U.S. patent application number 12/592378 was filed with the patent office on 2011-06-23 for ephrin-b receptor protein involved in carcinoma.
This patent application is currently assigned to Oxford Glycosciences (UK) LTD. Invention is credited to Robert Simon Boyd, Graham Charles Fletcher, Lindsey Jane Hudson, Sonal Patel, Jonathan Alexander Terrett.
Application Number | 20110150877 12/592378 |
Document ID | / |
Family ID | 9934469 |
Filed Date | 2011-06-23 |
United States Patent
Application |
20110150877 |
Kind Code |
A1 |
Boyd; Robert Simon ; et
al. |
June 23, 2011 |
EPHRIN-B RECEPTOR PROTEIN INVOLVED IN CARCINOMA
Abstract
The present invention provides a polypeptide (CCMP-1) of use in
the diagnosis, screening, treatment and prophylaxis of carcinoma.
Also provided are compositions comprising the protein, vaccines and
antibodies that are immunospecific for the protein.
Inventors: |
Boyd; Robert Simon;
(Abingdon, GB) ; Fletcher; Graham Charles;
(Abingdon, GB) ; Hudson; Lindsey Jane; (Abingdon,
GB) ; Patel; Sonal; (Abingdon, GB) ; Terrett;
Jonathan Alexander; (Abingdon, GB) |
Assignee: |
Oxford Glycosciences (UK)
LTD
|
Family ID: |
9934469 |
Appl. No.: |
12/592378 |
Filed: |
November 24, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10510524 |
Apr 27, 2005 |
7655426 |
|
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PCT/GB03/01593 |
Apr 9, 2003 |
|
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12592378 |
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Current U.S.
Class: |
424/136.1 ;
424/130.1; 424/184.1; 436/501; 514/19.3 |
Current CPC
Class: |
C07K 14/71 20130101;
C07K 16/28 20130101; G01N 2333/52 20130101; C07K 2317/34 20130101;
G01N 2800/52 20130101; G01N 33/57415 20130101; C07K 16/30 20130101;
G01N 2500/04 20130101; C07K 2317/77 20130101; G01N 33/57419
20130101; A61P 43/00 20180101; A61P 35/00 20180101; G01N 33/6863
20130101 |
Class at
Publication: |
424/136.1 ;
436/501; 514/19.3; 424/130.1; 424/184.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; G01N 33/53 20060101 G01N033/53; A61K 38/16 20060101
A61K038/16; A61K 39/00 20060101 A61K039/00; A61P 35/00 20060101
A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 9, 2002 |
GB |
0208089.3 |
Claims
1. A method of screening for and/or diagnosis of carcinoma in a
subject, and/or monitoring the effectiveness of carcinoma therapy,
which comprises the step of detecting and/or quantifying in a
biological sample obtained from said subject: (i) a CCMP-1
polypeptide which: a) comprises or consists of the amino acid
sequence of SEQ ID NO:1; b) is a derivative having one or more
amino acid substitutions, modifications, deletions or insertions
relative to the amino acid sequence of SEQ ID NO: 1 which retains
the activity of CCMP-1; or c) is a fragment of a polypeptide having
the amino acid sequence of SEQ ID NO: 1, which is at least ten
amino acids long and has at least 70% homology over the length of
the fragment; or (ii)) a nucleic acid molecule which: d) comprises
or consists of the DNA sequence of SEQ ID NO: 2 or its RNA
equivalent; e) has a sequence which is complementary to the
sequences of d); f) has a sequence which codes for a CCMP-1
polypeptide as defined in a) to c); g) has a sequence which shows
substantial identity with any of those of d), e) and f); or h) is a
fragment of d), e), f) or g), which is at least ten nucleotides in
length.
2. The method of claim 1, wherein the level of said polypeptide or
said nucleic acid is compared to a previously determined reference
range or control.
3. The method according to claim 1, wherein the step of detecting
comprises: (a) contacting the sample with a capture reagent that is
specific for a polypeptide as defined in claim 1 (i); and (b)
detecting whether binding has occurred between the capture reagent
and said polypeptide in the sample.
4. The method according to claim 3, wherein step (b) comprises
detecting the captured polypeptide using a directly or indirectly
labelled detection reagent.
5. The method according to claim 3, wherein the capture reagent is
immobilised on a solid phase.
6. (canceled)
7. The method according to claim 1, wherein the polypeptide is
detected and/or quantified using an antibody that recognises a
CCMP-1 polypeptide as defined in claim 1 (i).
8. (canceled)
9. A diagnostic kit comprising a capture reagent specific for a
CCMP-1 polypeptide as defined in claim 1 (i), reagents and
instructions for use.
10. A method for treating carcinoma, comprising administering a
medicament comprising a material selected from the group consisting
of: (i) a CCMP-1 polypeptide as defined in claim 1 (i); and (ii) a
nucleic acid molecule as defined in claim 1 (ii).
11. A method for treating carcinoma, comprising administering a
medicament, said medicament comprising an antibody as defined in
claim 6 or 8.
12. The method of claim 10 (i) or (ii), wherein the medicament is a
vaccine.
13. A method of screening for anti-carcinoma agents that interact
with a CCMP-1 polypeptide as defined in claim 1 (i), said method
comprising: (a) contacting said polypeptide with a candidate agent;
and (b) determining whether or not the candidate agent interacts
with said polypeptide.
14. The method according to claim 13, wherein the determination of
interaction between the candidate agent and CCMP-1 polypeptide
comprises quantitatively detecting binding of the candidate agent
and said polypeptide.
15. A method of screening for anti-carcinoma agents that modulate
(a) the expression or activity of a CCMP-1 polypeptide as defined
in claim 1 (i), or (b) the expression of a nucleic acid molecule as
defined in claim 1 (ii), comprising (i) comparing the expression or
activity of said polypeptide, or the expression of said nucleic
acid molecule, in the presence of a candidate agent with the
expression or activity of said polypeptide, or the expression of
said nucleic acid molecule, in the absence of the candidate agent
or in the presence of a control agent; and (ii) determining whether
the candidate agent causes the expression or activity of said
polypeptide, or the expression of said nucleic acid molecule, to
change.
16. The method of claim 15, wherein the expression or activity
level of said polypeptide, or the expression level of said nucleic
acid molecule is compared with a predetermined reference range.
17. The method of claim 15, wherein part (ii) additionally
comprises selecting an agent which modulates the expression or
activity of said polypeptide, or the expression of said nucleic
acid molecule for further testing, or therapeutic or prophylactic
use as an anti-carcinoma agent.
18. An agent identified by the method of claim 13, which causes the
expression or activity of said polypeptide, or the expression of
said nucleic acid molecule, to change.
19. A method for treating carcinoma comprising administering a
medicament which comprises an agent which interacts with, or
modulates the expression or activity of a CCMP-1 polypeptide or the
expression of a CCMP-1 nucleic acid as defined in claim 1.
20. The method of claim, wherein the carcinoma is breast cancer or
colon cancer.
21. The method of claim 1, wherein the carcinoma is breast cancer
or colon cancer.
22. The method of claim 10, wherein the carcinoma is breast cancer
or colon cancer.
Description
[0001] The present application is a division of U.S. Ser. No.
10/510,524, filed on Apr. 27, 2005, which is a national stage
application claiming the priority of PCT Application No.
PCT/GB03/01593, filed Apr. 9, 2003, which in turn, claims priority
from Great Britain Application Serial No. 0208089.3, filed Apr. 9,
2002. Applicants claim the benefits of 35 U.S.C. .sctn.120 as to
the PCT application and priority under 35 U.S.C. .sctn.119 as to
the Great Britain application, and the entire disclosures of both
applications are incorporated herein by reference in their
entireties.
[0002] The present invention relates to the use of the protein
CCMP-1/ephrin B-type 3 receptor, compositions comprising the
protein, including vaccines and antibodies that are immunospecific
for the protein, in the diagnosis, prophylaxis and treatment of
carcinoma.
[0003] Excluding skin cancers, colorectal cancer is the third most
common cancer diagnosed in men and women in the United States.
Survival rates for those people whose colorectal cancer is found
and treated in an early stage are good, but only 37% of colorectal
cancers are found at that early stage. Once the cancer has spread
to nearby organs or lymph nodes, the 5-year relative survival rate
significantly decreases. Carcinoembryonic antigen (CEA) and CA 19-9
are substances produced by cells of most colon and rectal cancers
and released into the bloodstream. These markers, however, can be
high for reasons other than cancer, or can be normal in a person
who has cancer. Thus, a need exists for more specific markers of
colorectal cancer.
[0004] There are no generally approved immunotherapeutic drugs for
the treatment of colorectal cancer, despite the fact that
immunotherapy may offer the greatest potential after surgical
resection in the adjuvant setting. Edrecolomab (monoclonal antibody
17-1A, or Panorex.TM.), however, is an adjunctive therapy for
colorectal cancer which is in clinical trials in the UK and the US,
and which has already been approved in Germany. Identification of
new suitable targets or antigens for immunotherapy of colorectal
cancer is therefore highly important.
[0005] Breast cancer is one of the leading causes of cancer death
for women in the Western world. The major challenges in breast
cancer treatment are to improve early detection rates, to find new
non-invasive markers that can be used to follow disease progression
and identify relapse, and to find improved and less toxic
therapies, especially for more advanced disease where 5 year
survival is still very poor. There is a great need to identify
targets which are more specific to the cancer cells, ideally ones
which are expressed on the surface of the tumour cells so that they
can be attacked bypproaches such as immunotherapeutics and targeted
toxins.
[0006] Tumour specific proteins have been identified for a number
of cancer types using techniques such as differential screening of
cDNAs (Hubert, R. S., et al., 1999, Proc. Natl. Acad, Sci. USA 96,
14523-14528) and the purification of cell-surface proteins that are
recognised by tumour-specific antibodies (Catimel, B., et al.,
1996, J. Biol. Chem. 271, 25664-25670). More recently, DNA `chips`
containing up to 10,000 expressed sequence elements have been used
to characterise tumour cell gene expression Dhanasekaran, S. M., et
al., 2001, Nature 412, 822-826). However, there are several reasons
why the numerous and extensive previous transcriptomic analysis of
cancers may not have revealed all, or even most, tumour associated
proteins. These include:
[0007] (i) a lack of correlation between transcript and
disease-associated protein levels; particularly common for membrane
proteins that often have a long half-life and as such do not have a
high mRNA turnover. Therefore, whilst the difference in protein
levels between normal and cancerous cells are consistent it is
often difficult to associate changes in the mRNA for a given
membrane protein with the cancerous state;
[0008] (ii) translocation of a protein in the disease state rather
than simply differential levels of the transcript, for example,
erbB2/HER2-neu, shows much greater plasma-membrane localisation in
cancer cells than normal breast cells, and the transcription
factors oestrogen receptor and STAT3 translocate to the nucleus to
exert their tumourigenic effects; and
[0009] (iii) novel/uncharacterised genes are not highly represented
within the `closed system` of a cDNA array where there are
restrictions on the number of expressed sequence elements per chip
and the knowledge and availability of DNA clones.
[0010] As an alternative approach to identifying tumour antigens,
the present inventors used proteomics to characterise the
complement of proteins in cell membranes isolated from carcinoma
cell lines.
[0011] CCMP-1 is a receptor tyrosine kinase also known as ephrin
B-type receptor 3. At the gene level, cDNA profiling indicates that
CCMP-1 is one of fifteen genes overexpressed in prostate cancer
tissue, however RT-PCR and Northern blot analysis verified these
results in 40% and 88% of genes, respectively, demonstrating the
need to better validate quantitative differences in gene expression
revealed by array-based techniques (Chaib H. et al., 2001,
Neoplasia 3: 43-52). Moderate to low levels of CCMP-1 transcripts
have also been demonstrated in three out of four small cell lung
carcinoma cell lines (Tang, X. et al., 1998, Proc. Natl. Acad. Sci.
USA 95:9779-9784) and differential expression of the ligands for
ephrin B-type receptors has been demonstrated with ephrin-B2 having
higher expression in the colon carcinoma specimens studied than in
adjacent normal mucosa. The ligand ephrin-B2 and ephrin B-type
receptor 4 were most frequently expressed on the luminal surface of
colon carcinoma epithelium (Liu, W. et al., 2002, Cancer 94:
934-939).
[0012] WO 01/57188 provides a large number of cDNA sequences and
their encoded proteins. These include a sequence, identified as
sequence ID NO:2273, which corresponds to CCMP-1. The sequence was
indicated as equivalent to any of the other 1350 sequences
identified, as being useful in preventing, treating or ameliorating
a condition, such as arthritis or cancer. DE4233782 provides cDNA
amplified from RNA isolated from embryonic tissue which encodes a
polypeptide 99% identical to CCMP-1. However, these disclosures do
not identify CCMP-1 as being localised to the peripheral membrane,
nor useful for a therapeutic approach to colorectal cancer or
breast cancer.
[0013] The prior art does not show a cancer-associated alteration
of the CCMP-1 protein and therefore, does not show the usefulness
of CCMP-1 in a therapeutic approach to carcinoma, more specifically
to tumour cells derived from epithelial cells typically found in
the lining of body organs for example, but not limited to, breast,
prostate, colon, pancreas, prostate adenocarcinoma, tongue squamous
cell carcinoma, larynx squamous cell carcinoma, pharynx squamous
cell carcinoma, parotid carcinoma, lung squamous cell carcinoma,
and a brain astrocytoma, as well as kidney carcinomas and renal
cell carcinomas, bladder carcinoma and a papillary
cystadenocarcinoma. The term `carcinoma` includes a malignant new
growth that arises from epithelium, found in skin or, more
commonly, the lining of body organs, for example: breast, prostate,
lung, stomach or bowel. Carcinomas tend to infiltrate into adjacent
tissue and spread (metastasise) to distant organs, for example: to
bone, liver, lung or the brain.
[0014] The present invention is based on the finding of
cancer-associated alterations in the expression of CCMP-1 protein
in carcinoma. Thus, the invention provides the use of CCMP-1 for
the diagnosis and treatment of carcinoma, for example, but not
limited to, breast and colorectal carcinoma.
[0015] Thus, in one aspect, the present invention provides a method
of screening for and/or diagnosis of carcinoma in a subject, and/or
monitoring the effectiveness of carcinoma therapy, which comprises
the step of detecting and/or quantifying in a biological sample
obtained from said subject:
[0016] (i) a CCMP-1 polypeptide which:
[0017] a) comprises or consists of the amino acid sequence of SEQ
ID NO:1;
[0018] b) is a derivative having one or more amino acid
substitutions, modifications, deletions or insertions relative to
the amino acid sequence of SEQ ID NO: 1 which retains the activity
of CCMP-1; or
[0019] c) is a fragment of a polypeptide having the amino acid
sequence of SEQ ID NO: 1, which is at least ten amino acids long
and has at least 70% homology over the length of the fragment.
[0020] Hereinafter, such polypeptides are referred to as CCMP-1
polypeptides. In order to more fully appreciate the present
invention polypeptides within the scope of a)-c), above, will now
be discussed in greater detail. It will be apparent to one skilled
in the art that peptides for use in the invention include CCMP-1,
derivatives, fragments and modified forms (e.g. analogues)
thereof.
[0021] Polypeptides within the Scope of a)
[0022] A polypeptide within the scope of a), may consist of the
particular amino acid sequence given in FIG. 1 (SEQ ID NO:1) or may
have an additional N-terminal and/or an additional C-terminal amino
acid sequence relative to said sequence. Additional sequences may
be provided in order to alter the characteristics of a particular
polypeptide. This can be useful in improving recombinant expression
or regulation of expression in particular expression systems. For
example, an additional sequence may provide some protection against
proteolytic cleavage. It is often advantageous to include an
additional amino acid sequence which contains secretory or leader
sequences, a pre-, pro- or prepro-protein sequence, or a sequence
which aids in purification such as an affinity tag, for example,
but without limitation, multiple histidine residues, a FLAG tag, HA
tag or myc tag. An additional sequence which may provide stability
during recombinant production may also be used. Such sequences may
be optionally removed as required by incorporating a cleavable
sequence as an additional sequence or part thereof. Thus, a
polypeptide may be fused to other moieties including other
polypeptides. Such additional sequences and affinity tags are well
known in the art.
[0023] Thus, additional sequences can also be useful in altering
the properties of a polypeptide to aid in identification or
purification. For example, a fusion protein may be provided in
which a polypeptide is linked to a moiety capable of being isolated
by affinity chromatography. The moiety may be an antigen or an
epitope and the affinity column may comprise immobilised antibodies
or immobilised antibody fragments which bind to said antigen or
epitope (desirably with a high degree of specificity). The fusion
protein can usually be eluted from the column by addition of an
appropriate buffer.
[0024] Additional N-terminal or C-terminal sequences may, however,
be present simply as a result of a particular technique used to
obtain a polypeptide and need not provide any particular
advantageous characteristic to the polypeptide. Such polypeptides
are within the scope of the present invention.
[0025] Whatever additional N-terminal or C-terminal sequence is
present, it is preferred that the resultant polypeptide should
exhibit the immunological or biological activity of the polypeptide
having the amino acid sequence shown in FIG. 1 (SEQ ID NO:1).
[0026] Polypeptides within the Scope of b)
[0027] Turning now to the polypeptides defined in b) above, it will
be appreciated by the person skilled in the art that these
polypeptides are derivatives (or variants) of the polypeptide given
in a) above. Such derivatives preferably exhibit the immunological
or biological activity of the polypeptide of SEQ ID NO:1.
Alternatively, the biological activity of the polypeptide may be
altered. As such, it will be appreciated by one skilled in the art
that derivatives can include post-translational modifications, for
example but without limitation, phosphorylation, glycosylation and
farnesylation.
[0028] Alterations in the amino acid sequence of a protein can
occur which do not affect the function of a protein. These include
amino acid deletions, insertions and substitutions and can result
from alternative splicing and/or the presence of multiple
translational start sites and/or stop sites. Polymorphisms may
arise as a result of the infidelity of the translational process.
Thus changes in amino acid sequence which do not affect the
protein's biological or immunological function may be
tolerated.
[0029] The skilled person will appreciate that various changes can
often be made to the amino acid sequence of a polypeptide which has
a particular activity to produce derivatives (sometimes known as
variants or "muteins") having at least a proportion of said
activity, and preferably having a substantial proportion of said
activity. Such derivatives of the polypeptides described in a)
above are within the scope of the present invention and include
allelic and non-allelic derivatives.
[0030] An example of a derivative of the polypeptide for use in the
present invention is a polypeptide as defined in a) above, apart
from the substitution of one or more amino acids with one or more
other amino acids. Amino acid substitutions may be conservative or
semi-conservative as known in the art and preferably do not
significantly affect the desired activity of the polypeptide.
Substitutions may be naturally occurring or may be introduced for
example using mutagenesis (e.g. Hutchinson et al., 1978, J. Biol.
Chem. 253:6551).
[0031] Thus, the amino acids glycine, alanine, valine, leucine and
isoleucine can often be substituted for one another (amino acids
having aliphatic side chains). Of these possible substitutions, it
is preferred that glycine and alanine are used to substitute for
one another (since they have relatively short side chains) and that
valine, leucine and isoleucine are used to substitute for one
another (since they have larger aliphatic side chains which are
hydrophobic).
[0032] Other amino acids which can often be substituted for one
another include:
[0033] phenylalanine, tyrosine and tryptophan (amino acids having
aromatic side chains);
[0034] lysine, arginine and histidine (amino acids having basic
side chains);
[0035] aspartate and glutamate (amino acids having acidic side
chains);
[0036] asparagine and glutamine (amino acids having amide side
chains); and
[0037] cysteine and methionine (amino acids having
sulphur-containing side chains); and
[0038] aspartic acid and glutamic acid can substitute for
phospho-serine and phospho-threonine, respectively (amino acids
with acidic side chains).
[0039] Amino acid deletions or insertions may also be made relative
to the amino acid sequence given in a) above. Thus, for example,
amino acids which do not have a substantial effect on the
biological and/or immunological activity of the polypeptide, or at
least which do not eliminate such activity, may be deleted. Such
deletions can be advantageous since the overall length and the
molecular weight of a polypeptide can be reduced whilst still
retaining activity. This can enable the amount of polypeptide
required for a particular purpose to be reduced, for example,
dosage levels can be reduced.
[0040] In a particular embodiment, the substituted amino acid(s) do
significantly affect the activity of the CCMP-1 polypeptide and may
be selected specifically to render dominant negative activity upon
the peptide. In another embodiment, the substituted amino acid(s)
may be selected specifically to render the polypeptide
constitutively active.
[0041] Polypeptides comprising amino acid insertions relative to
the sequence given in a) above are also included within the scope
of the invention. Such changes may alter the properties of a
polypeptide used in the present invention (e.g. to assist in
identification, purification or expression, as explained above in
relation to fusion proteins). Said amino acid changes can be made
using any suitable technique e.g. by using site-directed
mutagenesis (Hutchinson et al., 1978, J. Biol. Chem. 253:6551).
[0042] It should be appreciated that amino acid substitutions or
insertions to the polypeptide for use in the present invention can
be made using naturally occurring or non-naturally occurring amino
acids. Whether or not natural or synthetic amino acids are used, it
is preferred that only L-amino acids are present.
[0043] Whatever amino acid changes are made (whether by means of
substitution, modification, insertion or deletion), preferred
polypeptides for use in the present invention have at least 50%
sequence identity with a polypeptide as defined in a) above, more
preferably the degree of sequence identity is at least 75%, at
least 80%, at least 85%, at least 90% or at least 95%. Sequence
identities of at least 90%, at least 95% or at least 98% are most
preferred.
[0044] The term identity can be used to describe the similarity
between two polypeptide sequences. The degree of amino acid
sequence identity can be calculated using a program such as
"bestfit" (Smith and Waterman, Advances in Applied Mathematics,
482-489 (1981)) to find the best segment of similarity between any
two sequences. The alignment is based on maximising the score
achieved using a matrix of amino acid similarities, such as that
described by Schwarz and Dayhof (1979) Atlas of Protein Sequence
and Structure, Dayhof, M. O., Ed pp 353-358.
[0045] A software package well known in the art for carrying out
this procedure is the CLUSTAL program. It compares the amino acid
sequences of two polypeptides and finds the optimal alignment by
inserting spaces in either sequence as appropriate. The amino acid
identity or similarity (identity plus conservation of amino acid
type) for an optimal alignment can also be calculated using a
software package such as BLASTX. This program aligns the largest
stretch of similar sequence and assigns a value to the fit. For any
one pattern comparison, several regions of similarity may be found,
each having a different score. One skilled in the art will
appreciate that two polypeptides of different lengths may be
compared over the entire length of the longer fragment.
Alternatively small regions may be compared. Normally sequences of
the same length are compared for a useful comparison to be
made.
[0046] Where high degrees of sequence identity are present there
will be relatively few differences in amino acid sequence. Thus for
example they may be less than 20, less than 10, or even less than 5
differences.
[0047] Polypeptides within the Scope of c)
[0048] As discussed supra, it is often advantageous to reduce the
length of a polypeptide, provided that the resultant reduced length
polypeptide still has a desired activity or can give rise to useful
antibodies. Feature c) therefore covers fragments of polypeptides
a) or b) above for use in the present invention.
[0049] The skilled person can determine whether or not a particular
fragment has activity using the techniques disclosed above.
Preferred fragments are at least 10 amino acids long. They may be
at least 20, at least 50, at least 100 amino acids or more long.
Any given fragment of a polypeptide may or may not possess a
functional activity of the parent polypeptide.
[0050] A `derivative` of a polypeptide includes a polypeptide that
comprises an amino acid sequence of a parent polypeptide that has
been altered by the introduction of amino acid residue
substitutions, deletions or additions, and/or amino acid
modifications such as but not limited to, phosphorylation and
glycosylation. A derivative also encompasses homologues, analogues
and orthologues of a parent polypeptide. The derivative polypeptide
preferably possesses a similar or identical function to the parent
polypeptide.
[0051] One means for detecting/quantifying a CCMP-1 polypeptide of
use in the methods of screening and diagnosis disclosed herein,
involves the use of an antibody. Thus, CCMP-1 polypeptides also
find use in raising antibodies. Preferably, the antibody is used
for detecting and/or quantifying the amount of a polypeptide as
defined in the first aspect of the invention in a biological sample
obtained from said subject.
[0052] In one embodiment, binding of antibody in tissue sections
can be used to detect aberrant CCMP-1 polypeptide localisation or
an aberrant level of a CCMP-1 polypeptide. In a specific
embodiment, an antibody recognising a CCMP-1 polypeptide can be
used to assay a patient tissue (e.g. a breast biopsy) for the level
of the CCMP-1 polypeptide where an aberrant level of the CCMP-1
polypeptide is indicative of carcinoma. An "aberrant level"
includes a level that is increased or decreased compared with the
level in a subject free from carcinoma or a reference level.
[0053] In a further aspect, the method of detecting/quantifying the
presence of a CCMP-1 polypeptide comprises detecting the captured
polypeptide using a directly or indirectly labelled detection
reagent, e.g. a detectable marker such as, without limitation, a
chemiluminescent, enzymatic, fluorescent, or radioactive
moiety.
[0054] The methods of diagnosis according to the present invention
may be performed using a number of methods known to those skilled
in the art, including, without limitation, immunoprecipitation
followed by sodium dodecyl sulfate polyacrylamide gel
electrophoresis, 2-dimensional gel electrophoresis, competitive and
non-competitive assay systems using techniques such as Western
blots, immunocytochemistry, immunohistochemistry, immunoassays,
e.g. radioimmunoassays, ELISA (enzyme linked immunosorbent assay),
"sandwich" immunoassays, immunoprecipitation assays, precipitin
reactions, gel diffusion precipitin reactions, immunodiffusion
assays, agglutination assays, complement-fixation assays,
immunoradiometric assays, fluorescent immunoassays and protein A
immunoassays.
[0055] In the context of the present invention, the biological
sample can be obtained from any source, such as a serum sample or a
tissue sample, e.g. breast tissue. When looking for evidence of
metastasis, one would look at major sites of metastasis such as
lymph nodes, liver, lung and/or bone and brain.
[0056] The invention also provides diagnostic kits, comprising a
capture reagent (e.g. an antibody) against a CCMP-1 polypeptide as
defined above. In addition, such a kit may optionally comprise one
or more of the following:
[0057] (1) instructions for using the capture reagent for
diagnosis, prognosis, therapeutic monitoring or any combination of
these applications;
[0058] (2) a labelled binding partner to the capture reagent;
[0059] (3) a solid phase (such as a reagent strip) upon which the
capture reagent is immobilised; and
[0060] (4) a label or insert indicating regulatory approval for
diagnostic, prognostic or therapeutic use or any combination
thereof.
[0061] If no labelled binding partner to the capture reagent is
provided, the anti-CCMP-1 polypeptide capture reagent itself can be
labelled with a detectable marker (see above).
[0062] As will be discussed below, CCMP-1 polypeptides are of use
in an immunotherapeutic approach to carcinoma. The skilled person
will appreciate that for the preparation of one or more such
polypeptides, the preferred approach will be based on recombinant
DNA techniques. In addition, nucleic acid molecules encoding the
polypeptides or fragments thereof may be used in their own right.
Thus, the invention also provides a method of screening for and/or
diagnosis of carcinoma in a subject, and/or monitoring the
effectiveness of carcinoma therapy which comprises the step of
detecting and/or quantifying a nucleic acid molecule in a
biological sample obtained from said subject, wherein the nucleic
acid molecule:
[0063] d) comprises or consists of the DNA sequence of SEQ ID NO: 2
or its RNA equivalent;
[0064] e) has a sequence which is complementary to the sequence of
d);
[0065] f) has a sequence which codes for a CCMP-1 polypeptide as
defined in a) to c) above;
[0066] g) has a sequence which shows substantial identity with any
of those of d), e) and f); or
[0067] h) is a fragment of d), e), f) or g), which is at least 30
nucleotides in length.
[0068] Such nucleic acid molecules described above are hereinafter
referred to as CCMP-1 nucleic acids. It is preferred if sequences
which show substantial identity with any of those of d) to f) have
e.g. at least 50%, at least 75%, at least 80%, at least 85%, at
least 90% or 95% and most preferably at least 98% sequence
identity. The `bestfit` program (Smith and Waterman, Advances in
applied Mathematics, 482-489 (1981)) is one example of a type of
computer software used to find the best segment of similarity
between two nucleic acid sequences, whilst the GAP program enables
sequences to be aligned along their whole length and finds the
optimal alignment by inserting spaces in either sequence as
appropriate.
[0069] Unless the context indicates otherwise, CCMP-1 nucleic acid
molecules may have one or more of the following
characteristics:
[0070] 1) they may be DNA or RNA;
[0071] 2) they may be single or double stranded;
[0072] 3) they may be provided in recombinant form, e.g. covalently
linked to a 5' and/or a 3' flanking sequence to provide a molecule
which does not occur in nature;
[0073] 4) they may be provided without 5' and/or 3' flanking
sequences which normally occur in nature;
[0074] 5) they may be provided in substantially pure form. Thus
they may be provided in a form which is substantially free from
contaminating proteins and/or from other nucleic acids; and
[0075] 6) they may be provided with introns or without introns
(e.g. as cDNA).
[0076] These CCMP-1 nucleic acid molecules are now discussed in
greater detail. The term `RNA equivalent` above, indicates that a
given RNA molecule has a sequence which is complementary to that of
a given DNA molecule, allowing for the fact that in RNA `U`
replaces `T` in the genetic code. The nucleic acid molecule may be
in isolated, recombinant or chemically synthetic form.
[0077] In addition to CCMP-1 nucleic acid molecules coding for
CCMP-1 polypeptides, referred to herein as "coding" nucleic acid
molecules, complementary nucleic acids are also included. Thus, for
example, both strands of a double stranded nucleic acid molecule
are included in the present invention (whether or not they are
associated with one another). Also included are mRNA molecules and
complementary DNA molecules (e.g. cDNA molecules).
[0078] The use of nucleic acid molecules which can hybridise to any
of the CCMP-1 nucleic acid molecules discussed above is also
covered by the present invention. Such nucleic acid molecules are
referred to herein as "hybridising" nucleic acid molecules.
Hybridising nucleic acid molecules can be useful as probes or
primers, for example, or in hybridisation assays.
[0079] Hybridisation assays can be used for detection, prognosis,
diagnosis, or monitoring of conditions, disorders, or disease
states, associated with aberrant expression of genes encoding a
CCMP-1 polypeptide, or for differential diagnosis of patients with
signs or symptoms suggestive of carcinoma. In particular, such a
hybridisation assay can be carried out by a method comprising
contacting a patient sample containing nucleic acid with a nucleic
acid probe capable of hybridising to a CCMP-1 DNA or RNA that
encodes a CCMP-1 polypeptide, under conditions such that
hybridisation can occur, and detecting or measuring any resulting
hybridisation. Accordingly, such a hybridisation assay
comprises:
[0080] i) contacting a biological sample, obtained from a subject,
containing nucleic acid with a nucleic acid probe capable of
hybridising to a CCMP-1 nucleic acid molecule, under conditions
such that hybridisation can occur; and
[0081] ii) detecting or measuring any resulting hybridisation.
[0082] Desirably such hybridising molecules are at least 10
nucleotides in length and preferably are at least 25 or at least 50
nucleotides in length. The hybridising nucleic acid molecules
preferably hybridise to nucleic acids within the scope of d), e),
f), g) or h) above, specifically.
[0083] Desirably the hybridising molecules will hybridise to such
molecules under stringent hybridisation conditions. One example of
stringent hybridisation conditions is where attempted hybridisation
is carried out at a temperature of from about 35.degree. C. to
about 65.degree. C. using a salt solution which is about 0.9M.
However, the skilled person will be able to vary such conditions as
appropriate in order to take into account variables such as probe
length, base composition, type of ions present, etc. For a high
degree of selectivity, relatively stringent conditions are used to
form the duplexes, such as low salt or high temperature conditions.
As used herein, "highly stringent conditions" means hybridisation
to filter-bound DNA in 0.5 M NaHPO.sub.4, 7% sodium dodecyl
sulphate (SDS), 1 mM EDTA at 65.degree. C., and washing in
0.1.times.SSC/0.1% SDS at 68.degree. C. (Ausubel F. M. et al.,
eds., 1989, Current Protocols in Molecular Biology, Vol. 1, Green
Publishing Associates, Inc., and John Wiley & Sons, Inc., New
York, at p. 2.10.3). For some applications, less stringent
conditions for duplex formation are required. As used herein
"moderately stringent conditions" means washing in
0.2.times.SSC/0.1% SDS at 42.degree. C. (Ausubel et al., 1989,
supra). Hybridisation conditions can also be rendered more
stringent by the addition of increasing amounts of formamide, to
destabilise the hybrid duplex. Thus, particular hybridisation
conditions can be readily manipulated, and will generally be chosen
depending on the desired results. In general, convenient
hybridisation temperatures in the presence of 50% formamide are:
42.degree. C. for a probe which is 95 to 100% identical to the
fragment of a gene encoding a polypeptide as defined herein,
37.degree. C. for 90 to 95% identity and 32.degree. C. for 70 to
90% identity.
[0084] The invention also provides a kit comprising a nucleic acid
probe capable of hybridising to CCMP-1 RNA encoding a CCMP-1
polypeptide. In a specific embodiment, a kit comprises in one or
more containers a pair of primers (e.g. each in the size range of
6-30 nucleotides, more preferably 10-30 nucleotides and still more
preferably 10-20 nucleotides) that under appropriate reaction
conditions can prime amplification of at least a portion of a
nucleic acid encoding a polypeptide as defined herein, such as by
polymerase chain reaction (see e.g. Innis et al., 1990, PCR
Protocols, Academic Press, Inc., San Diego, Calif.), ligase chain
reaction (see EP320,308) use of Q.beta. replicase, cyclic probe
reaction, or other methods known in the art.
[0085] A hybridising nucleic acid molecule of use in the present
invention may have a high degree of sequence identity along its
length with a nucleic acid molecule within the scope of d)-h) above
(e.g. at least 50%, at least 75%, at least 80%, at least 85%, at
least 90%, 95%, or at least 98% sequence identity). As will be
appreciated by the skilled person, the higher the sequence identity
a given single stranded nucleic acid molecule has with another
nucleic acid molecule, the greater the likelihood that it will
hybridise to a nucleic acid molecule which is complementary to that
other nucleic acid molecule under appropriate conditions.
[0086] If desired, a gene encoding a CCMP-1 polypeptide, a related
gene, or related CCMP-1 nucleic acid sequence or sub-sequence,
including complementary sequences, can also be used in
hybridisation assays. A CCMP-1 nucleotide encoding a CCMP-1
polypeptide, or sub-sequences thereof comprising at least 8
nucleotides, can be used as a hybridisation probe.
[0087] In another aspect, the present invention provides a method
for the prophylaxis and/or treatment of carcinoma in a subject,
which comprises administering to said subject a therapeutically
effective amount of at least one CCMP-1 polypeptide.
[0088] In a yet another aspect, the present invention provides the
use of at least one CCMP-1 polypeptide in the preparation of a
pharmaceutical composition for use in the prophylaxis and/or
treatment of carcinoma. The subject may be a mammal and is
preferably a human.
[0089] In a particular embodiment, a CCMP-1 polypeptide is fused to
another polypeptide, such as the protein transduction domain of the
HIV/Tat protein, which facilitates the entry of the fusion protein
into a cell (Asoh, S. et al., 2002, Proc. Natl. Acad. Sci. USA,
99:17107-17112), is provided for use in the manufacture of a
pharmaceutical composition for the treatment of carcinoma.
[0090] Recombinant CCMP-1 polypeptides may be prepared by processes
well known in the art from genetically engineered host cells
comprising expression systems. Accordingly, the present invention
also relates to expression systems which comprise a CCMP-1
polypeptide or CCMP-1 nucleic acid, to host cells which are
genetically engineered with such expression systems and to the
production of CCMP-1 polypeptides by recombinant techniques.
Cell-free translation systems can also be employed to produce
recombinant polypeptides (e.g. rabbit reticulocyte lysate, wheat
germ lysate, SP6/T7 in vitro T&T and RTS100 E. Coli HY
transcription and translation kits from Roche Diagnostics Ltd.,
Lewes, UK and the TNT Quick coupled Transcription/Translation
System from Promega UK, Southampton, UK.
[0091] For recombinant CCMP-1 polypeptide production, host cells
can be genetically engineered to incorporate expression systems or
portions thereof for CCMP-1 nucleic acids.
[0092] Such incorporation can be performed using methods well known
in the art, such as, calcium phosphate transfection, DEAD-dextran
mediated transfection, transvection, microinjection, cationic
lipid-mediated transfection, electroporation, transduction, scrape
loading, ballistic introduction or infection (see e.g. Davis et
al., Basic Methods in Molecular Biology, 1986 and Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 2.sup.nd Ed., Cold Spring
Harbour laboratory Press, Cold Spring Harbour, N.Y., 1989).
[0093] Representative examples of host cells include bacterial
cells e.g. E. Coli, Streptococci, Staphylococci, Streptomyces and
Bacillus subtilis cells; fungal cells, such as yeast cells and
Aspergillus cells; insect cells such as Drosophila S2 and
Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127,
3T3, HEK293, BHK and Bowes melanoma cells; and plant cells.
[0094] A wide variety of expression systems can be used, such as
and without limitation, chromosomal, episomal and virus-derived
systems, e.g. vectors derived from bacterial plasmids, from
bacteriophage, from transposons, from yeast episomes, from
insertion elements, from yeast chromosomal elements, from viruses
such as baculoviruses, papova viruses such as SV40, vaccinia
viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and
retroviruses, and vectors derived from combinations thereof, such
as those derived from plasmid and bacteriophage genetic elements,
such as cosmids and phagemids. The expression systems may contain
control regions that regulate as well as engender expression.
Generally, any system or vector which is able to maintain,
propagate or express a nucleic acid to produce a polypeptide in a
host may be used. The appropriate CCMP-1 nucleic acid sequence may
be inserted into an expression system by any variety of well-known
and routine techniques, such as those set forth in Sambrook et al.,
supra. Appropriate secretion signals may be incorporated into the
CCMP-1 polypeptide to allow secretion of the translated protein
into the lumen of the endoplasmic reticulum, the periplasmic space
or the extracellular environment. These signals may be endogenous
to the CCMP-1 polypeptide or they may be heterologous signals.
[0095] In one embodiment, CCMP-1 polypeptides are provided in
isolated form and include CCMP-1 polypeptides that have been
purified to at least some extent and may be fused to other
moieties. CCMP-1 polypeptides can be produced using recombinant
methods, synthetically produced or produced by a combination of
these methods. In particular, fusions of the CCMP-1 polypeptides
with localisation-reporter proteins such as the Green Fluorescent
Protein (U.S. Pat. Nos. 5,625,048, 5,777,079, 6,054,321 and
5,804,387) or the DsRed fluorescent protein (Matz, et al., 1999,
Nature Biotech. 17:969-973) are specifically contemplated. CCMP-1
polypeptides may be provided in substantially pure form, that is to
say free, to a substantial extent, from other proteins. Thus, a
CCMP-1 polypeptide may be provided in a composition in which it is
the predominant component present (i.e. it is present at a level of
at least 50%; preferably at least 75%, at least 80%, at least 85%,
at least 90%, or at least 95%; when determined on a weight/weight
basis excluding solvents or carriers).
[0096] If a CCMP-1 polypeptide is to be expressed for use in
cell-based screening assays as discussed later, it is preferred
that the polypeptide be produced at the cell surface. In this
event, the cells may be harvested prior to use in the screening
assay. If the CCMP-1 polypeptide is secreted into the medium, the
medium can be recovered in order to isolate said polypeptide. If
produced intracellularly, the cells must first be lysed before the
CCMP-1 polypeptide is recovered.
[0097] CCMP-1 polypeptides can be recovered and purified from
recombinant cell cultures by well-known methods including, ammonium
sulphate or ethanol precipitation, acid extraction, anion or cation
exchange chromatography, phosphocellulose chromatography, affinity
chromatography, hydrophobic interaction chromatography,
hydroxylapatite chromatography, molecular sieving chromatography,
centrifugation methods, electrophoresis methods and lectin
chromatography. In one embodiment, a combination of these methods
is used. In another embodiment, high performance liquid
chromatography is used. In a further embodiment, an antibody which
specifically binds to a CCMP-1 polypeptide can be used to deplete a
sample comprising a CCMP-1 polypeptide of said polypeptide or to
purify said polypeptide. Techniques well-known in the art, may be
used for refolding to regenerate native or active conformations of
the CCMP-1 polypeptides when the polypeptides have been denatured
during isolation and or purification.
[0098] In a further aspect, the present invention provides a method
for the prophylaxis and/or treatment of carcinoma in a subject,
which comprises administering to said subject a therapeutically
effective amount of at least one CCMP-1 nucleic acid.
[0099] In yet another embodiment, the present invention provides
the use of at least one CCMP-1 nucleic acid in the preparation of a
pharmaceutical composition for use in the treatment of
carcinoma.
[0100] In a specific embodiment, hybridising nucleic acid molecules
are used as anti-sense molecules, to alter the expression of CCMP-1
polypeptides by binding to complementary CCMP-1 nucleic acids and
can be used in the treatment or prevention of carcinoma. An
anti-sense nucleic acid includes a CCMP-1 nucleic acid capable of
hybridising by virtue of some sequence complementarity to a portion
of an RNA (preferably mRNA) encoding a CCMP-1 polypeptide. The
anti-sense nucleic acid can be complementary to a coding and/or
non-coding region of an mRNA encoding such a polypeptide. Most
preferably, expression of a CCMP-1 polypeptide is inhibited by use
of anti-sense nucleic acids. Thus, the present invention provides
the therapeutic or prophylactic use of nucleic acids comprising at
least eight nucleotides that are anti-sense to a gene or cDNA
encoding a CCMP-1 polypeptide.
[0101] In one embodiment, the CCMP-1 nucleic acid is administered
via gene therapy (see for example Hoshida, T. et al., 2002,
Pancreas, 25:111-121; Ikuno, Y. 2002, Invest. Ophthalmol. Vis. Sci.
2002 43:2406-2411; Bollard, C., 2002, Blood 99:3179-3187; Lee E.,
2001, Mol. Med. 7:773-782). Gene therapy refers to administration
to a subject of an expressed or expressible nucleic acid. Any of
the methods for gene therapy available in the art can be used
according to the present invention. In one aspect, the CCMP-1
nucleic acid can be administered as a pharmaceutical composition,
said nucleic acid being part of an expression vector that expresses
a CCMP-1 polypeptide or chimeric protein thereof in a suitable
host. In particular, such a nucleic acid has a promoter operably
linked to the polypeptide coding region, said promoter being
inducible or constitutive (and, optionally, tissue-specific). In
another particular embodiment, a CCMP-1 nucleic acid molecule is
used in which the coding sequences and any other desired sequences
are flanked by regions that promote homologous recombination at a
desired site in the genome, thus providing for intrachromosomal
expression of the nucleic acid (Koller & Smithies, 1989, Proc.
Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature
342:435-438).
[0102] Delivery of the CCMP-1 nucleic acid into a patient may be
direct, in which case the patient is directly exposed to the
nucleic acid or nucleic acid-carrying vector; this approach is
known as in vivo gene therapy. Alternatively, delivery of the
nucleic acid into the patient may be indirect, in which case cells
are first transformed with the nucleic acid in vitro and then
transplanted into the patient; this approach is known as ex vivo
gene therapy.
[0103] Symptoms of carcinoma may be ameliorated by decreasing the
level or activity of a CCMP-1 polypeptide by using gene sequences
encoding a polypeptide as defined herein in conjunction with
well-known gene "knock-out," ribozyme or triple helix methods to
decrease gene expression of the polypeptide. In this approach,
ribozyme or triple helix molecules are used to modulate the
activity, expression or synthesis of the gene, and thus to
ameliorate the symptoms of carcinoma. Such molecules may be
designed to reduce or inhibit expression of a mutant or non-mutant
target gene. Techniques for the production and use of such
molecules are well known to those of skill in the art.
[0104] Endogenous CCMP-1 polypeptide expression can also be reduced
by inactivating or "knocking out" the gene encoding the
polypeptide, or the promoter of such a gene, using targeted
homologous recombination (e.g. see Smithies, et al., 1985, Nature
317:230-234; Thomas & Capecchi, 1987, Cell 51:503-512; Thompson
et al., 1989, Cell 5:313-321; and Zijistra et al., 1989, Nature
342:435-438). For example, a mutant gene encoding a non-functional
polypeptide (or a completely unrelated DNA sequence) flanked by DNA
homologous to the endogenous gene (either the coding regions or
regulatory regions of the gene encoding the polypeptide) can be
used, with or without a selectable marker and/or a negative
selectable marker, to transfect cells that express the target gene
in viva Insertion of the DNA construct, via targeted homologous
recombination, results in inactivation of the target gene.
[0105] CCMP-1 nucleic acids may be obtained using standard cloning
and screening techniques, from a cDNA library derived from mRNA in
human cells, using expressed sequence tag (EST) analysis (Adams, M.
et al., 1991, Science, 252:1651-1656; Adams, M. et al., 1992,
Nature 355:632-634; Adams, M. et al., 1995, Nature, 377:Suppl:
3-174). CCMP-1 nucleic acids can also be obtained from natural
sources such as genomic DNA libraries or can be synthesized using
well known and commercially available techniques. The CCMP-1
nucleic acids comprising coding sequence for CCMP-1 polypeptides
can be used for the recombinant production of said polypeptides.
The CCMP-1 nucleic acids may include the coding sequence for the
mature polypeptide, by itself; or the coding sequence for the
mature polypeptide in reading frame with other coding sequences,
such as those encoding a leader or secretory sequence, a pre-, pro-
or prepro-protein sequence, a cleavable sequence or other fusion
peptide portions, such as an affinity tag or an additional sequence
conferring stability during production of the polypeptide.
Preferred affinity tags include multiple histidine residues (for
example see Gentz et al., 1989, Proc. Natl. Acad. Sci USA
86:821-824), a FLAG tag, HA tag or myc tag. The CCMP-1 nucleic
acids may also contain non-coding 5' and 3' sequences, such as
transcribed, non-translated sequences, splicing and polyadenylation
signals, ribosome binding sites and sequences that stabilize
mRNA.
[0106] In the preparation of genomic libraries, DNA fragments are
generated, some of which will encode parts or the whole of a
polypeptide as defined herein. The DNA may be cleaved at specific
sites using various restriction enzymes. Alternatively, one may use
DNAse in the presence of manganese to fragment the DNA, or the DNA
can be physically sheared, as for example, by sonication. The DNA
fragments can then be separated according to size by standard
techniques, including but not limited to agarose and polyacrylamide
gel electrophoresis, column chromatography and sucrose gradient
centrifugation. The DNA fragments can then be inserted into
suitable vectors, including but not limited to plasmids, cosmids,
bacteriophages lambda or T.sub.4, and yeast artificial chromosomes
(YACs) (see, for example, Sambrook et al., 1989, Molecular Cloning,
A Laboratory Manual, 1D Ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y.; Glover, D. M. (ed.), 1985, DNA Cloning: A
Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II;
Ausubel F. M. et al., eds., 1989, Current Protocols in Molecular
Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley
& sons, Inc., New York). The genomic library may be screened by
nucleic acid hybridisation to labelled probe (Benton & Davis,
1977, Science 196:180; Grunstein & Hogness, 1975, Proc. Natl.
Acad. Sci. U.S.A. 72:3961).
[0107] Manipulation of the DNA encoding a protein is a particularly
powerful technique for both modifying proteins and for generating
large quantities of protein for purification purposes. This may
involve the use of PCR techniques to amplify a desired nucleic acid
sequence. Thus the sequence data provided herein can be used to
design primers for use in PCR so that a desired sequence can be
targeted and then amplified to a high degree.
[0108] Typically, primers will be at least five nucleotides long
and will generally be at least ten nucleotides long (e.g. fifteen
to twenty-five nucleotides long). In some cases, primers of at
least thirty or at least thirty-five nucleotides in length may be
used.
[0109] As a further alternative, chemical synthesis which may be
automated may be used. Relatively short sequences may be chemically
synthesised and ligated together to provide a longer sequence.
[0110] CCMP-1 polypeptide derivatives as referred to in part b)
above can be created by introducing one or more nucleotide
substitutions, additions or deletions into the nucleotide sequence
of a CCMP-1 nucleic acid such that one or more amino acid
substitutions, additions or deletions are introduced into the
encoded protein. Standard techniques known to those of skill in the
art can be used to introduce mutations, including, for example,
site-directed mutagenesis and PCR-mediated mutagenesis. Preferably,
conservative amino acid substitutions are made at one or more
predicted non-essential amino acid residues.
[0111] A CCMP-1 nucleic acid encoding a CCMP-1 polypeptide,
including homologues and orthologues from species other than human,
may be obtained by a process which comprises the steps of screening
an appropriate library under stringent hybridisation conditions
with a labelled probe having the sequence of a CCMP-1 nucleic acid
as described in d)-h) above, and isolating full-length cDNA and
genomic clones containing said nucleic acid sequence. Such
hybridisation techniques are well-known in the art and examples of
hybridisation conditions are described, supra.
[0112] One skilled in the art will understand that, in many cases,
an isolated cDNA sequence will be incomplete, in that the region
coding for the polypeptide is cut short at the 5' end of the cDNA.
This is a consequence of reverse transcriptase, an enzyme with
inherently low processivity (a measure of the ability of the enzyme
to remain attached to the template during the polymerization
reaction), failing to complete a DNA copy of the mRNA template
during 1.sup.st strand cDNA synthesis.
[0113] Methods to obtain full length cDNAs or to extend short cDNAs
are well known in the art, for example RACE (Rapid amplification of
cDNA ends; e.g. Frohman et al., 1988, Proc. Natl. Acad. Sci USA
85:8998-9002). Recent modifications of the technique, exemplified
by the Marathon.TM. technology (Clontech Laboratories Inc.) have
significantly simplified the search for longer cDNAs. This
technology uses cDNAs prepared from mRNA extracted from a chosen
tissue followed by the ligation of an adaptor sequence onto each
end. PCR is then carried out to amplify the missing 5' end of the
cDNA using a combination of gene specific and adaptor specific
oligonucleotide primers. The PCR reaction is then repeated using
nested primers which have been designed to anneal with the
amplified product, typically an adaptor specific primer that
anneals further 3' in the adaptor sequence and a gene specific
primer that anneals further 5' in the known gene sequence. The
products of this reaction can then be analysed by DNA sequencing
and a full length cDNA constructed either by joining the product
directly to the existing cDNA to give a complete sequence, or
carrying out a separate full length PCR using the new sequence
information for the design of the 5' primer.
[0114] A further aspect of the invention relates to a vaccine
composition of use in the treatment of carcinoma. Thus, a CCMP-1
polypeptide or CCMP-1 nucleic acid may be useful as antigenic
material, and may be used in the production of vaccines for
treatment or prophylaxis of carcinoma. Such material can be
"antigenic" and/or "immunogenic". Generally, "antigenic" is taken
to mean that the protein or nucleic acid is capable of being used
to raise antibodies or indeed is capable of inducing an antibody
response in a subject. "Immunogenic" is taken to mean that the
protein or nucleic acid is capable of eliciting a protective immune
response in a subject. Thus, in the latter case, the CCMP-1
polypeptide or nucleic acid may be capable of not only generating
an antibody response but, in addition, non-antibody based immune
responses.
[0115] It is well known that is possible to screen an antigenic
protein or polypeptide to identify epitopic regions, i.e. those
regions which are responsible for the protein or polypeptide's
antigenicity or immunogenicity. Amino acid and peptide
characteristics well known to the skilled person can be used to
predict the antigenic index (a measure of the probability that a
region is antigenic) of a CCMP-1 polypeptide. For example, but
without limitation, the `Peptidestructure` program (Jameson and
Wolf, 1988, CABIOS, 4(1):181) and a technique referred to as
`Threading` (Altuvia Y. et al., 1995, J. Mol. Biol. 249:244) can be
used. Thus, the CCMP-1 polypeptides may include one or more such
epitopes or be sufficiently similar to such regions so as to retain
their antigenic/immunogenic properties. Methods well known to the
skilled person can be used to test fragments and/or homologues
and/or derivatives of a polypeptide for antigenicity. Thus, the
fragments for use in the present invention may include one or more
such epitopic regions or be sufficiently similar to such regions to
retain their antigenic/immunogenic properties. Thus, for fragments
for use according to the present invention the degree of identity
is perhaps irrelevant, since they may be 100% identical to a
particular part of a protein or polypeptide, homologue or
derivative as described herein. The key issue may be that the
fragment retains the antigenic/immunogenic properties of the
protein from which it is derived.
[0116] CCMP-1 derivatives preferably possess at least a degree of
the antigenicity and/or immunogenicity of the protein or
polypeptide from which they are derived.
[0117] Thus, in a further aspect, the present invention provides
the use of a CCMP-1 polypeptide or a CCMP-1 nucleic acid in the
production of a pharmaceutical composition for the treatment or
prophylaxis of carcinoma, wherein the composition is a vaccine. The
vaccine optionally comprises one or more suitable adjuvants.
Examples of adjuvants well-known in the art include inorganic gels,
such as aluminium hydroxide, and water-in-oil emulsions, such as
incomplete Freund's adjuvant. Other useful adjuvants will be well
known to the skilled person.
[0118] Since a polypeptide or a nucleic acid may be broken down in
the stomach, the vaccine composition is preferably administered
parenterally (e.g. subcutaneous, intramuscular, intravenous or
intradermal injection).
[0119] Accordingly, the present invention provides:
[0120] (a) the use of a CCMP-1 polypeptide or CCMP-1 nucleic acid
in the preparation of an immunogenic composition, preferably a
vaccine for the treatment of carcinoma;
[0121] (b) the use of such an immunogenic composition in inducing
an immune response in a subject; and
[0122] (c) a method for the treatment or prophylaxis of carcinoma
in a subject, or of vaccinating a subject against carcinoma which
comprises the step of administering to the subject an effective
amount of a CCMP-1 polypeptide or CCMP-1 nucleic acid, preferably
as a vaccine.
[0123] In a further aspect, the present invention provides a method
for the treatment and/or prophylaxis of carcinoma in a subject
comprising administering to said subject, a therapeutically
effective amount of at least one antibody that binds to a CCMP-1
polypeptide.
[0124] Most preferred are antibodies that bind specifically to
CCMP-1 polypeptides. In one embodiment, antibodies which
specifically bind to CCMP-1 polypeptides may be used to inhibit the
activity of said polypeptides, or target therapeutic agents to the
tumour.
[0125] Thus, in yet another aspect, the present invention provides
the use of an antibody which binds to at least one CCMP-1
polypeptide in the preparation of a pharmaceutical composition for
use in the prophylaxis and/or treatment of carcinoma. In
particular, the preparation of vaccines and/or compositions
comprising or consisting of antibodies is a preferred embodiment of
this aspect of the invention.
[0126] Accordingly, a CCMP-1 polypeptide may be used as an
immunogen to generate antibodies which immunospecifically bind such
an immunogen. Antibodies of the invention include, but are not
limited to polyclonal, monoclonal, bispecific, humanised or
chimeric antibodies, single chain antibodies, Fab fragments and
F(ab') fragments, fragments produced by a Fab expression library,
anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments
of any of the above. The term "antibody" as used herein includes
immunoglobulin molecules and immunologically-active portions of
immunoglobulin molecules, i.e. molecules that contain an antigen
binding site that specifically binds an antigen. An antibody of the
invention recognises a CCMP-1 polypeptide. Preferably, an antibody
of the invention specifically binds to a CCMP-1 polypeptide. The
immunoglobulin molecules of the invention can be of any class (e.g.
IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulin
molecule.
[0127] In the production of antibodies, screening for the desired
antibody can be accomplished by techniques known in the art, e.g.
ELISA (enzyme-linked immunosorbent assay). For example, to select
antibodies which recognise a specific domain of a CCMP-1
polypeptide, one may assay generated hybridomas for a product which
binds to a polypeptide fragment containing such domain. For
selection of an antibody that specifically binds a first
polypeptide homologue but which does not specifically bind to (or
binds less avidly to) a second polypeptide homologue, one can
select on the basis of positive binding to the first polypeptide
homologue and a lack of binding to (or reduced binding to) the
second polypeptide homologue.
[0128] For preparation of monoclonal antibodies (mAbs) directed
toward a CCMP-1 polypeptide, any technique which provides for the
production of antibody molecules by continuous cell lines in
culture may be used. For example, the hybridoma technique
originally developed by Kohler and Milstein (1975, Nature
256:495-497), as well as the trioma technique, the human B-cell
hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72),
and the EBV-hybridoma technique to produce human monoclonal
antibodies (Cole et al., 1985, in Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of
any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any
subclass thereof. The hybridoma producing the mAbs in the invention
may be cultivated in vitro or in vivo. In an additional embodiment
of the invention, monoclonal antibodies can be produced in
germ-free animals utilising technology known in the art.
[0129] The monoclonal antibodies include, but are not limited to,
human monoclonal antibodies and chimeric monoclonal antibodies
(e.g. human-mouse chimeras). A chimeric antibody is a molecule in
which different portions are derived from different animal species,
such as those having a human immunoglobulin constant region and a
variable region derived from a murine mAb (see, e.g. U.S. Pat. No.
4,816,567; and U.S. Pat. No. 4,816,397) Humanised antibodies are
antibody molecules from non-human species having one or more
complementarity determining regions (CDRs) from the non-human
species and a framework region from a human immunoglobulin molecule
(see, e.g. U.S. Pat. No. 5,585,089).
[0130] Chimeric and humanised monoclonal antibodies can be produced
by recombinant DNA techniques known in the art, for example using
methods described in WO 87/02671; EP184,187; EP171,496; EP173,494;
WO 86/01533; U.S. Pat. No. 4,816,567; EP125,023; Better et al.,
1988, Science 240:1041-1043; Liu et al., 1987, Proc. Natl. Acad.
Sci. USA 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526;
Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura
et al., 1987, Canc. Res. 47:999-1005; Wood et al., 1985, Nature
314:446-449; Shaw et al., 1988, J. Natl. Cancer Inst. 80:1553-1559;
Morrison, 1985, Science 229:1202-1207; Oi et at, 1986,
Bio/Techniques 4:214; U.S. Pat. No. 5,225,539; Jones et at, 1986,
Nature 321:552-525; Verhoeyan et at (1988) Science 239:1534; and
Beidler et at, 1988, J. Immunol. 141:4053-4060.
[0131] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Such antibodies can be
produced using transgenic mice which are incapable of expressing
endogenous immunoglobulin heavy and light chain genes, but which
can express human heavy and light chain genes. The transgenic mice
are immunised in the normal fashion with a selected antigen, e.g.
all or a portion of a CCMP-1 polypeptide. Monoclonal antibodies
directed against the antigen can be obtained using conventional
hybridoma technology. The human immunoglobulin transgenes harboured
by the transgenic mice rearrange during B cell differentiation, and
subsequently undergo class switching and somatic mutation. Thus,
using such a technique, it is possible to produce therapeutically
useful IgG, IgA, IgM and IgE antibodies. For an overview of this
technology for producing human antibodies (see Lonberg &
Huszar, 1995, Int. Rev. Immunol. 13:65-93). For a detailed
discussion of this technology for producing human antibodies and
human monoclonal antibodies and protocols for producing such
antibodies, see, e.g. U.S. Pat. Nos. 5,625,126; 5,633,425;
5,569,825; 5,661,016; and 5,545,806. In addition, companies such as
Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.)
can be engaged to provide human antibodies directed against a
selected antigen using technology similar to that described
above.
[0132] Completely human antibodies which recognise a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach, a selected non-human monoclonal
antibody, e.g. a mouse antibody, is used to guide the selection of
a completely human antibody recognising the same epitope (Jespers
et al., 1994, Bio/technology 12:899-903).
[0133] The antibodies in the present invention can also be
generated using various phage display methods known in the art. In
phage display methods, functional antibody domains are displayed on
the surface of phage particles which carry the polynucleotide
sequences encoding them. In a particular, such phage can be
utilised to display antigen binding domains expressed from a
repertoire or combinatorial antibody library (e.g. human or
murine). Phage expressing an antigen binding domain that binds the
antigen of interest can be selected or identified with antigen,
e.g. using labelled antigen or antigen bound or captured to a solid
surface or bead. Phage used in these methods are typically
filamentous phage including fd and M13 binding domains expressed
from phage with Fab, Fv or disulphide stabilised Fv antibody
domains recombinantly fused to either the phage gene III or gene
VIII protein. Phage display methods that can be used to make the
antibodies of the present invention include those disclosed in
Brinkman et al., 1995, J. Immunol. Methods 182: 41-50; Ames et al.,
1995, J. Immunol. Methods 184:177-186; Kettleborough et al., 1994,
Eur. J. Immunol. 24:952-958; Persic et al, 1997, Gene 187 9-18;
Burton et al., 994, Advances in Immunology 57:191-280; EP0589877;
WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO
95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;
5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;
5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and
5,969,108.
[0134] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g. as described in detail below. For
example, techniques to recombinantly produce Fab, Fab' and F(ab')2
fragments can also be employed using methods known in the art such
as those disclosed in WO 92/22324; Mullinax et al., 1992,
Bio/Techniques 12(6):864-869; and Sawai et al., 1995, AJRI
34:26-34; and Better et al., 1988, Science 240:1041-1043.
[0135] Examples of techniques which can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in
Enzymology 203:46-88 (1991); Shu et al., 1993, PNAS 90:7995-7999;
and Skerra et al., 1988, Science 240:1038-1040.
[0136] The invention further provides bispecific antibodies, which
can be made by methods known in the art. Traditional production of
full length bispecific antibodies is based on the coexpression of
two immunoglobulin heavy chain-light chain pairs, where the two
chains have different specificities (Milstein et al., 1983, Nature
305:537-539). Because of the random assortment of immunoglobulin
heavy and light chains, these hybridomas (quadromas) produce a
potential mixture of 10 different antibody molecules, of which only
one has the correct bispecific structure. Purification of the
correct molecule, which is usually done by affinity chromatography
steps, is rather cumbersome, and the product yields are low.
Similar procedures are disclosed in WO 93/08829, and in Traunecker
et al., 1991, EMBO J. 10:3655-3659.
[0137] According to a different and more preferred approach,
antibody variable domains with the desired binding specificities
(antibody-antigen combining sites) are fused to immunoglobulin
constant domain sequences. The fusion preferably is with an
immunoglobulin heavy chain constant domain, comprising at least
part of the hinge, CH2, and CH3 regions. It is preferred to have
the first heavy-chain constant region (CH1) containing the site
necessary for light chain binding, present in at least one of the
fusions. DNAs encoding the immunoglobulin heavy chain fusions and,
if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable
host organism. This provides for great flexibility in adjusting the
mutual proportions of the three polypeptide fragments in
embodiments when unequal ratios of the three polypeptide chains
used in the construction provide the optimum yields. It is,
however, possible to insert the coding sequences for two or all
three polypeptide chains in one expression vector when the
expression of at least two polypeptide chains in equal ratios
results in high yields or when the ratios are of no particular
significance.
[0138] In a preferred embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm, and a hybrid immunoglobulin
heavy chain-light chain pair (providing a second binding
specificity) in the other arm. This asymmetric structure
facilitates the separation of the desired bispecific compound from
unwanted immunoglobulin chain combinations, as the presence of an
immunoglobulin light chain in only one half of the bispecific
molecule provides for a facile way of separation. This approach is
disclosed in WO 94/04690. For further details for generating
bispecific antibodies (see, for example, Suresh et al., 1986,
Methods in Enzymology 121:210).
[0139] The invention provides functionally-active fragments,
derivatives or analogues of the anti-polypeptide immunoglobulin
molecules. "Functionally-active" means that the fragment,
derivative or analogue is able to elicit anti-anti-idiotype
antibodies (i.e. tertiary antibodies) that recognise the same
antigen that is recognised by the antibody from which the fragment,
derivative or analogue is derived. Specifically, in a preferred
embodiment, the antigenicity of the idiotype of the immunoglobulin
molecule may be enhanced by deletion of framework and CDR sequences
that are C-terminal to the CDR sequence that specifically
recognises the antigen. To determine which CDR sequences bind the
antigen, synthetic peptides containing the CDR sequences can be
used in binding assays with the antigen by any binding assay method
known in the art.
[0140] The present invention provides antibody fragments such as,
but not limited to, F(ab')2 fragments and Fab fragments. Antibody
fragments which recognise specific epitopes may be generated by
known techniques. F(ab')2 fragments consist of the variable region,
the light chain constant region and the CH1 domain of the heavy
chain and are generated by pepsin digestion of the antibody
molecule. Fab fragments are generated by reducing the disulphide
bridges of the F(ab')2 fragments. The invention also provides heavy
chain and light chain dimers of the antibodies of the invention, or
any minimal fragment thereof such as Fvs or single chain antibodies
(SCAs) (e.g. as described in U.S. Pat. No. 4,946,778; Bird, 1988,
Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA
85:5879-5883; and Ward et al., 1989, Nature 334:544-54), or any
other molecule with the same specificity as the antibody of the
invention. Single chain antibodies are formed by linking the heavy
and light chain fragments of the Fv region via an amino acid
bridge, resulting in a single chain polypeptide. Techniques for the
assembly of functional Fv fragments in E. coli may be used (Skerra
et al., 1988, Science 242:1038-1041).
[0141] In other embodiments, the invention provides fusion proteins
of the immunoglobulins of the invention (or functionally active
fragments thereof), for example in which the immunoglobulin is
fused via a covalent bond (e.g. a peptide bond), at either the
N-terminus or the C-terminus to an amino acid sequence of another
protein (or portion thereof, preferably at least 10, 20 or 50 amino
acid portion of the protein) that is not the immunoglobulin.
Preferably the immunoglobulin, or fragment thereof, is covalently
linked to the other protein at the N-terminus of the constant
domain. As stated above, such fusion proteins may facilitate
purification, increase half-life in vivo, and enhance the delivery
of an antigen across an epithelial barrier to the immune
system.
[0142] The immunoglobulins of the invention include analogues and
derivatives that are either modified, i.e. by the covalent
attachment of any type of molecule as long as such covalent
attachment that does not impair immunospecific binding. For
example, but not by way of limitation, the derivatives and
analogues of the immunoglobulins include those that have been
further modified, e.g. by glycosylation, acetylation, pegylation,
phosphylation, amidation, derivatisation by known
protecting/blocking groups, proteolytic cleavage, linkage to a
cellular ligand or other protein, etc. Any of numerous chemical
modifications may be carried out by known techniques, including,
but not limited to specific chemical cleavage, acetylation,
formylation, etc. Additionally, the analogue or derivative may
contain one or more non-classical amino acids.
[0143] The foregoing antibodies can be used in methods known in the
art relating to the localisation and activity of the polypeptides
used in the invention, e.g. for imaging or radio-imaging these
proteins, measuring levels thereof in appropriate physiological
samples, in diagnostic methods, etc. and for radiotherapy.
[0144] The antibodies of the invention can be produced by any
method known in the art for the synthesis of antibodies, in
particular, by chemical synthesis or by recombinant expression, and
are preferably produced by recombinant expression technique.
[0145] Recombinant expression of antibodies, or fragments,
derivatives or analogues thereof, requires construction of a
nucleic acid that encodes the antibody. If the nucleotide sequence
of the antibody is known, a nucleic acid encoding the antibody may
be assembled from chemically synthesised oligonucleotides (e.g. as
described in Kutmeier et al., 1994, Bio/Techniques 17:242), which,
briefly, involves the synthesis of overlapping oligonucleotides
containing portions of the sequence encoding antibody, annealing
and ligation of those oligonucleotides, and then amplification of
the ligated oligonucleotides by PCR.
[0146] Alternatively, the nucleic acid encoding the antibody may be
obtained by cloning the antibody. If a clone containing the nucleic
acid encoding the particular antibody is not available, but the
sequence of the antibody molecule is known, a nucleic acid encoding
the antibody may be obtained from a suitable source (e.g. an
antibody cDNA library, or cDNA library generated from any tissue or
cells expressing the antibody) by PCR amplification using synthetic
primers hybridisable to the 3' and 5' ends of the sequence or by
cloning using an oligonucleotide probe specific for the particular
gene sequence.
[0147] If an antibody molecule that specifically recognises a
particular antigen is not available (or a source for a cDNA library
for cloning a nucleic acid encoding such an antibody), antibodies
specific for a particular antigen may be generated by any method
known in the art, for example, by immunising an animal, such as a
rabbit, to generate polyclonal antibodies or, more preferably, by
generating monoclonal antibodies. Alternatively, a clone encoding
at least the Fab portion of the antibody may be obtained by
screening Fab expression libraries (e.g. as described in Huse et
al., 1989, Science 246:1275-1281) for clones of Fab fragments that
bind the specific antigen or by screening antibody libraries (See,
e.g. Clackson et al., 1991, Nature 352:624; Hane et al., 1997,
Proc. Natl. Acad. Sci. USA 94:4937).
[0148] Once a nucleic acid encoding at least the variable domain of
the antibody molecule is obtained, it may be introduced into a
vector containing the nucleotide sequence encoding the constant
region of the antibody molecule (see, e.g. WO 86/05807; WO
89/01036; and U.S. Pat. No. 5,122,464). Vectors containing the
complete light or heavy chain for co-expression with the nucleic
acid to allow the expression of a complete antibody molecule are
also available. Then, the nucleic acid encoding the antibody can be
used to introduce the nucleotide substitution(s) or deletion(s)
necessary to substitute (or delete) the one or more variable region
cysteine residues participating in an intrachain disulphide bond
with an amino acid residue that does not contain a sulphydryl
group. Such modifications can be carried out by any method known in
the art for the introduction of specific mutations or deletions in
a nucleotide sequence, for example, but not limited to, chemical
mutagenesis, in vitro site directed mutagenesis (Hutchinson et al.,
1978, J. Biol. Chem. 253:6551), PCR based methods, etc.
[0149] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad.
Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda
et al., 1985, Nature 314:452-454) by splicing genes from a mouse
antibody molecule of appropriate antigen specificity together with
genes from a human antibody molecule of appropriate biological
activity can be used. As described supra, a chimeric antibody is a
molecule in which different portions are derived from different
animal species, such as those having a variable region derived from
a murine mAb and a human antibody constant region, e.g. humanised
antibodies.
[0150] Once a nucleic acid encoding an antibody molecule has been
obtained, the vector for the production of the antibody molecule
may be produced by recombinant DNA technology using techniques well
known in the art. Thus, methods for preparing the antibodies used
in the invention by expressing nucleic acid containing the antibody
molecule sequences are described herein. Methods which are well
known to those skilled in the art can be used to construct
expression vectors containing an antibody molecule coding sequences
and appropriate transcriptional and translational control signals.
These methods include, for example, in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. See, for example, the techniques described in
Sambrook et al (1990, Molecular Cloning, A Laboratory Manual, 2d
Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) and
Ausubel et al. (eds., 1998, Current Protocols in Molecular Biology,
John Wiley & Sons, NY).
[0151] The expression vector is transferred to a host cell by
conventional techniques and the transfected cells are then cultured
by conventional techniques to produce an antibody of the
invention.
[0152] The host cells used to express a recombinant antibody of the
invention may be either bacterial cells such as Escherichia coli,
or, preferably, eukaryotic cells, especially for the expression of
whole recombinant antibody molecule. In particular, mammalian cells
such as Chinese hamster ovary cells (CHO), in conjunction with a
vector such as the major intermediate early gene promoter element
from human cytomegalovirus is an effective expression system for
antibodies (Foecking et al., 1985, Gene 45:101; Cockett et al,
1990, BioTechnology 8:2).
[0153] A variety of host-expression vector systems may be utilised
to express an antibody molecule of the invention. Such
host-expression systems represent vehicles by which the coding
sequences of interest may be produced and subsequently purified,
but also represent cells which may, when transformed or transfected
with the appropriate nucleotide coding sequences, express the
antibody molecule of the invention in situ. These include but are
not limited to microorganisms such as bacteria (e.g. E. coli, B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid
DNA or cosmid DNA expression vectors containing antibody coding
sequences; yeast (e.g. Saccharomyces, Pichia) transformed with
recombinant yeast expression vectors containing antibody coding
sequences; insect cell systems infected with recombinant virus
expression vectors (e.g. baculovirus) containing the antibody
coding sequences; plant cell systems infected with recombinant
virus expression vectors (e.g. cauliflower mosaic virus, CaMV;
tobacco mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g. Ti plasmid) containing antibody coding
sequences; or mammalian cell systems (e.g. COS, CHO, BHK, 293, 3T3
cells) harbouring recombinant expression constructs containing
promoters derived from the genome of mammalian cells (e.g.
metallothionein promoter) or from mammalian viruses (e.g. the
adenovirus late promoter; the vaccinia virus 7.5K promoter).
[0154] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
antibody molecule being expressed. For example, when a large
quantity of such a protein is to be produced, for the generation of
pharmaceutical compositions comprising an antibody molecule,
vectors which direct the expression of high levels of fusion
protein products that are readily purified may be desirable. Such
vectors include, but are not limited, to the E. coli expression
vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the
antibody coding sequence may be ligated individually into the
vector in frame with the lac Z coding region so that a fusion
protein is produced; pIN vectors (Inouye & Inouye, 1985,
Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J.
Biol. Chem. 24:5503-5509), and the like pGEX vectors may also be
used to express foreign polypeptides as fusion proteins with
glutathione S-transferase (GST). In general, such fusion proteins
are soluble and can easily be purified from lysed cells by
adsorption and binding to a matrix glutathione-agarose beads
followed by elution in the presence of free glutathione. The pGEX
vectors are designed to include thrombin or factor Xa protease
cleavage sites so that the cloned target gene product can be
released from the GST moiety.
[0155] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The antibody
coding sequence may be cloned individually into non-essential
regions (for example, the polyhedrin gene) of the virus and placed
under control of an AcNPV promoter (for example, the polyhedrin
promoter). In mammalian host cells, a number of viral-based
expression systems (e.g. an adenovirus expression system) may be
utilised.
[0156] As discussed above, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g. glycosylation) and processing (e.g. cleavage)
of protein products may be important for the function of the
protein.
[0157] For long-term, high-yield production of recombinant
antibodies, stable expression is preferred. For example, cells
lines that stably express an antibody of interest can be produced
by transfecting the cells with an expression vector comprising the
nucleotide sequence of the antibody and the nucleotide sequence of
a selectable (e.g. neomycin or hygromycin), and selecting for
expression of the selectable marker. Such engineered cell lines may
be particularly useful in screening and evaluation of compounds
that interact directly or indirectly with the antibody
molecule.
[0158] The expression levels of the antibody molecule can be
increased by vector amplification (for a review, see Bebbington and
Hentschel, The use of vectors based on gene amplification for the
expression of cloned genes in mammalian cells in DNA cloning, Vol.
3, Academic Press, New York, 1987). When a marker in the vector
system expressing antibody is amplifiable, increase in the level of
inhibitor present in culture of host cell will increase the number
of copies of the marker gene. Since the amplified region is
associated with the antibody gene, production of the antibody will
also increase (Crouse et al., 1983, Mol. Cell Biol. 3:257).
[0159] The host cell may be co-transfected with two expression
vectors for use within the invention, the first vector encoding a
heavy chain derived polypeptide and the second vector encoding a
light chain derived polypeptide. The two vectors may contain
identical selectable markers which enable equal expression of heavy
and light chain polypeptides. Alternatively, a single vector may be
used which encodes both heavy and light chain polypeptides. In such
situations, the light chain should be placed before the heavy chain
to avoid an excess of toxic free heavy chain (Proudfoot, 1986,
Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197).
The coding sequences for the heavy and light chains may comprise
cDNA or genomic DNA.
[0160] Once the antibody molecule of the invention has been
recombinantly expressed, it may be purified by any method known in
the art for purification of an antibody molecule, for example, by
chromatography (e.g. ion exchange chromatography, affinity
chromatography such as with protein A or specific antigen, and
sizing column chromatography), centrifugation, differential
solubility, or by any other standard technique for the purification
of proteins.
[0161] Alternatively, any fusion protein may be readily purified by
utilising an antibody specific for the fusion protein being
expressed. For example, a system described by Janknecht et al.
allows for the ready purification of non-denatured fusion proteins
expressed in human cell lines (Janknecht et al., 1991, Proc. Natl.
Acad. Sci. USA 88:8972-897). In this system, the gene of interest
is subcloned into a vaccinia recombination plasmid such that the
open reading frame of the gene is translationally fused to an
amino-terminal tag consisting of six histidine residues. The tag
serves as a matrix binding domain for the fusion protein. Extracts
from cells infected with recombinant vaccinia virus are loaded onto
Ni.sup.2+ nitriloacetic acid-agarose columns and histidine-tagged
proteins are selectively eluted with imidazole-containing
buffers.
[0162] In a preferred embodiment, antibodies of the invention or
fragments thereof are conjugated to a diagnostic or therapeutic
moiety. The antibodies can be used for diagnosis or to determine
the efficacy of a given treatment regimen. Detection can be
facilitated by coupling the antibody to a detectable substance.
Examples of detectable substances include various enzymes,
prosthetic groups, fluorescent materials, luminescent materials,
bioluminescent materials, radioactive nuclides, positron emitting
metals (for use in positron emission tomography), and
non-radioactive paramagnetic metal ions (see generally U.S. Pat.
No. 4,741,900 for metal ions which can be conjugated to antibodies
for use as diagnostics according to the present invention).
Suitable enzymes include horseradish peroxidase, alkaline
phosphatase, beta-galactosidase, or acetylcholinesterase; suitable
prosthetic groups include streptavidin, avidin and biotin; suitable
fluorescent materials include umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride and phycoerythrin; suitable
luminescent materials include luminol; suitable bioluminescent
materials include luciferase, luciferin, and aequorin; and suitable
radioactive nuclides include .sup.125I, .sup.131I, .sup.111In and
.sup.99Tc.
[0163] Antibodies of the invention or fragments thereof can be
conjugated to a therapeutic agent or drug moiety to modify a given
biological response. The therapeutic agent or drug moiety is not to
be construed as limited to classical chemical therapeutic agents.
For example, the drug moiety may be a protein or polypeptide
possessing a desired biological activity. Such proteins may
include, for example, a toxin such as abrin, ricin A, pseudomonas
exotoxin, or diphtheria toxin; a protein such as tumor necrosis
factor, .alpha.-interferon, 62-interferon, nerve growth factor,
platelet derived growth factor, tissue plasminogen activator, a
thrombotic agent or an anti-angiogenic agent, e.g. angiostatin or
endostatin; or, a biological response modifier such as a
lymphokine, interleukin-1 (IL-1), interleukin-2 (IL-2),
interleukin-6 (IL-6), granulocyte macrophage colony stimulating
factor (GM-CSF), granulocyte colony stimulating factor (G-CSF),
nerve growth factor (NGF) or other growth factor. Other therapeutic
moieties may include radionuclides such as .sup.111 In and 90Y;
antibiotics, e.g. calicheamicin; or drugs such as but not limited
to, alkylphosphocholines, topoisomerase I inhibitors, taxoids and
suramin.
[0164] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g. Amon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56, Alan R. Liss, Inc. 1985; Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery, 2nd Edit.,
Robinson et al., eds., 1987, pp. 623-53, Marcel Dekker, Inc.;
Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications; Pinchera et al. eds., 1985, pp. 475-506; "Analysis,
Results, And Future Prospective Of The Therapeutic Use Of
Radiolabelled Antibody In Cancer Therapy", in Monoclonal Antibodies
For Cancer Detection And Therapy, Baldwin et al., eds., pp. 303-16,
Academic Press 1985; Thorpe et al., 1982, Immunol. Rev., 62:119-58;
and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83,
67-123.
[0165] Alternatively, an antibody can be conjugated to a second
antibody to form an antibody heteroconjugate as described in U.S.
Pat. No. 4,676,980.
[0166] An antibody with or without a therapeutic moiety conjugated
to it, can be used as a therapeutic agent that is administered
alone or in combination with cytotoxic factor(s) and/or
cytokine(s).
[0167] A further aspect of the invention provides methods of
screening for active agents that modulate (upregulate or
downregulate) a characteristic of, e.g. the expression or the
enzymatic or binding activity, of a CCMP-1 polypeptide. Agents
identified through the screening methods of the invention are
potential therapeutics for use in the treatment of carcinoma.
[0168] The invention provides methods for identifying active agents
that bind to a CCMP-1 polypeptide or have a stimulatory or
inhibitory effect on the expression or activity of a CCMP-1
polypeptide. Examples of candidate agents, include, but are not
limited to, nucleic acids (e.g. DNA and RNA), carbohydrates,
lipids, proteins, peptides, peptidomimetics, antibodies, agonists,
antagonists, small molecules and other drugs. Active agents can be
obtained using any of the numerous suitable approaches in
combinatorial library methods known in the art, including:
biological libraries; spatially addressable parallel solid phase or
solution phase libraries; synthetic library methods requiring
deconvolution; the "one-bead one-compound" library method; and
synthetic library methods using affinity chromatography selection.
The biological library approach is limited to peptide libraries,
while the other four approaches are applicable to peptide,
non-peptide oligomer or small molecule libraries of compounds (Lam,
1997, Anticancer Drug Des. 12:145; U.S. Pat. No. 5,738,996; and
U.S. Pat. No. 5,807,683).
[0169] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al., 1993, Proc.
Natl. Acad. Sci. USA 90:6909; Erb et al., 1994, Proc. Natl. Acad.
Sci. USA 91:11422; Zuckermann et al., 1994, J. Med. Chem. 37:2678;
Cho et al., 1993, Science 261:1303; Carrell et al., 1994, Angew.
Chem. Int. Ed. Engl. 33:2059; Carell et al., 1994, Angew. Chem.
Int. Ed. Engl. 33:2061; and Gallop et al., 1994, J. Med. Chem.
37:1233.
[0170] Libraries of compounds may be presented, e.g. presented in
solution (e.g. Houghten, 1992, Bio/Techniques 13:412-421), or on
beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature
364:555-556), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat.
Nos. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al.,
1992, Proc. Natl. Acad. Sci. USA 89:1865-1869) or phage (Scott and
Smith, 1990, Science 249:386-390; Devlin, 1990, Science
249:404-406; Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA
87:6378-6382; and Felici, 1991, J. Mol. Biol. 222:301-310).
[0171] In one embodiment, agents that interact with (i.e. bind to)
a CCMP-1 polypeptide are identified in a cell-based assay system.
In accordance with this embodiment, cells expressing a CCMP-1
polypeptide are contacted with a candidate agent and the ability of
the candidate agent to interact with the CCMP-1 polypeptide is
determined. Preferably, the ability of a candidate agent to
interact with a CCMP-1 polypeptide is compared to a reference range
or control. In another embodiment, a first and second population of
cells expressing a CCMP-1 polypeptide are contacted with a
candidate agent or a control agent and the ability of the candidate
agent to interact with the polypeptide is determined by comparing
the difference in interaction between the candidate agent and
control agent. If desired, this assay may be used to screen a
plurality (e.g. a library) of candidate agents. The cell, for
example, can be of prokaryotic origin (e.g. E. coli) or eukaryotic
origin (e.g. yeast or mammalian). Further, the cells can express
the CCMP-1 polypeptide endogenously or be genetically engineered to
express the polypeptide. In some embodiments, the CCMP-1
polypeptide or the candidate agent is labelled, for example with a
radioactive label (such as .sup.32P, .sup.35S or 125I) or a
fluorescent label (such as fluorescein isothiocyanate, rhodamine,
phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde or
fluorescamine) to enable detection of an interaction between a
polypeptide and a candidate agent. The ability of the candidate
agent to interact directly or indirectly with the CCMP-1
polypeptide can be determined by methods known to those of skill in
the art. For example, the interaction between a candidate agent and
a polypeptide can be determined by flow cytometry, a scintillation
assay, immunoprecipitation or Western blot analysis.
[0172] In another embodiment, agents that interact with (i.e. bind
to) a CCMP-1 polypeptide are identified in a cell-free assay system
where a native or recombinant CCMP-1 polypeptide is contacted with
a candidate agent and the ability of the candidate agent to
interact with the polypeptide is determined. Preferably, the
ability of a candidate agent to interact with a CCMP-1 polypeptide
is compared to a reference range or control. Alternatively, a first
and second sample comprising native or recombinant CCMP-1
polypeptide are contacted with a candidate agent or a control agent
and the ability of the candidate agent to interact with the
polypeptide is determined by comparing the difference in
interaction between the candidate agent and control agent. If
desired, this assay may be used to screen a plurality (e.g. a
library) of candidate agents. Preferably, the CCMP-1 polypeptide is
first immobilized, by, for example, contacting the CCMP-1
polypeptide with an immobilized antibody which specifically
recognizes and binds it, or by contacting a purified preparation of
polypeptide with a surface designed to bind proteins. The CCMP-1
polypeptide may be partially or completely purified (e.g. partially
or completely free of other polypeptides) or part of a cell lysate.
Further, the CCMP-1 polypeptide may be a fusion protein comprising
the CCMP-1 polypeptide or a biologically active portion thereof and
a domain such as glutathionine-S-transferase. Alternatively, the
CCMP-1 polypeptide can be biotinylated using techniques well known
to those of skill in the art (e.g. biotinylation kit, Pierce
Chemicals; Rockford, Ill.). The ability of a candidate agent to
interact with a CCMP-1 polypeptide can be can be duplicated by
methods known to those of skill in the art.
[0173] In one embodiment, a CCMP-1 polypeptide is used as a "bait
protein" in a two-hybrid assay or three hybrid assay to identify
other proteins that bind to or interact with the CCMP-1 polypeptide
(see e.g. U.S. Pat. No. 5,283,317; Zervos et al., 1993, Cell
72:223-232; Madura et al. 1993, J. Biol. Chem. 268:12046-12054;
Bartel et al., 1993, Bio/Techniques 14:920-924; Iwabuchi et al.,
1993, Oncogene 8:1693-1696; and WO 94/10300). As those skilled in
the art will appreciate, such binding proteins are also likely to
be involved in the propagation of signals by a CCMP-1 polypeptide.
For example, they may be upstream or downstream elements of a
signalling pathway involving a CCMP-1 polypeptide. Alternatively,
polypeptides that interact with a CCMP-1 polypeptide can be
identified by isolating a protein complex comprising a CCMP-1
polypeptide (i.e. a CCMP-1 polypeptide which interacts directly or
indirectly with one or more other polypeptides) and identifying the
associated proteins using methods known in the art such as mass
spectrometry or Western blotting (for examples see Blackstock, W.
& Weir, M. 1999, Trends in Biotechnology, 17:121-127; Rigaut,
G. 1999, Nature Biotechnology, 17:1030-1032; Husi, H. 2000, Nature
Neurosci. 3:661-669; Ho, Y. et al, 2002, Nature, 415:180-183;
Gavin, A. et al., 2002, Nature, 415: 141-147).
[0174] In all cases, the ability of the candidate agent to interact
directly or indirectly with the CCMP-1 polypeptide can be
determined by methods known to those of skill in the art. For
example but without limitation, the interaction between a candidate
agent and a CCMP-1 polypeptide can be determined by flow cytometry,
a scintillation assay, an activity assay, mass spectrometry,
microscopy, immunoprecipitation or Western blot analysis.
[0175] In another embodiment, agents that competitively interact
with (i.e. bind to) a CCMP-1 polypeptide are identified in a
competitive binding assay. In accordance with this embodiment,
cells expressing the polypeptide are contacted with a candidate
agent and an agent known to interact with the polypeptide; the
ability of the candidate agent to competitively interact with the
polypeptide is then determined. Alternatively, agents that
competitively interact with (i.e. bind to) a CCMP-1 polypeptide are
identified in a cell-free assay system by contacting the
polypeptide with a candidate agent and an agent known to interact
with the polypeptide. As stated above, the ability of the candidate
agent to interact with a CCMP-1 polypeptide can be determined by
methods known to those of skill in the art. These assays, whether
cell-based or cell-free, can be used to screen a plurality (e.g. a
library) of candidate agents.
[0176] In another embodiment, active agents that modulate (i.e.
upregulate or down-regulate) the expression of a CCMP-1 polypeptide
are identified in a cell-based assay. Accordingly, a population of
cells expressing a CCMP-1 polypeptide or nucleic acid are contacted
with a candidate agent and the ability of the candidate agent to
alter expression of the CCMP-1 polypeptide or CCMP-1 nucleic acid
is determined by comparison to a reference range or control. In
another embodiment, a first and second population of cells
expressing a CCMP-1 polypeptide or CCMP-1 nucleic acid are
contacted with a candidate agent or a control agent and the ability
of the candidate agent to alter the expression of the CCMP-1
polypeptide or CCMP-1 nucleic acid is determined by comparing the
difference in the level of expression of the CCMP-1 polypeptide or
CCMP-1 nucleic acid between the first and second populations of
cells. In a further embodiment, the expression of the CCMP-1
polypeptide or CCMP-1 nucleic acid in the first population may be
further compared to a reference range or control. The candidate
agent can then be identified as a modulator of the expression of
the CCMP-1 polypeptide or nucleic acid based on this comparison.
For example, when expression of the CCMP-1 polypeptide or mRNA
encoding said polypeptide is significantly greater in the presence
of the candidate agent than in its absence, the candidate agent is
identified as a stimulator of expression of the CCMP-1 polypeptide
or mRNA encoding said polypeptide. Alternatively, when expression
of the CCMP-1 polypeptide or mRNA encoding the polypeptide is
significantly less in the presence of the candidate agent than in
its absence, the candidate agent is identified as an inhibitor of
the expression of the CCMP-1 polypeptide or mRNA encoding the
polypeptide. The level of expression of a CCMP-1 polypeptide, or
the mRNA that encodes it, can be determined by methods known to
those of skill in the art based on the present description. For
example, CCMP-1 mRNA expression can be assessed by Northern blot
analysis or RT-PCR, and protein levels can be assessed by Western
blot analysis or other means known in the art.
[0177] In another embodiment, active agents that modulate the
activity of a CCMP-1 polypeptide are identified by contacting a
preparation containing the CCMP-1 polypeptide, or cells (e.g.
prokaryotic or eukaryotic cells) expressing the CCMP-1 polypeptide
with a candidate agent or a control agent and determining the
ability of the candidate agent to modulate (e.g. stimulate or
inhibit) the activity of the CCMP-1 polypeptide. The activity of a
CCMP-1 polypeptide can be assessed by detecting its effect on a
"downstream effector" for example, but without limitation,
induction of a cellular signal transduction pathway of the
polypeptide (e.g. intracellular Ca2+, diacylglycerol, IP.sub.3,
cAMP, or other intermediate), detecting catalytic or enzymatic
activity of the CCMP-1 polypeptide on a suitable substrate,
detecting the induction of a reporter gene (e.g. a regulatory
element that is responsive to a CCMP-1 polypeptide and is operably
linked to a nucleic acid encoding a detectable marker, e.g.
luciferase), or detecting a cellular response, for example,
cellular differentiation, or cell proliferation as the case may be,
based on the present description, techniques known to those of
skill in the art can be used for measuring these activities (see,
e.g. U.S. Pat. No. 5,401,639). The candidate agent can then be
identified as a modulator of the activity of a CCMP-1 polypeptide
by comparing the effects of the candidate agent to the control
agent. Suitable control agents include PBS and normal saline
(NS).
[0178] In another embodiment, a cell-based assay system is used to
identify active agents that bind to or modulate the activity of a
protein, such as an enzyme, or a biologically active portion
thereof, which is responsible for the production or degradation of
the CCMP-1 polypeptide or is responsible for the post-translational
modification of the CCMP-1 polypeptide. In a primary screen, a
plurality (e.g. a library) of agents are contacted with cells that
naturally or recombinantly express: (i) a CCMP-1 polypeptide; and
(ii) a protein that is responsible for processing of the CCMP-1
polypeptide in order to identify compounds that modulate the
production, degradation, or post-translational modification of said
CCMP-1 polypeptide. If desired, active agents identified in the
primary screen can then be assayed in a secondary screen against
cells naturally or recombinantly expressing the CCMP-1 polypeptide.
The ability of the candidate agent to modulate the production,
degradation or post-translational modification of a CCMP-1
polypeptide can be determined by methods known to those of skill in
the art, including without limitation, a kinase assay, a
phosphatase assay, flow cytometry, a scintillation assay,
immunoprecipitation and Western blot analysis.
[0179] In yet another embodiment, cells expressing a CCMP-1
polypeptide are contacted with a plurality of candidate agents. The
ability of such an agent to modulate the production, degradation or
post-translational modification of a CCMP-1 polypeptide can be
determined by methods known to those of skill in the art, as
described above.
[0180] In another embodiment, active agents that modulate (i.e.
upregulate or down-regulate) the expression, activity or both the
expression and activity of a CCMP-1 polypeptide are identified in
an animal model. Examples of suitable animals include, but are not
limited to, mice, rats, rabbits, monkeys, guinea pigs, dogs and
cats. Preferably, the animal used represents a model of carcinoma.
Accordingly, a first and second group of mammals are administered
with a candidate agent or a control agent and the ability of the
candidate agent to modulate the expression of the CCMP-1
polypeptide or nucleic acid is determined by comparing the
difference in the level of expression between the first and second
group of mammals. Where desired, the expression levels of the
CCMP-1 polypeptide or nucleic acid in the first and second groups
of mammals can be compared to the level of a CCMP-1 polypeptide or
nucleic acid in a control group of mammals. The candidate agent or
a control agent can be administered by means known in the art (e.g.
orally, rectally or parenterally such as intraperitoneally or
intravenously). Changes in the expression of a CCMP-1 polypeptide
or nucleic acid can be assessed by the methods outlined above. In a
particular embodiment, a therapeutically effective amount of an
agent can be determined by monitoring an amelioration or
improvement in disease symptoms, to delay onset or slow progression
of the disease, for example but without limitation, a reduction in
tumour size. Techniques known to physicians familiar with carcinoma
can be used to determine whether a candidate agent has altered one
or more symptoms associated with the disease.
[0181] The present invention also provides assays for use in drug
discovery in order to identify or verify the efficacy of compounds
for treatment or prevention of carcinoma. Test compounds can be
assayed for their ability to modulate levels of a CCMP-1
polypeptide in a subject having carcinoma. Compounds able to
modulate levels of a CCMP-1 polypeptide in a subject having
carcinoma towards levels found in subjects free from carcinoma or
to produce similar changes in experimental animal models of
carcinoma can be used as lead compounds for further drug discovery,
or used therapeutically. Such assays can also be used to screen
candidate drugs, in clinical monitoring or in drug development,
where abundance of a CCMP-1 polypeptide can serve as a surrogate
marker for clinical disease.
[0182] One skilled in the art will also appreciate that a CCMP-1
polypeptide may also be used in a method for the structure-based
design of an agent, in particular a small molecule which acts to
modulate (e.g. stimulate or inhibit) the activity of said
polypeptide, said method comprising:
[0183] 1) determining the three-dimensional structure of said
polypeptide;
[0184] 2) deducing the three-dimensional structure within the
polypeptide of the likely reactive or binding site(s) of the
agent;
[0185] 3) synthesising candidate agents that are predicted to react
or bind to the deduced reactive or binding site; and
[0186] 4) testing whether the candidate agent is able to modulate
the activity of said polypeptide.
[0187] It will be appreciated that the method described above is
likely to be an iterative process.
[0188] This invention further provides agents identified by the
above-described screening assays, CCMP-1 polypeptides and CCMP-1
nucleic acids and uses thereof for treatments as described herein.
Hereinafter, the agents, CCMP-1 polypeptides, antibodies thereto
and CCMP-1 nucleic acids of use in treatment are referred to as
`active agents`. The term `treatment` includes either therapeutic
or prophylactic therapy. When a reference is made herein to a
method of treating or preventing a disease or condition using a
particular active agent or combination of agents, it is to be
understood that such a reference is intended to include the use of
that active agent or combination of agents in the preparation of a
medicament for the treatment or prevention of the disease or
condition.
[0189] Accordingly, the present invention provides a method for the
prophylaxis and/or treatment of carcinoma, which comprises
administering to said subject a therapeutically effective amount of
at least one active agent of the invention.
[0190] In yet another aspect, the present provides a pharmaceutical
composition comprising at least one active agent of the invention,
optionally together with one or more pharmaceutically acceptable
excipients, carriers or diluents.
[0191] In one embodiment, one or more active agents are
administered alone or in combination with one or more additional
treatments or therapeutic compounds for carcinoma. Examples of such
treatments include, surgery and radiation therapy. Examples of
therapeutic compounds include but are not limited to
cyclophosphamide (Cytoxan.TM.); methotrexate (Methotrexate.TM.);
5-fluorouracil (5-FU); paclitaxel (Taxol); docetaxel
(Taxotere.TM.); vincristine (Oncovin.TM.); vinblastine
(Velban.TM.); vinorelbine (Navelbine.TM.); doxorubicin
(Adriamycin); tamoxifen (Nolvadex.TM.); toremifene (Fareston.TM.);
megestrol acetate (Megace.TM.); anastrozole (Arimidex.TM.);
goserelin (Zoladex.TM.); anti-HER2 monoclonal antibody
(Herceptin.TM.); capecitabine (Xeloda.TM.) and raloxifene
hydrochloride (Evista.TM.).
[0192] In order to use active agents of the invention in therapy
(human or veterinary), they will normally be formulated into a
pharmaceutical composition in accordance with standard
pharmaceutical practice, e.g. by admixing the active agent and a
pharmaceutically acceptable carrier. Thus, according to a further
aspect of the invention there is provided a pharmaceutical
composition comprising at least one active agent of the invention
and a pharmaceutically acceptable carrier. The pharmaceutical
compositions are particularly useful in the prevention or treatment
of carcinoma. In one aspect, the pharmaceutical composition is for
use as a vaccine and so any additional components will be
acceptable for vaccine use. In addition, the skilled person will
appreciate that one or more suitable adjuvants may be added to such
vaccine preparations.
[0193] Active agents of the invention may be administered to a
subject by any of the routes conventionally used for drug
administration, for example they may be administered parenterally,
orally, topically (including buccal, sublingual or transdermal) or
by inhalation. The most suitable route for administration in any
given case will depend on the particular active agent, the
carcinoma involved, the subject, and the nature and severity of the
disease and the physical condition of the subject.
[0194] The active agents may be administered in combination, e.g.
simultaneously, sequentially or separately, with one or more other
therapeutically active, e.g. anti-tumour, compounds.
[0195] The dosage to be administered of an active agent will vary
according to the particular active agent, the carcinoma involved,
the subject, and the nature and severity of the disease and the
physical condition of the subject, and the selected route of
administration; the appropriate dosage can be readily determined by
a person skilled in the art. For the treatment of carcinoma and
tumours in humans and animals, the dosage may range from 0.01 mg/kg
to 750 mg/kg. For prophylactic use in human and animals, the dosage
may range from 0.01 mg/kg to 100 mg/kg.
[0196] The compositions may contain from 0.1% by weight, preferably
from 10-60% by weight, of the active agent of the invention,
depending on the method of administration.
[0197] Pharmaceutical compositions may be conveniently presented in
unit dose forms containing a predetermined amount of an active
agent of the invention per dose. Such a unit may contain for
example but without limitation, 750 mg/kg to 0.01 mg/kg depending
on the condition being treated, the route of administration and the
age, weight and condition of the subject. Preferred unit dosage
compositions are those containing a daily dose or sub-dose, as
recited above, or an appropriate fraction thereof, of the active
agent.
[0198] It will be recognized by one of skill in the art that the
optimal quantity and spacing of individual dosages of an active
agent of the invention will be determined by the nature and extent
of the condition being treated, the form, route and site of
administration, and the particular subject being treated, and that
such optimums can be determined by conventional techniques. It will
also be appreciated by one of skill in the art that the optimal
course of treatment, i.e. the number of doses of an active agent of
the invention given per day for a defined number of days, can be
ascertained by those skilled in the art using conventional course
of treatment determination tests.
[0199] Dosage regimens are adjusted to provide the optimum desired
response. For example, a single bolus may be administered, several
divided doses may be administered over time or the dose may be
proportionally reduced or increased as indicated by the exigencies
of the therapeutic situation.
[0200] Pharmaceutically acceptable carriers for use in the
invention may take a wide variety of forms depending, e.g. on the
route of administration.
[0201] Compositions for oral administration may be liquid or solid.
Oral liquid preparations may be in the form of, for example,
aqueous or oily suspensions, solutions, emulsions, syrups or
elixirs, or may be presented as a dry product for reconstitution
with water or other suitable vehicle before use. Oral liquid
preparations may contain suspending agents, for example sorbitol,
methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose,
carboxymethyl cellulose, aluminium stearate gel or hydrogenated
edible fats, emulsifying agents, for example lecithin, sorbitan
monooleate, or acacia; water; non-aqueous vehicles (which may
include edible oils), for example almond oil, oily esters such as
glycerine, propylene glycol, or ethyl alcohol; preservatives, for
example methyl or propyl p-hydroxybenzoate or sorbic acid;
flavoring agents, preservatives, coloring agents and the like may
be used.
[0202] In the case of oral solid preparations such as powders,
capsules and tablets, carriers such as starches, sugars,
microcrystalline cellulose, diluents, granulating agents,
lubricants, binders, disintegrating agents, and the like may be
included. Because of their ease of administration, tablets and
capsules represent the most advantageous oral dosage unit form in
which case solid pharmaceutical carriers are generally employed. In
addition to the common dosage forms set out above, active agents of
the invention may also be administered by controlled release means
and/or delivery devices. Tablets and capsules may comprise
conventional carriers or excipients such as binding agents for
example, syrup, acacia, gelatin, sorbitol, tragacanth, or
polyvinylpyrrolidone; fillers, for example lactose, sugar,
maize-starch, calcium phosphate, sorbitol or glycine; tableting
lubricants, for example magnesium stearate, talc, polyethylene
glycol or silica; disintegrants, for example potato starch; or
acceptable wetting agents such as sodium lauryl sulphate. The
tablets may be coated by standard aqueous or non-aqueous techniques
according to methods well known in normal pharmaceutical
practice.
[0203] Pharmaceutical compositions of the present invention
suitable for oral administration may be presented as discrete units
such as capsules, cachets or tablets, each containing a
predetermined amount of the active agent, as a powder or granules,
or as a solution or a suspension in an aqueous liquid, a
non-aqueous liquid, an oil-in-water emulsion or a water-in-oil
liquid emulsion. Such compositions may be prepared by any of the
methods of pharmacy but all methods include the step of bringing
into association the active agent with the carrier, which
constitutes one or more necessary ingredients. In general, the
compositions are prepared by uniformly and intimately admixing the
active agent with liquid carriers or finely divided solid carriers
or both, and then, if necessary, shaping the product into the
desired presentation. For example, a tablet may be prepared by
compression or moulding, optionally with one or more accessory
ingredients.
[0204] Compressed tablets may be prepared by compressing, in a
suitable machine, the active agent in a free-flowing form such as a
powder or granules, optionally mixed with a binder, lubricant,
inert diluent, surface active or dispersing agent. Moulded tablets
may be made by moulding, in a suitable machine, a mixture of the
powdered compound moistened with an inert liquid diluent.
Desirably, each tablet contains from about 1 mg to about 500 mg of
the active agent and each cachet or capsule contains from about 1
to about 500 mg of the active agent.
[0205] Compositions comprising an active agent of the invention may
also be prepared in powder or liquid concentrate form. Conventional
water soluble excipients, such as lactose or sucrose, may be
incorporated in the powders to improve their physical properties.
Thus, particularly suitable powders of this invention comprise 50
to 100% w/w, and preferably 60 to 80% w/w of the combination and 0
to 50% w/w and preferably 20 to 40% w/w of conventional excipients.
When used in a veterinary setting such powders may be added to
animal feedstuffs, for example by way of an intermediate premix, or
diluted in animal drinking water.
[0206] Liquid concentrates of this invention for oral
administration suitably contain a water-soluble compound
combination and may optionally include a veterinarily acceptable
water miscible solvent, for example polyethylene glycol, propylene
glycol, glycerol, glycerol formal or such a solvent mixed with up
to 30% v/v of ethanol. The liquid concentrates may be administered
to the drinking water of animals.
[0207] Pharmaceutical compositions suitable for parenteral
administration may be prepared as solutions or suspensions of the
active agents of the invention in water suitably mixed with a
surfactant such as hydroxypropylcellulose. Dispersions can also be
prepared in glycerol, liquid polyethylene glycols, and mixtures
thereof in oils. Under ordinary conditions of storage and use,
these preparations contain a preservative to prevent the growth of
microorganisms.
[0208] The pharmaceutical forms suitable for injectable use include
aqueous or non-aqueous sterile injection solutions which may
contain anti-oxidants, buffers, bacteriostats and solutes which
render the composition isotonic with the blood of the intended
recipient, and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents. Extemporaneous
injection solutions, dispersions and suspensions may be prepared
from sterile powders, granules and tablets.
[0209] The compositions may be presented in unit-dose or multi-dose
containers, for example in sealed ampoules and vials and to enhance
stability, may be stored in a freeze-dried (lyophilized) condition
requiring only the addition of the sterile liquid carrier, for
example water for injections, immediately prior to use. The sterile
liquid carrier may be supplied in a separate vial or ampoule and
can be a solvent or dispersion medium containing, for example,
water, ethanol, polyol (e.g. glycerol, propylene glycol and liquid
polyethylene glycol), suitable mixtures thereof, and vegetable
oils. Advantageously, agents such as a local anaesthetic,
preservative and buffering agents can be included the sterile
liquid carrier.
[0210] In certain embodiments, the active agents of the invention
can be formulated to ensure proper distribution in vivo, for
example, in liposomes. For methods of manufacturing liposomes, see,
e.g. U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The
liposomes may comprise one or more moieties'which are selectively
transported into specific cells or organs, thus enhance targeted
drug delivery (see, e.g. Ranade, V. 1989, J. Clin. Pharmacol. 29:
685).
[0211] Exemplary targeting moieties include folate or biotin (see,
e.g., U.S. Pat. No. 5,416,016); mannosides (Umezawa et al., 1988,
Biochem. Biophys. Res. Comm. 153:1038); antibodies (Bloeman, P. et
al., 1995, FEBS Lett. 357:140; Owais, M. et al. (1995) Antimicrob.
Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe
et al. (1995) Am. J. Physiol. 1233: 134), different species of
which may comprise the compositions of the inventions, as well as
components of the invented molecules; psi 20 (Schreier et al.
(1994) J. Biol. Chem. 269: 9090); see also Keinanen, K. &
Laukkanen, M., 1994, FEBS Lett. 346: 123; Killion, J. & Fidler,
I., 1994, Immunomethods 4: 273. In one embodiment of the invention,
the active agents of the invention are formulated in liposomes; in
a more preferred embodiment, the liposomes include a targeting
moiety. In a most preferred embodiment, the therapeutic compounds
in the liposomes are delivered by bolus injection to a site
proximal to the tumour.
[0212] Pharmaceutical compositions adapted for topical
administration may be formulated as ointments, creams, suspensions,
lotions, powders, solutions, pastes, gels, impregnated dressings,
sprays, aerosols or oils, transdermal devices, dusting powders, and
the like. These compositions may be prepared via conventional
methods containing the active agent. Thus, they may also comprise
compatible conventional carriers and additives, such as
preservatives, solvents to assist drug penetration, emollients in
creams or ointments and ethanol or oleyl alcohol for lotions. Such
carriers may be present as from about 1% up to about 98% of the
composition. More usually they will form up to about 80% of the
composition. As an illustration only, a cream or ointment is
prepared by mixing sufficient quantities of hydrophilic material
and water, containing from about 5-10% by weight of the compound,
in sufficient quantities to produce a cream or ointment having the
desired consistency.
[0213] Pharmaceutical compositions adapted for transdermal
administration may be presented as discrete patches intended to
remain in intimate contact with the epidermis of the recipient for
a prolonged period of time. For example, the active agent may be
delivered from the patch by iontophoresis.
[0214] For applications to external tissues, for example the mouth
and skin, the compositions are preferably applied as a topical
ointment or cream. When formulated in an ointment, the active agent
may be employed with either a paraffinic or a water-miscible
ointment base. Alternatively, the active agent may be formulated in
a cream with an oil-in-water cream base or a water-in-oil base.
[0215] Pharmaceutical compositions adapted for topical
administration in the mouth include lozenges, pastilles and mouth
washes.
[0216] Pharmaceutical compositions adapted for topical
administration to the eye include eye drops wherein the active
agent is dissolved or suspended in a suitable carrier, especially
an aqueous solvent. They also include topical ointments or creams
as above.
[0217] Pharmaceutical compositions suitable for rectal
administration wherein the carrier is a solid are most preferably
presented as unit dose suppositories. Suitable carriers include
cocoa butter or other glyceride or materials commonly used in the
art, and the suppositories may be conveniently formed by admixture
of the combination with the softened or melted carrier(s) followed
by chilling and shaping moulds. They may also be administered as
enemas.
[0218] Pharmaceutical compositions adapted for vaginal
administration may be presented as pessaries, tampons, creams,
gels, pastes, foams or spray compositions. These may comprise
emollients or bases as commonly used in the art.
[0219] In view of the importance of CCMP-1 in carcinoma, the
following form additional aspects of the present invention:
[0220] i) a method of screening for compounds that modulate, e.g.
up-regulate or down-regulate, the expression of a CCMP-1
polypeptide.
[0221] ii) a method for monitoring/assessing carcinoma treatment in
a patient, which comprises the step of determining the presence or
absence and/or quantifying a CCMP-1 polypeptide in a biological
sample obtained from said patient;
[0222] iii) a method for the identification of carcinoma cells in a
biological sample obtained from a subject, which comprises the step
of determining the presence or absence and/or quantifying at least
one CCMP-1 polypeptide;
[0223] (iv) methods of treating carcinoma, comprising administering
to a patient a therapeutically effective amount of an active agent
that modulates' (e.g. upregulates or downregulates) or complements
the expression or the biological activity (or both), or interaction
of a CCMP polypeptide or CCMP-1 nucleic acid in patients having
carcinoma,
[0224] (v) the use of a CCMP-1 polypeptide or CCMP-1 nucleic acid
in the manufacture of a medicament in order to (a) prevent the
onset or development of carcinoma; (b) prevent the progression of
carcinoma; or (c) ameliorate the symptoms of carcinoma.
[0225] (vi) the use of an antibody that recognises a CCMP-1
polypeptide in the manufacture of a medicament for the treatment of
carcinoma;
[0226] (vii) the use of an antibody conjugated to a therapeutic
moiety, said antibody recognising a CCMP-1 polypeptide, in the
manufacture of a medicament for the treatment of carcinoma;
[0227] (viii) a method of treatment of carcinoma in a subject,
which comprises administering to said subject a therapeutically
effective amount of an antibody recognising a CCMP-1
polypeptide;
[0228] (ix) an antibody recognising a CCMP-1 polypeptide for use in
the treatment of carcinoma;
[0229] (x) a method of treatment of carcinoma in a subject, which
comprises administering to said subject a therapeutically effective
amount of an agent which interacts with or modulates the expression
or activity of a CCMP-1 polypeptide or CCMP-1 nucleic acid; and
[0230] (xi) an agent which interacts with or modulates the
expression or activity of a CCMP-1 polypeptide or CCMP-1 nucleic
acid for use in the treatment of carcinoma.
[0231] Preferred features of each aspect of the invention are as
for each of the other aspects mutatis mutandis. The documents
including patents and patent applications mentioned herein are
incorporated to the fullest extent permitted by law.
DESCRIPTION OF THE FIGURES
[0232] FIG. 1 shows the nucleotide and predicted amino acid
sequences of CCMP-1. The tandem mass spectrum is in bold and is
underlined.
[0233] FIG. 2 shows tissue distribution of CCMP-1 mRNA. Levels of
mRNA in normal tissues (open bars) and normal colon tissues
(spotted bars), colon adenocarcinoma tissues (solid bars) and colon
carcinoma cell lines (hatched bars) were quantified by real time
RT-PCR. mRNA levels are expressed along the y-axis as the number of
copies ng.sup.-1 cDNA.
[0234] FIG. 3 shows the expression of CCMP-1 in normal breast
tissues (open bars), tumour breast tissues (hatched bars) and
breast cancer cell lines (solid bars). Levels of CCMP-1 mRNA in
matched normal and tumour tissues from seven breast cancer patients
were measured by real time RT-PCR. mRNA levels are expressed as the
number of copies ng-1 cDNA.
EXAMPLE 1
Identification of CCMP-1 in Colon Adenocarcinoma Cell Lines
[0235] CCMP-1 was isolated from colorectal cancer cell membranes
and breast cancer cell membranes, purified by 1D gel
electrophoresis and characterised by mass spectrometry before being
cloned.
[0236] Proteins in colon adenocarcinoma cell line membranes were
separated by SDS-PAGE and analysed.
[0237] Crude Fractionation of Adherent Colon Adenocarcinoma Cell
Lines
[0238] 1a--Cell Culture
[0239] Cell lines LoVo (ECACC 87060101) and LS174T (ECACC 87060401)
were cultured in Hams F12 or EMEM plus 1% NEM media respectively,
supplemented with 10% foetal calf serum, 2 mM glutamine, 1%
penicillin and 1% streptomycin. The cells were grown at 37.degree.
C. in a humidified atmosphere of 95% air and 5% carbon dioxide.
[0240] 1b--Cell Fractionation and Plasma Membrane Generation
[0241] Purified membrane preparations were isolated from a pool of
colon adenocarcinoma cell lines. Adherent cells (2.times.10.sup.8)
were washed three times with PBS and scraped using a plastic cell
lifter. Cells were centrifuged at 1000.times.g for 5 min at
4.degree. C. and the cell pellet was resuspended in homogenization
buffer (250 mM Sucrose, 10 mM HEPES, 1 mM EDTA, 1 mM Vanadate and
0.02% azide, protease inhibitors). Cells were fractionated using a
ball bearing homogeniser (8.002 mm ball, HGM Lab equipment) until
approx. 95% of cells were broken. Membranes were fractionated using
the method described by Pasquali et al. The fractionated cells were
centrifuged at 3000.times.g for 10 min at 4.degree. C. and the
postnuclear supernatant was layered onto a 60% sucrose cushion and
centrifuged at 100 000.times.g for 45 min. The membranes were
collected using a pasteur pipette and layered on a preformed 15 to
60% sucrose gradient and spun at 100 000.times.g for 17 hr.
Proteins from the fractionated sucrose gradient were run on a 4-20%
1D gel (Novex) and subject to western blotting; those fractions
containing alkaline phosphatase and transferrin immunoreactivity
but not oxidoreductase II or calnexin immunoreactivity were pooled
and represented the plasma membrane fraction.
[0242] 1c--Preparation of Plasma Membrane Fractions for 1D-Gel
Analysis
[0243] Plasma membrane fractions were pooled and diluted at least
four times with 10 mM HEPES, 1 mM EDTA 1 mM Vanadate, 0.02% Azide.
The diluted sucrose fraction was added to a SW40 or SW60 tube and
centrifuged at 100 000.times.g for 45 min with slow acceleration
and deceleration. The supernatant was removed from the membrane
pellet and the pellet washed three times with PBS-CM. The membrane
pellet was solubulized in 2% SDS in 63 mM Tris HCl, pH 7.4. A
protein assay was performed followed by the addition of
mercaptoethanol (2% final), glycerol (10%) and bromopheneol blue
(0.0025% final) was added. A final protein concentration of 1
microgram/microlitre was used for 1D-gel loading.
[0244] 1d-1D--Gel Technology
[0245] Protein or membrane pellets were solubilised in 1D-sample
buffer (approximately 1 mg/ml) and the mixture heated to 95.degree.
C. for 5 min.
[0246] Samples were separated using 1D-gel electrophoresis on
pre-cast 8-16% gradient gels purchased from Bio-Rad (Bio-Rad
Laboratories, Hemel Hempstead, UK). A sample containing 30-50
micrograms of the protein mixtures obtained from a detergent
extract were applied to the stacking gel wells using a
micro-pipette. A well containing molecular weight (10, 15, 25, 37,
50, 75, 100, 150 and 250 kDa) was included for calibration by
interpolation of the separating gel after imaging. Separation of
the proteins was performed by applying a current of 30 mA to the
gel for approximately 5 hr or until the bromophenol blue marker dye
had reached the bottom of the gel.
[0247] After electrophoresis the gel plates were prised open, the
gel placed in a tray of fixer (10% acetic acid, 40% ethanol, 50%
water) and shaken overnight. The gel was then primed for 30 min by
shaking in a primer solution (7.5% acetic acid, 0.05% SDS in
Milli-Q water) followed by incubation with a fluorescent dye (0.06%
OGS dye in 7.5% acetic acid) with shaking for 3 hr. A preferred
fluorescent dye is disclosed in U.S. Pat. No. 6,335,446. Sypro Red
(Molecular Probes, Inc., Eugene, Oreg.) is a suitable alternative
dye for this purpose.
[0248] A digital image of the stained gel was obtained by scanning
on a Storm Scanner (Molecular Dynamics Inc, USA) in the blue
fluorescence mode. The captured image was used to determine the
area of the gel to excise for in-gel proteolysis.
[0249] 1e--Recovery and Analysis of Selected Proteins
[0250] Each vertical lane of the gel was excised using either a
stainless steel scalpel blade or a PEEK gel cutter (OGS) that cuts
sequentially down the length of the gel lane with no attempt at
collecting specific protein bands. The protein CCMP-1 had an
apparent MW of 17.9 kDa.
[0251] Proteins were processed using in-gel digestion with trypsin
(Modified trypsin, Promega, Wisconsin, USA) to generate tryptic
digest peptides. Recovered samples were divided into two. Prior to
MALDI analysis samples were desalted and concentrated using C18 Zip
Tips.TM. (Millipore, Bedford, Mass.). Samples for tandem mass
spectrometry were purified using a nano LC system (LC Packings,
Amsterdam, The Netherlands) incorporating C18 SPE material.
Recovered peptide pools were analysed by MALDI-TOF-mass
spectrometry (Voyager STR, Applied Biosystems, Framingham, Mass.)
using a 337 nm wavelength laser for desorption and the reflectron
mode of analysis. Pools were also analyzed by nano-LC tandem mass
spectrometry (LC/MS/MS) using a Micromass Quadrupole Time-of-Flight
(Q-TOF) mass spectrometer (Micromass, Altrincham, UK). For partial
amino acid sequencing and identification of colon carcinoma
membrane proteins uninterpreted tandem mass spectra of tryptic
peptides were searched against a database of public domain proteins
constructed of protein entries in the non-redundant database held
by the National Centre for Biotechnology Information (NCBI) which
is accessible at http://www.ncbi.nim.nih.gov/using the SEQUEST
search program (Eng et al., 1994, J. Am. Soc. Mass Spectrom.
5:976-989), version v.C.1. Criteria for database identification
included: the cleavage specificity of trypsin; the detection of a
suite of a, b and y ions in peptides returned from the database,
and a mass increment for all Cys residues to account for
carbamidomethylation. Following identification of proteins through
spectral-spectral correlation using the SEQUEST program, masses
detected in MALDI-TOF mass spectra were assigned to tryptic digest
peptides within the proteins identified. In cases where no amino
acid sequences could be identified through searching with
uninterpreted MS/MS spectra of tryptic digest peptides using the
SEQUEST program, tandem mass spectra of the peptides were
interpreted manually, using methods known in the art (in the case
of interpretation of low-energy fragmentation mass spectra of
peptide ions see Gaskell et al., 1992, Rapid Commun. Mass Spectrom.
6:658-662). The method described in WO 02/21139 was also used to
interpret mass spectra.
[0252] One tandem spectrum (see FIG. 1) and mass 1672.9 Da was
found to match GenBank Accession No. X75208.1 and SwissProt
Accession No. P54753 which correspond to CCMP-1.
EXAMPLE 2
Expression of CCMP-1 mRNA in Human Tissues
[0253] Real time quantitative RT-PCR (Heid, et al., 1996, Genome
Res. 6: 986-994; Morrison, et al, 1988, Bio/Techniques 24: 954-958)
was used to analyse the distribution of CCMP-1 mRNA in normal colon
and colon tumour tissues and adenocarcinoma cell lines (see FIG. 2)
and normal breast tissue removed during breast reduction
mammoplasty and breast cancer tissues removed during surgery and
mammary carcinoma cell lines (see FIG. 3). Ethical approval for the
normal and tumour breast samples was obtained at surgery
(University of Oxford, UK). The primers used for PCR were as
follows: sense, 5' actgatcctcgagtggagtgag 3' (SEQ ID NO:3),
antisense, 5' cacctcaaaggtgtagcgcgtg 3' (SEQ ID NO:4). Reactions
containing 10 ng cDNA, prepared as described above, SYBR green
sequence detection reagents (PE Biosystems) and sense and antisense
primers were assayed on an ABI7700 sequence detection system (PE
Biosystems). The PCR conditions were 1 cycle at 50.degree. C. for 2
min, 1 cycle at 95.degree. C. for 10 min, and 40 cycles of
95.degree. C. for 15 s/65.degree. C. for 1 min. The accumulation of
PCR product was measured in real time as the increase in SYBR green
fluorescence, and the data were analysed using the Sequence
Detector program v1.6.3 (PE Biosystems). Standard curves relating
initial template copy number to fluorescence and amplification
cycle were generated using the amplified PCR product as a template,
and were used to calculate CCMP-1 copy number in each sample.
[0254] Expression of CCMP-1 in human tissue showed that the highest
levels of expression were found in mammary gland, brain, salivary
gland and lymph node. CCMP-1 mRNA was also detected in the prostate
cancer cell lines PC3 and PC3M: expression in PC3 cells was
elevated in comparison to normal prostate (not shown). Little or no
CCMP-1 mRNA was detected in the other cell lines or normal tissues
examined. The highest levels of CCMP-1 expression were observed in
the colon carcinoma cell line LS174T (3000 copies ng.sup.-1 cDNA;
FIG. 2), with lower levels of expression in the LoVo cell line
(1000-1100 copies ng.sup.-1 cDNA; FIG. 2). CCMP-1 mRNA was also
detected in mammary carcinoma cell lines BT-20 (ATCC HTB-19),
BT-474 (HTB-20), MCF-7 (ATCC HTB-22; ECACC 86012803), T47D,
MDA-MB-468 (ATCC HTB-132) and MDA-MB-453 (ATCC HTB-131) with a
range of 500-4400 copies ng.sup.-1 cDNA (see FIG. 3).
[0255] To examine whether the observed elevation in CCMP-1
expression in some colon and breast carcinoma lines is reiterated
in clinical samples, the expression of mRNA in matched normal and
tumour tissue samples from thirteen colon cancer patients was also
measured (FIG. 2; samples 1080 through 1402 and 14 through 16,
where N normal tissue and T=tumour tissue) and seven breast cancer
patients (FIG. 3, where N=normal tissue and T=tumour tissue).
CCMP-1 expression was increased in nine of the colon tumour
samples, relative to their matched normal tissues: increases in
expression levels ranged from approximately 1.6- to 16-fold (FIG.
2) and in all seven breast tumour samples and breast cell lines
(FIG. 3). Note the different y-axis scales between FIGS. 2 and
3.
[0256] Thus, CCMP-1 shows a restricted pattern of expression in
normal human tissues, and is elevated in some colon and breast
tumours, suggesting that this protein a potential therapeutic
target.
EXAMPLE 3
Cloning and Expression of CCMP-1
[0257] Full length CCMP-1 was amplified from a pool of cDNAs
(colon, HT29 cells, mammary, LS174T cells) using forward primer:
ctcggctcctagagctgcc (SEQ ID NO:5); reverse primer:
ctgcttggcatccctgcac (SEQ ID NO:6) and the following PCR conditions:
94.degree. C. for 3 min-1 cycle; 94.degree. C. for 30 s, 55.degree.
C. for 30 s, 72.degree. C. for 3 min-40 cycles and 72.degree. C.
for 3 min-1 cycle. The PCR product was amplified with Stratagene
Pfu enzyme. The PCR product coding for CCMP-1 was initially cloned
into pCR4 (Invitrogen) and then re-amplified and cloned into
pcDNA3.1 (Invitrogen) with a C-terminal V5-hexaHlS tag at
KpnI/BamHI sites.
[0258] CCMP-1 cloned into pcDNA3.1 neomycin vector (Invitrogen) was
transfected into HEK 293 cells (GeneJuice, Novagen). The cells were
selected in 0.8 mg/ml Geneticin (antibiotic G418, Sigma) and
resistant cells were pooled and used as a mixed clone pool. HEK 293
cells (primary human embryo kidney cells, ATCC no: CRL-1537) were
grown in Dulbecco's medium NUT mix F12, 10% Fetal Calf serum, 2 mM
glutamine.
[0259] A polyclonal antibody was raised against CCMP-1 using the
services of Abcam Ltd., Cambridge, UK. The antibody was raised in
rabbits immunized with two specific peptides whose sequences were
chosen for synthesis based on plots of hydrophobicity,
antigenicity, surface probability, and low identity to other known
protein family members. Peptides were synthesized using Fmoc
chemistry with a cysteine residue added to the end to enable
specific thiol reactive coupling of Keyhole limpet Hemocyanin prior
to immunization. The CCMP-1 peptides used were: GQYSRPAEFETTSER
(SEQ ID NO:7) and CATGHEPAAKESQ (SEQ ID NO:8).
[0260] Western blotting with the antisera raised recognized a band
of approximately 110 kDa in HEK 293 cell lysate from the mixed
clone pool which over-expressed CCMP-1 but not the parental HEK 293
cells. This recombinant protein also expressed a V5 tag which is
recognized by a specific monoclonal antibody (Invitrogen). Western
Blotting with the anti-V5 tag antibody also recognized a single
band of approximately 110 kDa in HEK 293 cell lysate expressing
CCMP-1.
[0261] These antisera also recognized a protein of identical
molecular weight in LS174T cell lysates (human colon
adenocarcinoma, adherent, ATCC CL-188).
[0262] Measurement of Tyrosine Phosphorylation of CCMP-1
[0263] Parental HEK 293 cells and HEK 293 cells over-expressing
recombinant CCMP-1 with a V5 tag were grown as described above.
Cells were treated for various times in the presence or absence of
500 ng/ml recombinant LERK8-Fc chimera protein (also known as
Ephrin B3 which is a ligand for CCMP-1; R&D Sytems). LERK8-Fc
is a soluble ligand comprising the extracellular domain of LERK8
fused to the Fc region of a human IgG molecule via a polypeptide
linker; this permits the LERK8-Fc protein to be detected using
standard anti-human secondary antibodies. Cells were harvested and
lysed in buffer (20 mM-Tris HCL pH 7.6, 1 mM-EDTA, 150 mM-NaCl, 10
mM-NaF, 1 mM-sodium orthovanadate), mixed and left on ice for 20
min. The cell lysate was then spun at 10,000.times.g and the
supernatant separated by 1D-SDS PAGE electrophoresis. The cell
lysate was then subjected to Western Blotting using RC20
anti-phosphotyrosine antibody (Transduction laboratories).
[0264] It was found that HEK 293 cells over-expressing CCMP-1
showed increased phosphorylation upon treatment with the LERK8
ligand (500 ng/ml) compared to untreated cells grown in serum or in
serum free conditions. Tyrosine phosphorylation was highest between
30 min and 2 hr, but remained increased at 6 hr and 24 hr compared
to untreated cells. LERK8 treatment also resulted in a decrease in
CCMP-1 protein levels as seen by Western blotting using an anti-V5
antibody. Western blots were also stained with Ponceau S and showed
that LERK8 treated cells had equivalent protein loadings to the
untreated cells.
[0265] Phosphotyrosine Immunoprecipitation in LS174T Cells
[0266] To demonstrate CCMP-1 phosphorylation in LS174T cells which
express endogenous CCMP-1, lysates were prepared as described
above, from cells which were treated with LERK8 ligand (500 ng/ml).
Lysates were incubated for 1 hr at 4.degree. C. with 10.mu.1 of
PY-20H anti-phosphotyrosine antiserum (Transduction laboratories).
The antibody was then captured using protein A Sepharose beads
(Pierce). The beads were washed three times with PBS and bound
protein eluted by the addition of 20.mu.1 of 1D-SDS-PAGE sample
buffer lysis buffer. The immunoprecipitate was separated by
1D-SDS-PAGE electrophoresis and subjected to Western blotting using
anti-CCMP-1 antisera. These data indicated that LERK8 ligand
treatment resulted in an increase in tyrosine phosphorylation in
LS174T cells from 10 min to 2 hr.
EXAMPLE 4
Immunocytochemical Analysis of HEK 293 Cells Expressing Recombinant
CCMP-1
[0267] Parental HEK 293 cells and HEK 293 cells over-expressing
recombinant CCMP-1 with a V5 tag were plated overnight, fixed in 4%
paraformaldehyde/PBS and then blocked and permeabilised by
incubation in 10% Donkey Serum/0.3% Triton X-100/PBS for one hr. To
detect CCMP-1 via the C-terminal V5 tag, cells were probed using
1:500 anti-V5 antibody (invitrogen) in 2.5% donkey serum/0.075%
Triton X-100/PBS, washed 3 times in PBS then incubated for another
hour in biotinylated anti-mouse secondary antibody (Jackson
Laboratories) in 2.5% donkey serum/0.075% Triton X-100/PBS. To
detect CCMP-1 using the binding properties of CCMP-1 for its
ligand, LERK8, cells were incubated in 500 ng/ml LERK8-Fc in 2.5%
donkey serum/0.075% Triton X-100/PBS, washed 3 times in PBS then
incubated for a further 1 hr in biotinylated anti-human secondary
antibody (Jackson Laboratories). Cells were then washed 3 times in
PBS then incubated with Extravadin Cy-3 (Sigma).
[0268] Immunocytochemical staining using anti-V5 tag antibody
clearly demonstrated the localisation of recombinant CCMP-1 protein
to the cell membrane of the transfected HEK 293 cells. In these
transfected cells the staining was most intense at sites of
cell-cell contact and on the outer membrane of the cell with no
obvious nuclear, perinuclear or cytosolic staining. No staining in
the parental HEK 293 cells indicated that the antibody was binding
specifically to the tagged recombinant protein in the transfected
cells. Using the LERK-Fc ligand as a probe similar staining
patterns were seen in the transfected HEK 293 cells, with minimal
background in the non-transfected cells.
[0269] Taken together, these results demonstrate that i)
recombinant CCMP-1 is expressed and localised to the cell membrane;
and ii) the recombinant CCMP-1 is folded properly, in that it can
bind to LERK8; and iii) LERK8-Fc can be used to follow CCMP-1
localisation.
EXAMPLE 5
LERK8 Binding to CCMP-1 Induces CCMP-1 Internalisation
[0270] CCMP-1 transfected HEK 293 cells were plated overnight and
incubated under standard tissue culture conditions in the presence
or absence of 500 ng/ml LERK8-Fc ligand for 2 hr prior to fixation
in paraformaldehyde/PBS. Cells were blocked and permeabilised by
incubation in 10% Donkey Serum/0.3% Triton X-100/PBS for an hour
before sequential incubation in 500 ng/ml LERK8-Fc (R & D
systems), then biotinylated anti-human secondary antibody (Jackson
Laboratories) and finally Extravadin Cy-3 (Sigma) all diluted in
2.5% donkey serum/0.075% Triton X-100/PBS and with three washes in
PBS in between each incubation.
[0271] CCMP-1 in untreated CCMP-1 transfected HEK 293 cells was
localised to the cell membrane. The localisation of CCMP-1 in the
HEK 293 cells is most intense at the end of lamellipodia. In LERK8
treated HEK 293 cells, two hours of incubation with LERK-8 Fc
resulted in the loss of essentially all of the cell membrane
staining. Intense, granular cytoplasmic staining was instead
present, indicative of internalisation.
[0272] Flow Cytometry
[0273] CCMP-1 transfected HEK 293 cells and LS174T cells were
plated overnight and incubated under standard tissue culture
conditions in the presence or absence of 500 ng/ml LERK8-Fc ligand
for 10 min, 2 hr or 24 hr, respectively. Cells were dissociated
from the tissue culture plate and 500,000 cells per condition
transferred into a 96 well plate for subsequent processing at
4.degree. C. After washing (by spinning and then re-suspension) in
flow buffer (1% BSA/0.1% azide/PBS) cells were incubated in 500
ng/ml LERK8-Fc in the same buffer for 30 min. Cells were washed 3
times in flow buffer and then incubated in phycoerythrin (PE)
conjugated anti-human secondary antibody (AbCam, cat no 7006)
diluted 1:50 in flow buffer for 30 min in the dark. After 1 wash in
flow buffer and 2 washes in Isoton Flow Cytometry Solution (Beckman
Coulter) the mean fluorescence of a pool of cells was obtained by
analysis using a Beckman Coulter Epics XL.MCL Flow Cytometer.
[0274] The addition of LERK8 to the HEK 293 cells in culture
resulted in a rapid decrease in the mean cell fluorescence
consistent with internalisation of the CCMP-1 protein. The drop in
mean cell fluorescence caused by incubation with LERK8 that is seen
in LS174T cells indicates that these cell also express CCMP-1 on
the cell membrane and hence that endogenous CCMP-1 is expressed on
the surface of human derived carcinoma cell lines. A decrease in
mean cell fluorescence was also seen in LS174T cells when taken in
conjunction with the immunocytochemistry results is consistent with
internalisation of the endogenous CCMP-1 in these cells.
[0275] The above findings suggests that antibody binding to CCMP-1
may result in internalisation of the antibody which may include an
antibody which is conjugated to a toxin, cytokine or
radionucleotide. Thus CCMP-1 represents a good target for
immunotherapy-based approaches to the treatment of carcinoma.
EXAMPLE 6
LERK8 Induces Alterations in Cell Morphology and Cell-Cell
Interactions
[0276] CCMP-1 transfected HEK 293 cells and parental HEK 293 cells
were cultured for 24 hr and followed by overnight incubation in the
presence or absence of 500 ng/ml LERK8-Fc.
[0277] Untreated parental HEK 293 cells grew in loose groups of
cells with cell projections growing out of the cells resulting in a
`jagged` border. Both LERK-8 treated parental cells and untreated
CCMP-1 transfected cells had the same morphology. Overnight
incubation of the CCMP-1 transfected HEK 293 cells, however,
resulted in the retraction of cell projections and the movement of
cells into tight clumps, with maximal cell-cell contact and a
`smooth` border.
[0278] A time course study of the rate of change in morphology
induced by LERK8-Fc demonstrated that retraction of the cell
projections could be seen as early as 20 min after the addition of
LERK8, with complete retraction of the projections after 40 min.
The movement of cells into clumps was initiated after 40 min of
LERK8 treatment and small, smooth-bordered clumps of cells had
formed after 60 min of LERK8 treatment. Over the next 5 hr these
groups of cells moved together to form larger associations of
cells.
EXAMPLE 7
Immunohistochemical Analysis of Tumour Tissues
[0279] A polyclonal antibody was raised against CCMP-1 using the
services of Covalab, France. The antibody was raised in rabbits
immunized with two specific peptides whose sequences were chosen
for synthesis based on plots of hydrophobicity, antigenicity,
surface probability, and low identity to other known protein family
members. Peptide was synthesized using Fmoc chemistry with a
cysteine residue added to the end to enable specific thiol reactive
coupling of Keyhole limpet Hemocyanin prior to immunization. The
CCMP-1 peptide used was: GQYSRPAEFETTSERGS (SEQ ID NO:9).
[0280] To demonstrate the specificity of the antibody raised
against the above peptide, Western blotting analysis of CCMP-1 and
other ephrin B receptor proteins was performed. Lysate from
parental HEK 293 cells was prepared. 5.mu.g of lysate was spiked
with 1.mu.g of recombinant CCMP-1, or 1.mu.g murine ephrin B1, B2,
B3, B4 or B6, or murine ephrin A1-8 receptor proteins (R & D
systems) followed by separation by 1D-SDS-PAGE. The gel was
transferred to PVDF membrane and probed with the polyclonal
antibody raised against the peptide described above (SEQ ID NO:9).
Protein was detected using streptavidin-HRP in conjugation with a
biotinylated secondary antibody. The results indicated that the
antibody recognized only recombinant CCMP-1 and murine ephrin B3
receptor protein (R & D systems), but not the other ephrin B or
ephrin A receptor family members.
[0281] Immunohistochemical analysis was carried out on
formalin-fixed paraffin-embedded tissue microarrays containing 1 mm
sections of breast carcinoma tissue from 55 donors as well as 20
sections of various normal tissues (Clinomics Laboratories Inc.,
165 Tor Court, Pittsfield, Mass. 01201). Slides were deparafinised
by 2.times.5 min washes in xylene then rehydrated through
successive graded ethanol solutions and washed for 5 min in PBS.
Antigen retrieval was achieved by immersing the slides in DAKO
antigen retrieval solution and microwaving for 10 min at full power
(950 W). In addition, detection with the antibody was improved by
protease treatment of the tissue with Autozyme (AbCam) for 5 min at
37.degree. C. The tissue was blocked in 10% donkey serum/PBS for 1
hr before addition of 1.5.mu.g/ml primary polyclonal antibody (in
2.5% donkey serum/PBS). Following 3 washes in PBS the tissue
sections were incubated with biotin-conjugated secondary antibodies
(Biotin-SP-conjugated AffiniPure Donkey anti-guinea pig, Jackson
ImmunoResearch) diluted at 1:200 (2.5.mu.g/ml in 2.5% donkey
serum/PBS) for 1 hr. Slides were washed 3 times in PBS and the
tissue incubated with Streptavidin-HRP (Jackson Immunoresearch)
diluted 1:100 (5.mu.g/ml in 2.5% donkey serum/PBS), followed by
3.times.5 min washes in PBS. Antibody signal was detected using DAB
substrate solution (Dako Ltd.) according to the manufacturers'
instructions. An adjacent tissue array was counterstained for
hematoxylin and eosin (Dako Ltd.) and images were captured by a
digital camera attached to a light microscope.
[0282] Results of immunohistochemical analysis of CCMP-1 on the
breast tumour microarray demonstrated that CCMP-1 was present in
breast carcinoma, breast adencarcinoma and breast infiltrating
ductal carcinoma tissue sections. In each case, expression was
generally restricted to the cancerous epithelial cells of the
tumour tissue and showed a predominantly cell membrane
localisation.
[0283] Results of immunohistochemical analysis of CCMP-1 on the
colon cancer microarray and microarrays comprising prostate
adenocarcinoma, tongue squamous cell carcinoma, larynx squamous
cell carcinoma, pharynx squamous cell carcinoma, parotid carcinoma,
lung squamous cell carcinoma, and a brain astrocytoma, as well as
kidney carcinomas and renal cell carcinomas, bladder carcinoma and
a papillary cystadenocarcinoma all showed expression of CCMP-1
predominantly localised to cancerous epithelial cells.
EXAMPLE 8
Induction of Colony Formation by CCMP-1
[0284] HEK293 cells expressing recombinant CCMP-1 in the presence
and absence of 500 ng/ml LERK8-Fc, HEK 293 parental cells or HEK293
cells transfected with empty pcDNA3.1 vector (vector control cells)
were assessed for colony forming ability.
[0285] 6-well tissue culture plates were coated in 0.9% agar and
left overnight at 37.degree. C. in an incubator. 1,000 cells in 0.5
ml of cell media (HEK 293 pcDNA3.1 vector control cells, HEK 293
parental cells and CCMP-1 expressing HEK 293 cells) were added to 2
ml of methylcellulose-based media (ClonaCell.TM.-TCS, StemCell
Technologies Inc). This mixture was inverted 7 times and left to
stand for 10 min. The 2 ml of media containing cells was then
aliquoted into each well and the tissue culture plates were
incubated at 37.degree. C. 5% CO.sub.2 for 7 days. The number of
colonies for each condition comprising more than one cell was
scored.
[0286] 1 Results Cells Number of colonies HEK 293 parental 18 HEK
293 pcDNA3.1 vector control 24 CCMP-1 overexpressing HEK 293 157
CCMP-1 overexpressing HEK 293 plus LERK8-Fc 244 CCMP-1 expression
in HEK 293 cells increases both the number and the size of colonies
formed suggesting that CCMP-1 over-expression increases
tumourigenic potential.
TABLE-US-00001 Sequence CWU 1 9 1 998 PRT Homo sapiens 1 Met Ala
Arg Ala Arg Pro Pro Pro Pro Pro Ser Pro Pro Pro Gly Leu 1 5 10 15
Leu Pro Leu Leu Pro Pro Leu Leu Leu Leu Pro Leu Leu Leu Leu Pro 20
25 30 Ala Gly Cys Arg Ala Leu Glu Glu Thr Leu Met Asp Thr Lys Trp
Val 35 40 45 Thr Ser Glu Leu Ala Trp Thr Ser His Pro Glu Ser Gly
Trp Glu Glu 50 55 60 Val Ser Gly Tyr Asp Glu Ala Met Asn Pro Ile
Arg Thr Tyr Gln Val 65 70 75 80 Cys Asn Val Arg Glu Ser Ser Gln Asn
Asn Trp Leu Arg Thr Gly Phe 85 90 95 Ile Trp Arg Arg Asp Val Gln
Arg Val Tyr Val Glu Leu Lys Phe Thr 100 105 110 Val Arg Asp Cys Asn
Ser Ile Pro Asn Ile Pro Gly Ser Cys Lys Glu 115 120 125 Thr Phe Asn
Leu Phe Tyr Tyr Glu Ala Asp Ser Asp Val Ala Ser Ala 130 135 140 Ser
Ser Pro Phe Trp Met Glu Asn Pro Tyr Val Lys Val Asp Thr Ile 145 150
155 160 Ala Pro Asp Glu Ser Phe Ser Arg Leu Asp Ala Gly Arg Val Asn
Thr 165 170 175 Lys Val Arg Ser Phe Gly Pro Leu Ser Lys Ala Gly Phe
Tyr Leu Ala 180 185 190 Phe Gln Asp Gln Gly Ala Cys Met Ser Leu Ile
Ser Val Arg Ala Phe 195 200 205 Tyr Lys Lys Cys Ala Ser Thr Thr Ala
Gly Phe Ala Leu Phe Pro Glu 210 215 220 Thr Leu Thr Gly Ala Glu Pro
Thr Ser Leu Val Ile Ala Pro Gly Thr 225 230 235 240 Cys Ile Pro Asn
Ala Val Glu Val Ser Val Pro Leu Lys Leu Tyr Cys 245 250 255 Asn Gly
Asp Gly Glu Trp Met Val Pro Val Gly Ala Cys Thr Cys Ala 260 265 270
Thr Gly His Glu Pro Ala Ala Lys Glu Ser Gln Cys Arg Pro Cys Pro 275
280 285 Pro Gly Ser Tyr Lys Ala Lys Gln Gly Glu Gly Pro Cys Leu Pro
Cys 290 295 300 Pro Pro Asn Ser Arg Thr Thr Ser Pro Ala Ala Ser Ile
Cys Thr Cys 305 310 315 320 His Asn Asn Phe Tyr Arg Ala Asp Ser Asp
Ser Ala Asp Ser Ala Cys 325 330 335 Thr Thr Val Pro Ser Pro Pro Arg
Gly Val Ile Ser Asn Val Asn Glu 340 345 350 Thr Ser Leu Ile Leu Glu
Trp Ser Glu Pro Arg Asp Leu Gly Val Arg 355 360 365 Asp Asp Leu Leu
Tyr Asn Val Ile Cys Lys Lys Cys His Gly Ala Gly 370 375 380 Gly Ala
Ser Ala Cys Ser Arg Cys Asp Asp Asn Val Glu Phe Val Pro 385 390 395
400 Arg Gln Leu Gly Leu Ser Glu Pro Arg Val His Thr Ser His Leu Leu
405 410 415 Ala His Thr Arg Tyr Thr Phe Glu Val Gln Ala Val Asn Gly
Val Ser 420 425 430 Gly Lys Ser Pro Leu Pro Pro Arg Tyr Ala Ala Val
Asn Ile Thr Thr 435 440 445 Asn Gln Ala Ala Pro Ser Glu Val Pro Thr
Leu Arg Leu His Ser Ser 450 455 460 Ser Gly Ser Ser Leu Thr Leu Ser
Trp Ala Pro Pro Glu Arg Pro Asn 465 470 475 480 Gly Val Ile Leu Asp
Tyr Glu Met Lys Tyr Phe Glu Lys Ser Glu Gly 485 490 495 Ile Ala Ser
Thr Val Thr Ser Gln Met Asn Ser Val Gln Leu Asp Gly 500 505 510 Leu
Arg Pro Asp Ala Arg Tyr Val Val Gln Val Arg Ala Arg Thr Val 515 520
525 Ala Gly Tyr Gly Gln Tyr Ser Arg Pro Ala Glu Phe Glu Thr Thr Ser
530 535 540 Glu Arg Gly Ser Gly Ala Gln Gln Leu Gln Glu Gln Leu Pro
Leu Ile 545 550 555 560 Val Gly Ser Ala Thr Ala Gly Leu Val Phe Val
Val Ala Val Val Val 565 570 575 Ile Ala Ile Val Cys Leu Arg Lys Gln
Arg His Gly Ser Asp Ser Glu 580 585 590 Tyr Thr Glu Lys Leu Gln Gln
Tyr Ile Ala Pro Gly Met Lys Val Tyr 595 600 605 Ile Asp Pro Phe Thr
Tyr Glu Asp Pro Asn Glu Ala Val Arg Glu Phe 610 615 620 Ala Lys Glu
Ile Asp Val Ser Cys Val Lys Ile Glu Glu Val Ile Gly 625 630 635 640
Ala Gly Glu Phe Gly Glu Val Cys Arg Gly Arg Leu Lys Gln Pro Gly 645
650 655 Arg Arg Glu Val Phe Val Ala Ile Lys Thr Leu Lys Val Gly Tyr
Thr 660 665 670 Glu Arg Gln Arg Arg Asp Phe Leu Ser Glu Ala Ser Ile
Met Gly Gln 675 680 685 Phe Asp His Pro Asn Ile Ile Arg Leu Glu Gly
Val Val Thr Lys Ser 690 695 700 Arg Pro Val Met Ile Leu Thr Glu Phe
Met Glu Asn Cys Ala Leu Asp 705 710 715 720 Ser Phe Leu Arg Leu Asn
Asp Gly Gln Phe Thr Val Ile Gln Leu Val 725 730 735 Gly Met Leu Arg
Gly Ile Ala Ala Gly Met Lys Tyr Leu Ser Glu Met 740 745 750 Asn Tyr
Val His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Asn Ser 755 760 765
Asn Leu Val Cys Lys Val Ser Asp Phe Gly Leu Ser Arg Phe Leu Glu 770
775 780 Asp Asp Pro Ser Asp Pro Thr Tyr Thr Ser Ser Leu Gly Gly Lys
Ile 785 790 795 800 Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile Ala Tyr
Arg Lys Phe Thr 805 810 815 Ser Ala Ser Asp Val Trp Ser Tyr Gly Ile
Val Met Trp Glu Val Met 820 825 830 Ser Tyr Gly Glu Arg Pro Tyr Trp
Asp Met Ser Asn Gln Asp Val Ile 835 840 845 Asn Ala Val Glu Gln Asp
Tyr Arg Leu Pro Pro Pro Met Asp Cys Pro 850 855 860 Thr Ala Leu His
Gln Leu Met Leu Asp Cys Trp Val Arg Asp Arg Asn 865 870 875 880 Leu
Arg Pro Lys Phe Ser Gln Ile Val Asn Thr Leu Asp Lys Leu Ile 885 890
895 Arg Asn Ala Ala Ser Leu Lys Val Ile Ala Ser Ala Gln Ser Gly Met
900 905 910 Ser Gln Pro Leu Leu Asp Arg Thr Val Pro Asp Tyr Thr Thr
Phe Thr 915 920 925 Thr Val Gly Asp Trp Leu Asp Ala Ile Lys Met Gly
Arg Tyr Lys Glu 930 935 940 Ser Phe Val Ser Ala Gly Phe Ala Ser Phe
Asp Leu Val Ala Gln Met 945 950 955 960 Thr Ala Glu Asp Leu Leu Arg
Ile Gly Val Thr Leu Ala Gly His Gln 965 970 975 Lys Lys Ile Leu Ser
Ser Ile Gln Asp Met Arg Leu Gln Met Asn Gln 980 985 990 Thr Leu Pro
Val Gln Val 995 2 3805 DNA Homo sapiens 2 ggctcggctc ctagagctgc
cacggccatg gccagagccc gcccgccgcc gccgccgtcg 60 ccgccgccgg
ggcttctgcc gctgctccct ccgctgctgc tgctgccgct gctgctgctg 120
cccgccggct gccgggcgct ggaagagacc ctcatggaca caaaatgggt aacatctgag
180 ttggcgtgga catctcatcc agaaagtggg tgggaagagg tgagtggcta
cgatgaggcc 240 atgaatccca tccgcacata ccaggtgtgt aatgtgcgcg
agtcaagcca gaacaactgg 300 cttcgcacgg ggttcatctg gcggcgggat
gtgcagcggg tctacgtgga gctcaagttc 360 actgtgcgtg actgcaacag
catccccaac atccccggct cctgcaagga gaccttcaac 420 ctcttctact
acgaggctga cagcgatgtg gcctcagcct cctccccctt ctggatggag 480
aacccctacg tgaaagtgga caccattgca cccgatgaga gcttctcgcg gctggatgcc
540 ggccgtgtca acaccaaggt gcgcagcttt gggccacttt ccaaggctgg
cttctacctg 600 gccttccagg accagggcgc ctgcatgtcg ctcatctccg
tgcgcgcctt ctacaagaag 660 tgtgcatcca ccaccgcagg cttcgcactc
ttccccgaga ccctcactgg ggcggagccc 720 acctcgctgg tcattgctcc
tggcacctgc atccctaacg ccgtggaggt gtcggtgcca 780 ctcaagctct
actgcaacgg cgatggggag tggatggtgc ctgtgggtgc ctgcacctgt 840
gccaccggcc atgagccagc tgccaaggag tcccagtgcc gcccctgtcc ccctgggagc
900 tacaaggcga agcagggaga ggggccctgc ctcccatgtc cccccaacag
ccgtaccacc 960 tccccagccg ccagcatctg cacctgccac aataacttct
accgtgcaga ctcggactct 1020 gcggacagtg cctgtaccac cgtgccatct
ccaccccgag gtgtgatctc caatgtgaat 1080 gaaacctcac tgatcctcga
gtggagtgag ccccgggacc tgggtgtccg ggatgacctc 1140 ctgtacaatg
tcatctgcaa gaagtgccat ggggctggag gggcctcagc ctgctcacgc 1200
tgtgatgaca acgtggagtt tgtgcctcgg cagctgggcc tgtcggagcc ccgggtccac
1260 accagccatc tgctggccca cacgcgctac acctttgagg tgcaggcggt
caacggtgtc 1320 tcgggcaaga gccctctgcc gcctcgttat gcggccgtga
atatcaccac aaaccaggct 1380 gccccgtctg aagtgcccac actacgcctg
cacagcagct caggcagcag cctcacccta 1440 tcctgggcac ccccagagcg
gcccaacgga gtcatcctgg actacgagat gaagtacttt 1500 gagaagagcg
agggcatcgc ctccacagtg accagccaga tgaactccgt gcagctggac 1560
gggcttcggc ctgacgcccg ctatgtggtc caggtccgtg cccgcacagt agctggctat
1620 gggcagtaca gccgccctgc cgagtttgag accacaagtg agagaggctc
tggggcccag 1680 cagctccagg agcagcttcc cctcatcgtg ggctccgcta
cagctgggct tgtcttcgtg 1740 gtggctgtcg tggtcatcgc tatcgtctgc
ctcaggaagc agcgacacgg ctctgattcg 1800 gagtacacgg agaagctgca
gcagtacatt gctcctggaa tgaaggttta tattgaccct 1860 tttacctacg
aggaccctaa tgaggctgtt cgggagtttg ccaaggagat cgacgtgtcc 1920
tgcgtcaaga tcgaggaggt gatcggagct ggggaatttg gggaagtgtg ccgtggtcga
1980 ctgaaacagc ctggccgccg agaggtgttt gtggccatca agacgctgaa
ggtgggctac 2040 accgagaggc agcggcggga cttcctaagc gaggcctcca
tcatgggtca gtttgatcac 2100 cccaatataa tccggctcga gggcgtggtc
accaaaagtc ggccagttat gatcctcact 2160 gagttcatgg aaaactgcgc
cctggactcc ttcctccggc tcaacgatgg gcagttcacg
2220 gtcatccagc tggtgggcat gttgcggggc attgctgccg gcatgaagta
cctgtccgag 2280 atgaactatg tgcaccgcga cctggctgct cgcaacatcc
ttgtcaacag caacctggtc 2340 tgcaaagtct cagactttgg cctctcccgc
ttcctggagg atgacccctc cgatcctacc 2400 tacaccagtt ccctgggcgg
gaagatcccc atccgctgga ctgccccaga ggccatagcc 2460 tatcggaagt
tcacttctgc tagtgatgtc tggagctacg gaattgtcat gtgggaggtc 2520
atgagctatg gagagcgacc ctactgggac atgagcaacc aggatgtcat caatgccgtg
2580 gagcaggatt accggctgcc accacccatg gactgtccca cagcactgca
ccagctcatg 2640 ctggactgct gggtgcggga ccggaacctc aggcccaaat
tctcccagat tgtcaatacc 2700 ctggacaagc tcatccgcaa tgctgccagc
ctcaaggtca ttgccagcgc tcagtctggc 2760 atgtcacagc ccctcctgga
ccgcacggtc ccagattaca caaccttcac gacagttggt 2820 gattggctgg
atgccatcaa gatggggcgg tacaaggaga gcttcgtcag tgcggggttt 2880
gcatcttttg acctggtggc ccagatgacg gcagaagacc tgctccgtat tggggtcacc
2940 ctggccggcc accagaagaa gatcctgagc agtatccagg acatgcggct
gcagatgaac 3000 cagacgctgc ctgtgcaggt ctgacaccgg ctcccacggg
gaccctgagg accgtgcagg 3060 gatgccaagc agccggctgg actttcggac
tcttggactt ttggatgcct ggccttaggc 3120 tgtggcccag aagctggaag
tttgggaaag gcccaagctg ggacttctcc aggcctgtgt 3180 tccctcccca
ggaagtgcgc cccaaacctc ttcatattga agatggatta ggagaggggg 3240
tgatgacccc tccccaagcc cctcagggcc cagaccttcc tgctctccag caggggatcc
3300 ccacaacctc acacttgtct gttcttcagt gctggaggtc ctggcagggt
caggctgggg 3360 taagccgggg ttccacaggg cccagccctg gcaggggtct
ggccccccag gtaggcggag 3420 agcagtccct ccctcaggaa ctggaggagg
ggactccagg aatggggaaa tgtgacacca 3480 ccatcctgaa gccagcttgc
acctccagtt tgcacaggga tttgtcctgg gggctgaggg 3540 ccctgtcccc
acccccgccc ttggtgctgt cataaaaggg caggcagggg caggctgagg 3600
agttgcccgt tgccccccag agactgactc tcagagccag agatgggatg tgtgagtgtg
3660 tgtgtgtgtg tgtgcgcgcg cgcgcgcgtg tgtgtgtgca cgcactggcc
tgcacagaga 3720 gcatgggtga gcgtgtaaaa gcttggccct gtgccctaca
gtggggacag ctgggccgac 3780 agcagaataa aggcaataag atgaa 3805 3 22
DNA Homo sapiens 3 actgatcctc gagtggagtg ag 22 4 22 DNA Homo
sapiens 4 cacctcaaag gtgtagcgcg tg 22 5 19 DNA Homo sapiens 5
ctcggctcct agagctgcc 19 6 19 DNA Homo sapiens 6 ctgcttggca
tccctgcac 19 7 15 PRT Homo sapiens 7 Gly Gln Tyr Ser Arg Pro Ala
Glu Phe Glu Thr Thr Ser Glu Arg 1 5 10 15 8 13 PRT Homo sapiens 8
Cys Ala Thr Gly His Glu Pro Ala Ala Lys Glu Ser Gln 1 5 10 9 17 PRT
Homo sapiens 9 Gly Gln Tyr Ser Arg Pro Ala Glu Phe Glu Thr Thr Ser
Glu Arg Gly 1 5 10 15 Ser
Sequence CWU 1
1
91998PRTHomo sapiens 1Met Ala Arg Ala Arg Pro Pro Pro Pro Pro Ser
Pro Pro Pro Gly Leu1 5 10 15Leu Pro Leu Leu Pro Pro Leu Leu Leu Leu
Pro Leu Leu Leu Leu Pro 20 25 30Ala Gly Cys Arg Ala Leu Glu Glu Thr
Leu Met Asp Thr Lys Trp Val 35 40 45Thr Ser Glu Leu Ala Trp Thr Ser
His Pro Glu Ser Gly Trp Glu Glu 50 55 60Val Ser Gly Tyr Asp Glu Ala
Met Asn Pro Ile Arg Thr Tyr Gln Val65 70 75 80Cys Asn Val Arg Glu
Ser Ser Gln Asn Asn Trp Leu Arg Thr Gly Phe 85 90 95Ile Trp Arg Arg
Asp Val Gln Arg Val Tyr Val Glu Leu Lys Phe Thr 100 105 110Val Arg
Asp Cys Asn Ser Ile Pro Asn Ile Pro Gly Ser Cys Lys Glu 115 120
125Thr Phe Asn Leu Phe Tyr Tyr Glu Ala Asp Ser Asp Val Ala Ser Ala
130 135 140Ser Ser Pro Phe Trp Met Glu Asn Pro Tyr Val Lys Val Asp
Thr Ile145 150 155 160Ala Pro Asp Glu Ser Phe Ser Arg Leu Asp Ala
Gly Arg Val Asn Thr 165 170 175Lys Val Arg Ser Phe Gly Pro Leu Ser
Lys Ala Gly Phe Tyr Leu Ala 180 185 190Phe Gln Asp Gln Gly Ala Cys
Met Ser Leu Ile Ser Val Arg Ala Phe 195 200 205Tyr Lys Lys Cys Ala
Ser Thr Thr Ala Gly Phe Ala Leu Phe Pro Glu 210 215 220Thr Leu Thr
Gly Ala Glu Pro Thr Ser Leu Val Ile Ala Pro Gly Thr225 230 235
240Cys Ile Pro Asn Ala Val Glu Val Ser Val Pro Leu Lys Leu Tyr Cys
245 250 255Asn Gly Asp Gly Glu Trp Met Val Pro Val Gly Ala Cys Thr
Cys Ala 260 265 270Thr Gly His Glu Pro Ala Ala Lys Glu Ser Gln Cys
Arg Pro Cys Pro 275 280 285Pro Gly Ser Tyr Lys Ala Lys Gln Gly Glu
Gly Pro Cys Leu Pro Cys 290 295 300Pro Pro Asn Ser Arg Thr Thr Ser
Pro Ala Ala Ser Ile Cys Thr Cys305 310 315 320His Asn Asn Phe Tyr
Arg Ala Asp Ser Asp Ser Ala Asp Ser Ala Cys 325 330 335Thr Thr Val
Pro Ser Pro Pro Arg Gly Val Ile Ser Asn Val Asn Glu 340 345 350Thr
Ser Leu Ile Leu Glu Trp Ser Glu Pro Arg Asp Leu Gly Val Arg 355 360
365Asp Asp Leu Leu Tyr Asn Val Ile Cys Lys Lys Cys His Gly Ala Gly
370 375 380Gly Ala Ser Ala Cys Ser Arg Cys Asp Asp Asn Val Glu Phe
Val Pro385 390 395 400Arg Gln Leu Gly Leu Ser Glu Pro Arg Val His
Thr Ser His Leu Leu 405 410 415Ala His Thr Arg Tyr Thr Phe Glu Val
Gln Ala Val Asn Gly Val Ser 420 425 430Gly Lys Ser Pro Leu Pro Pro
Arg Tyr Ala Ala Val Asn Ile Thr Thr 435 440 445Asn Gln Ala Ala Pro
Ser Glu Val Pro Thr Leu Arg Leu His Ser Ser 450 455 460Ser Gly Ser
Ser Leu Thr Leu Ser Trp Ala Pro Pro Glu Arg Pro Asn465 470 475
480Gly Val Ile Leu Asp Tyr Glu Met Lys Tyr Phe Glu Lys Ser Glu Gly
485 490 495Ile Ala Ser Thr Val Thr Ser Gln Met Asn Ser Val Gln Leu
Asp Gly 500 505 510Leu Arg Pro Asp Ala Arg Tyr Val Val Gln Val Arg
Ala Arg Thr Val 515 520 525Ala Gly Tyr Gly Gln Tyr Ser Arg Pro Ala
Glu Phe Glu Thr Thr Ser 530 535 540Glu Arg Gly Ser Gly Ala Gln Gln
Leu Gln Glu Gln Leu Pro Leu Ile545 550 555 560Val Gly Ser Ala Thr
Ala Gly Leu Val Phe Val Val Ala Val Val Val 565 570 575Ile Ala Ile
Val Cys Leu Arg Lys Gln Arg His Gly Ser Asp Ser Glu 580 585 590Tyr
Thr Glu Lys Leu Gln Gln Tyr Ile Ala Pro Gly Met Lys Val Tyr 595 600
605Ile Asp Pro Phe Thr Tyr Glu Asp Pro Asn Glu Ala Val Arg Glu Phe
610 615 620Ala Lys Glu Ile Asp Val Ser Cys Val Lys Ile Glu Glu Val
Ile Gly625 630 635 640Ala Gly Glu Phe Gly Glu Val Cys Arg Gly Arg
Leu Lys Gln Pro Gly 645 650 655Arg Arg Glu Val Phe Val Ala Ile Lys
Thr Leu Lys Val Gly Tyr Thr 660 665 670Glu Arg Gln Arg Arg Asp Phe
Leu Ser Glu Ala Ser Ile Met Gly Gln 675 680 685Phe Asp His Pro Asn
Ile Ile Arg Leu Glu Gly Val Val Thr Lys Ser 690 695 700Arg Pro Val
Met Ile Leu Thr Glu Phe Met Glu Asn Cys Ala Leu Asp705 710 715
720Ser Phe Leu Arg Leu Asn Asp Gly Gln Phe Thr Val Ile Gln Leu Val
725 730 735Gly Met Leu Arg Gly Ile Ala Ala Gly Met Lys Tyr Leu Ser
Glu Met 740 745 750Asn Tyr Val His Arg Asp Leu Ala Ala Arg Asn Ile
Leu Val Asn Ser 755 760 765Asn Leu Val Cys Lys Val Ser Asp Phe Gly
Leu Ser Arg Phe Leu Glu 770 775 780Asp Asp Pro Ser Asp Pro Thr Tyr
Thr Ser Ser Leu Gly Gly Lys Ile785 790 795 800Pro Ile Arg Trp Thr
Ala Pro Glu Ala Ile Ala Tyr Arg Lys Phe Thr 805 810 815Ser Ala Ser
Asp Val Trp Ser Tyr Gly Ile Val Met Trp Glu Val Met 820 825 830Ser
Tyr Gly Glu Arg Pro Tyr Trp Asp Met Ser Asn Gln Asp Val Ile 835 840
845Asn Ala Val Glu Gln Asp Tyr Arg Leu Pro Pro Pro Met Asp Cys Pro
850 855 860Thr Ala Leu His Gln Leu Met Leu Asp Cys Trp Val Arg Asp
Arg Asn865 870 875 880Leu Arg Pro Lys Phe Ser Gln Ile Val Asn Thr
Leu Asp Lys Leu Ile 885 890 895Arg Asn Ala Ala Ser Leu Lys Val Ile
Ala Ser Ala Gln Ser Gly Met 900 905 910Ser Gln Pro Leu Leu Asp Arg
Thr Val Pro Asp Tyr Thr Thr Phe Thr 915 920 925Thr Val Gly Asp Trp
Leu Asp Ala Ile Lys Met Gly Arg Tyr Lys Glu 930 935 940Ser Phe Val
Ser Ala Gly Phe Ala Ser Phe Asp Leu Val Ala Gln Met945 950 955
960Thr Ala Glu Asp Leu Leu Arg Ile Gly Val Thr Leu Ala Gly His Gln
965 970 975Lys Lys Ile Leu Ser Ser Ile Gln Asp Met Arg Leu Gln Met
Asn Gln 980 985 990Thr Leu Pro Val Gln Val 99523805DNAHomo sapiens
2ggctcggctc ctagagctgc cacggccatg gccagagccc gcccgccgcc gccgccgtcg
60ccgccgccgg ggcttctgcc gctgctccct ccgctgctgc tgctgccgct gctgctgctg
120cccgccggct gccgggcgct ggaagagacc ctcatggaca caaaatgggt
aacatctgag 180ttggcgtgga catctcatcc agaaagtggg tgggaagagg
tgagtggcta cgatgaggcc 240atgaatccca tccgcacata ccaggtgtgt
aatgtgcgcg agtcaagcca gaacaactgg 300cttcgcacgg ggttcatctg
gcggcgggat gtgcagcggg tctacgtgga gctcaagttc 360actgtgcgtg
actgcaacag catccccaac atccccggct cctgcaagga gaccttcaac
420ctcttctact acgaggctga cagcgatgtg gcctcagcct cctccccctt
ctggatggag 480aacccctacg tgaaagtgga caccattgca cccgatgaga
gcttctcgcg gctggatgcc 540ggccgtgtca acaccaaggt gcgcagcttt
gggccacttt ccaaggctgg cttctacctg 600gccttccagg accagggcgc
ctgcatgtcg ctcatctccg tgcgcgcctt ctacaagaag 660tgtgcatcca
ccaccgcagg cttcgcactc ttccccgaga ccctcactgg ggcggagccc
720acctcgctgg tcattgctcc tggcacctgc atccctaacg ccgtggaggt
gtcggtgcca 780ctcaagctct actgcaacgg cgatggggag tggatggtgc
ctgtgggtgc ctgcacctgt 840gccaccggcc atgagccagc tgccaaggag
tcccagtgcc gcccctgtcc ccctgggagc 900tacaaggcga agcagggaga
ggggccctgc ctcccatgtc cccccaacag ccgtaccacc 960tccccagccg
ccagcatctg cacctgccac aataacttct accgtgcaga ctcggactct
1020gcggacagtg cctgtaccac cgtgccatct ccaccccgag gtgtgatctc
caatgtgaat 1080gaaacctcac tgatcctcga gtggagtgag ccccgggacc
tgggtgtccg ggatgacctc 1140ctgtacaatg tcatctgcaa gaagtgccat
ggggctggag gggcctcagc ctgctcacgc 1200tgtgatgaca acgtggagtt
tgtgcctcgg cagctgggcc tgtcggagcc ccgggtccac 1260accagccatc
tgctggccca cacgcgctac acctttgagg tgcaggcggt caacggtgtc
1320tcgggcaaga gccctctgcc gcctcgttat gcggccgtga atatcaccac
aaaccaggct 1380gccccgtctg aagtgcccac actacgcctg cacagcagct
caggcagcag cctcacccta 1440tcctgggcac ccccagagcg gcccaacgga
gtcatcctgg actacgagat gaagtacttt 1500gagaagagcg agggcatcgc
ctccacagtg accagccaga tgaactccgt gcagctggac 1560gggcttcggc
ctgacgcccg ctatgtggtc caggtccgtg cccgcacagt agctggctat
1620gggcagtaca gccgccctgc cgagtttgag accacaagtg agagaggctc
tggggcccag 1680cagctccagg agcagcttcc cctcatcgtg ggctccgcta
cagctgggct tgtcttcgtg 1740gtggctgtcg tggtcatcgc tatcgtctgc
ctcaggaagc agcgacacgg ctctgattcg 1800gagtacacgg agaagctgca
gcagtacatt gctcctggaa tgaaggttta tattgaccct 1860tttacctacg
aggaccctaa tgaggctgtt cgggagtttg ccaaggagat cgacgtgtcc
1920tgcgtcaaga tcgaggaggt gatcggagct ggggaatttg gggaagtgtg
ccgtggtcga 1980ctgaaacagc ctggccgccg agaggtgttt gtggccatca
agacgctgaa ggtgggctac 2040accgagaggc agcggcggga cttcctaagc
gaggcctcca tcatgggtca gtttgatcac 2100cccaatataa tccggctcga
gggcgtggtc accaaaagtc ggccagttat gatcctcact 2160gagttcatgg
aaaactgcgc cctggactcc ttcctccggc tcaacgatgg gcagttcacg
2220gtcatccagc tggtgggcat gttgcggggc attgctgccg gcatgaagta
cctgtccgag 2280atgaactatg tgcaccgcga cctggctgct cgcaacatcc
ttgtcaacag caacctggtc 2340tgcaaagtct cagactttgg cctctcccgc
ttcctggagg atgacccctc cgatcctacc 2400tacaccagtt ccctgggcgg
gaagatcccc atccgctgga ctgccccaga ggccatagcc 2460tatcggaagt
tcacttctgc tagtgatgtc tggagctacg gaattgtcat gtgggaggtc
2520atgagctatg gagagcgacc ctactgggac atgagcaacc aggatgtcat
caatgccgtg 2580gagcaggatt accggctgcc accacccatg gactgtccca
cagcactgca ccagctcatg 2640ctggactgct gggtgcggga ccggaacctc
aggcccaaat tctcccagat tgtcaatacc 2700ctggacaagc tcatccgcaa
tgctgccagc ctcaaggtca ttgccagcgc tcagtctggc 2760atgtcacagc
ccctcctgga ccgcacggtc ccagattaca caaccttcac gacagttggt
2820gattggctgg atgccatcaa gatggggcgg tacaaggaga gcttcgtcag
tgcggggttt 2880gcatcttttg acctggtggc ccagatgacg gcagaagacc
tgctccgtat tggggtcacc 2940ctggccggcc accagaagaa gatcctgagc
agtatccagg acatgcggct gcagatgaac 3000cagacgctgc ctgtgcaggt
ctgacaccgg ctcccacggg gaccctgagg accgtgcagg 3060gatgccaagc
agccggctgg actttcggac tcttggactt ttggatgcct ggccttaggc
3120tgtggcccag aagctggaag tttgggaaag gcccaagctg ggacttctcc
aggcctgtgt 3180tccctcccca ggaagtgcgc cccaaacctc ttcatattga
agatggatta ggagaggggg 3240tgatgacccc tccccaagcc cctcagggcc
cagaccttcc tgctctccag caggggatcc 3300ccacaacctc acacttgtct
gttcttcagt gctggaggtc ctggcagggt caggctgggg 3360taagccgggg
ttccacaggg cccagccctg gcaggggtct ggccccccag gtaggcggag
3420agcagtccct ccctcaggaa ctggaggagg ggactccagg aatggggaaa
tgtgacacca 3480ccatcctgaa gccagcttgc acctccagtt tgcacaggga
tttgtcctgg gggctgaggg 3540ccctgtcccc acccccgccc ttggtgctgt
cataaaaggg caggcagggg caggctgagg 3600agttgcccgt tgccccccag
agactgactc tcagagccag agatgggatg tgtgagtgtg 3660tgtgtgtgtg
tgtgcgcgcg cgcgcgcgtg tgtgtgtgca cgcactggcc tgcacagaga
3720gcatgggtga gcgtgtaaaa gcttggccct gtgccctaca gtggggacag
ctgggccgac 3780agcagaataa aggcaataag atgaa 3805322DNAHomo sapiens
3actgatcctc gagtggagtg ag 22422DNAHomo sapiens 4cacctcaaag
gtgtagcgcg tg 22519DNAHomo sapiens 5ctcggctcct agagctgcc
19619DNAHomo sapiens 6ctgcttggca tccctgcac 19715PRTHomo sapiens
7Gly Gln Tyr Ser Arg Pro Ala Glu Phe Glu Thr Thr Ser Glu Arg1 5 10
15813PRTHomo sapiens 8Cys Ala Thr Gly His Glu Pro Ala Ala Lys Glu
Ser Gln1 5 10917PRTHomo sapiens 9Gly Gln Tyr Ser Arg Pro Ala Glu
Phe Glu Thr Thr Ser Glu Arg Gly1 5 10 15Ser
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References