr Thr Ala Ser Gly Leu Leu Glu Ala Ser Thr Pro
65 70 75 80
Val His Ser Ser Thr Gly Ser Pro His Thr Thr Leu Ser Pro Ala Gly
85 90 95
Ser Thr Thr Arg Gln Gly Glu Ser Thr Thr Phe Gln Ser Trp Pro Asn
100 105 110
Ser Lys Asp Thr Thr Pro Ala Pro Pro Thr Thr Thr Ser Ala Phe Val
115 120 125
Glu Leu Ser Thr Thr Ser His Gly Ser Pro Ser Ser Thr Pro Thr Thr
130 135 140
His Phe Ser Ala 5er Ser Thr Thr Leu Gly Arg Ser Glu Glu Ser Thr
145 150 155 160
Thr Val His Ser Ser Pro Val Ala Thr Ala Thr Thr Pro Ser Pro Ala
165 170 175
Arg Ser Thr Thr Ser Gly Leu Val Glu Glu Ser Thr Thr Tyr His Ser
180 185 190
Ser Pro Gly Ser Thr Gln Thr Met His Phe Pro Glu Ser Asp Thr Thr
195 200 205
Ser Gly Arg Gly Glu Glu Ser Thr Thr Ser His Ser Ser Thr Thr His
210 215 220
7
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
Thr Ile Ser Ser Ala Pro Ser Thr Thr Ser Ala Leu Val Glu Glu Pro
225 230 235 290
Thr Ser Tyr His Ser Ser Pro Gly Ser Thr Ala Thr Thr His Phe Pro
245 250 255
Asp Ser Ser Thr Thr Ser Gly Arg Ser Glu Glu Ser Thr Ala Ser His
260 265 270
Ser Asn Gln Asp Ala Thr Gly Thr Ile Val Leu Pro Ala Arg Ser Thr
275 280 285
Thr Ser Val Leu Leu Gly Glu Ser Thr Thr Ser Pro Ile Ser Ser Gly
290 295 300
Ser Met Glu Thr Thr Ala Leu Pro Gly Ser Thr Thr Thr Pro Gly Leu
305 310 315 320
Ser Glu Lys Ser Thr Thr Phe His Ser Ser Pro Arg Ser Pro Ala Thr
325 330 335
Thr Leu Ser Pro Ala Ser Thr Thr Ser Ser Gly Val Ser Glu Glu Ser
340 345 350
Thr Thr Ser His Ser Arg Pro Gly Ser Thr His Thr Thr Ala Phe Pro
355 360 365
Asp Ser Thr Thr Thr Pro Gly Leu Ser Arg His Ser Thr Thr Ser His
370 375 380
Ser Ser Pro Gly Ser Thr Asp Thr Thr Leu Leu Pro Ala Ser Thr Thr
385 390 395 400
Thr Ser Gly Pro Ser Gln Glu Ser Thr Thr Ser His Ser Ser Pro Gly
405 410 415
Ser Thr Asp Thr Ala Leu Ser Pro Gly Ser Thr Thr Ala Leu Ser Phe
420 425 430
Gly Gln Glu Ser Thr Thr Phe His Ser Ser Pro Gly Ser Thr His Thr
435 440 445
Thr Leu Phe Pro Asp Ser Thr Thr Ser Ser Gly Ile Val Glu Ala Ser
450 455 460
Thr Arg Val His Ser Ser Thr Gly Ser Pro Arg Thr Thr Leu Ser Pro
465 470 475 480
8
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
Ala Ser Ser Thr Ser Pro Gly Leu Gln Gly Glu Ser Thr Ala Phe Gln
485 490 495
Thr His Pro Ala Ser Thr His Thr Thr Pro Ser Thr Pro Ser Thr Ala
500 505 510
Thr Ala Pro Val Glu Glu Ser Thr Thr Tyr His Arg Ser Pro Ser Ser
515 520 525
Thr Pro Thr Thr His Phe Pro Ala Ser Ser Thr Thr Ser Gly His Ser
530 535 540
Glu Lys Ser Thr Ile Phe His Ser Ser Pro Asp Ala Ser Gly Thr Thr
545 550 555 560
Pro Ser Ser Ala His Ser Thr Thr Ser Gly Arg Gly Glu Ser Thr Thr
565 570 575
Ser Arg Ile Ser Pro Gly Ser Thr Glu Ile Thr Thr Leu Pro Gly Ser
580 585 590
Thr Thr Thr Pro Gly Leu Ser Glu Ala Ser Thr Thr Phe Tyr Ser Ser
595 600 605
Pro Arg Ser Pro Thr Thr Thr Leu Ser Pro Ala Ser Met Thr Ser Leu
610 615 620
Gly Val Gly Glu Glu Ser Thr Thr Ser Arg Ser Gln Pro Gly Ser Thr
625 630 635 640
His Ser Thr Val Ser Pro Ala Ser Thr Thr Thr Pro Gly Leu Ser Glu
645 650 655
Glu Ser Thr Thr Val Tyr Ser Ser Ser Pro Gly Ser Thr Glu Thr Thr
660 665 670
Val Phe Pro Arg Ser Thr Thr Thr Ser Val Arg G1y Glu Glu Pro Thr
675 680 685
Thr Phe His Ser Arg Pro Ala Ser Thr His Thr Thr Leu Phe Thr Glu
690 695 700
Asp Ser Thr Thr Ser Gly Leu Thr Glu Glu Ser Thr Ala Phe Pro Gly
705 710 715 720
Ser Pro Ala Ser Thr Gln Thr Gly Leu Pro Ala Thr Leu Thr Thr Ala
725 730 735
9
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
Asp Leu Gly Glu Glu Ser Thr Thr Phe Pro Ser Ser Ser Gly Sex Thr
790 745 750
Gly Thr Thr Leu Ser Pro Ala Arg Ser Thr Thr Ser Gly Leu Val Gly
755 760 765
Glu Ser Thr Pro Ser Arg Leu Ser Pro Ser Ser Thr G1u Thr Thr Thr
770 775 780
Leu Pro Gly Ser Pro Thr Thr Pro Ser Leu Ser Glu Lys Ser Thr Thr
785 790 795 800
Phe Tyr Thr Ser Pro Arg Ser Pro Asp Ala Thr Leu Ser Pro Ala Thr
805 810 815
Thr Thr Ser Ser Gly Val Ser Glu Glu Ser Ser Thr Ser His Ser Gln
820 825 830
Pro Gly Ser Thr His Thr Thr Ala Phe Pro Asp Ser Thr Thr Thr Ser
835 890 895
Gly Leu Ser Gln Glu Pro Lys Thr Ser His Ser Ser Gln Gly Ser Thr
850 855 860
Glu Ala Thr Leu Ser Pro Gly Ser Thr Thr Ala Ser Ser Leu Gly Gln
865 870 875 880
Gln Ser Thr Thr Phe His Ser Ser Pro Gly Asp Thr Glu Thr Thr Leu
885 890 895
Leu Pro Asp Asp Thr Ile Thr Ser Gly Leu Val Glu Ala Ser Thr Pro
900 905 910
Thr His Ser Ser Thr Gly Ser Leu His Thr Thr Leu Thr Pro Ala Ser
915 920 925
Ser Thr Ser Ala Gly Leu G1n Glu Glu Ser Thr Thr Phe Gln Ser Trp
930 935 990
Pro Ser Ser Ser Asp Thr Thr Pro Ser Pro Pro Gly Pro
945 950 955
<210> 4
<211> 28
<212> PRT
<213> Homo sapiens
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
<220>
<221> REPEAT
<222> (1)..(281
<223> MUC12 consensus tandenn repeat sequence
<400> 4
Ser Gly Leu Ser Gln Glu Ser Thr Thr Phe His Ser Ser Pro Gly Ser
1 5 10 15
Thr Glu Thr Thr Leu Ser Pro Ala Ser Thr Thr Thr
20 25
<210> 5
<211> 2095
<212> DNA
<213> Homo Sapiens
<220>
<221> CDS
<222> (3)..(1757)
<400> 5
as aca ctc tca cct gcc agc atg aga agc tcc agc atc agt gga gaa 47
Thr Leu Ser Pro Ala Ser f4et Arg Ser Ser Ser Ile Ser Gly Glu
1 5 10 15
ccc acc agc ttg tat agc caa gca gag tca aca cac aca aca gcg ttc 95
Pro Thr Ser Leu Tyr Ser Gln Ala Glu Ser Thr His Thr Thr Ala Phe
20 25 30
cct gcc agc acc acc acc tca ggc ctc agt cag gaa tca aca act ttc 143
Pro Ala Ser Thr Thr Thr Ser Gly Leu Ser Gln Glu Ser Thr Thr Phe
35 40 45
cac agt aag cca ggc tca act gag aca aca ctg tcc cct ggc agc atc 191
His Ser Lys Pro Gly Ser Thr Glu Thr Thr Leu Ser Pro Gly Ser Ile
50 55 60
aca act tca tct ttt get caa gaa ttt acc acc cct cat agc caa cca 239
Thr Thr Ser Ser Phe Ala Gln Glu Phe Thr Thr Pro His Ser Gln Pro
65 70 75
ggc tca get ctg tca aca gtg tca cct gcc agc acc aca gtg cca ggc 287
Gly Ser Ala Leu Ser Thr Val Ser Pro Ala Ser Thr Thr Val Pro Gly
80 85 90 95
ctt agt gag gaa tct acc acc ttc tac agc agc c<:a ggc tca act gaa 335
11
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
Leu Ser Glu Glu Ser Thr Thr Phe Tyr Ser Ser Pro Gly Ser Thr Glu
100 105 110
acc aca gcg ttt tct cac agc aac aca atg tcc att cat agt caa caa 383
Thr Thr Ala Phe Ser His Ser Asn Thr Met Ser Ile His Ser Gln Gln
115 120 125
tct aca ccc ttc cct gac agc cca ggc ttc act cac aca gtg tta cct 431
Ser Thr Pro Phe Pro Asp Ser Pro Gly Phe Thr His Thr Val Leu Pro
130 135 140
gcc acc ctc aca acc aca gac att ggt cag gaa tca aca gcc ttc cac 479
Ala Thr Leu Thr Thr Thr Asp Ile Gly Gln Glu Ser Thr Ala Phe His
145 150 155
agc agc tca gac gca act gga aca aca ccc tta cct gcc cgc tcc aca 527
Ser Ser Ser Asp Ala Thr Gly Thr Thr Pro Leu Pro Ala Arg Ser Thr
160 165 170 175
gcc tca gac ctt gtt gga gaa cct aca act ttc tac atc agc cca tcc 575
Ala Ser Asp Leu Val Gly Glu Pro Thr Thr Phe Tyr Ile Ser Pro Ser
180 185 190
cct act tac aca aca ctc ttt cct gcg agt tcc agc aca tca ggc ctc 623
Pro Thr Tyr Thr Thr Leu Phe Pro Ala Ser Ser Ser Thr Ser Gly Leu
195 200 205
act gag gaa tct acc acc ttc cac acc agt cca agc ttc act tct aca 671.
Thr Glu Glu Ser Thr Thr Phe His Thr Ser Pro Ser Phe Thr Ser Thr
210 215 220
att gtg tct act gaa agc ctg gaa acc tta gca cca ggg ttg tgc cag 719
Ile Val Ser Thr Glu Ser Leu Glu Thr Leu Ala Pro Gly Leu Cys Gln
225 230 235
gaa gga caa att tgg aat gga aaa caa tgc gtc tgt ccc caa ggc tac 767
Glu Gly Gln Ile Trp Asn Gly Lys Gln Cys Val Cys Pro Gln Gly Tyr
240 245 250 255
gtt ggt tac cag tgc ttg tcc cct ctg gaa tcc ttc cct gta gaa acc 815
Val Gly Tyr Gln Cys Leu Ser Pro Leu Glu Ser Phe Pro Val Glu Thr
260 265 270
ccg gaa aaa ctc aac gcc act tta ggt atg aca gtg aaa gtg act tac 863
Pro Glu Lys Leu Asn Ala Thr Leu Gly Met Thr Val Lys Val Thr Tyr
275 280 285
aga aat ttc aca gaa aag atg aat gac gca tcc tcc cag gaa tac cag 911
12
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
Arg Asn Phe Thr Glu Lys Met Asn Asp Ala Ser Ser Gln Glu Tyr Gln
290 295 300
aac ttc agt acc ctc ttc aag aat cgg atg gat gtc gtt ttg aag ggc 959
Asn Phe Ser Thr Leu Phe Lys Asn Arg Met Asp Val Val Leu Lys Gly
305 310 315
gac aat ctt cct cag tat aga ggg gtg aac att cgg aga ttg ctc aac 1007
Asp Asn Leu Pro Gln Tyr Arg Gly Val Asn Ile Rrg Arg Leu Leu Asn
320 325 330 335
ggt agc atc gtg gtc aag aac gat gtc atc ctg gag gca gac tac act 1055
Gly Ser Ile Val Val Lys Asn Asp Val Ile Leu Glu Ala Asp Tyr Thr
390 345 350
tta gag tat gag gaa ctg ttt gaa aac ctg gca gag att gta aag gcc 1103
Leu Glu Tyr Glu Glu Leu Phe Glu Asn Leu Ala Glu Ile Val Lys Ala
355 360 365
aag att atg aat gaa act aga aca act ctt ctt gat cct gat tcc tgc 1151
Lys Ile Met Asn Glu Thr Arg Thr Thr Leu Leu Asp Pro Asp Ser Cys
370 375 380
aga aag gcc ata ctg tgc tat agt gaa gag gac act ttc gtg gat tca 1199
Arg Lys Ala Ile Leu Cys Tyr 5er Glu Glu Asp Thr Phe Val Asp Ser
385 390 395
tcg gtg act ccg ggc ttt gac ttc cag gag caa tgc acc cag aag get 1297
Ser Val Thr Pro Gly Phe Asp Phe Gln Glu Gln Cys Thr Gln Lys Ala
400 405 910 415
gcc gaa gga tat acc cag ttc tac tat gtg gat gtc ttg gat ggg aag 1295
Ala Glu Gly Tyr Thr Gln Phe Tyr Tyr Val Asp Val Leu Asp Gly Lys
420 425 430
ctg gcc tgt gtg aac aag tgc acc aaa gga acg aag tcg caa atg aac 1343
Leu Ala Cys Val Asn Lys Cys Thr Lys Gly Thr Lys Ser Gln Met Asn
435 490 495
tgt aac ctg ggc aca tgt cag~ ctg caa cgc agt ggc ccc cgc tgc ctg 1391
Cys Asn Leu Gly Thr Cys Gln. Leu Gln Arg Ser Gly Pro Arg Cys Leu
450 455 960
tgc cca aat acg aac aca cac: tgg tac tgg gga gag acc tgt gaa ttc 1439
Cys Pro Asn Thr Asn Thr His Trp Tyr Trp Gly Glu Thr Cys Glu Phe
465 470 475
aac atc gcc aag agc ctc gtc~ tat ggg atc gtg ggg get gtg atg gcg 1487
13
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
Asn Ile Ala Lys Ser Leu Val Tyr Gly Ile Val Gly Ala Val Met Ala
480 485 490 495
gtg ctg ctg ctc gca ttg atc atc cta atc atc tta ttc agc cta tcc 1535
Val Leu Leu Leu Ala Leu Ile Ile Leu Ile Ile Leu Phe Ser Leu Ser
500 505 510
cag aga aaa cgg cac agg gaa cag tat gat gtg cct caa gag tgg cga 1583
Gln Arg Lys Arg His Arg Glu Gln Tyr Asp Val Pro Gln Glu Trp Arg
515 520 525
aag gaa ggc acc cct ggc atc ttc cag aag acg gcc atc tgg gaa gac 1631
Lys Glu Gly Thr Pro Gly Ile Phe Gln Lys Thr Ala Ile Trp Glu Asp
530 S35 540
cag aat ctg agg gag agc aga ttc ggc ctt gag aac gcc tac aac aac 16?9
Gln Asn Leu Arg Glu Ser Arg Phe Gly Leu Glu Asn Ala Tyr Asn Asn
545 550 555
ttc cgg ccc acc ctg gag act gtt gac tct ggc aca gag ctc cac atc 1727
Phe Arg Pro Thr Leu Glu Thr Val Asp Ser Gly Thr Glu Leu His Ile
560 565 570 575
cag agg ccg gag atg gta gca tcc act gtg tgagccaacg ggggcctccc 1777
Gln Arg Pro Glu Met Val Ala Ser Thr Val
580 585
accctcatct agctctgttc aggagagctg caaacacaga gcccaccaca agcctccggg 1837
gcgggtcaag aggagaccga agtcaggccc tgaagccggt cctgctctga gctgacagac 1897
ttggccagtc ccctgcctgt gctcctgctg gggaaggctg ggggctgtaa gcctctccat 1957
ccgggagctt ccagactccc agaagcctcg gcacccctgt ctcctcctgg gtggctcccc 2017
actctggaat ttccctacca ataaaagcaa atctgaaagc tcaaaaaaaa aaaaaaaaaa 2077
aaaaaaaaaa aaaaaaaa 2095
<210>6
<211>585
<212>PRT
<213>Homo Sapiens
<400> 6
Thr Leu Ser Pro Ala Ser Met Arg Ser Ser Ser I1e Ser Gly Glu Pro
1 5 i0 15
14
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579 _
Thr Ser Leu Tyr Ser Gln Ala Glu Ser Thr His Thr Thr Ala Phe Pro
20 25 30
Ala Ser Thr Thr Thr Ser Gly Leu Ser Gln Glu Ser Thr Thr Phe His
35 40 45
Ser Lys Pro Gly Ser Thr Glu Thr Thr Leu Ser Pro Gly Ser Ile Thr
50 55 60
Thr Ser Ser Phe Ala Gln Glu Phe Thr Thr Pro His ser Gln Pro Gly
65 70 75 80
Ser Ala Leu Ser Thr Val Ser Pro Ala Ser Thr Thr Val Pro Gly Leu
85 90 95
Ser Glu Glu Ser Thr Thr Phe Tyr Ser Ser Pro Gly Ser Thr Glu Thr
100 105 110
Thr Ala Phe Ser His Ser Asn Thr Met Ser Ile His Ser Gln Gln Ser
115 120 125
Thr Pro Phe Pro Asp Ser Pro Gly Phe Thr His Thr Val Leu Pro Ala
130 135 140
Thr Leu Thr Thr Thr Asp Ile Gly Gln Glu Ser Thr Ala Phe His Ser
145 150 155 160
Ser Ser Asp Ala Thr Gly Thr Thr Pro Leu Pro Ala Arg Ser Thr Ala
165 170 175
Ser Asp Leu Val Gly Glu Pro Thr Thr Phe Tyr Ile Ser Pro Ser Pro
180 185 190
Thr Tyr Thr Thr Leu Phe Pro Ala Ser Ser Ser Thr Ser Gly Leu Thr
195 200 205
Glu Glu Ser Thr Thr Phe His Thr Ser Pro Ser Phe Thr Ser Thr Ile
210 215 220
Val Ser Thr Glu Ser Leu Glu Thr Leu Ala Pro Gly Leu Cys Gln Glu
225 230 235 240
Gly Gln Ile Trp Asn Gly Lys Gln Cys Val Cys Pro Gln Gly Tyr Val
245 250 255
Gly Tyr Gln Cys Leu Ser Pro Leu Glu Ser Phe Pro Val Glu Thr Pro
260 265 270
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
Glu Lys Leu Asn Ala Thr Leu Gly Met Thr Val Lys Val Thr Tyr Arg
275 280 285
Asn Phe Thr Glu Lys Met Asn Asp Ala Ser Ser Gln Glu Tyr Gln Asn
290 295 300
Phe Ser Thr Leu Phe Lys Asn Arg Met Asp Val Val Leu Lys Gly Asp
305 310 315 320
Asn Leu Pro Gln Tyr Arg Gly Val Asn Ile Arg Arg Leu Leu Asn Gly
325 330 335
Ser Ile Val Val Lys Asn Asp Val Ile Leu Glu Ala Asp Tyr Thr Leu
340 345 350
Glu Tyr Glu Glu Leu Phe Glu Asn Leu Ala Glu Ile Val Lys Ala Lys
355 360 365
Ile Met Asn Glu Thr Arg Thr Thr Leu Leu Asp Pro Asp Ser Cys Arg
370 375 380
Lys Ala Ile Leu Cys Tyr ser Glu Glu Asp Thr Phe Val Asp Ser Ser
385 390 395 400
Val Thr Pro Gly Phe Asp Phe Gln Glu Gln Cys Thr Gln Lys Ala Ala
405 410 415
Glu Gly Tyr Thr Gln Phe Tyr Tyr Val Asp Val Leu Asp Gly Lys Leu
42p 425 430
Ala Cys Val Asn Lys Cys Thr Lys Gly Thr Lys Ser Gln Met Asn Cys
435 440 445
Asn Leu Gly Thr Cys Gln Leu Gln Arg Ser Gly Pro Arg Cys Leu Cys
450 455 460
Pro Asn Thr Asn Thr His Trp Tyr Trp Gly Glu Thr Cys Glu Phe Asn
965 970 475 480
Ile Ala Lys Ser Leu Val Tyr Gly Ile Val Gly Ala Val Met Ala Val
485 490 495
Leu Leu Leu Ala Leu Ile Ile Leu Ile Ile Leu Phe Ser Leu Ser Gln
500 505 510
Arg Lys Arg His Arg Glu Gln. Tyr Asp Val Pro Gln Glu Trp Rrg Lys
515 520 525
16
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
Glu Gly Thr Pro Gly Ile Phe Gln Lys Thr Ala Ile Trp Glu Asp Gln
530 535 540
Rsn Leu Arg Glu Ser Arg Phe Gly Leu Glu Asn Ala Tyr Asn Asn Phe
545 550 555 560
Arg Pro Thr Leu Glu Thr Val Asp Ser Gly Thr Glu Leu His Ile Gln
565 570 575
Arg Pro Glu Met Val Ala Ser Thr Val
580 585
<210> 7
<211> 10
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Random lOmer
PCR primer
<400> 7
acttcgccac 10
<210> 8
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:MUC 12 PCR
forward primer
<400> B
tgaagggcga caatcttcct c 21
<210> 9
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:MUCl2 reverse
PCR primer
17
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
<400> 9
tacacgaggc tcttggcgat gttg 24
<210> 10
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Mucll forward
PCR primer
<400> 10
caggcgtcag tcaggaatct acag 24
<210> 11
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Mucll reverse
PCR primer
<400> 11
gaggctgtgg tgttgtcagg taag 24
<210> 12
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
Beta2-microglobulin forward PCR primer
<400> 12
tgaattgcta tgtgtctggg t 21
<210> 13
<211> 21
<212> DNA
<213> Artificial Sequence
18
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
<220>
<223> Description of Artificial Sequence: Beta
2-microglobulin reverse PCR primer
<400> 13
cctccatgat gctgcttaca t 21
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: MUC12 forward
primer for verification of contiguous sequence
<400> 14
agccaaccag gctcagctct 20
<210> 15
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: MUC12 reverse
primer for verification of contiguous sequence
<400> 15
gctcacacag tggatgctac c 21
<210> 16
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Clone Ii5
reverse PCR primer
<900> 16
gggaacactg tggtttcagt tgag 24
19
CA 02333853 2001-O1-16
WO 00/04142 PCT/AU99/00579
<210>17
<211>17
<212>PRT
<213>Homo Sapiens
<220>
<221> PEPTIDE
<222> (1)..(17)
<223> MUC11 immunizing peptide
<400> 17
Cys Phe His Ser Arg Pro Ala Ser Thr His Thr Thr Leu Phe Thr Glu
1 5 10 15
~P
<210>18
<211>17
<212>PRT
<213>Homo Sapiens
<220>
<221> PEPTIDE
<222> (1)..(17)
<223> MUC12 immunizing peptide
<400> 18
Thr Tyr Arg Asn Phe Thr Glu Lys Met Asn Asp Ala Ser Ser Gln Glu
1 5 10 15
Cys
