Note: Descriptions are shown in the official language in which they were submitted.
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NOVEL METHODS OF DIAGNOSING COLORECTAL CANCER, COMPOSITIONS, AND
METHODS OF SCREENING FOR COLORECTAL CANCER MODULATORS
FIELD OF THE INVENTION
The invention relates to the identification of expression profiles and the
nucleic acids involved in
colorectal cancer, and to the use of such expression profiles and nucleic
acids in diagnosis and
prognosis of colorectal cancer. The invention further relates to methods for
identifying and using
candidate agents and/or targets which modulate colorectal cancer.
BACKGROUND OF THE INVENTION
Colorectal cancer is a significant cancer in Western populations. It develops
as the result of a
pathologic transformation of normal colon epithelium to an invasive cancer.
There have been a
number of recently characterized genetic alterations that have been implicated
in colorectal cancer,
including mutations in two classes of genes, tumor-suppressor genes and proto-
oncogenes, with
recent work suggesting that mutations in DNA repair genes may also be involved
in tumorigenesis.
For example, inactivating mutations of both alleles of the adenomatous
polyposis coli (APC) gene, a
tumor suppressor gene, appears to be one of the earliest events in colorectal
cancer, and may even
be the initiating event. Other genes implicated in colorectal cancer include
the MCC gene, the p53
gene, the DCC (deleted in colorectal carcinoma) gene and other chromosome 18q
genes, and genes
in the TGF-[3 signaling pathway. For a review, see Molecular Biology of
Colorectal Cancer, pp238-
299, in Curr. Probl. Cancer, Sept/Oct 1997.
Imaging of colorectal cancer for diagnosis has been problematic and limited.
In addition, dissemination
of tumor cells (metastases) to locoregional lymph nodes is an important
prognostic factor; five year
survival rates drop from 80 percent in patients with no lymph node metastases
to 45 to 50 percent in
those patients who do have lymph node metastases. A recent report showed that
micrometastases
can be detected from lymph nodes using reverse transcriptase-PCR methods based
on the presence
of mRNA for carcinoembryonic antigen, which has previously been shown to be
present in the vast
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majority of colorectal cancers but not in normal tissues. Liefers et al., New
England J. of Med.
339(4):223 (1998).
Thus, methods that can be used for diagnosis and prognosis of colorectal
cancer would be desirable.
Accordingly, provided herein are methods that can be used in diagnosis and
prognosis of colorectal
cancer. Further provided are methods that can be used to screen candidate
bioactive agents for the
ability to modulate colorectal cancer. Additionally, provided herein are
molecular targets for
therapeutic intervention in colorectal and other cancers.
SUMMARY OF THE INVENTION
The present invention provides methods for screening for compositions which
modulate colorectal
cancer. Also provided herein are methods of inhibiting proliferation of cell,
preferably a colorectal
cancer cell. Methods of treatment of cancer, as well as compositions, are also
provided herein.
In one aspect, a method of screening drug candidates comprises providing a
cell that expresses an
expression profile gene or fragments thereof. Preferred embodiments of the
expression profile gene
are genes which are differentially expressed in cancer cells, preferably
colorectal cancer cells,
compared to other cells. Preferred embodiments of expression profile genes
used in the methods
herein include but are not limited to the group consisting of CZAB, BCX2,
CBC2, CBC1, CBC3, CJAB,
CJA9, CGA7, BCNS, CQA1, BCN7, CQA2, CGAB, CAA7 and CAA9; fragments of the
proteins of this
group are also preferred. It is understood that molecules for use in the
present invention may be from
any figure or any subset of fisted molecules. Therefore, for example, any one
or more of the genes
listed above can be used in the methods herein. In another embodiment, a
nucleic acid is selected
from Figures 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. Preferred
nucleic acids are in Figure 12,
more preferably Figure 13, and most preferably in Figure 14. The method
further includes adding a
drug candidate to the cell and determining the effect of the drug candidate on
the expression of the
expression profile gene.
In one embodiment, the method of screening drug candidates includes comparing
the level of
expression in the absence of the drug candidate to the level of expression in
the presence of the drug
candidate, wherein the concentration of the drug candidate can vary when
present, and wherein the
comparison can occur after addition or removal of the drug candidate. In a
preferred embodiment, the
cell expresses at least two expression profile genes. The profile genes may
show an increase or
decrease.
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Also provided herein is a method of screening for a bioactive agent capable of
binding to a colorectal
cancer modulator protein (CCMP), the method comprising combining the CCMP and
a candidate
bioactive agent, and determining the binding of the candidate agent to the
CCMP. Preferably the
CCMP is a protein or fragment thereof selected from the group consisting of
CZAB, BCX2, CBC2,
CBC1, CBC3, CJAB, CJA9, CGA7, BCNS, CQA1, BCN7, CQA2, CGAB, CAA7 and CAA9. In
another
embodiment, the protein is encoded by a nucleic acid selected from Figures 1,
2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13 or 14. Preferred nucleic acids are in Figure 12, more preferably
Figure 13, and most
preferably in Figure 14.
Further provided herein is a method for screening for a bioactive agent
capable of modulating the
activity of a CCMP. In one embodiment, the method comprises combining the CCMP
and a candidate
bioactive agent, and determining the effect of the candidate agent on the
bioactivity of the CCMP.
Preferably the CCMP is a protein or fragment thereof selected from the group
consisting of CZAB,
BCX2, CBC2, CBC1, CBC3, CJAB, CJA9, CGA7, BCNS, CQA1, BCN7, CQA2, CGAB, CAA7
and
CAA9. In another embodiment, the protein is encoded by a nucleic acid selected
from Figures 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. Preferred nucleic acids are in Figure
12, more preferably Figure
13, and most preferably in Figure 14.
Also provided is a method of evaluating the effect of a candidate colorectal
cancer drug comprising
administering the drug to a transgenic animal expressing or over-expressing
the CCMP, or an animal
lacking the CCMP, for example as a result of a gene knockout.
Additionally, provided herein is a method of evaluating the effect of a
candidate colorectal cancer drug
comprising administering the drug to a patient and removing a cell sample from
the patient. The
expression profile of the cell is then determined. This method may further
comprise comparing the
expression profile to an expression profile of a healthy individual.
Moreover, provided herein is a biochip comprising a nucleic acid segment which
encodes a colorectal
cancer protein, preferably selected from the group consisting of CZAR, BCX2,
CBC2, CBC1, CBC3,
CJAB, CJA9, CGA7, BCNS, CQA1, BCN7, CQA2, CGAB, CAA7 and CAA9, or a fragment
thereof,
wherein the biochip comprises fewer than 1000 nucleic acid probes. Preferably
at least two nucleic
acid segments are included. In another embodiment, the nucleic acid selected
from Figures 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. Preferred nucleic acids are in Figure 12,
more preferably Figure 13,
and most preferably in Figure 14.
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Furthermore, a method of diagnosing a disorder associated with colorectal
cancer is provided. The '
method comprises determining the expression of a gene which encodes a
colorectal cancer protein
preferably selected from the group consisting of CZAB, BCX2, CBC2, CBC1, CBC3,
CJAB, CJA9,
CGA7, BCNS, CQA1, BCN7, CQA2, CGAB, CAA7 and CAA9 or a fragment thereof in a
first tissue
type of a first individual, and comparing the distribution to the expression
of the gene from a second
normal tissue type from the first individual or a second unaffected
individual. In another embodiment,
the protein is encoded by a nucleic acid selected from Figures 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13 or
14. Preferred nucleic acids are in Figure 12, more preferably Figure 13, and
most preferably in Figure
14. A difference in the expression indicates that the first individual has a
disorder associated with
colorectal cancer.
In another aspect, the present invention provides an antibody which
specifically binds to a colorectal
cancer protein, preferably selected from the group consisting of CZAB, BCX2,
CBC2, CBC1, CBC3,
CJAB, CJA9, CGA7, BCNS, CQA1, BCN7, CQA2, CGAB, CAA7 and CAA9, or a fragment
thereof. In
another embodiment, the protein is encoded by a nucleic acid selected from
Figures 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13 or 14. Preferred nucleic acids are in Figure 12, more
preferably Figure 13, and
most preferably in Figure 14. In a preferred embodiment, the fragment of CAA9
is selected from
CAA9p1; CAA9p2, CAA9p3, CAA9p4, CAA9p4MAPS, CAA9p5 and CAA9p5MAPS. Other
preferred
fragments for the breast cancer proteins are shown in the figures. Preferably
the antibody is a
monoclonal antibody. The antibody can be a fragment of an antibody such as a
single stranded
antibody as further described herein, or can be conjugated to another
molecule. In one embodiment,
the antibody is a humanized antibody.
In one embodiment a method for screening for a bioactive agent capable of
interfering with the binding
of a colorectal cancer protein (CCMP) or a fragment thereof and an antibody
which binds to said
CCMP or fragment thereof. In a preferred embodiment, the method comprises
combining a CCMP or
fragment thereof, a candidate bioactive agent and an antibody which binds to
said CCMP or fragment
thereof. The method further includes determining the binding of said CCMP or
fragment thereof and
said antibody. Wherein there is a change in binding, an agent is identified as
an interfering agent.
The interfering agent can be an agonist or an antagonist. Preferably, the
antibody as well as the agent
inhibits colorectal cancer.
In a further aspect, a method for inhibiting colorectal cancer is provided. In
one embodiment, the
method comprises administering to a cell a composition comprising an antibody
to a colorectal
modulating protein, preferably selected from the group consisting of CZAB,
BCX2, CBC2, CBC1,
CBC3, CJAB, CJA9, CGA7, BCNS, COA1, BCN7, CQA2, CGAB, CAA7 and CAA9, or a
fragment
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thereof. In another embodiment, the protein is encoded by a nucleic acid
selected from Figures 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. Preferred nucleic acids are in
Figure 12, more preferably
Figure 13, and most preferably in Figure 14. The method can be performed in
vitro or in vivo,
preferably in vivo to an individual. In a preferred embodiment the method of
inhibiting colorectal
cancer is provided to an individual with cancer. As described herein, methods
of inhibiting colorectal
cancer can be performed by administering an inhibitor of colorectal cancer
protein activity, including
antisense molecules, and preferably small molecules.
Also provided herein are methods eliciting an immune response in an
individual. In one embodiment a
method provided herein comprises administering to an individual a composition
comprising a
colorectal modulating protein, preferably selected from the group consisting
of CZAB, BCX2, CBC2,
CBC1, CBC3, CJAB, CJA9, CGA7, BCNS, CQA1, BCN7, CQA2, CGAB, CAA7 and CAA9, or
a
fragment thereof. In another embodiment, the protein is encoded by a nucleic
acid selected from
Figures 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. Preferred nucleic
acids are in Figure 12, more
preferably Figure 13, and most preferably in Figure 14. In another aspect,
said composition comprises
a nucleic acid comprising a sequence encoding a colorectal cancer modulating
protein, preferably
selected from the group consisting of CZAB, BCX2, CBC2, CBC1, CBC3, CJAB,
CJA9, CGA7, BCN5,
CQA1, BCN7, CQA2, CGAB, CAA7 and CAA9, or a fragment thereof. In another
embodiment, the
nucleic acid is selected from Figures 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13 or 14. Preferred nucleic
acids are in Figure 12, more preferably Figure 13, and most preferably in
Figure 14.
Further provided herein are compositions capable of eliciting an immune
response in an individual. In
one embodiment, a composition provided herein comprises a colorectal cancer
modulating protein,
preferably selected from the group consisting of CZAB, BCX2, CBC2, CBC1, CBC3,
CJAB, CJA9,
CGA7, BCNS, CQA1, BCN7, CQA2, CGA8, CAA7 and CAA9, or a fragment thereof, and
a
pharmaceutically acceptable carrier. In another embodiment, the protein is
encoded by a nucleic acid
selected from Figures 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.
Preferred nucleic acids are in
Figure 12, more preferably Figure 13, and most preferably in Figure 14. In
another embodiment, said
composition comprises a nucleic acid comprising a sequence encoding a
colorectal cancer
modulating protein, preferably selected from the group consisting of CZAB,
BCX2, CBC2, CBC1,
CBC3, CJAB, CJA9, CGA7, BCNS, CQA1, BCN7, CQA2, CGAB, CAA7 and CAA9, or a
fragment
thereof, and a pharmaceutically acceptable carrier. In another embodiment, the
nucleic acid is
selected from Figures 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.
Preferred nucleic acids are in
Figure 12, more preferably Figure 13, and most preferably in Figure 14.
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A method of neutralizing the effect of a colorectal cancer protein, preferably
selected from the group
consisting of CZA8, BCX2, CBC2, CBC1, CBC3, CJAB, CJA9, CGA7, BCNS, CQA1,
BCN7, CQA2,
CGAB, CAA7 and CAA9, or a fragment thereof, comprising contacting an agent
specific for said
protein with said protein in an amount sufficient fio effect neutralization.
In another embodiment, the
protein is encoded by a nucleic acid selected from Figures 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13 or 14.
Preferred nucleic acids are in Figure 12, more preferably Figure 13, and most
preferably in Figure 94.
fn another aspect of the invention, a method of treating an individual for
colorectal cancer is provided.
In one embodiment, the method comprises administering to said individual an
inhibitor of CJAB. In
another embodiment, the method comprises administering to a patient having
colorectal cancer an
antibody to CJA8 conjugated to a therapeutic moiety. Such a therapeutic moiety
can be a cytotoxic
agent or a radioisotope.
Also provided herein is a method for determining the prognosis of an
individual with colorectal cancer
comprising determining the level of CJA8 in a sample, wherein a high level of
CJA8 indicates a poor
prognosis.
Novel sequences are also provided herein. Other aspects of the invention will
become apparent to the
skilled artisan by the following description of the invention.
DETAILED DESCRIPTION OF THE FIGURES
Figure 1 provides the Accession numbers for genes, including expression
sequence tags,
(incorporated in their entirety here and throughout the application where
Accession numbers are
provided), upregulated in tumor tissue compared to normal colon tissue.
Figure 2 provides the Accession numbers for genes, including expression
sequence tags, upregulated
in tumor tissue compared to normal colon tissue.
Figure 3 provides the Accession numbers for genes, including expression
sequence tags, upregulated
in tumor tissue compared to normal colon tissue.
Figure 4 provides the Accession numbers for genes, including expression
sequence tags, upregulated
in tumor tissue compared to normal colon tissue.
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Figure 5 provides the Accession numbers for genes, including expression
sequence tags,
downregulated in tumor tissue compared to normal colon tissue.
Figure 6 provides the Accession numbers for genes, including expression
sequence tags,
downregulated in tumor tissue compared to normal colon tissue.
Figure 7 provides the Accession numbers for genes, including expression
sequence tags,
downregulated in tumor tissue compared to normal colon tissue.
Figure 8 provides the Accession numbers for genes, including expression
sequence tags, upregulated
in tumor tissue compared to normal colon tissue. Open reading frames in the
sequences have been
characterized as having a signal sequence (SS), a transmembrane domain (TM) or
other.
Figure 9 provides the Accession numbers for genes, including expression
sequence tags, upregulated
in tumor tissue compared to normal colon tissue. Open reading frames in the
sequences have been
characterized as having a signal sequence (SS), a transmembrane domain (TM) or
other.
Figure 10 provides the Accession numbers for genes, including expression
sequence tags,
upregulated in tumor tissue compared to normal colon tissue. Open reading
frames have been
characterized as having a signal sequence (SS), a transmembrane domain (TM) or
other.
Figure 11 provides the Accession numbers for genes, including expression
sequence tags,
upregulated in tumor tissue compared to normal colon tissue. Open reading
frames have been
characterized as having a signal sequence (SS), a transmembrane domain (TM) or
other.
Figure 12 provides the Accession numbers for genes, including expression
sequence tags,
upregulated in tumor tissue compared to normal colon tissue. Open reading
frames have been
characterized as having a signal sequence (SS), a transmembrane domain (TM) or
other.
Figure 13 provides the Accession numbers for genes or fragments thereof,
including descriptions of
the gene or encoded protein, upregulated in tumor tissue compared to normal
colon tissue.
Figure 14 provides a list of proteins, including Accession numbers for nucleic
acid sequences
associated with the encoding genes thereof, upregulated in tumor tissue
compared to normal colon
tissue.
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Figure 15 shows an embodiment of a nucleic acid which includes a sequence
which encodes a
colorectal protein provided herein, CAA2. The start and stop codon are shaded.
The sequence within
the two cross marks indicates a preferred novel fragment of CAA2 provided
herein, referred to herein
as the "CAA2 5' end". Preferred embodiments of CAA2 include at least a portion
of the CAA2 5'. The
sequence in bold and indicated with a bar at the bottom right beginning with
"GGC" and ending with
"AAA" can be found in Accession no. AA505133.
Figure 16 shows an embodiment of a nucleic acid encoding CAA2, wherein the
start and stop codons
are shaded.
Figure 17 shows an embodiment of an amino acid sequence of CAA2. Preferred
fragments include at
least about 10 amino acids in the N-terminal end. The N-terminus as defined
herein includes an
embodiment beginning at the first amino acid until about any one of the three
amino acids marked with
a dot above them. In another embodiment, the N-terminus of CAA2 is defined as
the amino acid
sequence encoded by the CAA2 5' end.
Figure 18 shows the amino acid sequence of CAA2p1, a preferred CAA2 fragment
provided herein.
Figure 19 shows the amino acid sequence of CAA2p2, a preferred CAA2 fragment
provided herein.
Figure 20 shows an alignment of the human and mouse CAA2 polypeptides provided
herein. The
mouse polypeptide contains at least some of the sequence of each of the
following Accession
numbers: AA386837; AI508773; AA505293; and AA636546.
Figure 21 shows the relative amount of expression of CAA2 in various samples
of colon cancer tissue
(dark bars) and many normal tissue types (light bars).
Figure 22 shows an embodiment of a colorectal cancer nucleic acid, CAA9 mRNA.
The start and stop
codons are underlined.
Figure 23 shows the open reading frame of the CAA9 gene wherein the start and
stop codons are
underlined.
Figure 24 shows an embodiment of the amino acid sequence of a colorectal
cancer protein, CAA9,
wherein putative transmembrane sequences are underlined. In one embodiment,
CAA9 or fragments
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of CAA9 are soluble, therefore, the transmembrane domains are deleted,
inactivated, and/or the
peptide is truncated (with or without re-ligation) to form soluble CAA9.
Figure 25 shows embodiments of colorectal cancer proteins (also termed
colorectal cancer modulator
proteins). Specifically, Figure 25 shows CAA9p1, CAA9p2, CAA9p3, CAA9p4,
CAA9p4MAPS,
CAA9p5 and CAA9p5MAPS and their respective solubilities.
Figure 26 shows the relative amount of CAA9 expression in several different
samples of colon cancer
tissue (dark bars) and normal tissues. (light bars).
Figure 27 shows the nucleic acid sequence for the gene encoding CGA7. Start
(ATG) and stop (TAG)
codons are indicated by shaded boxes. In bold is the sequence of Accession No.
AA331393.
Underlined is the consensus sequence derived from the compilation and
alignment of published est
sequences.
Figures 28A and 28B show the alignment summary and descriptions, respectively,
of the various est's
(by accession number) compiled to generate the consensus sequence of figure 1.
Figure 29 shows the amino acid sequence of CGA7.
Figures 30A and 30B show the relative expression of CGA7 in normal tissue and
colon cancer tissue,
respectively.
Figure 31 shows the nucleic acid sequence for the mRNA encoding CGAB. Start
(ATG) and stop
(TAG) codons are indicated by shaded boxes. In bold is the sequence of
Accession No. AA2786503.
Underlined is the consensus sequence derived from the compilation and
alignment of published est
sequences.
Figures 32A and 32B show the alignment summary and descriptions, respectively,
of the various est's
(by accession number) compiled to generate the consensus sequence of figure 1.
Figure 33 shows the amino acid sequence of CGA8.
Figure 34 shows the relative expression of CGA8 in breast cancer tissue, colon
cancer tissue, normal
tissue and fetal tissue.
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Figure 35 shows the sequence for the mRNA encoding CJAB. Start (ATG) and stop
(TAA) colons
are indicated by shaded boxes.
Figure 36 shows the amino acid sequence for CJAB. A putative transmembrane
region is designated
by shading. A mouse homolog of this human protein is found at Accession Number
AAF21308.1.
Figure 37 shows the relative amount of expression of CJA8 in several different
samples of colon
tissues (dark bars) and normal tissues (light bars).
Figure 38 shows the relative amount of expression of BCN7 in several different
samples of colon
tissues (dark bars) and normal tissues (light bars), as determined using the
sequence of Accession
Number N22107 as a probe.
Figure 39 shows an embodiment of a nucleic acid which includes a sequence
which encodes a
colorectal cancer protein provided herein, BCN7.
Figure 40 shows the sequence for the mRNA encoding CZA8. Start (ATG) and stop
(TGA) colons
are indicated by underlining.
Figure 41 shows the sequence for the mRNA encoding BCX2. Start (ATG) and stop
(TGA) colons
are indicated by underlining.
Figure 42 shows the sequence for the mRNA encoding CBC2. Start (ATG) and stop
(TAA) colons
are indicated by underlining.
Figure 43 shows the sequence for the mRNA encoding CBC1. Start (ATG) and stop
(TGA) colons
are indicated by underlining.
Figure 44 shows the sequence for the mRNA encoding CBC3. Start (ATG) and stop
(TGA) colons
are indicated by underlining.
Figure 45 shows the sequence for the mRNA encoding BCN5. Start (ATG) and stop
(TAA) colons
are indicated by underlining.
Figure 46 shows an embodiment of a nucleic acid which includes a sequence
which encodes a
colorectal cancer protein provided herein, CJA9.
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Figure 47 shows an embodiment of a nucleic acid which includes a sequence
which encodes a
colorectal cancer protein provided herein, COA1.
Figure 48 shows an embodiment of a nucleic acid which includes a sequence
which encodes a
colorectal cancer protein provided herein, CQA2.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel methods for diagnosis and prognosis
evaluation for colorectal
cancer (CRC), as well as methods for screening for compositions which modulate
CRC. In one
aspect, the expression levels of genes are determined in different patient
samples for which either
diagnosis or prognosis information is desired, to provide expression profiles.
An expression profile of
a particular sample is essentially a "fingerprint" of the state of the sample;
while two states may have
any particular gene similarly expressed, the evaluation of a number of genes
simultaneously allows
the generation of a gene expression profile that is unique to the state of the
cell. That is, normal tissue
may be distinguished from CRC tissue, and within CRC tissue, different
prognosis states (good or
poor long term survival prospects, for example) may be determined. By
comparing expression profiles
of colon tissue in known different states, information regarding which genes
are important (including
both up- and down-regulation of genes) in each of these states is obtained.
The identification of
sequences that are differentially expressed in CRC versus normal colon tissue,
as well as differential
expression resulting in different prognostic outcomes, allows the use of this
information in a number of
ways. For example, the evaluation of a particular treatment regime may be
evaluated: does a
chemotherapeutic drug act to improve the long-term prognosis in a particular
patient. Similarly,
diagnosis may be done or confirmed by comparing patient samples with the known
expression
profiles. Furthermore, these gene expression profiles (or individual genes)
allow screening of drug
candidates with an eye to mimicking or altering a particular expression
profile; for example, screening
can be done for drugs that suppress the CRC expression profile or convert a
poor prognosis profile to
a better prognosis profile. This may be done by making biochips comprising
sets of the important
CRC genes, which can then be used in these screens. These methods can also be
done on the
protein basis; that is, protein expression levels of the CRC proteins can be
evaluated for diagnostic
and prognostic purposes or to screen candidate agents. In addition, the CRC
nucleic acid sequences
can be administered for gene therapy purposes, including the administration of
antisense nucleic
acids, or the CRC proteins (including antibodies and other modulators thereof)
administered as
therapeutic drugs.
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Thus the present invention provides nucleic acid and protein sequences that
are differentially
expressed in colorectal cancer, CRC, herein termed "CRC sequences". As
outlined below, CRC
sequences include those that are up-regulated (i.e. expressed at a higher
level) in CRC, as well as
those that are down-regulated (i.e. expressed at a lower level) in CRC. In a
preferred embodiment,
the CRC sequences are from humans; however, as will be appreciated by those in
the art, CRC
sequences from other organisms may be useful in animal models of disease and
drug evaluation;
thus, other CRC sequences are provided, from vertebrates, including mammals,
including rodents
(rats, mice, hamsters, guinea pigs, etc.), primates, farm animals (including
sheep, goats, pigs, cows,
horses, etc). CRC sequences from other organisms may be obtained using the
techniques outlined
below.
CRC sequences can include both nucleic acid and amino acid sequences. In a
preferred
embodiment, the CRC sequences are recombinant nucleic acids. By the term
"recombinant nucleic
acid" herein is meant nucleic acid, originally formed in vitro, in general, by
the manipulation of nucleic
acid by polymerases and endonucleases, in a form not normally found in nature.
Thus an isolated
nucleic acid, in a linear form, or an expression vector formed in vitro by
ligating DNA molecules that
are not normally joined, are both considered recombinant for the purposes of
this invention. It is
understood that once a recombinant nucleic acid is made and reintroduced into
a host cell or
organism, it will replicate non-recombinantly, i.e. using the in vivo cellular
machinery of the host cell
rather than in vitro manipulations; however, such nucleic acids, once produced
recombinantly,
although subsequently replicated non-recombinantly, are still considered
recombinant for the purposes
of the invention.
Similarly, a "'recombinant protein" is a protein made using recombinant
techniques, i.e. through the
expression of a recombinant nucleic acid as depicted above. A recombinant
protein is distinguished
from naturally occurring protein by at least one or more characteristics. For
example, the protein may
be isolated or purified away from some or ail of the proteins and compounds
with which it is normally
associated in its wild type host, and thus may be substantially pure. For
example, an isolated protein
is unaccompanied by at least some of the material with which it is normally
associated in its natural
state, preferably constituting at least about 0.5%, more preferably at least
about 5% by weight of the
total protein in a given sample. A substantially pure protein comprises at
least about 75% by weight of
the total protein, with at least about 80% being preferred, and at least about
90% being particularly
preferred. The definition includes the production of a CRC protein from one
organism in a different
organism or host cell. Alternatively, the protein may be made at a
significantly higher concentration
than is normally seen, through the use of an inducible promoter or high
expression promoter, such
that the protein is made at increased concentration levels. Alternatively, the
protein may be in a form
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not normally found in nature, as in the addition of an epitope tag or amino
acid substitutions, insertions
and deletions, as discussed below.
In a preferred embodiment, the CRC sequences are nucleic acids. As will be
appreciated by those in
the art and is more fully outlined below, CRC sequences are useful in a
variety of applications,
including diagnostic applications, which will detect naturally occurring
nucleic acids, as well as
screening applications; for example, biochips comprising nucleic acid probes
to the CRC sequences
can be generated. In the broadest sense, then, by "nucleic acid" or
"oligonucleotide" or grammatical
equivalents herein means at least two nucleotides covalently linked together.
A nucleic acid of the
present invention will generally contain phosphodiester bonds, although in
some cases, as outlined
below, nucleic acid analogs are included that may have alternate backbones,
comprising, for
example, phosphoramidate (Beaucage et al., Tetrahedron 49(10):1925 (1993) and
references therein;
Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem.
81:579 (1977); Letsinger et
al., Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984),
Letsinger et al., J. Am.
Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripts 26:14191986)),
phosphorothioate
(Mag et al., Nucleic Acids Res. 79:1437 (1991 ); and U.S. Patent No.
5,644,048), phosphorodithioate
(Briu et al., J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite
linkages (see Eckstein,
Oligonucleotides and Analogues: A Practical Approach, Oxford University
Press), and peptide nucleic
acid backbones and linkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992);
Meier et al., Chem.
Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et
al., Nature 380:207
(1996), all of which are incorporated by reference). Other analog nucleic
acids include those with
positive backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995);
non-ionic backbones
(U.S. Patent Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863;
Kiedrowshi et al.,
Angew. Chem. Intl. Ed. English 30:423 (1991 ); Letsinger et al., J. Am. Chem.
Soc. 110:4470 (1988);
Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3,
ASC Symposium Series
580, "'Carbohydrate Modifications in Antisense Research", Ed. Y.S. Sanghui and
P. Dan Cook;
Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et
al., J. Biomolecular NMR
34:17 (1994); Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones,
including those described
in U.S. Patent Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC
Symposium Series 580,
"Carbohydrate Modifications in Antisense Research", Ed. Y.S. Sanghui and P.
Dan Cook. Nucleic
acids containing one or more carbocyclic sugars are also included within one
definition of nucleic
acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp169-176). Several nucleic
acid analogs are
described in Rawls, C & E News June 2, 1997 page 35. All of these references
are hereby expressly
incorporated by reference. These modifications of the ribose-phosphate
backbone may be done for a
variety of reasons, for example to increase the stability and half-life of
such molecules in physiological
environments or as probes on a biochip.
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As will be appreciated by those in the art, all of these nucleic acid analogs
may find use in the present
invention. In addition, mixtures of naturally occurring nucleic acids and
analogs can be made;
alternatively, mixtures of different nucleic acid analogs, and mixtures of
naturally occurring nucleic
acids and analogs may be made.
Particularly preferred are peptide nucleic acids (PNA) which includes peptide
nucleic acid analogs.
These backbones are substantially non-ionic under neutral conditions, in
contrast to the highly
charged phosphodiester backbone of naturally occurring nucleic acids. This
results in two
advantages. First, the PNA backbone exhibits improved hybridization kinetics.
PNAs have larger
changes in the melting temperature (Tm) for mismatched versus perfectly
matched basepairs. DNA
and RNA typically exhibit a 2-4°C drop in Tm for an internal mismatch.
With the non-ionic PNA
backbone, the drop is closer to 7-9°C. Similarly, due to their non-
ionic nature, hybridization of the
bases attached to these backbones is relatively insensitive to salt
concentration. In addition, PNAs
are not degraded by cellular enzymes, and thus can be more stable.
The nucleic acids may be single stranded or double stranded, as specified, or
contain portions of both
double stranded or single stranded sequence. As will be appreciated by those
in the art, the depiction
of a single strand ("Watson") also defines the sequence of the other strand
("Crick"); thus the
sequences described herein also includes the complement of the sequence. The
nucleic acid may be
DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains
any combination of
deoxyribo- and ribo-nucleotides, and any combination of bases, including
uracil, adenine, thymine,
cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine,
etc. As used herein, the
term "nucleoside" includes nucleotides and nucleoside and nucleotide analogs,
and modified
nucleosides such as amino modified nucleosides. In addition, "nucleoside"
includes non-naturally
occurring analog structures. Thus for example the individual units of a
peptide nucleic acid, each
containing a base, are referred to herein as a nucleoside.
A CRC sequence can be initially identified by substantial nucleic acid and/or
amino acid sequence
homology to the CRC sequences outlined herein. Such homology can be based upon
the overall
nucleic acid or amino acid sequence, and is generally determined as outlined
below, using either
homology programs or hybridization conditions.
The CRC sequences of the invention can be identified as follows. Samples of
normal and tumor
tissue are applied to biochips comprising nucleic acid probes. The samples are
first microdissected, if
applicable, and treated as is know in the art for the preparation of mRNA.
Suitable biochips are
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commercially available, for example from Affymetrix. Gene expression profiles
as described herein
are generated, and the data analyzed.
In a preferred embodiment, the genes showing changes in expression as between
normal and
disease states are compared to genes expressed in other normal tissues,
including, but not limited to
lung, heart, brain, liver, breast, kidney, muscle, prostate, small intestine,
large intestine, spleen, bone,
and placenta. In a preferred embodiment, those genes identified during the CRC
screen that are
expressed in any significant amount in other tissues are removed from the
profile, although in some
embodiments, this is not necessary. That is, when screening for drugs, it is
preferable that the target
be disease specific, to minimize possible side effects.
In a preferred embodiment, CRC sequences are those that are up-regulated in
CRC; that is, the
expression of these genes is higher in colorectal carcinoma as compared to
normal colon tissue. "Up-
regulation" as used herein means at least about a two-fold change, preferably
at least about a three
fold change, with at least about five-fold or higher being preferred. All
accession numbers herein are
for the GenBank sequence database and the sequences of the accession numbers
are hereby
expressly incorporated by reference. GenBank is known in the art, see, e.g.,
Benson, DA, et al.,
Nucleic Acids Research 26:1-7 (1998) and http://www.ncbi.nlm.nih.gov/. In
addition, these genes
were found to be expressed in a limited amount or not at all in heart, brain,
lung, liver, breast, kidney,
prostate, small intestine and spleen.
In a preferred embodiment, CRC sequences are those that are down-regulated in
CRC; that is, the
expression of these genes is lower in colorectal carcinoma as compared to
normal colon tissue.
"Down-regulation" as used herein means at least about a two-fold change,
preferably at least about a
three fold change, with at least about five-fold or higher being preferred.
CRC proteins of the present invention may be classified as secreted proteins,
transmembrane
proteins or intracellular proteins. In a preferred embodiment the CRC protein
is an intracellular
protein. Intracellular proteins may be found in the cytoplasm andlor in the
nucleus. Intracellular
proteins are involved in all aspects of cellular function and replication
(including, for example, signaling
pathways); aberrant expression of such proteins results in unregulated or
disregulated cellular
processes. For example, many intracellular proteins have enzymatic activity
such as protein kinase
activity, protein phosphatase activity, protease activity, nucleotide cyclase
activity, polymerise activity
and the like. Intracellular proteins also serve as docking proteins that are
involved in organizing
complexes of proteins, or targeting proteins to various subcellular
localizations, and are involved in
maintaining the structural integrity of organelles.
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An increasingly appreciated concept in characterizing intracellular proteins
is the presence in the
proteins of one or more motifs for which defined functions have been
attributed. In addition to the
highly conserved sequences found in the enzymatic domain of proteins, highly
conserved sequences
have been identified in proteins that are involved in protein-protein
interaction. For example, Src-
homology-2 (SH2) domains bind tyrosine-phosphorylated targets in a sequence
dependent manner.
PTB domains, which are distinct from SH2 domains, also bind tyrosine
phosphorylated targets. SH3
domains bind to proline-rich targets. In addition, PH domains,
tetratricopeptide repeats and WD
domains to name only a few, have been shown to mediate protein-protein
interactions. Some of these
may also be involved in binding to phospholipids or other second messengers.
As will be appreciated
by one of ordinary skill in the art, these motifs can be identified on the
basis of primary sequence;
thus, an analysis of the sequence of proteins may provide insight into both
the enzymatic potential of
the molecule andlor molecules with which the protein may associate.
In a preferred embodiment, the CRC sequences are transmembrane proteins.
Transmembrane
proteins are molecules that span the phospholipid bilayer of a cell. They may
have an intracellular
domain, an extracellular domain, or both. The intracellular domains of such
proteins may have a
number of functions including those already described for intracellular
proteins. For example, the
intracellular domain may have enzymatic activity and/or may serve as a binding
site for additional
proteins. Frequently the intracellular domain of transmembrane proteins serves
both roles. For
example certain receptor tyrosine kinases have both protein kinase activity
and SH2 domains. In
addition, autophosphorylation of tyrosines on the receptor molecule itself,
creates binding sites for
additional SH2 domain containing proteins.
Transmembrane proteins may contain from one to many transmembrane domains. For
example,
receptor tyrosine kinases, certain cytokine receptors, receptor guanylyl
cyclases and receptor
serine/threonine protein kinases contain a single transmembrane domain.
However, various other
proteins including channels and adenylyl cyclases contain numerous
transmembrane domains. Many
important cell surface receptors are classified as "seven transmembrane
domain" proteins, as they
contain 7 membrane spanning regions. Important transmembrane protein receptors
include, but are
not limited to insulin receptor, insulin-like growth factor receptor, human
growth hormone receptor,
glucose transporters, transferrin receptor, epidermal growth factor receptor,
low density lipoprotein
receptor, epidermal growth factor receptor, leptin receptor, interleukin
receptors, e.g. IL-1 receptor,
IL-2 receptor, etc.
Characteristics of transmembrane domains include approximately 20 consecutive
hydrophobic amino
acids that may be followed by charged amino acids. Therefore, upon analysis of
the amino acid
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sequence of a particular protein, the localization and number of transmembrane
domains within the
protein may be predicted.
The extracelluiar domains of transmembrane proteins are diverse; however,
conserved motifs are
found repeatedly among various extracellular domains. Conserved structure
and/or functions have
been ascribed to different extracellular motifs. For example, cytokine
receptors are characterized by a
cluster of cysteines and a WSXWS (W= tryptophan, S= serine, X=any amino acid)
motif.
Immunoglobuiin-like domains are highly conserved. Mucin-like domains may be
involved in cell
adhesion and leucine-rich repeats participate in protein-protein interactions.
Many extracellular domains are involved in binding to other molecules. In one
aspect, extracellular
domains are receptors. Factors that bind the receptor domain include
circulating ligands, which may
be peptides, proteins, or small molecules such as adenosine and the like. For
example, growth
factors such as EGF, FGF and PDGF are circulating growth factors that bind to
their cognate
receptors to initiate a variety of cellular responses. Other factors include
cytokines, mitogenic factors,
neurotrophic factors and the like. Extracellular domains also bind to cell-
associated molecules. In this
respect, they mediate cell-cell interactions. Cell-associated ligands can be
tethered to the cell for
example via a glycosylphosphatidylinositol (GPI) anchor, or may themselves be
transmembrane
proteins. Extracellular domains also associate with the extracellular matrix
and contribute to the
maintenance of the cell structure.
CRC proteins that are transmembrane are particularly preferred in the present
invention as they are
good targets for immunotherapeutics, as are described herein. In addition, as
outlined below,
transmembrane proteins can be also useful in imaging modalities.
It will also be appreciated by those in the art that a transmembrane protein
can be made soluble by
removing transmembrane sequences, for example through recombinant methods.
Furthermore,
transmembrane proteins that have been made soluble can be made to be secreted
through
recombinant means by adding an appropriate signal sequence.
In a preferred embodiment, the CRC proteins are secreted proteins; the
secretion of which can be
either constitutive or regulated. These proteins have a signal peptide or
signal sequence that targets
the molecule to the secretory pathway. Secreted proteins are involved in
numerous physiological
events; by virtue of their circulating nature, they serve to transmit signals
to various other cell types.
The secreted protein may function in an autocrine manner (acting on the cell
that secreted the factor),
a paracrine manner (acting on cells in close proximity to the cell that
secreted the factor) or an
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endocrine manner (acting on cells at a distance). Thus secreted molecules find
use in modulating or
altering numerous aspects of physiology. CRC proteins that are secreted
proteins are particularly
preferred in the present invention as they serve as good targets for
diagnostic markers, for example
for blood tests.
A CRC sequence is initially identified by substantial nucleic acid andlor
amino acid sequence
homology to the CRC sequences outlined herein. Such homology can be based upon
the overall
nucleic acid or amino acid sequence, and is generally determined as outlined
below, using either
homology programs or hybridization conditions.
As used herein, a nucleic acid is a "CRC nucleic acid" if the overall homology
of the nucleic acid
sequence to the nucleic acid sequences encoding the amino acid sequences of
the figures is
preferably greater than about 75%, more preferably greater than about 80%,
even more preferably
greater than about 85% and mosf preferably greater than 90%. In some
embodiments the homology
will be as high as about 93 to 95 or 98%. Homology in this context means
sequence similarity or
identity, with identity being preferred. A preferred comparison for homology
purposes is to compare
the sequence containing sequencing errors to the correct sequence. This
homology will be
determined using standard techniques known in the art, including, but not
limited to, the local
homology algorithm of Smith ~ Wate,rman, Adv. Appl. Math. 2:482 (1981 ), by
the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biool. 48:443 (1970), by the search
for similarity method of
Pearson & Lipman, PNAS USA 85:2444 (1988), by computerized implementations of
these algorithms
(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,
Genetics
Computer Group, 575 Science Drive, Madison, WI), the Best Fit sequence program
described by
Devereux et al., Nucl. Acid Res. 12:387-395 (1984), preferably using the
default settings, or by
inspection.
In a preferred embodiment, the sequences which are used to determine sequence
identity or similarity
are selected from the sequences set forth in the figures, preferably those
represented in Figure 12,
more preferably those represented in Figures 13A and 13B, still more
preferably those of Figures 14-
20, 22-25, 27-29, 31-33, 35-37 and 39-48, and fragments thereof. In one
embodiment the sequences
utilized herein are those set forth in the figures. In another embodiment, the
sequences are naturally
occurring allelic variants of the sequences set forth in the figures. In
another embodiment, the
sequences are sequence variants as further described herein.
One example of a useful algorithm is PILEUP. PILEUP creates a multiple
sequence alignment from a
group of related sequences using progressive, pairwise alignments. It can also
plot a tree showing the
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WO 00/55633 PCT/US00/07044
clustering relationships used to create the alignment. PILEUP uses a
simplification of the progressive
alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987); the
method is similar to that
described by Higgins & Sharp CABIOS 5:151-153 (1989). Useful PILEUP parameters
including a
default gap weight of 3.00, a default gap length weight of 0.10, and weighted
end gaps.
Another example of a useful algorithm is the BLAST algorithm, described in
Altschul et al., J. Mol. Biol.
215, 403-410, (1990) and Kariin et al., PNAS USA 90:5873-5787 (1993). A
particularly useful BLAST
program is the WU-BLAST-2 program which was obtained from Altschul et al.,
Methods in
Enzymology, 266: 460-480 (1996); http://blast.wustl/edu/blast/ REACRCE.html].
WU-BLAST-2 uses
several search parameters, most of which are set to the default values. The
adjustable parameters
are set with the following values: overlap span =1, overlap fraction = 0.125,
word threshold (T) = 11.
The HSP S and HSP S2 parameters are dynamic values and are established by the
program itself
depending upon the composition of the particular sequence and composition of
the particular
database against which the sequence of interest is being searched; however,
the values may be
adjusted to increase sensitivity. A % amino acid sequence identity value is
determined by the number
of matching identical residues divided by the total number of residues of the
"longer" sequence in the
aligned region. The "longer" sequence is the one having the most actual
residues in the aligned
region (gaps introduced by WU-Blast-2 to maximize the alignment score are
ignored).
Thus, "percent (%) nucleic acid sequence identity" is defined as the
percentage of nucleotide residues
in a candidate sequence that are identical with the nucleotide residues of the
sequences of the figures.
A preferred method utilizes the BLASTN module of WU-BLAST-2 set to the default
parameters, with
overlap span and overlap fraction set to 1 and 0.125, respectively.
The alignment may include the introduction of gaps in the sequences to be
aligned. In addition, for
sequences which contain either more or fewer nucleosides than those of the
figures, it is understood
that the percentage of homology will be determined based on the number of
homologous nucleosides
in relation to the total number of nucleosides. Thus, for example, homology of
sequences shorter than
those of the sequences identified herein and as discussed below, will be
determined using the number
of nucleosides in the shorter sequence.
In one embodiment, the nucleic acid homology is determined through
hybridization studies. Thus, for
example, nucleic acids which hybridize under high stringency to the nucleic
acid sequences which
encode the peptides identified in the figures, or their complements, are
considered a CRC sequence.
High stringency conditions are known in the art; see for example Maniatis et
al., Molecular Cloning: A
Laboratory Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology,
ed. Ausubel, et al.,
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both of which are hereby incorporated by reference. Stringent conditions are
sequence-dependent
and will be different in different circumstances. Longer sequences hybridize
specifically at higher
temperatures. An extensive guide to the hybridization of nucleic acids is
found in Tijssen, Techniques
in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes,
"Overview of principles
of hybridization and the strategy of nucleic acid assays" (1993). Generally,
stringent conditions are
selected to be about 5-10°C lower than the thermal melting point (Tm)
for the specific sequence at a
defined ionic strength pH. The Tm is the temperature (under defined ionic
strength, pH and nucleic
acid concentration) at which 50% of the probes complementary to the target
hybridize to the target
sequence at equilibrium (as the target sequences are present in excess, at Tm,
50% of the probes are
occupied at equilibrium). Stringent conditions will be those in which the salt
concentration is less than
about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration
(or other salts) at pH
7.0 to 8.3 and the temperature is at least about 30°C for short probes
(e.g. 10 to 50 nucleotides) and
at least about 60°C for long probes (e.g. greater than 50 nucleotides).
Stringent conditions may also
be achieved with the addition of destabilizing agents such as formamide.
In another embodiment, less stringent hybridization conditions are used; for
example, moderate or low
stringency conditions may be used, as are known in the art; see Maniatis and
Ausubel, supra, and
Tijssen, supra.
In addition, the CRC nucleic acid sequences of the invention are fragments of
larger genes, i.e. they
are nucleic acid segments. "Genes" in this context includes coding regions,
non-coding regions, and
mixtures of coding and non-coding regions. Accordingly, as will be appreciated
by those in the art,
using the sequences provided herein, additional sequences of the CRC genes can
be obtained, using
techniques well known in the art for cloning either longer sequences or the
full length sequences; see
Maniatis et al., and Ausubel, et al., supra, hereby expressly incorporated by
reference.
Once the CRC nucleic acid is identified, it can be cloned and, if necessary,
its constituent parts
recombined to form the entire CRC nucleic acid. Once isolated from its natural
source, e.g., contained
within a plasmid or other vector or excised therefrom as a linear nucleic acid
segment, the
recombinant CRC nucleic acid can be further-used as a probe to identify and
isolate other CRC
nucleic acids, for example additional coding regions. It can also be used as a
"precursor" nucleic acid
to make modified or variant CRC nucleic acids and proteins.
The CRC nucleic acids of the present invention are used in several ways. In a
first embodiment,
nucleic acid probes to the CRC nucleic acids are made and attached to biochips
to be used in
screening and diagnostic methods, as outlined below, or for administration,
for example for gene
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therapy and/or antisense applications. Alternatively, the CRC nucleic acids
that include coding regions
of CRC proteins can be put into expression vectors for the expression of CRC
proteins, again either
for screening purposes or for administration to a patient.
In a preferred embodiment, nucleic acid probes to CRC nucleic acids (both the
nucleic acid
sequences encoding peptides outlined in the figures and/or the complements
thereof) are made. The
nucleic acid probes attached to the biochip are designed to be substantially
complementary to the
CRC nucleic acids, i.e. the target sequence (either the target sequence of the
sample or to other
probe sequences, for example in sandwich assays), such that hybridization of
the target sequence and
the probes of the present invention occurs. As outlined below, this
complementarity need not be
perfect; there may be any number of base pair mismatches which will interfere
with hybridization
between the target sequence and the single stranded nucleic acids of the
present invention. However,
if the number of mutations is so great that no hybridization can occur under
even the least stringent of
hybridization conditions, the sequence is not a complementary target sequence.
Thus, by
"substantially complementary" herein is meant that the probes are sufficiently
complementary to the
target sequences to hybridize under normal reaction conditions, particularly
high stringency conditions,
as outlined herein.
A nucleic acid probe is generally single stranded but can be partially single
and partially double
stranded. The strandedness of the probe is dictated by the structure,
composition, and properties of
the target sequence. In general, the nucleic acid probes range from about 8 to
about 100 bases long,
with from about 10 to about 80 bases being preferred, and from about 30 to
about 50 bases being
particularly preferred. That is, generally whole genes are not used. In some
embodiments, much
longer nucleic acids can be used, up to hundreds of bases.
In a preferred embodiment, more than one probe per sequence is used, with
either overlapping
probes or probes to different sections of the target being used. That is, two,
three, four or more
probes, with three being preferred, are used to build in a redundancy for a
particular target. The
probes can be overlapping (i.e. have some sequence in common), or separate.
As will be appreciated by those in the art, nucleic acids can be attached or
immobilized to a solid
support in a wide variety of ways. By "immobilized" and grammatical
equivalents herein is meant the
association or binding between the nucleic acid probe and the solid support is
sufficient to be stable
under the conditions of binding, washing, analysis, and removal as outlined
below. The binding can be
covalent or non-covalent. By "non-covalent binding" and grammatical
equivalents herein is meant one
or more of either electrostatic, hydrophilic, and hydrophobic interactions.
Included in non-covalent
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binding is the covalent attachment of a molecule, such as, streptavidin to the
support and the non-
covalent binding of the biotinylated probe to the streptavidin. By "covalent
binding" and grammatical
equivalents herein is meant that the two moieties, the solid support and the
probe, are attached by at
least one bond, including sigma bonds, pi bonds and coordination bonds.
Covalent bonds can be
formed directly between the probe and the solid support or can be formed by a
cross linker or by
inclusion of a specific reactive group on either the solid support or the
probe or both molecules.
Immobilization may also involve a combination of covalent and non-covalent
interactions.
In general, the probes are attached to the biochip in a wide variety of ways,
as will be appreciated by
those in the art. As described herein, the nucleic acids can either be
synthesized first, with
subsequent attachment to the biochip, or can be directly synthesized on the
biochip.
The biochip comprises a suitable solid substrate. By "substrate" or "solid
support" or other
grammatical equivalents herein is meant any material that can be modified to
contain discrete
individual sites appropriate for the attachment or association of the nucleic
acid probes and is
amenable to at least one detection method. As will be appreciated by those in
the art, the number of
possible substrates are very large, and include, but are not limited to, glass
and modified or
functionalized glass, plastics (including acrylics, polystyrene and copolymers
of styrene and other
materials, polypropylene, polyethylene, polybutylene, polyurethanes, TefIonJ,
etc.), polysaccharides,
nylon or nitrocellulose, resins, silica or silica-based materials including
silicon and modified silicon,
carbon, metals, inorganic glasses, plastics, etc. In general, the substrates
allow optical detection and
do not appreciably fluoresce. A preferred substrate is described in copending
application entitled
Reusable Low Fluorescent Plastic Biochip, U.S. Application Serial No.
09/270,214, filed March 15,
1999, herein incorporated by reference in its entirety.
Generally the substrate is planar, although as will be appreciated by those in
the art, other
configurations of substrates may be used as well. For example, the probes may
be placed on the
inside surface of a tube, for flow-through sample analysis to minimize sample
volume. Similarly, the
substrate may be flexible, such as a flexible foam, including closed cell
foams made of particular
plastics.
In a preferred embodiment, the surface of the biochip and the probe may be
derivatized with chemical
functional groups for subsequent attachment of the two. Thus, for example, the
biochip is derivatized
with a chemical functional group including, but not limited to, amino groups,
carboxy groups, oxo
groups and thiol groups, with amino groups being particularly preferred. Using
these functional
groups, the probes can be attached using functional groups on the probes. For
example, nucleic
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acids containing amino groups can be attached to surfaces comprising amino
groups, for example
using linkers as are known in the art; for example, homo-or hetero-
bifunctional linkers as are well
known (see 1994 Pierce Chemical Company catalog, technical section on cross-
linkers, pages
155-200, incorporated herein by reference). In addition, in some cases,
additional linkers, such as
alkyl groups (including substituted and heteroalkyl groups) may be used.
In this embodiment, the oligonucleotides are synthesized as is known in the
art, and then attached to
the surface of the solid support. As will be appreciated by those skilled in
the art, either the 5' or 3'
terminus maybe attached to the solid support, or attachment may be via an
internal nucleoside.
In an additional embodiment, the immobilization to the solid support may be
very strong, yet non-
covalent. For example, biotinylated oligonucleotides can be made, which bind
to surfaces covalently
coated with streptavidin, resulting in attachment.
Alternatively, the oligonucleotides may be synthesized on the surface, as is
known in the art. For
example, photoactivation techniques utilizing photopolymerization compounds
and techniques are
used. In a preferred embodiment, the nucleic acids can be synthesized in situ,
using well known
photolithographic techniques, such as those described in WO 95/25116; WO
95/35505; U.S. Patent
Nos. 5,700,637 and 5,445,934; and references cited within, all of which are
expressly incorporated by
reference; these methods of attachment form the basis of the Affimetrix
GeneChipT"' technology.
In a preferred embodiment, CRC nucleic acids encoding CRC proteins are used to
make a variety of
expression vectors to express CRC proteins which can then be used in screening
assays, as
described below. The expression vectors may be either self-replicating
extrachromosomal vectors or
vectors which integrate into a host genome. Generally, these expression
vectors include
transcriptional and translational regulatory nucleic acid operably linked to
the nucleic acid encoding the
CRC protein. The term "control sequences" refers to DNA sequences necessary
for the expression of
an operably linked coding sequence in a particular host organism. The control
sequences that are
suitable for prokaryotes, for example, include a promoter, optionally an
operator sequence, and a
ribosome binding site. Eukaryotic cells are known to utilize promoters,
polyadenylation signals, and
enhancers.
Nucleic acid is "operably linked" when it is placed into a functional
relationship with another nucleic
acid sequence. For example, DNA for a presequence or secretory leader is
operably linked to DNA
for a polypeptide if it is expressed as a preprotein that participates in the
secretion of the polypeptide;
a promoter or enhancer is operably linked to a coding sequence if it affects
the transcription of the
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sequence; or a ribosome binding site is operably linked to a coding sequence
if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the DNA
sequences being linked are
contiguous, and, in the case of a secretory leader, contiguous and in reading
phase. However,
enhancers do not have to be contiguous. Linking is accomplished by ligation at
convenient restriction
sites. If such sites do not exist, the synthetic oligonucleotide adaptors or
linkers are used in
accordance with conventional practice. The transcriptional and translational
regulatory nucleic acid
will generally be appropriate to the host cell used to express the CRC
protein; for example,
transcriptional and translationai regulatory nucleic acid sequences from
Bacillus are preferably used to
express the CRC protein in Bacillus. Numerous types of appropriate expression
vectors, and suitable
regulatory sequences are known in the art for a variety of host cells.
In general, the transcriptional and translational regulatory sequences may
include, but are not limited
to, promoter sequences, ribosomal binding sites, transcriptional start and
stop sequences,
translational start and stop sequences, and enhancer or activator sequences.
In a preferred
embodiment, the regulatory sequences include a promoter and transcriptional
start and stop
sequences.
Promoter sequences encode either constitutive or inducible promoters. The
promoters may be either
naturally occurring promoters or hybrid promoters. Hybrid promoters, which
combine elements of
more than one promoter, are also known in the art, and are useful in the
present invention.
In addition, the expression vector may comprise additional elements. For
example, the expression
vector may have two replication systems, thus allowing it to be maintained in
two organisms, for
example in mammalian or insect cells for expression and in a procaryotic host
for cloning and
amplification. Furthermore, for integrating expression vectors, the expression
vector contains at least
one sequence homologous to the host cell genome, and preferably two homologous
sequences which
flank the expression construct. The integrating vector may be directed to a
specific locus in the host
cell by selecting the appropriate homologous sequence for inclusion in the
vector. Constructs for
integrating vectors are well known in the art.
In addition, in a preferred embodiment, the expression vector contains a
selectable marker gene to
allow the selection of transformed host cells. Selection genes are well known
in the art and will vary
with the host cell used.
The CRC proteins of the present invention are produced by culturing a host
cell transformed with an
expression vector containing nucleic acid encoding a CRC protein, under the
appropriate conditions to
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induce or cause expression of the CRC protein. The conditions appropriate for
CRC protein
expression will vary with the choice of the expression vector and the host
cell, and will be easily
ascertained by one skilled in the art through routine experimentation. For
example, the use of
constitutive promoters in the expression vector will require optimizing the
growth and proliferation of
the host cell, while the use of an inducible promoter requires the appropriate
growth conditions for
induction. In addition, in some embodiments, the timing of the harvest is
important. For example, the
baculoviral systems used in insect cell expression are lytic viruses, and thus
harvest time selection
can be crucial for product yield.
Appropriate host cells include yeast, bacteria, archaebacteria, fungi, and
insect and animal cells,
including mammalian cells. Of particular interest are Drosophila melangaster
cells, Saccharomyces
cerevisiae and other yeasts, E. coli, Bacillus subtilis, Sf9 cells, C129
cells, 293 cells, Neurospora,
BHK, CHO, COS, HeLa cells, THP1 cell line (a macrophage cell line) and human
cells and cell lines.
In a preferred embodiment, the CRC proteins are expressed in mammalian cells.
Mammalian
expression systems are also known in the art, and include retroviral systems.
A preferred expression
vector system is a retroviral vector system such as is generally described in
PCT/US97i01019 and
PCT/US97/01048, both of which are hereby expressly incorporated by reference.
Of particular use as
mammalian promoters are the promoters from mammalian viral genes, since the
viral genes are often
highly expressed and have a broad host range. Examples include the SV40 early
promoter, mouse
mammary tumor virus LTR promoter, adenovirus major late promoter, herpes
simplex virus promoter,
and the CMV promoter. Typically, transcription termination and polyadenylation
sequences
recognized by mammalian cells are regulatory regions located 3' to the
translation stop codon and
thus, together with the promoter elements, flank the coding sequence. Examples
of transcription
terminator and polyadenlytion signals include those derived form SV40.
The methods of introducing exogenous nucleic acid into mammalian hosts, as
well as other hosts, is
well known in the art, and will vary with the host cell used. Techniques
include dextran-mediated
transfection, calcium phosphate precipitation, polybrene mediated
transfection, protoplast fusion,
electroporation, viral infection, encapsulation of the polynucleotide(s) in
liposomes, and direct
microinjection of the DNA into nuclei.
In a preferred embodiment, CRC proteins are expressed in bacterial systems.
Bacterial expression
systems are well known in the art. Promoters from bacteriophage may also be
used and are known in
the art. In addition, synthetic promoters and hybrid promoters are also
useful; for example, the tac
promoter is a hybrid of the trp and lac promoter sequences. Furthermore, a
bacterial promoter can
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include naturally occurring promoters of non-bacterial origin that have the
ability to bind bacterial RNA
polymerase and initiate transcription. In addition to a functioning promoter
sequence, an efficient
ribosome binding site is desirable. The expression vector may also include a
signal peptide sequence
that provides for secretion of the CRC protein in bacteria. The protein is
either secreted into the
growth media (gram-positive bacteria) or into the periplasmic space, located
between the inner and
outer membrane of the cell (gram-negative bacteria). The bacterial expression
vector may also
include a selectable marker gene to allow for the selection of bacterial
strains that have been
transformed. Suitable selection genes include genes which render the bacteria
resistant to drugs such
as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and
tetracycline. Selectable
markers also include biosynthetic genes, such as those in the histidine,
tryptophan and leucine
biosynthetic pathways. These components are assembled into expression vectors.
Expression
vectors for bacteria are well known in the art, and include vectors for
Bacillus subtilis, E. coli,
Streptococcus cremoris, and Streptococcus lividans, among others. The
bacterial expression vectors
are transformed into bacterial host cells using techniques well known in the
art, such as calcium
chloride treatment, electroporation, and others.
In one embodiment, CRC proteins are produced in insect cells. Expression
vectors for the
transformation of insect cells, and in particular, baculovirus-based
expression vectors, are well known
in the art.
In a preferred embodiment, CRC protein is produced in yeast cells. Yeast
expression systems are
well known in the art, and include expression vectors for Saccharomyces
cerevisiae, Candida albicans
and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis,
Pichia guillerimondii and
P. pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica.
The CRC protein may also be made as a fusion protein, using techniques well
known in the art. Thus,
for example, for the creation of monoclonal antibodies, if the desired epitope
is small, the CRC protein
may be fused to a carrier protein to form an immunogen. Alternatively, the CRC
protein may be made
as a fusion protein to increase expression, or for other reasons. For example,
when the CRC protein
is a CRC peptide, the nucleic acid encoding the peptide may be linked to other
nucleic acid for
expression purposes.
In one embodiment, the CRC nucleic acids, proteins and antibodies of the
invention are labeled. By
"labeled" herein is meant that a compound has at least one element, isotope or
chemical compound
attached to enable the detection of the compound. In general, labels fall into
three classes: a) isotopic
labels, which may be radioactive or heavy isotopes; b) immune labels, which
may be antibodies or
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antigens; and c) colored or fluorescent dyes. The labels may be, incorporated
into the CRC nucleic
acids, proteins and antibodies at any position. For example, the label should
be capable of producing,
either directly or indirectly, a detectable signal. The detectable moiety may
be a radioisotope, such as
sH ,aC azP 35S or'z51, a fluorescent or chemiluminescent compound, such as
fluorescein
isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline
phosphatase, beta-
galactosidase or horseradish peroxidase. Any method known in the art for
conjugating the antibody to
the label may be employed, including those methods described by Hunter et al.,
Nature, 144:945
(1962); David et al., Biochemistry, 13:1014 (1974); Pain et al., J. Immunol.
Meth., 40:219 (1981 ); and
Nygren, J. Histochem. and Cytochem., 30:407 (1982).
Accordingly, the present invention also provides CRC protein sequences. A CRC
protein of the
present invention may be identified in several ways. "Protein" in this sense
includes proteins,
polypeptides, and peptides. As will be appreciated by those in the art, the
nucleic acid sequences of
the invention can be used to generate protein sequences. There are a variety
of ways to do this,
including cloning the entire gene and verifying its frame and amino acid
sequence, or by comparing it
to known sequences to search for homology to provide a frame, assuming the CRC
protein has
homology to some protein in the database being used. Generally, the nucleic
acid sequences are
input into a program that will search all three frames for homology. This is
done in a preferred
embodiment using the following NCBI Advanced BLAST parameters. The program is
blastx or blastn.
The database is nr. The input data is as "Sequence in FASTA format". The
organism list is "none".
The "expect" is 10; the filter is default. The "descriptions" is 500, the
"alignments" is 500, and the
"alignment view" is pairwise. The "Query Genetic Codes" is standard (1 ). The
matrix is BLOSUM62;
gap existence cost is 11, per residue gap cost is 1; and the lambda ratio is
.85 default. This results
in the generation of a putative protein sequence.
Also included within one embodiment of CRC proteins are amino acid variants of
the naturally
occurring sequences, as determined herein. Preferably, the variants are
preferably greater than about
75% homologous to the wild-type sequence, more preferably greater than about
80%, even more
preferably greater than about 85% and most preferably greater than 90%. In
some embodiments the
homology will be as high as about 93 to 95 or 98%. As for nucleic acids,
homology in this context
means sequence similarity or identity, with identity being preferred. This
homology will be determined
using standard techniques known in the art as are outlined above for the
nucleic acid homologies.
CRC proteins of the present invention may be shorter or longer than the wild
type amino acid
sequences. Thus, in a preferred embodiment, included within the definition of
CRC proteins are
portions or fragments of the wild type sequences. herein. In addition, as
outlined above, the CRC
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nucleic acids of the invention may be used to obtain additional coding
regions, and thus additional
protein sequence, using techniques known in the art.
In a preferred embodiment, the CRC proteins are derivative or variant CRC
proteins as compared to
the wild-type sequence. That is, as outlined more fully below, the derivative
CRC peptide will contain
at least one amino acid substitution, deletion or insertion, with amino acid
substitutions being
particularly preferred. The amino acid substitution, insertion or deletion may
occur at any residue
within the CRC peptide.
Also included in an embodiment of CRC proteins of the present invention are
amino acid sequence
variants. These variants fall into one or more of three classes:
substitutional, insertional or deletional
variants. These variants ordinarily are prepared by site specific mutagenesis
of nucleotides in the
DNA encoding the CRC protein, using cassette or PCR mutagenesis or other
techniques well known
in the art, to produce DNA encoding the variant, and thereafter expressing the
DNA in recombinant
cell culture as outlined above. However, variant CRC protein fragments having
up to about 100-150
residues may be prepared by in vitro synthesis using established techniques.
Amino acid sequence
variants are characterized by the predetermined nature of the variation, a
feature that sets them apart
from naturally occurring allelic or interspecies variation of the CRC protein
amino acid sequence. The
variants typically exhibit the same qualitative biological activity as the
naturally occurring analogue,
although variants can also be selected which have modified characteristics as
will be more fully
outlined below.
While the site or region for introducing an amino acid sequence variation is
predetermined, the
mutation per se need not be predetermined. For example, in order to optimize
the performance of a
mutation at a given site, random mutagenesis may be conducted at the target
codon or region and the
expressed CRC variants screened for the optimal combination of desired
activity. Techniques for
making substitution mutations at predetermined sites in DNA having a known
sequence are well
known, for example, M13 primer mutagenesis and PCR mutagenesis. Screening of
the mutants is
done using assays of CRC protein activities.
Amino acid substitutions are typically of single residues; insertions usually
will be on the order of from
about 1 to 20 amino acids, although considerably larger insertions may be
tolerated. Deletions range
from about 1 to about 20 residues, although in some cases deletions may be
much larger.
Substitutions, deletions, insertions or any combination thereof may be used to
arrive at a final
derivative. Generally these changes are done on a few amino acids to minimize
the alteration of the
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WO 00/55633 PCT/US00/07044
molecule. However, larger changes may be tolerated in certain circumstances.
When small
alterations in the characteristics of the CRC protein are desired,
substitutions are generally made in
accordance with the following chart:
Chart I
Original Residue Exemplary Substitutions
Ala Ser
Arg Lys
Asn Gln, His
Asp Glu
Cys Ser
Gln Asn
Glu Asp
Giy Pro
His Asn, Gln
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe Met, Leu, Tyr
Ser Thr
Thr Ser
Trp Tyr
Tyr Trp, Phe
Val Ile, Leu
Substantial changes in function or immunological identity are made by
selecting substitutions that are
less conservative than those shown in Chart I. For example, substitutions may
be made which more
significantly affect: the structure of the polypeptide backbone in the area of
the alteration, for example
the alpha-helical or beta-sheet structure; the charge or hydrophobicity of the
molecule at the target
site; or the bulk of the side chain. The substitutions which in general are
expected to produce the
greatest changes in the polypeptide's properties are those in which (a) a
hydrophilic residue, e.g. seryl
or threonyl is substituted for (or by) a hydrophobic residue, e.g. leucyl,
isoleucyl, phenylalanyl, valyl or
alanyl; (b) a cysteine or proline is substituted for (or by) any other
residue; (c) a residue having an
electropositive side chain, e.g. lysyl, arginyl, or histidyl, is substituted
for (or by) an electronegative
residue, e.g. glutamyl or aspartyl; or (d) a residue having a bulky side
chain, e.g, phenylalanine, is
substituted for (or by) one not having a side chain, e.g. glycine.
The variants typically exhibit the same qualitative biological activity and
will elicit the same immune
response as the naturally-occurring analogue, although variants also are
selected to modify the
characteristics of the CRC proteins as needed. Alternatively, the variant may
be designed such that
the biological activity of the CRC protein is altered. For example,
glycosylation sites may be altered or
removed.
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Covalent modifications of CRC polypeptides are included within the scope of
this invention. One type
of covalent modification includes reacting targeted amino acid residues of a
CRC polypeptide with an
organic derivatizing agent that is capable of reacting with selected side
chains or the N-or C-terminal
residues of a CRC polypeptide. Derivatization with bifunctional agents is
useful, for instance, for
crosslinking CRC to a water-insoluble support matrix or surface for use in the
method for purifying
anti-CRC antibodies or screening assays, as is more fully described below.
Commonly used
crosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,
glutaraldehyde, N-hydroxy-
succinimide esters, for example, esters with 4-azidosalicylic acid,
homobifunctional imidoesters,
including disuccinimidyl esters such as 3,3'-
dithiobis(succinimidylpropionate), bifunctional maleimides
such as bis-N-maleimido-1,8-octane and agents such as methyl-3-[(p-
azidophenyl)dithio]propioimi-
date.
Other modifications include deamidation of glutaminyl and asparaginyl residues
to the corresponding
glutamyl and aspartyl residues, respectively, hydroxylation of proline and
lysine, phosphorylation of
hydroxyl groups of seryl, threonyl or tyrosyl residues, methylation of the oc-
amino groups of lysine,
arginine, and histidine side chains [T.E. Creighton, Proteins: Structure and
Molecular Properties, W.H.
Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal
amine, and amidation
of any C-terminal carboxyl group.
Another type of covalent modification of the CRC polypeptide included within
the scope of this
invention comprises altering the native glycosylation pattern of the
polypeptide. "Altering the native
glycosylation pattern" is intended for purposes herein to mean deleting one or
more carbohydrate
moieties found in native sequence CRC polypeptide, and/or adding one or more
glycosylation sites
that are not present in the native sequence CRC polypeptide.
Addition of glycosylation sites to CRC polypeptides may be accomplished by
altering the amino acid
sequence thereof. The alteration may be made, for example, by the addition of,
or substitution by, one
or more serine or threonine residues to the native sequence CRC polypeptide
(for O-linked
glycosylation sites). The CRC amino acid sequence may optionally be altered
through changes at the
DNA level, particularly by mutating the DNA encoding the CRC polypeptide at
preselected bases such
that codons are generated that will translate into the desired amino acids.
Another means of increasing the number of carbohydrate moieties on the CRC
polypeptide is by
chemical or enzymatic coupling of glycosides to the polypeptide. Such methods
are described in the
art, e.g., in WO 87105330 published 11 September 1987, and in Aplin and
Wriston, CRC Crit. Rev.
Biochem., pp. 259-306 (1981).
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Removal of carbohydrate moieties present on the CRC polypeptide may be
accomplished chemically
or enzymatically or by mutational substitution of codons encoding for amino
acid residues that serve
as targets for glycosylation. Chemical deglycosylation techniques are known in
the art and described,
for instance, by Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987)
and by Edge et al., Anal.
Biochem., 118:131 (1981 ). Enzymatic cleavage of carbohydrate moieties on
polypeptides can be
achieved by the use of a variety of endo-and exo-glycosidases as described by
Thotakura et al., Meth.
Enzymol., 138:350 (1987).
Another type of covalent modification of CRC comprises linking the CRC
polypeptide to one of a
variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene
glycol, or
polyoxyalkylenes, in the manner set forth in U:S. Patent Nos. 4,640,835;
4,496,689; 4,301,144;
4,670,417; 4,791,192 or4,179,337.
CRC polypeptides of the present invention may also be modified in a way to
form chimeric molecules
comprising a CRC polypeptide fused to another, heterologous polypeptide or
amino acid sequence. In
one embodiment, such a chimeric molecule comprises a fusion of a CRC
polypeptide with a tag
polypeptide which provides an epitope to which an anti-tag antibody can
selectively bind. The epitope
tag is generally placed at the amino-or carboxyl-terminus of the CRC
polypeptide. The presence of
such epitope-tagged forms of a CRC polypeptide can be detected using an
antibody against the tag
polypeptide. Also, provision of the epitope tag enables the CRC polypeptide to
be readily purified by
affinity purification using an anti-tag antibody or another type of affinity
matrix that binds to the epitope
tag. In an alternative embodiment, the chimeric molecule may comprise a fusion
of a CRC
polypeptide with an immunoglobulin or a particular region of an
immunvglobulin. For a bivalent form
of the chimeric molecule, such a fusion could be to the Fc region of an IgG
molecule.
Various tag polypeptides and their respective antibodies are well known in the
art. Examples include
poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the
flu HA tag polypeptide and its
antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc
tag and the 8F9, 3C7,
6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular
Biology, 5:3610-3616
(1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody
[Paborsky et al.,
Protein Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include the
Flag-peptide [Hope et
al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et
al., Science, 255:192-194
(1992)]; tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266:15163-
15166 (1991 )]; and the T7
gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci.
USA, 87:6393-6397 (1990)].
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Also included with the definition of CRC protein in one embodiment are other
CRC proteins of the
CRC family, and CRC proteins from other organisms, which are cloned and
expressed as outlined
below. Thus, probe or degenerate polymerase chain reaction (PCR) primer
sequences may be used
to find other related CRC proteins from humans or other organisms. As will be
appreciated by those in
the art, particularly useful probe and/or PCR primer sequences include the
unique areas of the CRC
nucleic acid sequence. As is generally known in the art, preferred PCR primers
are from about 15 to
about 35 nucleotides in length, with from about 20 to about 30 being
preferred, and may contain
inosine as needed. The conditions for the PCR reaction are well known in the
art.
In addition, as is outlined herein, CRC proteins can be made that are longer
than those depicted in the
figures, for example, by the elucidation of additional sequences, the addition
of epitope or purification
tags, the addition of other fusion sequences, etc.
CRC proteins may also be identified as being encoded by CRC nucleic acids.
Thus, CRC proteins are
encoded by nucleic acids that will hybridize to the sequences of the sequence
listings, or their
complements, as outlined herein.
In a preferred embodiment, when the CRC protein is to be used to generate
antibodies, for example
for immunotherapy, the CRC protein should share at least one epitope or
determinant with the full
length protein. By "epitope" or "determinant" herein is meant a portion of a
protein which will generate
and/or bind an antibody or T-cell receptor in the context of MHC. Thus, in
most instances, antibodies
made to a smaller CRC protein will be able to bind to the full length protein.
In a preferred
embodiment, the epitope is unique; that is, antibodies generated to a unique
epitope show little or no
cross-reactivity. In a preferred embodiment, the epitope is selected from
CAA2p1 and CAA2p2. In
another preferred embodiment, the epitope is selected from CAA9p1, CAA9p2,
CAA9p3, CAAQ9p4,
CAA9p4MAPS, CAA89p5 and CAA9p5MAPS.
In one embodiment, the term "antibody" includes antibody fragments, as are
known in the art,
including Fab, Fab2, single chain antibodies (Fv for example), chimeric
antibodies, etc., either
produced by the modification of whole antibodies or those synthesized de novo
using recombinant
DNA technologies.
Methods of preparing polyclonal antibodies are known to the skilled artisan.
Polyclonal antibodies can
be raised in a mammal, for example, by one or more injections of an immunizing
agent and, if desired,
an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected
in the mammal by
multiple subcutaneous or intraperitoneal injections. The immunizing agent may
include the CAA2 or
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fragment thereof or a fusion protein thereof. It may be useful to conjugate
the immunizing agent to a
protein known to be immunogenic in the mammal being immunized. Examples of
such immunogenic
proteins include but are not limited to keyhole limpet hemocyanin, serum
albumin, bovine
thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may
be employed include
Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,
synthetic trehalose
dicorynomycolate). The immunization protocol may be selected by one skilled in
the art without undue
experimentation.
The antibodies may, alternatively, be monoclonal antibodies. Monoclonal
antibodies may be prepared
using hybridoma methods, such as those described by Kohler and Milstein,
Nature, 256:495 (1975).
In a hybridoma method, a mouse, hamster, or other appropriate host animal, is
typically immunized
with an immunizing agent to elicit lymphocytes that produce or are capable of
producing antibodies
that will specifically bind to the immunizing agent. Alternatively, the
lymphocytes may be immunized in
vitro. The immunizing agent will typically include the CAA2 polypeptide or
fragment thereof or a fusion
protein thereof. Generally, either peripheral blood lymphocytes ("PBLs") are
used if cells of human
origin are desired, or spleen cells or lymph node cells are used if non-human
mammalian sources are
desired. The lymphocytes are then fused with an immortalized cell line using a
suitable fusing agent,
such as polyethylene glycol, to form a hybridoma cell [coding, Monoclonal
Antibodies: Principles and
Practice, Academic Press, {1986) pp. 59-103]. Immortalized cell lines are
usually transformed
mammalian cells, particularly myeloma cells of rodent, bovine and human
origin. Usually, rat or
mouse myeloma cell lines are employed. The hybridoma cells may be cultured in
a suitable culture
medium that preferably contains one or more substances that inhibit the growth
or survival of the
unfused, immortalized cells. For example, if the parental cells lack the
enzyme hypoxanthine guanine
phosphoribosyl transferase {HGPRT or HPRT), the culture medium for the
hybridomas typically will
include hypoxanthine, aminopterin, and thymidine {"HAT medium"), which
substances prevent the
growth of HGPRT-deficient cells.
In one embodiment, the antibodies are bispecific antibodies. Bispecific
antibodies are monoclonal,
preferably human or humanized, antibodies that have binding specificities for
at least two different
antigens. In the present case, one of the binding specificities is for a CRC
protein or a fragment
thereof, the other one is for any other antigen, and preferably for a cell-
surface protein or receptor or
receptor subunit, preferably one that is tumor specific.
In a preferred embodiment, the antibodies to CRC are capable of reducing or
eliminating the biological
function of CRC, as is described below. That is, the addition of anti-CRC
antibodies (either polyclonal
or preferably monoclonal) to CRC (or cells containing CRC) may reduce or
eliminate the CRC activity.
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Generally, at least a 25% decrease in activity is preferred, with at least
about 50% being particularly
preferred and about a 95-100% decrease being especially preferred.
In a preferred embodiment the antibodies to the CRC proteins are humanized
antibodies. Humanized
forms of non-human (e.g., murine) antibodies are chimeric molecules of
immunoglobulins,
immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or
other antigen-binding
subsequences of antibodies) which contain minimal sequence derived from non-
human
immunoglobulin. Humanized antibodies include human immunoglobulins (recipient
antibody) in which
residues form a complementary determining region (CDR) of the recipient are
replaced by residues
from a CDR of a non-human species (donor antibody) such as mouse, rat or
rabbit having the desired
specificity, affinity and capacity. In some instances, Fv framework residues
of the human
immunoglobulin are replaced by corresponding non-human residues. Humanized
antibodies may also
comprise residues which are found neither in the recipient antibody nor in the
imported CDR or
framework sequences. In general, the humanized antibody will comprise
substantially all of at least
one, and typically two, variable domains, in which all or substantially all of
the CDR regions correspond
to those of a non-human immunoglobulin and all or substantially all of the FR
regions are those of a
human immunoglobulin consensus sequence. The humanized antibody optimally also
wifl comprise at
least a portion of an immunoglobulin constant region (Fc), typically that of a
human immunoglobulin
[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-
329 (1988); and Presta,
Curr. Op. Struct. Biol., 2:593-596 (1992)].
Methods for humanizing non-human antibodies are well known in the art.
Generally, a humanized
antibody has one or more amino acid residues introduced into it from a source
which is non-human.
These non-human amino acid residues are often referred to as import residues,
which are typically
taken from an import variable domain. Humanization can be essentially
performed following the
method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986);
Riechmann et al., Nature,
332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by
substituting rodent CDRs
or CDR sequences for the corresponding sequences of a human antibody.
Accordingly, such
humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567),
wherein substantially less
than an intact human variable domain has been substituted by the corresponding
sequence from a
non-human species. In practice, humanized antibodies are typically human
antibodies in which some
CDR residues and possibly some FR residues are substituted by residues from
analogous sites in
rodent antibodies.
Human antibodies can also be produced using various techniques known in the
art, including phage
display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991 );
Marks et al., J. Mol. Biol.,
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222:581 (1991 )]. The techniques of Cole et al. and Boerner et al. are also
available for the preparation
of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer
Therapy, Alan R.
Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147 1 :86-95 (1991 )].
Similarly, human antibodies
can be made by introducing of human immunoglobulin loci into transgenic
animals, e.g., mice in which
the endogenous immunoglobulin genes have been partially or completely
inactivated. Upon
challenge, human antibody production is observed, which closely resembles that
seen in humans in all
respects, including gene rearrangement, assembly, and antibody repertoire.
This approach is
described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425;
5,661,016, and in the following scientific publications: Marks et al.,
Bio/Technoloay 10, 779-783
(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368, 812-
13 (1994); Fishwild et
al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology
14, 826 (1996);
Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).
By immunotherapy is meant treatment of CRC with an antibody raised against CRC
proteins. As
used herein, immunotherapy can be passive or active. Passive immunotherapy as
defined herein is
the passive transfer of antibody to a recipient (patient). Active immunization
is the induction of
antibody and/or T-cell responses in a recipient (patient). Induction of an
immune response is the
result of providing the recipient with an antigen to which antibodies are
raised. As appreciated by one
of ordinary skill in the art, the antigen may be provided by injecting a
polypeptide against which
antibodies are desired to be raised into a recipient, or contacting the
recipient with a nucleic acid
capable of expressing the antigen and under conditions for expression of the
antigen.
In a preferred embodiment the CRC proteins against which antibodies are raised
are secreted
proteins as described above. Without being bound by theory, antibodies used
for treatment, bind and
prevent the secreted protein from binding to its receptor, thereby
inactivating the secreted CRC
protein.
In another preferred embodiment, the CRC protein to which antibodies are
raised is a transmembrane
protein. Without being bound by theory, antibodies used for treatment, bind
the extracellular domain
of the CRC protein and prevent it from binding to other proteins, such as
circulating ligands or cell-
associated molecules. The antibody may cause down-regulation of the
transmembrane CRC protein.
As will be appreciated by one of ordinary skill in the art, the antibody may
be a competitive, non-
competitive or uncompetitive inhibitor of protein binding to the extracellular
domain of the CRC protein.
The antibody is also an antagonist of the CRC protein. Further, the antibody
prevents activation of the
transmembrane CRC protein. In one aspect, when the antibody prevents the
binding of other
molecules to the CRC protein, the antibody prevents growth of the cell. The
antibody also sensitizes
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the cell to cytotoxic agents, including, but not limited to TNF-a, TNF-b, IL-
1, INF-g and IL-2, or
chemotherapeutic agents including SFU, vinblastine, actinomycin D, cisplatin,
methotrexate, and the
like. In some instances the antibody belongs to a sub-type that activates
serum complement when
complexed with the transmembrane protein thereby mediating cytotoxicity. Thus,
CRC is treated by
administering to a patient antibodies directed against the transmembrane CRC
protein.
In another preferred embodiment, the antibody is conjugated to a therapeutic
moiety. In one aspect
the therapeutic moiety is a small molecule that modulates the activity of the
CRC protein. In another
aspect the therapeutic moiety modulates the activity of molecules associated
with or in close proximity
to the CRC protein. The therapeutic moiety may inhibit enzymatic activity such
as protease or protein
kinase activity associated with CRC.
fn a preferred embodiment, the therapeutic moiety may also be a cytotoxic
agent. In this method,
targeting the cytotoxic agent to tumor tissue or cells, results in a reduction
in the number of afflicted
cells, thereby reducing symptoms associated with CRC. Cytotoxic agents are
numerous and varied
and include, but are not limited to, cytotoxic drugs or toxins or active
fragments of such toxins.
Suitable toxins and their corresponding fragments include diptheria A chain,
exotoxin A chain, ricin A
chain, abrin A chain, curcin, crotin, phenomycin, enomycin and the like.
Cytotoxic agents also include
radiochemicals made by conjugating radioisotopes to antibodies raised against
CRC proteins, or
binding of a radionuclide to a chelating agent that has been covalently
attached to the antibody.
Targeting the therapeutic moiety to transmembrane CRC proteins not only serves
to increase the local
concentration of therapeutic moiety in the CRC afflicted area, but also serves
to reduce deleterious
side effects that may be associated with the therapeutic moiety.
In another preferred embodiment, the CRC protein against which the antibodies
are raised is an
intracellular protein. In this case, the antibody may be conjugated to a
protein which facilitates entry
into the cell. In one case, the antibody enters the cell by endocytosis. In
another embodiment, a
nucleic acid encoding the antibody is administered to the individual or cell.
Moreover, wherein the
CRC protein can be targeted within a cell, i.e., the nucleus, an antibody
thereto contains a signal for
that target localization, i.e., a nuclear localization signal.
The CRC antibodies of the invention specifically bind to CRC proteins. By
"specifically bind" herein is
meant that the antibodies bind to the protein with a binding constant in the
range of at least 10~- 10-6
M-', with a preferred range being 10-' - 10-9 M-'.
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In a preferred embodiment, the CRC protein is purified or isolated after
expression. CRC proteins
may be isolated or purified in a variety of ways known to those skilled in the
art depending on what
other components are present in the sample. Standard purification methods
include electrophoretic,
molecular, immunological and chromatographic techniques, including ion
exchange, hydrophobic,
affinity, and reverse-phase HPLC chromatography, and chromatofocusing. For
example, the CRC
protein may be purified using a standard anti-CRC antibody column.
Ultrafiltration and diafiltration
techniques, in conjunction with protein concentration, are also useful. For
general guidance in suitable
purification techniques, see Scopes, R., Protein Purification, Springer-
Verlag, NY (1982). The degree
of purification necessary will vary depending on the use of the CRC protein.
In some instances no
purification will be necessary.
Once expressed and purified if necessary, the CRC proteins and nucleic acids
are useful in a number
of applications.
In one aspect, the expression levels of genes are determined for different
cellular states in the CRC
phenotype; that is, the expression levels of genes in normal colon tissue and
in CRC tissue (and in
some cases, for varying severities of CRC that relate to prognosis, as
outlined below) are evaluated to
provide expression profiles. An expression profile of a particular cell state
or point of development is
essentially a "fingerprint" of the state; while two states may have any
particular gene similarly
expressed, the evaluation of a number of genes simultaneously allows the
generation of a gene
expression profile that is unique to the state of the cell. By comparing
expression profiles of cells in
different states, information regarding which genes are important (including
both up- and down-
regulation of genes) in each of these states is obtained. Then, diagnosis may
be done or confirmed:
does tissue from a particular patient have the gene expression profile of
normal or CRC tissue.
"Differential expression," or grammatical equivalents as used herein, refers
to both qualitative as well
as quantitative differences in the genes' temporal and/or cellular expression
patterns within and
among the cells. Thus, a differentially expressed gene can qualitatively have
its expression altered,
including an activation or inactivation, in, for example, normal versus CRC
tissue. That is, genes may
be turned on or turned off in a particular state, relative to another state.
As is apparent to the skilled
artisan, any comparison of two or more states can be made. Such a
qualitatively regulated gene will
exhibit an expression pattern within a state or cell type which is detectable
by standard techniques in
one such state or cell type, but is not detectable in both. Alternatively, the
determination is quantitative
in that expression is increased or decreased; that is, the expression of the
gene is either upregulated,
resulting in an increased amount of transcript, or downregulated, resulting in
a decreased amount of
transcript. The degree to which expression differs need only be large enough
to quantify via standard
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characterization techniques as outlined below, such as by use of Affymetrix
GeneChipTM expression
arrays, Lockhart, Nature Biotechnology, 14:1675-1680 (1996), hereby expressly
incorporated by
reference. Other techniques include, but are not limited to, quantitative
reverse transcriptase PCR,
Northern analysis and RNase protection. As outlined above, preferably the
change in expression (i.e.
upregulation or downregulation) is at least about 50%, more preferably at
least about 100%, more
preferably at least about 150%, more preferably, at least about 200%, with
from 300 to at least 1000%
being especially preferred.
As will be appreciated by those in the art, this may be done by evaluation at
either the gene transcript,
or the protein level; that is, the amount of gene expression may be monitored
using nucleic acid
probes to the DNA or RNA equivalent of the gene transcript, and the
quantification of gene expression
levels, or, alternatively, the final gene product itself (protein) can be
monitored, for example through
the use of antibodies to the CRC protein and standard immunoassays (ELISAs,e
tc.) or other
techniques, including mass spectroscopy assays, 2D gel electrophoresis assays,
etc. Thus, the
proteins corresponding to CRC genes, i.e. those identified as being important
in a CRC phenotype,
can be evaluated in a CRC diagnostic test.
In a preferred embodiment, gene expression monitoring is done and a number of
genes, i.e. an
expression profile, is monitored simultaneously, although multiple protein
expression monitoring can
be done as well. Similarly, these assays may be done on an individual basis as
well.
In this embodiment, the CRC nucleic acid probes are attached to biochips as
outlined herein for the
detection and quantification of CRC sequences in a particular cell. The assays
are further described
below in the example.
In a preferred embodiment nucleic acids encoding the CRC protein are detected.
Although DNA or
RNA encoding the CRC protein may be detected, of particular interest are
methods wherein the
mRNA encoding a CRC protein is detected. The presence of mRNA in a sample is
an indication that
the CRC gene has been transcribed to form the mRNA, and suggests that the
protein is expressed.
Probes to detect the mRNA can be any nucleotide/deoxynucleotide probe that is
complementary to
and base pairs with the mRNA and includes but is not limited to
oligonucleotides, cDNA or RNA.
Probes also should contain a detectable label, as defined herein. In one
method the mRNA is
detected after immobilizing the nucleic acid to be examined on a solid support
such as nylon
membranes and hybridizing the probe with the sample. Following washing to
remove the non-
specifically bound probe, the label is detected. In another method detection
of the mRNA is performed
in situ. In this method permeabilized cells or tissue samples are contacted
with a detectably labeled
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nucleic acid probe for sufficient time to allow the probe to hybridize with
the target mRNA. Following
washing to remove the non-specifically bound probe, the label is detected. For
example a digoxygenin
labeled riboprobe (RNA probe) that is complementary to the mRNA encoding a CRC
protein is
detected by binding the digoxygenin with an anti-digoxygenin secondary
antibody and developed with
nitro blue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate.
In a preferred embodiment, any of the three classes of proteins as described
herein (secreted,
transmembrane or intracellular proteins) are used in diagnostic assays. The
CRC proteins,
antibodies, nucleic acids, modified proteins and cells containing CRC
sequences are used.in
diagnostic assays. This can be done on an individual gene or corresponding
polypeptide level. In a
preferred embodiment, the expression profiles are used, preferably in
conjunction with high throughput
screening techniques to allow monitoring for expression profile genes and/or
corresponding
polypeptides.
As described and defined herein, CRC proteins, including intracellular,
transmembrane or secreted
proteins, find use as markers of CRC. Detection of these proteins in putative
CRC tissue or patients
allows for a determination or diagnosis of CRC. Numerous methods known to
those of ordinary skill in
the art find use in detecting CRC. In one embodiment, antibodies are used to
detect CRC proteins. A
preferred method separates proteins from a sample or patient by
electrophoresis on a gel (typically a
denaturing and reducing protein gel, but may be any other type of gel
including isoelectric focusing
gels and the like). Following separation of proteins, the CRC protein is
detected by immunoblotting
with antibodies raised against the CRC protein. Methods of immunoblotting are
well known to those of
ordinary skill in the art.
In another preferred method, antibodies to the CRC protein find use in in situ
imaging techniques. In
this method cells are contacted with from one to many antibodies to the CRC
protein(s). Following
washing to remove non-specific antibody binding, the presence of the antibody
or antibodies is
detected. In one embodiment the antibody is detected by incubating with a
secondary antibody that
contains a detectable label. In another method the primary antibody to the CRC
proteins) contains a
detectable label. In another preferred embodiment each one of multiple primary
antibodies contains a
distinct and detectable label. This method finds particular use in
simultaneous screening for a plurality
of CRC proteins. As will be appreciated by one of ordinary skill in the art,
numerous other histological
imaging techniques are useful in the invention.
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In a preferred embodiment the label is detected in a fluorometer which has the
ability to detect and
distinguish emissions of different wavelengths. In addition, a fluorescence
activated cell sorter (FACS)
can be used in the method.
In another preferred embodiment, antibodies find use in diagnosing CRC from
blood samples. As
previously described, certain CRC proteins are secreted/circulating molecules.
Blood samples,
therefore, are useful as samples to be probed or tested for the presence of
secreted CRC proteins.
Antibodies can be used to detect the CRC by any of the previously described
immunoassay
techniques including ELISA; immunoblotting (Western blotting),
immunoprecipitation, BIACORE
technology and the like, as will be appreciated by one of ordinary skill in
the art.
In a preferred embodiment, in situ hybridization of labeled CRC nucleic acid
probes to tissue arrays is
done. For example, arrays of tissue samples, including CRC tissue and/or
normal tissue, are made.
In situ hybridization as is known in the art can then be done.
It is understood that when comparing the fingerprints between an individual
and a standard, the skilled
artisan can make a diagnosis as well as a prognosis. It is further understood
that the genes which
indicate the diagnosis may differ from those which indicate the prognosis.
In a preferred embodiment, the CRC proteins, antibodies, nucleic acids,
modified proteins and cells
containing CRC sequences are used in prognosis assays. As above, gene
expression profiles can be
generated that correlate to CRC severity, in terms of long term prognosis.
Again, this may be done on
either a protein or gene level, with the use of genes being preferred. As
above, the CRC probes are
attached to biochips for the detection and quantification of CRC sequences in
a tissue or patient. The
assays proceed as outlined for diagnosis.
In a preferred embodiment, any of the three classes of proteins as described
herein are used in drug
screening assays. The CRC proteins, antibodies, nucleic acids, modified
proteins and cells containing
CRC sequences are used in drug screening assays or by evaluating the effect of
drug candidates on a
"gene expression profile" or expression profile of polypeptides. In a
preferred embodiment, the
expression profiles are used, preferably in conjunction with high throughput
screening techniques to
allow monitoring for expression profile genes after treatment with a candidate
agent, Zlokarnik, et al.,
Science 279, 84-8 (1998), Heid, 1996 #69.
In a preferred embodiment, the CRC proteins, antibodies, nucleic acids,
modified proteins and cells
containing the native or modified CRC proteins are used in screening assays.
That is, the present
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invention provides novel methods for screening for compositions which modulate
the CRC phenotype.
As above, this can be done on an individual gene level or by evaluating the
effect of drug candidates
on a "gene expression profile". In a preferred embodiment, the expression
profiles are used,
preferably in conjunction with high throughput screening techniques to allow
monitoring for expression
profile genes after treatment with a candidate agent, see Zlokarnik, supra.
Having identified the differentially expressed genes herein, a variety of
assays may be executed. In a
preferred embodiment, assays may be run on an individual gene or protein
level. That is, having
identified a particular gene as up regulated in CRC, candidate bioactive
agents may be screened to
modulate this gene's response; preferably to down regulate the gene, although
in some circumstances
to up regulate the gene. "Modulation" thus includes both an increase and a
decrease in gene
expression. The preferred amount of modulation will depend on the original
change of the gene
expression in normal versus tumor tissue, with changes of at least 10%,
preferably 50%, more
preferably 100-300%, and in some embodiments 300-1000% or greater. Thus, if a
gene exhibits a 4
fold increase in tumor compared to normal tissue, a decrease of about four
fold is desired; a 10 fold
decrease in tumor compared to normal tissue gives a 10 fold increase in
expression for a candidate
agent is desired.
As will be appreciated by those in the art, this may be done by evaluation at
either the gene or the
protein level; that is, the amount of gene expression may be monitored using
nucleic acid probes and
the quantification of gene expression levels, or, alternatively, the gene
product itself can be monitored,
for example through the use of antibodies to the CRC protein and standard
immunoassays.
In a preferred embodiment, gene expression monitoring is done and a number of
genes, i.e. an
expression profile, is monitored simultaneously, although multiple protein
expression monitoring can
be done as well.
In this embodiment, the CRC nucleic acid probes are attached to biochips as
outlined herein for the
detection and quantification of CRC sequences in a particular cell. The assays
are further described
below.
Generally, in a preferred embodiment, a candidate bioactive agent is added to
the cells prior to
analysis. Moreover, screens are provided to identify a candidate bioactive
agent which modulates
colorectal cancer, modulates CRC proteins, binds to a CRC protein, or
interferes between the binding
of a CRC protein and an antibody.
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The term "candidate bioactive agent" or "drug candidate" or grammatical
equivaients as used herein
describes any molecule, e.g., protein, oligopeptide, small organic molecule,
polysaccharide,
polynucleotide, etc., to be tested for bioactive agents that are capable of
directly or indirectly altering
either the CRC phenotype or the expression of a CRC sequence, including both
nucleic acid
sequences and protein sequences. In preferred embodiments, the bioactive
agents modulate the
expression profiles, or expression profile nucleic acids or proteins provided
herein. In a particularly
preferred embodiment, the candidate agent suppresses a CRC phenotype, for
example to a normal
colon tissue fingerprint. Similarly, the candidate agent preferably suppresses
a severe CRC
phenotype. Generally a plurality of assay mixtures are run in parallel with
different agent
concentrations to obtain a differential response to the various
concentrations. Typically, one of these
concentrations serves as a negative control, i.e., at zero concentration or
below the level of detection.
In one aspect, a candidate agent will neutralize the effect of a CRC protein.
By "neutralize" is meant
that activity of a protein is either inhibited or counter acted against so as
to have substantially no effect
on a cell.
Candidate agents encompass numerous chemical classes, though typically they
are organic
molecules, preferably small organic compounds having a molecular weight of
more than 100 and less
than about 2,500 daltons. Preferred small molecules are less than 2000, or
less than 1500 or less
than 1000 or less than 500 D. Candidate agents comprise functional groups
necessary for structural
interaction with proteins, particularly hydrogen bonding, and typically
include at least an amine,
carbonyl, hydroxyl or carboxyl group, preferably at least two of the
functional chemical groups. The
candidate agents often comprise cyclical carbon or heterocyclic structures
and/or aromatic or
polyaromatic structures substituted with one or more of the above functional
groups. Candidate
agents are also found among biomolecules including peptides, saccharides,
fatty acids, steroids,
purines, pyrimidines, derivatives, structural analogs or combinations thereof.
Particularly preferred are
peptides.
Candidate agents are obtained from a wide variety of sources including
libraries of synthetic or natural
compounds. For example, numerous means are available for random and directed
synthesis of a
wide variety of organic compounds and biomolecules, including expression of
randomized
oligonucleotides. Alternatively, libraries of natural compounds in the form of
bacterial, fungal, plant
and animal extracts are available or readily produced. Additionally, natural
or synthetically produced
libraries and compounds are readily modified through conventional chemical,
physical and
biochemical means. Known pharmacological agents may be subjected to directed
or random
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chemical modifications, such as acylation, alkylation, esterification,
amidification to produce structural
analogs.
In a preferred embodiment, the candidate bioactive agents are proteins. By
"protein" herein is meant
at least two covalently attached amino acids, which includes proteins,
polypeptides, oligopeptides and
peptides. The protein may be made up of naturally occurring amino acids and
peptide bonds, or
synthetic peptidomimetic structures. Thus "amino acid", or "peptide residue",
as used herein means
both naturally occurring and synthetic amino acids. For example, homo-
phenylalanine, citrulline and
noreleucine are considered amino acids for the purposes of the invention.
"Amino acid" also includes
imino acid residues such as proline and hydroxyproline. The side chains may be
in either the (R) or
the (S) configuration. In the preferred embodiment, the amino acids are in the
(S) or L-configuration.
If non-naturally occurring side chains are used, non-amino acid substituents
may be used, for example
to prevent or retard in vivo degradations.
fn a preferred embodiment, the candidate bioactive agents are naturally
occurring proteins or
fragments of naturally occurring proteins. Thus, for example, cellular
extracts containing proteins, or
random or directed digests of proteinaceous cellular extracts, may be used. In
this way libraries of
procaryotic and eucaryotic proteins may be made for screening in the methods
of the invention.
Particularly preferred in this embodiment are libraries of bacterial, fungal,
viral, and mammalian
proteins, with the latter being preferred, and human proteins being especially
preferred.
In a preferred embodiment, the candidate bioactive agents are peptides of from
about 5 to about 30
amino acids, with from about 5 to about 20 amino acids being preferred, and
from about 7 to about 15
being particularly preferred. The peptides may be digests of naturally
occurring proteins as is outlined
above, random peptides, or "biased" random peptides. By "randomized" or
grammatical equivalents
herein is meant that each nucleic acid and peptide consists of essentially
random nucleotides and
amino acids, respectively. Since generally these random peptides (or nucleic
acids, discussed below)
are chemically synthesized, they may incorporate any nucleotide or amino acid
at any position. The
synthetic process can be designed to generate randomized proteins or nucleic
acids, to allow the
formation of all or most of the possible combinations over the length of the
sequence, thus forming a
library of randomized candidate bioactive proteinaceous agents.
In one embodiment, the library is fully randomized, with no sequence
preferences or constants at any
position. In a preferred embodiment, the library is biased. That is, some
positions within the
sequence are either held constant, or are selected from a limited number of
possibilities. For
example, in a preferred embodiment, the nucleotides or amino acid residues are
randomized within a
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defined class, for example, of hydrophobic amino acids, hydrophilic residues,
sterically biased (either
small or large) residues, towards the creation of nucleic acid binding
domains, the creation of
cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines,
tyrosines or histidines for
phosphorylation sites, etc., or to purines, etc.
In a preferred embodiment, the candidate bioactive agents are nucleic acids,
as defined above.
As described above generally for proteins, nucleic acid candidate bioactive
agents may be naturally
occurring nucleic acids, random nucleic acids, or "biased" random nucleic
acids. For example, digests
of procaryotic or eucaryotic genomes may be used as is outlined above for
proteins.
In a preferred embodiment, the candidate bioactive agents are organic chemical
moieties, a wide
variety of which are available in the literature.
After the candidate agent has been added and the cells allowed to incubate for
some period of time,
the sample containing the target sequences to be analyzed is added to the
biochip. If required, the
target sequence is prepared using known techniques. For example, the sample
may be treated to
lyse the cells, using known lysis buffers, electroporation, etc., with
purification andlor amplification
such as PCR occurring as needed, as will be appreciated by those in the art.
For example, an in vitro
transcription with labels covalently attached to the nucleosides is done.
Generally, the nucleic acids
are labeled with biotin-FITC or PE, or with cy3 or cy5.
In a preferred embodiment, the target sequence is labeled with, for example, a
fluorescent, a
chemiluminescent, a chemical, or a radioactive signal, to provide a means of
detecting the target
sequence's specific binding to a probe. The label also can be an enzyme, such
as, alkaline
phosphatase or horseradish peroxidase, which when provided with an appropriate
substrate produces
a product that can be detected. Alternatively, the label can be a labeled
compound or small molecule,
such as an enzyme inhibitor, that binds but is not catalyzed or altered by the
enzyme. The label also
can be a moiety or compound, such as, an epitope tag or biotin which
specifically binds to streptavidin.
For the example of biotin, the streptavidin is labeled as described above,
thereby, providing a
detectable signal for the bound target sequence. As known in the art, unbound
labeled streptavidin is
removed prior to analysis.
As will be appreciated by those in the art, these assays can be direct
hybridization assays or can
comprise "sandwich assays", which include the use of multiple probes, as is
generally outlined in U.S.
Patent Nos. 5,681,702, 5,597,909, 5,545,730, 5,594,117, 5,591,584, 5,571,670,
5,580,731, 5,571,670,
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5,591,584, 5,624,802, 5,635,352, 5,594,118, 5,359,100, 5,124,246 and
5,681,697, all of which are
hereby incorporated by reference. In this embodiment, in general, the target
nucleic acid is prepared
as outlined above, and then added to the biochip comprising a plurality of
nucleic acid probes, under
conditions that allow the formation of a hybridization complex.
A variety of hybridization conditions may be used in the present invention,
including high, moderate
and low stringency conditions as outlined above. The assays are generally run
under stringency
conditions which allows formation of the label probe hybridization complex
only in the presence of
target. Stringency can be controlled by altering a step parameter that is a
thermodynamic variable,
including, but not limited to, temperature, formamide concentration, salt
concentration, chaotropic salt
concentration pH, organic solvent concentration, etc.
These parameters may also be used to control non-specific binding, as is
generally outlined in U.S.
Patent No. 5,681,697. Thus it may be desirable to perform certain steps at
higher stringency
conditions to reduce non-specific binding.
The reactions outlined herein may be accomplished in a variety of ways, as
will be appreciated by
those in the art. Components of the reaction may be added simultaneously, or
sequentially, in any
order, with preferred embodiments outlined below. In addition, the reaction
may include a variety of
other reagents may be included in the assays. These include reagents like
salts, buffers, neutral
proteins, e.g. albumin, detergents, etc which may be used to facilitate
optimal hybridization and
detection, and/or reduce non-specific or background interactions. Also
reagents that otherwise
improve the efficiency of the assay, such as protease inhibitors, nuclease
inhibitors, anti-microbial
agents, etc., may be used, depending on the sample preparation methods and
purity of the target.
Once the assay is run, the data is analyzed to determine the expression
levels, and changes in
expression levels as between states, of individual genes, forming a gene
expression profile.
The screens are done to identify drugs or bioactive agents that modulate the
CRC phenotype.
Specifically, there are several types of screens that can be run. A preferred
embodiment is in the
screening of candidate agents that can induce or suppress a particular
expression profile, thus
preferably generating the associated phenotype. That is, candidate agents that
can mimic or produce
an expression profile in CRC similar to the expression profile of normal colon
tissue is expected to
result in a suppression of the CRC phenotype. Thus, in this embodiment,
mimicking an expression
profile, or changing one profile to another, is the goal.
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In a preferred embodiment, as for the diagnosis and prognosis applications,
having identified the
differentially expressed genes important in any one state, screens can be run
to alter the expression of
the genes individually. That is, screening for modulation of regulation of
expression of a single gene
can be done; that is, rather than try to mimic all or part of an expression
profile, screening for
regulation of individual genes can be done. Thus, for example, particularly in
the case of target genes
whose presence or absence is unique between two states, screening is done for
modulators of the
target gene expression.
In a preferred embodiment, screening is done to alter the biological function
of the expression product
of the differentially expressed gene. Again, having identified the importance
of a gene in a particular
state, screening for agents that bind andlor modulate the biological activity
of the gene product can be
run as is more fully outlined below.
Thus, screening of candidate agents that modulate the CRC phenotype either at
the gene expression
level or the protein level can be done.
In addition screens can be done for novel genes that are induced in response
to a candidate agent.
After identifying a candidate agent based upon its ability to suppress a CRC
expression pattern
leading to a normal expression pattern, or modulate a single CRC gene
expression profile so as to
mimic the expression of the gene from normal tissue, a screen as described
above can be performed
to identify genes that are specifically modulated in response to the agent.
Comparing expression
profiles between normal tissue and agent treated CRC tissue reveals genes that
are not expressed in
normal tissue or CRC tissue, but are expressed in agent treated tissue. These
agent specific
sequences can be identified and used by any of the methods described herein
for CRC genes or
proteins. In particular these sequences and the proteins they encode find use
in marking or
identifying agent treated cells. In addition, antibodies can be raised against
the agent induced proteins
and used to target novel therapeutics to the treated CRC tissue sample.
Thus, in one embodiment, a candidate agent is administered to a population of
CRC cells, that thus
has an associated CRC expression profile. By "administration" or "contacting"
herein is meant that the
candidate agent is added to the cells in such a manner as to allow the agent
to act upon the cell,
whether by uptake and intracellular action, or by action at the cell surface.
In some embodiments,
nucleic acid encoding a proteinaceous candidate agent (i.e. a peptide) may be
put into a viral
construct such as a retroviral construct and added to the cell, such that
expression of the peptide
agent is accomplished; see PCT US97/01019, hereby expressly incorporated by
reference.
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Once the candidate agent has been administered to the cells, the cells can be
washed if desired and
are allowed to incubate under preferably physiological conditions for some
period of time. The cells
are then harvested and a new gene expression profile is generated, as outlined
herein.
Thus, for example, CRC tissue may be screened for agents that reduce or
suppress the CRC
phenotype. A change in at least one gene of the expression profile indicates
that the agent has an
effect on CRC activity. By defining such a signature for the CRC phenotype,
screens for new drugs
that alter the phenotype can be devised. With this approach, the drug target
need not be known and
need not be represented in the original expression screening platform, nor
does the level of transcript
for the target protein need to change.
In a preferred embodiment, as outlined above, screens may be done on
individual genes and gene
products (proteins). That is, having identified a particular differentially
expressed gene as important in
a particular state, screening of modulators of either the expression of the
gene or the gene product
itself can be done. The gene products of differentially expressed genes are
sometimes referred to
herein as "CRC proteins" or a "CCMP". In preferred embodiments, the CCMP is as
depicted in
Figures 17-20, 24, 25, 29, 33 and 36, more preferably the protein having the
sequence shown in
Figures 29 or 36 or encoded by the sequences of Figures 27, 36 and 39-48. The
CCMP may be a
fragment, or alternatively, be the full length protein to a fragment shown
herein. Preferably, the CCMP
is a fragment of approximately 14 to 24 amino acids long. More preferably the
fragment is a soluble
fragment.
In a preferred embodiment, the fragment is from CAA9. Preferably, the fragment
includes a non-
transmenbrane region. In a preferred embodiment, the CAA9 fragment has an N-
terminal Cys to aid
in solubility. Preferably, the fragment is selected from CAA9p1, Caa9p2,
CAA9p3, CAA9p4,
CAA9p4MAPS, CAA9p5 and CAA9p5MAPS.
In a preferred embodiment, the fragment is charged and from the c-terminus of
CAA2. In one
embodiment, the c-terminus of the fragment is kept as a free acid and the n-
terminus is a free amine
to aid in coupling, i.e., to cysteine. In another embodiment, the fragment is
an internal peptide
overlapping hydrophilic stretch of CAA2. In a preferred embodiment, the
termini is blocked.
Preferably, the fragment of CAA2 is selected from CAA2p1 or CAA2p2. In another
preferred
embodiment, the fragment is a novel fragment from the N-terminal. In one
embodiment, the fragment
excludes sequence outside of the N-terminal, in another embodiment, the
fragment includes at least a
portion of the N-terminal. "N-terminal" is used interchangeably herein with "N-
terminus" which is
further described above.
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In one embodiment the CRC proteins are conjugated to an immunogenic agent as
discussed herein.
In one embodiment the CRC protein is conjugated to BSA.
Thus, in a preferred embodiment, screening for modulators of expression of
specific genes can be
done. This will be done as outlined above, but in general the expression of
only one or a few genes
are evaluated.
In a preferred embodiment, screens are designed to first find candidate agents
that can bind to
differentially expressed proteins, and then these agents may be used in assays
that evaluate the
ability of the candidate agent to modulate differentially expressed activity.
Thus, as will be appreciated
by those in the art, there are a number of different assays which may be run;
binding assays and
activity assays.
In a preferred embodiment, binding assays are done. In general, purified or
isolated gene product is
used; that is, the gene products of one or more differentially expressed
nucleic acids are made. In
general, this is done as is known in the art. For example, antibodies are
generated to the protein gene
products, and standard immunoassays are run to determine the amount of protein
present.
Alternatively, cells comprising the CRC proteins can be used in the assays.
Thus, in a preferred embodiment, the methods comprise combining a CRC protein
and a candidate
bioactive agent, and determining the binding of the candidate agent to the CRC
protein. Preferred
embodiments utilize the human CRC protein, although other mammalian proteins
may also be used,
for example for the development of animal models of human disease. In some
embodiments, as
outlined herein, variant or derivative CRC proteins may be used.
Generally, in a preferred embodiment of the methods herein, the CRC protein or
the candidate agent
is non-diffusably bound to an insoluble support having isolated sample
receiving areas (e.g. a
microtiter plate, an array, etc.). The insoluble supports may be made of any
composition to which the
compositions can be bound, is readily separated from soluble material, and is
otherwise compatible
with the overall method of screening. The surface of such supports may be
solid or porous and of any
convenient shape. Examples of suitable insoluble supports include microtiter
plates, arrays,
membranes and beads. These are typically made of glass, plastic (e.g.,
polystyrene),
polysaccharides, nylon or nitrocellulose, teflonT"", etc. Microtiter plates
and arrays are especially
convenient because a large number of assays can be carried out simultaneously,
using small amounts
of reagents and samples. The particular manner of binding of the composition
is not crucial so long
as it is compatible with the reagents and overall methods of the invention,
maintains the activity of the
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composition and is nondiffusable. Preferred methods of binding include the use
of antibodies (which
do not sterically block either the ligand binding site or activation sequence
when the protein is bound to
the support), direct binding to "sticky" or ionic supports, chemical
crosslinking, the synthesis of the
protein or agent on the surface, etc. Following binding of the protein or
agent, excess unbound
material is removed by washing. The sample receiving areas may then be blocked
through incubation
with bovine serum albumin (BSA), casein or other innocuous protein or other
moiety.
In a preferred embodiment, the CRC protein is bound to the support, and a
candidate bioactive agent
is added to the assay. Alternatively, the candidate agent is bound to the
support and the CRC protein
is added. Novel binding agents include specific antibodies, non-natural
binding agents identified in
screens of chemical libraries, peptide analogs, etc. Of particular interest
are screening assays for
agents that have a low toxicity for human cells. A wide variety of assays may
be used for this purpose,
including labeled in vitro protein-protein binding assays, electrophoretic
mobility shift assays,
immunoassays for protein binding, functional assays (phosphorylation assays,
etc.) and the tike.
The determination of the binding of the candidate bioactive agent to the CRC
protein may be done in a
number of ways. In a preferred embodiment, the candidate bioactive agent is
labeled, and binding
determined directly. For example, this may be done by attaching all or a
portion of the CRC protein to
a solid support, adding a labeled candidate agent (for example a fluorescent
label), washing off
excess reagent, and determining whether the label is present on the solid
support. Various blocking
and washing steps may be utilized as is known in the art.
By "labeled" herein is meant that the compound is either directly or
indirectly labeled with a label which
provides a detectable signal, e.g. radioisotope, fluorescers, enzyme,
antibodies, particles such as
magnetic particles, chemiluminescers, or specific binding molecules, etc.
Specific binding molecules
include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc.
For the specific binding
members, the complementary member would normally be labeled with a molecule
which provides for
detection, in accordance with known procedures, as outlined above. The label
can directly or indirectly
provide a detectable signal.
In some embodiments, only one of the components is labeled. For example, the
proteins (or
proteinaceous candidate agents) may be labeled at tyrosine positions using
'251, or with fluorophores.
Alternatively, more than one component may be labeled with different labels;
using '251 for the proteins,
for example, and a fluorophor for the candidate agents.
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In a preferred embodiment, the binding of the candidate bioactive agent is
determined through the use
of competitive binding assays. In this embodiment, the competitor is a binding
moiety known to bind
to the target molecule (i.e. CRC), such as an antibody, peptide, binding
partner, ligand, etc. Under
certain circumstances, there may be competitive binding as between the
bioactive agent and the
binding moiety, with the binding moiety displacing the bioactive agent.
In one embodiment, the candidate bioactive agent is labeled. Either the
candidate bioactive agent, or
the competitor, or both, is added first to the protein for a time sufficient
to allow binding, if present.
Incubations may be performed at any temperature which facilitates optimal
activity, typically between 4
and 40°C. Incubation periods are selected for optimum activity, but may
also be optimized to facilitate
rapid high through put screening. Typically between 0.1 and 1 hour will be
sufficient. Excess reagent
is generally removed or washed away. The second component is then added, and
the presence or
absence of the labeled component is followed, to indicate binding.
In a preferred embodiment, the competitor is added first, followed by the
candidate bioactive agent.
Displacement of the competitor is an indication that the candidate bioactive
agent is binding to the
CRC protein and thus is capable of binding to, and potentially modulating, the
activity of the CRC
protein. In this embodiment, either component can be labeled. Thus, for
example, if the competitor is
labeled, the presence of label in the wash solution indicates displacement by
the agent. Alternatively,
if the candidate bioactive agent is labeled, the presence of the label on the
support indicates
displacement.
fn an alternative embodiment, the candidate bioactive agent is added first,
with incubation and
washing, followed by the competitor. The absence of binding by the competitor
may indicate that the
bioactive agent is bound to the CRC protein with a higher affinity. Thus, if
the candidate bioactive
agent is labeled, the presence of the label on the support, coupled with a
lack of competitor binding,
may indicate that the candidate agent is capable of binding to the CRC
protein.
In a preferred embodiment, the methods comprise differential screening to
identity bioactive agents
that are capable of modulating the activity of the CRC proteins. In this
embodiment, the methods
comprise combining a CRC protein and a competitor in a first sample. A second
sample comprises a
candidate bioactive agent, a CRC protein and a competitor. The binding of the
competitor is
determined for both samples, and a change, or difference in binding between
the two samples
indicates the presence of an agent capable of binding to the CRC protein and
potentially modulating
its activity. That is, if the binding of the competitor is different in the
second sample relative to the first
sample, the agent is capable of binding to the CRC protein.
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Alternatively, a preferred embodiment utilizes differential screening to
identify drug candidates that
bind to the native CRC protein, but cannot bind to modified CRC proteins. The
structure of the CRC
protein may be modeled, and used in rational drug design to synthesize agents
that interact with that
site. Drug candidates that affect CRC bioactivity are also identified by
screening drugs for the ability to
either enhance or reduce the activity of the protein.
Positive controls and negative controls may be used in the assays. Preferably
all control and test
samples are performed in at least triplicate to obtain statistically
significant results. Incubation of all
samples is for a time sufficient for the binding of the agent to the protein.
Following incubation, all
samples are washed free of non-specifically bound material and the amount of
bound, generally
labeled agent determined. For example, where a radiolabel is employed, the
samples may be
counted in a scintillation counter to determine the amount of bound compound.
A variety of other reagents may be included in the screening assays. These
include reagents like
salts, neutral proteins, e.g. albumin, detergents, etc which may be used to
facilitate optimal
protein-protein binding and/or reduce non-specific or background interactions.
Also reagents that
otherwise improve the efficiency of the assay, such as protease inhibitors,
nuclease inhibitors,
anti-microbial agents, etc., may be used. The mixture of components may be
added in any order that
provides for the requisite binding.
Screening for agents that modulate the activity of CRC proteins may also be
done. In a preferred
embodiment, methods for screening for a bioactive agent capable of modulating
the activity of CRC
proteins comprise the steps of adding a candidate bioactive agent to a sample
of CRC proteins, as
above, and determining an alteration in the biological activity of CRC
proteins. "Modulating the activity
of CRC" includes an increase in activity, a decrease in activity, or a change
in the type or kind of
activity present. Thus, in this embodiment, the candidate agent should both
bind to CRC proteins
(although this may not be necessary), and alter its biological or biochemical
activity as defined herein.
The methods include both in vitro screening methods, as are generally outlined
above, and in vivo
screening of cells for alterations in the presence, distribution, activity or
amount of CRC proteins.
Thus, in this embodiment, the methods comprise combining a CRC sample and a
candidate bioactive
agent, and evaluating the effect on CRC activity. By "CRC activity" or
grammatical equivalents herein
is meant one of the CRC's biological activities, including, but not limited
to, cell division, preferably in
colon tissue, cell proliferation, tumor growth, transformation of cells. In
one embodiment, CRC activity
includes activation of CZA8, BC7C2, CBC2, CBC1, CBC3, CJA9, BCNS, CQA1, BCN7,
CQA2, CJAB,
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CAA2, CAA9, CGA7 and/or CGA8*, preferably one of the CRC proteins listed in
Figure 14. An
inhibitor of CRC activity is the inhibition of any one or more CRC activities.
in a preferred embodiment, the activity of the CRC protein is increased; in
another preferred
embodiment, the activity of the CRC protein is decreased. Thus, bioactive
agents that are antagonists
are preferred in some embodiments, and bioactive agents that are agonists may
be preferred in other
embodiments.
In a preferred embodiment, the invention provides methods for screening for
bioactive agents capable
of modulating the activity of a CRC protein. The methods comprise adding a
candidate bioactive
agent, as defined above, to a cell comprising CRC proteins. Preferred cell
types include almost any
cell. The cells contain a recombinant nucleic acid that encodes a CRC protein.
In a preferred
embodiment, a library of candidate agents are tested on a plurality of cells.
In one aspect, the assays are evaluated in the presence or absence or previous
or subsequent
exposure of physiological signals, for example hormones, antibodies, peptides,
antigens, cytokines,
growth factors, action potentials, pharmacological agents including
chemotherapeutics, radiation,
carcinogenics, or other cells (i.e. cell-cell contacts). In another example,
the determinations are
determined at different stages of the cell cycle process.
In this way, bioactive agents are identified. Compounds with pharmacological
activity are able to
enhance or interfere with the activity of the CRC protein. In one embodiment,
"colorectal cancer
protein activity" as used herein includes at least one of the following:
colorectal cancer activity, binding
to CJAB, activation of CJA8 or activation of substrates of CJA8 by CJAB. In
one embodiment,
colorectal cancer activity is defined as the unregulated proliferation of
colon tissue, or the growth of
cancer in colon tissue. In one aspect, colorectal cancer activity as defined
herein is related to the
activity of CJA8 in the upregulation of CJA8 in colon cancer tissue.
In another embodiment, colorectal cancer protein activity includes at least
one of the following:
colorectal cancer activity, binding to one of CAA2, CAA9, CGA7 and CGAB,
activation of one of
CAA2, CAA9, CGA7, and CGA8 or activation of substrates of CAA2, CAA9, CGA7 or
CGA8 by CAA2,
CAA9, CGA7 or CGA8, respectively. fn one preferred embodiment, CAA2 comprises
its N-terminal
end. In one aspect, colorectal cancer activity as defined herein is related to
the activity of CAA2,
CAA9, CGA7 andlor CGA8 in the upregulation of CAA2, CAA9, CGA7 and/or CGAB,
respectively, in
colon cancer tissue.
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In one embodiment, a method of inhibiting colon cancer cell division is
provided. The method
comprises administration of a colorectal cancer inhibitor.
In another embodiment, a method of inhibiting tumor growth is provided. The
method comprises
administration of a colorectal cancer inhibitor.
In a further embodiment, methods of treating cells or individuals with cancer
are provided. The
method comprises administration of a colorectal cancer inhibitor.
In one embodiment, a colorectal cancer inhibitor is an antibody as discussed
above. In another
embodiment, the colorectal cancer inhibitor is an antisense molecule.
Antisense molecules as used
herein include antisense or sense oligonucleotides comprising a singe-stranded
nucleic acid sequence
(either RNA or DNA) capable of binding to target mRNA (sense) or DNA
(antisense) sequences for
colorectal cancer molecules. A preferred antisense molecule is for CZA8, BCX2,
CBC2, CBC1,
CBC3, CJAB, CJA9, BCNS, CQA1, BCN7, CQA2, CAA2, CAA9, CGA7 or CGAB, more
preferably for
the CRC sequences referenced in Figure 14, or for a ligand or activator
thereof. A most preferred
antisense molecule is for CJA8 or for a ligand or activator thereof. Antisense
or sense
oligonucleotides, according to the present invention, comprise a fragment
generally at least about 14
nucleotides, preferably from about 14 to 30 nucleotides. The ability to derive
an antisense or a sense
oligonucleotide, based upon a cDNA sequence encoding a given protein is
described in, for example,
Stein and Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al.
(BioTechniaues 6:958, 1988).
Antisense molecules may be introduced into a cell containing the target
nucleotide sequence by
formation of a conjugate with a ligand binding molecule, as described in WO
91/04753. Suitable
ligand binding molecules include, but are not limited to, cell surface
receptors, growth factors, other
cytokines, or other ligands that bind to cell surface receptors. Preferably,
conjugation of the ligand
binding molecule does not substantially interfere with the ability of the
ligand binding molecule to bind
to its corresponding molecule or receptor, or block entry of the sense or
antisense oligonucleotide or
its conjugated version into the cell. Alternatively, a sense or an antisense
oligonucleotide may be
introduced into a cell containing the target nucleic acid sequence by
formation of an oligonucleotide-
lipid complex, as described in WO 90/10448. It is understood that the use of
antisense molecules or
knock out and knock in models may also be used in screening assays as
discussed above, in addition
to methods of treatment.
The compounds having the desired pharmacological activity may be administered
in a physiologically
acceptable carrier to a host, as previously described. The agents may be
administered in a variety of
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ways, orally, parenterally e.g., subcutaneously, intraperitoneally,
intravascularly, etc. Depending upon
the manner of introduction, the compounds may be formulated in a variety of
ways. The concentration
of therapeutically active compound in the formulation may vary from about 0.1-
100 wt.%. The agents
may be administered alone or in combination with other treatments, i.e.,
radiation.
The pharmaceutical compositions can be prepared in various forms, such as
granules, tablets, pills,
suppositories, capsules, suspensions, salves, lotions and the like.
Pharmaceutical grade organic or
inorganic carriers and/or diluents suitable for oral and topical use can be
used to make up
compositions containingthe therapeutically-active compounds. Diluents known to
the art include
aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting
and emulsifying
agents, salts for varying the osmotic pressure or buffers for securing an
adequate pH value, and skin
penetration enhancers can be used as auxiliary agents.
Without being bound by theory, it appears that the various CRC sequences are
important in CRC.
Accordingly, disorders based on mutant or variant CRC genes may be determined.
In one
embodiment, the invention provides methods for identifying cells containing
variant CRC genes
comprising determining all or part of the sequence of at least one endogeneous
CRC genes in a cell.
As will be appreciated by those in the art, this may be done using any number
of sequencing
techniques. In a preferred embodiment, the invention provides methods of
identifying the CRC
genotype of an individual comprising determining all or part of the sequence
of at least one CRC gene
of the individual. This is generally done in at least one tissue of the
individual, and may include the
evaluation of a number of tissues or different samples of the same tissue. The
method may include
comparing the sequence of the sequenced CRC gene to a known CRC gene, i.e. a
wild-type gene.
The sequence of all or part of the CRC gene can then be compared to the
sequence of a known CRC
gene to determine if any differences exist. This can be done using any number
of known homology
programs, such as Bestfit, etc. In a preferred embodiment, the presence of a a
difference in the
sequence between the CRC gene of the patient and the known CRC gene is
indicative of a disease
state or a propensity for a disease state, as outlined herein.
In a preferred embodiment, the CRC genes are used as probes to determine the
number of copies of
the CRC gene in the genome.
In another preferred embodiment CRC genes are used as probed to determine the
chromosomal
localization of the CRC genes. Information such as chromosomal localization
finds use in providing a
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diagnosis or prognosis in particular when chromosomal abnormalities such as
translocations, and the
like are identified in CRC gene loci.
Thus, in one embodiment, methods of modulating CRC in cells or organisms are
provided. In one
embodiment, the methods comprise administering to a cell an anti-CRC antibody
that reduces or
eliminates the biological activity of an endogeneous CRC protein.
Alternatively, the methods comprise
administering to a cell or organism a recombinant nucleic acid encoding a CRC
protein. As will be
appreciated by those in the art, this may be accomplished in any number of
ways. In a preferred
embodiment, for example when the CRC sequence is down-regulated in CRC, the
activity of the CRC
gene is increased by increasing the amount of CRC in the cell, for example by
overexpressing the
endogeneous CRC or by administering a gene encoding the CRC sequence, using
known gene-
therapy techniques, for example. In a preferred embodiment, the gene therapy
techniques include the
incorporation of the erogenous gene using enhanced homologous recombination
(EHR), for example
as described in PCT/US93/03868, hereby incorporated by reference in its
entirety. Alternatively, for
example when the CRC sequence is up-regulated in CRC, the activity of the
endogeneous CRC gene
is decreased, for example by the administration of a CRC antisense nucleic
acid.
In one embodiment, the CRC proteins of the present invention may be used to
generate polyclonal
and monoclonal antibodies to CRC proteins, which are useful as described
herein. Similarly, the CRC
proteins can be coupled, using standard technology, to affinity chromatography
columns. These
columns may then be used to purify CRC antibodies. In a preferred embodiment,
the antibodies are
generated to epitopes unique to a CRC protein; that is, the antibodies show
little or no cross-reactivity
to other proteins. These antibodies find use in a number of applications. For
example, the CRC
antibodies may be coupled to standard affinity chromatography columns and used
to purify CRC
proteins. The antibodies may also be used as blocking polypeptides, as
outlined above, since they will
specifically bind to the CRC protein.
fn one embodiment, a therapeutically effective dose of a CRC or modulator
thereof is administered to
a patient. By "therapeutically effective dose" herein is meant a dose that
produces the effects for
which it is administered. The exact dose wilt depend on the purpose of the
treatment, and will be
ascertainable by one skilled in the art using known techniques. As is known in
the art, adjustments for
CRC degradation, systemic versus localized delivery, and rate of new protease
synthesis, as well as
the age, body weight, general health, sex, diet, time of administration, drug
interaction and the severity
of the condition may be necessary, and will be ascertainable with routine
experimentation by those
skilled in the art.
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A "patient" for the purposes of the present invention includes both humans and
other animals,
particularly mammals, and organisms. Thus the methods are applicable to both
human therapy and
veterinary applications. In the preferred embodiment the patient is a mammal,
and in the most
preferred embodiment the patient is human.
The administration of the CRC proteins and modulators of the present invention
can be done in a
variety of ways as discussed above, including, but not limited to, orally,
subcutaneously, intravenously,
intranasally, transdermally, intraperitoneally, intramuscularly,
intrapulmonary, vaginally, rectally, or
intraocularly. In some instances, for example, in the treatment of wounds and
inflammation, the CRC
proteins and modulators may be directly applied as a solution or spray.
The pharmaceutical compositions of the present invention comprise a CRC
protein in a form suitable
for administration to a patient. In the preferred embodiment, the
pharmaceutical compositions are in a
water soluble form, such as being present as pharmaceutically acceptable
salts, which is meant to
include both acid and base addition salts. "Pharmaceutically acceptable acid
addition salt" refers to
those salts that retain the biological effectiveness of the free bases and
that are not biologically or
otherwise undesirable, formed with inorganic acids such as hydrochloric acid,
hydrobromic acid,
sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids
such as acetic acid, propionic
acid, glycolic acid, pyruvic acid, oxalic acid, malefic acid, malonic acid,
succinic acid, fumaric acid,
tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid,
ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
"Pharmaceutically acceptable
base addition salts" include those derived from inorganic bases such as
sodium, potassium, lithium,
ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts
and the like.
Particularly preferred are the ammonium, potassium, sodium, calcium, and
magnesium salts. Salts
derived from pharmaceutically acceptable organic non-toxic bases include salts
of primary, secondary;
and tertiary amines, substituted amines including naturally occurring
substituted amines, cyclic amines
and basic ion exchange resins, such as isopropylamine, trimethylamine,
diethylamine, triethylamine,
tripropylamine, and ethanolamine.
The pharmaceutical compositions may also include one or more of the following:
carrier proteins such
as serum albumin; buffers; fillers such as microcrystalline cellulose,
lactose, corn and other starches;
binding agents; sweeteners and other flavoring agents; coloring agents; and
polyethylene glycol.
Additives are well known in the art, and are used in a variety of
formulations.
In a preferred embodiment, CRC proteins and modulators are administered as
therapeutic agents,
and can be formulated as outlined above. Similarly, CRC genes (including both
the full-length
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sequence, partial sequences, or regulatory sequences of the CRC coding
regions) can be
administered in gene therapy applications, as is known in the art. These CRC
genes can include
antisense applications, either as gene therapy (i.e. for incorporation into
the genome) or as antisense
compositions, as will be appreciated by those in the art.
In a preferred embodiment, CRC genes are administered as DNA vaccines, either
single genes or
combinations of CRC genes. Naked DNA vaccines are generally known in the art.
Brower, Nature
Biotechnology, 16:1304-1305 (1998).
In one embodiment, CRC genes of the present invention are used as DNA
vaccines. Methods for the
use of genes as DNA vaccines are well known to one of ordinary skill in the
art, and include placing a
CRC gene or portion of a CRC gene under the control of a promoter for
expression in a CRC patient.
The CRC gene used for DNA vaccines can encode full-length CRC proteins, but
more preferably
encodes portions of the CRC proteins including peptides derived from the CRC
protein. In a preferred
embodiment a patient is immunized with a DNA vaccine comprising a plurality of
nucleotide
sequences derived from a CRC gene. Similarly, it is possible to immunize a
patient with a plurality of
CRC genes or portions thereof as defined herein. Without being bound by
theory, expression of the
polypeptide encoded by the DNA vaccine, cytotoxic T-cells, helper T-cells and
antibodies are induced
which recognize and destroy or eliminate cells expressing CRC proteins.
In a preferred embodiment, the DNA vaccines include a gene encoding an
adjuvant molecule with the
DNA vaccine. Such adjuvant molecules include cytokines that increase the
immunogenic response to
the CRC polypeptide encoded by the DNA vaccine. Additional or alternative
adjuvants are known to
those of ordinary skill in the art and find use in the invention.
In another preferred embodiment CRC genes find use in generating animal models
of CRC. As is
appreciated by one of ordinary skill in the art, when the CRC gene identified
is repressed or
diminished in CRC tissue, gene therapy technology wherein antisense RNA
directed to the CRC gene
will also diminish or repress expression of the gene. An animal generated as
such serves as an
animal model of CRC that finds use in screening bioactive drug candidates.
Similarly, gene knockout
technology, for example as a result of homologous recombination with an
appropriate gene targeting
vector, will result in the absence of the CRC protein. When desired, tissue-
specific expression or
knockout of the CRC protein may be necessary.
It is also possible that the CRC protein is overexpressed in CRC. As such,
transgenic animals can be
generated that overexpress the CRC protein. Depending on the desired
expression level, promoters
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of various strengths can be employed to express the transgene. Also, the
number of copies of the
integrated transgene can be determined and compared for a determination of the
expression level of
the transgene. Animals generated by such methods find use as animal models of
CRC and are
additionally useful in screening for bioactive molecules to treat CRC.
It is understood that the examples described herein in no way serve to limit
the true scope of this
invention, but rather are presented for illustrative purposes. All references
and sequences of
accession numbers cited herein are incorporated by reference in their
entirety.
EXAMPLES
Example 1
Tissue Preparation, Labeling Chips, and Fincterprints
Purify total RNA from tissue using TRlzol Reagent
Estimate tissue weight. Homogenize tissue samples in 1 ml of TRlzol per 50mg
of tissue using a
Polytron 3100 homogenizer. The generator/probe used depends upon the tissue
size. A
generator that is too large for the amount of tissue to be homogenized will
cause a loss of sample
and lower RNA yield. Use the 20mm generator for tissue weighing more than
0.6g. If the working
volume is greater than 2m1, then homogenize tissue in a 15m1 polypropylene
tube (Falcon 2059).
Fill tube no greater than 10m1.
HOMOGENIZATION
Before using generator, it should have been cleaned after last usage by
running it through soapy
H20 and rinsing thoroughly. Run through with EtOH to sterilize. Keep tissue
frozen until ready.
Add TRlzol directly to frozen tissue then homogenize.
Following homogenization, remove insoluble material from the homogenate by
centrifugation at
7500 x g for 15 min. in a Sorvall superspeed or 12,000 x g for 10 min. in an
Eppendorf centrifuge
at 4°C. Transfer the cleared homogenate to a new tube(s). The samples
may be frozen now at -
60 to -70°C (and kept for at least one month) or you may continue with
the purification.
PHASE SEPARATION
Incubate the homogenized samples for 5 minutes at room temperature.
Add 0.2m1 of chloroform per 1 ml of TRlzol reagent used in the original
homogenization.
Cap tubes securely and shake tubes vigorously by hand (do not vortex) for 15
seconds.
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incubate samples at room temp, for 2-3 minutes. Centrifuge samples of 6500rpm
in a Sorvali
superspeed for 30 min. at 4°C. (You may spin at up to 12,000 x g for 10
min. but you risk
breaking your tubes in the centrifuge.)
RNA PRECIPITATION
Transfer the aqueous phase to a fresh tube. Save the organic phase if
isolation of DNA or protein
is desired. Add 0.5m1 of isopropyl alcohol per 1 ml of TRlzol reagent used in
the original
homogenization. Cap tubes securely and invert to mix. Incubate samples at room
temp. for 10
minutes. Centrifuge samples at 6500rpm in Sonrall for 20min. at 4°C.
RNA WASH
Pour off the supernate. Wash pellet with cold 75% ethanol. Use 1 ml of 75%
ethanol per 1 ml of
TRlzol reagent used in the initial homogenization. Cap tubes securely and
invert several times to
loosen pellet. (Do not vortex). Centrifuge at <8000rpm (<7500 x g) for 5
minutes at 4°C.
Pour off the wash. Carefully transfer pellet to an eppendorf tube (let it
slide down the tube into the
new tube and use a pipet tip to help guide it in if necessary). Depending on
the volumes you are
working with, you can decide what size tubes) you want to precipitate the RNA
in. When I tried
leaving the RNA in the large 15m1 tube, it took so long to dry (i.e. it did
not dry) that I eventually
had to transfer it to a smaller tube. Let pellet dry in hood. Resuspend RNA in
an appropriate
volume of DEPC HZO. Try for 2-5ug/ul. Take absorbance readings.
Purif~~oly A+ mRNA from total RNA or clean up total RNA with Qiagen' s
RNeas,r kit
Purification of poly A+ mRNA from total RNA. Heat oligotex suspension to
37°C and mix
immediately before adding to RNA. Incubate Elution Buffer at 70°C. Warm
up 2 x Binding Buffer
at 65°C if there is precipitate in the buffer. Mix total RNA with DEPC-
treated water, 2 x Binding
Buffer, and Oligotex according to Table 2 on page 16 of the Oligotex Handbook.
Incubate for 3
minutes at 65°C. Incubate for 10 minutes at room temperature.
Centrifuge for 2 minutes at 14,000 to 18,000 g. If centrifuge has a "soft
setting," then use it.
Remove supernatant without disturbing Oligotex pellet. A little bit of
solution can be left behind to
reduce the loss of Oligotex. Save sup until certain that satisfactory binding
and elution of poly A+
mRNA has occurred.
Gently resuspend in Wash Buffer OW2 and pipet onto spin column. Centrifuge the
spin column
at full speed (soft setting if possible) for 1 minute.
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Transfer spin column to a new collection tube and gently resuspend in Wash
Buffer OW2 and
centrifuge as describe herein.
Transfer spin column to a new tube and elute with 20 to 100 ul of preheated
(70°C) Elution Buffer.
Gently resuspend Oligotex resin by pipetting up and down. Centrifuge as above.
Repeat elution
with fresh elution buffer or use first eluate to keep the elution volume low.
Read absorbance, using diluted Elution Buffer as the blank.
Before proceeding with cDNA synthesis, the mRNA must be precipitated.
Some component leftover or in the Elution Buffer from the Oligotex
purification procedure will
inhibit downstream enzymatic reactions of the mRNA.
Ethanol Precipitation
Add 0.4 vol, of 7.5 M NH40Ac + 2.5 vol. of cold 100% ethanol. Precipitate at -
20°C 1 hour to
overnight (or 20-30 min. at -70°C). Centrifuge at 14,000-16,000 x g for
30 minutes at 4°C. Wash
pellet with 0.5m1 of 80%ethanol (-20°C) then centrifuge at 14,000-
16,000 x g for 5 minutes at room
temperature. Repeat 80% ethanol wash. Dry the last bit of ethanol from the
pellet in the hood.
(Do not speed vacuum). Suspend pellet in DEPC H20 at 1 ug/ul concentration.
Clean up total RNA using Oiaaen's RNeasy kit
Add no more than 100ug to an RNeasy column. Adjust sample to a volume of 100u1
with RNase-
free water. Add 350u1 Buffer RLT then 250u1 ethanol (100%) to the sample. Mix
by pipetting (do
not centrifuge) then apply sample to an RNeasy mini spin column. Centrifuge
for 15 sec at
>10,OOOrpm. If concerned about yield, re-apply flowthrough to column and
centrifuge again.
Transfer column to a new 2-ml collection tube. Add 500u1 Buffer RPE and
centrifuge for 15 sec
at >10,OOOrpm. Discard flowthrough. Add 500u1 Buffer RPE and centrifuge for 15
sec at
>10,OOOrpm. Discard flowthrough then centrifuge for 2 min at maximum speed to
dry column
membrane. Transfer column to a new 1.5-ml collection tube and apply 30-50u1 of
RNase-free
water directly onto column membrane. Centrifuge 1 min at >10,OOOrpm. Repeat
elution.
Take absorbance reading. If necessary, ethanol precipitate with ammonium
acetate and 2.5X
volume 100% ethanol.
Make cDNA using Gibco's "Superscript Choice System for cDNA S~,rnthesis" kit
First Strand cDNA Synthesis
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Use 5ug of total RNA or 1 ug of polyA+ mRNA as starting material. For total
RNA, use 2ul of
Superscript RT. For polyA+ mRNA, use 1 ul of Superscript RT. Final volume of
first strand
synthesis mix is 20u1. RNA must be in a volume no greater than 10u1. Incubate
RNA with 1 ul of
100pmol T7-T24 oligo for 10 min at 70C. On ice, add 7 ul of: 4ul 5X 1St Strand
Buffer, 2ul of
0.1 M DTT, and 1 ul of 10mM dNTP mix. Incubate at 37C for 2 min then add
Superscript RT
Incubate at 37C for 1 hour.
Second Strand Synthesis
Place 1St strand reactions on ice.
Add: 91u1 DEPC H20
30u1 5X 2"d Strand Buffer
3ul 10mM dNTP mix
1ul 10U/ul E.coii DNA Ligase
4ul 10U/ul E.coli DNA Polymerase
1 ul 2U/ul RNase H
Make the above into a mix if there are more than 2 samples. Mix and incubate 2
hours at 16C.
Add 2ul T4 DNA Polymerase. Incubate 5 min at 16C. Add 10u1 of 0.5M EDTA
Clean up cDNA
PhenoI:Chloroform:lsoamyl Alcohol (25:24:1 ) purification using Phase-Lock gel
tubes:
Centrifuge PLG tubes for 30 sec at maximum speed. Transfer cDNA mix to PLG
tube. Add equal
volume of phenol:chloroform:isamyl alcohol and shake vigorously (do not
vortex). Centrifuge 5
minutes at maximum speed. Transfer top aqueous solution to a new tube. Ethanol
precipitate:
add 7.5X 5M NH4Oac and 2.5X volume of 100% ethanol. Centrifuge immediately at
room temp.
for 20 min, maximum speed. Remove sup then wash pellet 2X with cold 80%
ethanol. Remove
as much ethanol wash as possible then let pellet air dry. Resuspend pellet in
3ul RNase-free
water.
In vitro Transcription (IVTLand labeling with biotin
Pipet 1.5u1 of cDNA into a thin-wall PCR tube.
Make NTP labeling mix:
Combine at room temperature: 2ul T7 10xATP (75mM) (Ambion)
2ul T7 10xGTP (75mM) (Ambion)
1.5u1 T7 10xCTP (75mM) (Ambion)
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1.5u1 T7 10xUTP (75mM) (Ambion)
3.75u1 10mM Bio-11-UTP (Boehringer-Mannheim/Roche or
Enzo)
3.75u1 10mM Bio-16-CTP (Enzo)
2ul 10x T7 transcription buffer (Ambion)
2ul 10x T7 enzyme mix (Ambion)
Final volume of total reaction is 20u1. Incubate 6 hours at 37C in a PCR
machine.
RNeasy clean-up of IVT product
Follow previous instructions for RNeasy columns or refer to Qiagen's RNeasy
protocol handbook.
cRNA will most likely need to be ethanol precipitated. Resuspend in a volume
compatible with
the fragmentation step.
Fragmentation
15 ug of labeled RNA is usually fragmented. Try to minimize the fragmentation
reaction volume;
a 10 ul volume is recommended but 20 ul is all right. Do not go higher than 20
ul because the
magnesium in the fragmentation buffer contributes to precipitation in the
hybridization buffer.
Fragment RNA by incubation at 94 C for 35 minutes in 1 x Fragmentation buffer.
x Fragmentation buffer:
200 mM Tris-acetate, pH 8.1
500 mM KOAc
150 mM MgOAc
The labeled RNA transcript can be analyzed before and after fragmentation.
Samples can be
heated to 65C for 15 minutes and electrophoresed on 1 % agarose/TBE gels to
get an
approximate idea of the transcript size range
Hybridization
200 ul (10ug cRNA) of a hybridization mix is put on the chip. If multiple
hybridizations are to be
done (such as cycling through a 5 chip set), then it is recommended that an
initial hybridization
mix of 300 ul or more be made.
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Hybrization Mix: fragment labeled RNA (50ng/ul final cone)
50 pM 948-b control oligo
1.5 pM BioB
pM BioC
25 pM BioD
100 pM CRE
0.1 mg/ml herring sperm DNA
0.5mg/ml acetylated BSA
to 300 ul with 1xMES hyb. buffer
The instruction manuals for the products used herein are incorporated herein
in their entirety.
Labelina Protocol Provided Herein
Hybridization reaction:
Start with non-biotinylated IVT (purified by RNeasy columns)
(see example 1 for steps from tissue to IVT)
IVT antisense RNA; 4 fig: ~I
Random Hexamers (1 ~g/~I): 4 ~I
H20: ~I
14 ~I
- Incubate 70°C, 10 min. Put on ice.
Reverse transcription:
5X First Strand 6
(BRL) buffer: III
0.1 M DTT: 3
III
50X dNTP mix: 0.6
ill
H20: 2.4
~I
Cy3 or Cy5 dUTP 3
(1 mM): ~I
SS RT II (BRL): 1
~!I
16 ~I
- Add to hybridization reaction.
- Incubate 30 min., 42°C.
- Add 1 ~I SSII and let go for another hour.
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Put on ice.
- 50X dNTP mix (25mM of cold dATP, dCTP, and dGTP, 10mM of dTTP: 25 ~I each of
100mM
dATP, dCTP, and dGTP; 10 ~I of 100mM dTTP to 15 ~I H20. dNTPs from Pharmacia)
RNA degradation:
86 ~1 HZO
- Add 1.5 ~I 1 M NaOH/ 2mM EDTA, incubate at 65°C, 10 min. 10 ~I 1 ON
NaOH
4 ~I 50mM EDTA
U-Con 30
500 ~I TE/sample spin at 70008 for 10 min, save flow through for purification
Qiagen purification:
-suspend u-con recovered material in 5001 buffer PB
-proceed w/ normal Qiagen protocol
DNAse digest:
- Add 1 ~I of 1/100 dil of DNAse/30u1 Rx and incubate at 37°C for 15
min.
-5 min 95°C to denature enzyme
Sample preparation:
- Add:
Cot-1 DNA: 10 ~I
50X dNTPs: 1 ~I
20X SSC: 2.3 ~I
Na pyro phosphate: 7.5 ~I
10mg/ml Herring sperm DNA 1ul of 1/10 dilution
21.8 final vol.
- Dry down in speed vac.
- Resuspend in 15 ~I H20.
- Add 0.38 ~I 10% SDS.
- Heat 95°C, 2 min.
- Slow cool at room temp. for 20 min.
Put on slide and hybridize overnight at 64°C.
Washing after the hybridization:
3X SSC/0.03% SDS: 2 min. 37.5 mls 20X SSC+0.75m1s 10% SDS in 250m1s H20
1X SSC: 5 min. 12.5 mls 20X SSC in 250m1s H20
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0.2X SSC: 5 min. 2.5 mls 20X SSC in 250m1s Hz0
Dry slides in centrifuge, 1000 RPM, 1 min.
Scan at appropiate PMT's and channels.
The results are shown in Figures 1 through 11. The lists of genes come from
colorectal tumors
from a variety of stages of the disease. The genes that are up regulated in
the tumors (overall)
were also found to be expressed at a limited amount or not at all in the body
map. The body map
for the colorectal project consists of ten tissues: Heart, Brain, Lung, Liver,
Breast, Kidney,
Prostrate! Small Intestine, Spleen, and Colon. The down regulated genes in
tumors (overall)
versus normal colon were not selected for their expression or lack of
expression in the body map.
As indicated, some of the Accession numbers include expression sequence tags
(ESTs). Thus, in
one embodiment herein, genes within an expression profile, also termed
expression profile genes,
include ESTs and are not necessarily full length. Figure 1 shows 51
upregulated genes; Figure 2
shows 194 upregulated genes; Figure 3 shows 1144 upregulated genes and Figure
4 shows 1815
upregulated genes. The genes shown in Figures 1 and 5 are particularly
preferred. Figure 5
shows 54 downregulated genes; Figure 6 shows 558 downregulated genes; and
Figure 7 shows
1923 downregulated genes; and Figures 8, 9, 10 and 11 provide the Accession
numbers for
genes, including expression sequence tags, upregulated in tumor tissue
compared to normal
colon tissue.
Example 2
Expression studies were performed herein.
As indicated in Figure 21, CAA2 is upregulated in colon cancer tissue. CAA2 is
found in
chromosome 15, cytoband 15q15-22, interval D15S146-D15S117. CAA2 has N-
myristoylation
sites and a C-terminal microbody targeting signal. The preferred fragments
shown in Figures 18
and 19 have a solubility of 1 mg/ 1 ml H20.
As indicated in Figure 26, CAA9 is upregulated in colon cancer tissue. CAA9 is
found in
chromosome 5, cytoband 5q23.3, interval D5S471-D5S393.
As indicated in Figures 30A and 30B, CGA7 is upregulated in colon cancer
tissue. CGA7 is found
in chromosome 2.
As indicated in Figure 34, CGA8 is upregulated in colon cancer tissue.
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As indicated in Figure 37, CJA8 is upregulated in colon cancer tissue. CJA8 is
found in
chromosome 11.
As indicated in Figure 38, BCN7 is upregulated in colon cancer tissue. BCN7 is
found in
chromosome 5, cytoband 5q22, interval D5S471-D5S393.
66
