U.S. patent application number 10/254289 was filed with the patent office on 2003-06-26 for novel methods of diagnosing colorectal cancer, compositions, and methods of screening for colorectal cancer modulators.
This patent application is currently assigned to Eos Biotechnology, Inc.. Invention is credited to Gish, Kurt C., Mack, David H., Wilson, Keith E..
Application Number | 20030118509 10/254289 |
Document ID | / |
Family ID | 27051084 |
Filed Date | 2003-06-26 |
United States Patent
Application |
20030118509 |
Kind Code |
A1 |
Mack, David H. ; et
al. |
June 26, 2003 |
Novel methods of diagnosing colorectal cancer, compositions, and
methods of screening for colorectal cancer modulators
Abstract
Described herein are methods that can be used for diagnosis and
prognosis of colorectal cancer. Also described herein are methods
that can be used to screen candidate bioactive agents for the
ability to modulate colorectal cancer. Additionally, methods and
molecular targets (genes and their products) for therapeutic
intervention in colorectal cancer are described.
Inventors: |
Mack, David H.; (Menlo Park,
CA) ; Gish, Kurt C.; (San Francisco, CA) ;
Wilson, Keith E.; (Redwood City, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Eos Biotechnology, Inc.
South San Francisco
CA
|
Family ID: |
27051084 |
Appl. No.: |
10/254289 |
Filed: |
September 24, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10254289 |
Sep 24, 2002 |
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09656002 |
Sep 6, 2000 |
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6455668 |
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09656002 |
Sep 6, 2000 |
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09525993 |
Mar 15, 2000 |
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09525993 |
Mar 15, 2000 |
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09493444 |
Jan 28, 2000 |
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Current U.S.
Class: |
424/9.2 ;
435/6.14; 435/7.23 |
Current CPC
Class: |
G01N 33/57419 20130101;
C07K 14/4748 20130101; C12Q 2600/158 20130101; C07K 14/47 20130101;
C12Q 2600/136 20130101; G01N 33/5011 20130101; A61K 38/00 20130101;
A61K 49/0008 20130101; A61K 39/00 20130101; C12Q 1/6886
20130101 |
Class at
Publication: |
424/9.2 ; 435/6;
435/7.23 |
International
Class: |
A61K 049/00; C12Q
001/68; G01N 033/574 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 15, 2000 |
PCT/US00/07044 |
Claims
We claim:
1. A method of screening drug candidates comprising: a) providing a
cell that expresses an expression profile gene encoding CJA8 or
fragment thereof; b) adding a drug candidate to said cell; and c)
determining the effect of said drug candidate on the expression of
said expression profile gene.
2. A method according to claim 1 wherein said determining comprises
comparing the level of expression in the absence of said drug
candidate to the level of expression in the presence of said drug
candidate.
3. A method of screening for a bioactive agent capable of binding
to CJA8 or a fragment thereof, said method comprising: a) combining
said CJA8 or a fragment thereof and a candidate bioactive agent;
and b) determining the binding of said candidate agent to said CJA8
or a fragment thereof.
4. A method for screening for a bioactive agent capable of
modulating the activity of CJA8, said method comprising: a)
combining CJA8 and a candidate bioactive agent; and b) determining
the effect of said candidate agent on the bioactivity of CJA8.
5. A method of evaluating the effect of a candidate colorectal
cancer drug comprising: a) administering said drug to a patient; b)
removing a cell sample from said patient; and c) determining the
expression of a gene encoding CJA8 or fragment thereof.
6. A method according to claim 5 further comprising comparing said
expression profile to an expression profile of a healthy
individual.
7. A method of diagnosing colorectal cancer comprising: a)
determining the expression of a gene encoding CJA8 or a fragment
thereof in a first colon tissue of a first individual; and b)
comparing said expression of said gene(s) from a second normal
colon tissue from said first individual or a second unaffected
individual; wherein a difference in said expression indicates that
the first individual has colorectal cancer.
8. An antibody which specifically binds to CJA8 or a fragment
thereof.
9. The antibody of claim 8, wherein said antibody is a monoclonal
antibody.
10. The antibody of claim 8, wherein said antibody is a humanized
antibody.
11. The antibody of claim 8, wherein said antibody is an antibody
fragment.
12. The antibody of claim 8, wherein said antibody modulates the
bioactivity of CJA8.
13. The antibody of claim 12, wherein said antibody is capable of
inhibiting the bioactivity or neutralizing the effect of CJA8.
14. A method for screening for a bioactive agent capable of
interfering with the binding of CJA8 or a fragment thereof and an
antibody which binds to CJA8 or fragment thereof, said method
comprising: a) combining CJA8 or fragment thereof, a candidate
bioactive agent and an antibody which binds to CJA8 or fragment
thereof; and b) determining the binding of CJA8 or fragment thereof
and said antibody.
15. A method according to claim 14, wherein said antibody is
capable of inhibiting or neutralizing the bioactivity of CJA8.
16. A method for inhibiting the activity of CJA8, said method
comprising binding an inhibitor to CJA8.
17. A method according to claim 16 wherein said inhibitor is an
antibody.
18. A method of neutralizing the effect of CJA8 or a fragment
thereof, comprising contacting an agent specific for said CJA8 or
fragment thereof with said CJA8 or fragment thereof in an amount
sufficient to effect neutralization.
19. A method of treating colorectal cancer comprising administering
to a patient an inhibitor of CJA8.
20. A method according to claim 19 wherein said inhibitor is an
antibody.
21. A method for localizing a therapeutic moiety to colorectal
cancer tissue comprising exposing said tissue to an antibody to
CJA8 or fragment thereof conjugated to said therapeutic moiety.
22. The method of claim 21, wherein said therapeutic moiety is a
cytotoxic agent.
23. The method of claim 21, wherein said therapeutic moiety is a
radioisotope.
24. A method of treating colorectal cancer comprising administering
to an individual having said colorectal cancer an antibody to CJA8
or fragment thereof conjugated to a therapeutic moiety.
25. The method of claim 24, wherein said therapeutic moiety is a
cytotoxic agent.
26 The method of claim 24, wherein said therapeutic moiety is a
radioisotope.
27. A method for inhibiting colorectal cancer in a cell, wherein
said method comprises administering to a cell a composition
comprising antisense molecules to a nucleic acid of FIG. 1.
28. A biochip comprising one or more nucleic acid segments encoding
CJA8 or a fragment thereof, wherein said biochip comprises fewer
than 1000 nucleic acid probes.
29. A method of eliciting an immune response in an individual, said
method comprising administering to said individual a composition
comprising CJA8 or a fragment thereof.
30. A method of eliciting an immune response in an individual, said
method comprising administering to said individual a composition
comprising a nucleic acid encoding CJA8 or a fragment thereof.
31. 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.
Description
FIELD OF THE INVENTION
[0001] 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
[0002] 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-.beta. signalling pathway. For a review, see Molecular Biology
of Colorectal Cancer, pp 238-299, in Curr. Probl. Cancer,
September/October 1997.
[0003] 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 majority of colorectal cancers but not in
normal tissues. Liefers et al., New England J. of Med. 339(4):223
(1998).
[0004] Thus, methods that can be used for diagnosis and prognosis
of colorectal cancer would be desirable. While academia and
industry has made an effort to identify novel sequences, there has
not been an equal effort exerted to identify the function of the
novel sequences in disease states of concern, such as cancer. For
example, databases show the sequence for accession numbers M411502
and AF216312, and the later has been identified as a type II
membrane serine protease, but there is no data correlating these
sequences with a disease state. 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
[0005] The present invention provides methods for screening for
compositions which modulate colorectal cancer. 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 as described
herein include the sequence comprising CJA8 or a fragment thereof.
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.
[0006] 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.
[0007] Also provided herein is a method of screening for a
bioactive agent capable of binding to a colorectal cancer
modulating protein (BCMP) or a fragment thereof, the method
comprising combining the BCMP or fragment thereof and a candidate
bioactive agent, and determining the binding of the candidate agent
to the BCMP or fragment thereof. In a preferred embodiment, the
BCMP is CJA8.
[0008] Further provided herein is a method for screening for a
bioactive agent capable of modulating the bioactivity of a BCMP or
a fragment thereof. In one embodiment, the method comprises
combining the BCMP or fragment thereof and a candidate bioactive
agent, and determining the effect of the candidate agent on the
bioactivity of the BCMP or the fragment thereof. In a preferred
embodiment, the BCMP is CJA8
[0009] Also provided herein 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 a BCMP or
a fragment thereof, or an animal lacking a BCMP for example as a
result of a gene knockout. In a preferred embodiment, the BCMP is
CJA8.
[0010] 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.
[0011] Furthermore, a method of diagnosing colorectal cancer is
provided. The method comprises determining the expression of a gene
which encodes CJA8 or a fragment thereof in a first tissue type of
a first individual, and comparing this to the expression of the
gene from a second unaffected individual. A difference in the
expression indicates that the first individual has colorectal
cancer.
[0012] In another aspect, the present invention provides an
antibody which specifically binds to CJA8, or a fragment thereof.
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.
[0013] In one embodiment a method for screening for a bioactive
agent capable of interfering with the binding of CJA8 or a fragment
thereof and an antibody which binds to said CJA8 or fragment
thereof is provided. In a preferred embodiment, the method
comprises combining CJA8 or a fragment thereof, a candidate
bioactive agent and an antibody which binds to said CJA8 or
fragment thereof. The method further includes determining the
binding of said CJA8 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.
[0014] In one aspect of the invention, a method for inhibiting the
activity of a colorectal cancer modulating protein are provided.
The method comprises binding an inhibitor to the protein. In a
preferred embodiment, the protein is CJA8.
[0015] In another aspect, the invention provides a method for
neutralizing the effect of a colorectal cancer modulating protein.
The method comprises contacting an agent specific for the protein
with the protein in an amount sufficient to effect neutralization.
In a preferred embodiment, the protein is CJA8.
[0016] In a further aspect, a method for treating or inhibiting
colorectal cancer is provided. In one embodiment, the method
comprises administering to a cell a composition comprising an
antibody to CJA8 or a fragment thereof. In one embodiment, the
antibody is conjugated to a therapeutic moiety.
[0017] Such therapeutic moieties include a cytotoxic agent and a
radioisotope. 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 such cancer.
[0018] As described herein, methods of treating or inhibiting
colorectal cancer can be performed by administering an inhibitor of
CJA8 activity to a cell or individual. In one embodiment, a CJA8
inhibitor is an antisense molecule to a nucleic acid encoding
CJA8.
[0019] Moreover, provided herein is a biochip comprising a nucleic
acid segment which encodes CJA8, or a fragment thereof, wherein the
biochip comprises fewer than 1000 nucleic acid probes. Preferably
at least two nucleic acid segments are included.
[0020] Also provided herein are methods of eliciting an immune
response in an individual. In one embodiment a method provided
herein comprises administering to an individual a composition
comprising CJA8 or a fragment thereof. In another aspect, said
composition comprises a nucleic acid comprising a sequence encoding
CJA8 or a fragment thereof.
[0021] Further provided herein are compositions capable of
eliciting an immune response in an individual. In one embodiment, a
composition provided herein comprises CJA8 or a fragment thereof
and a pharmaceutically acceptable carrier. In another embodiment,
said composition comprises a nucleic acid comprising a sequence
encoding CJA8 or a fragment thereof and a pharmaceutically
acceptable carrier.
[0022] Other aspects of the invention will become apparent to the
skilled artisan by the following description of the invention.
DETAILED DESCRIPTION OF THE FIGURES
[0023] FIG. 1 shows an embodiment of a nucleic acid (mRNA) which
includes a sequence which encodes a colorectal cancer protein
provided herein, CJA8. The start and stop codons are shaded.
[0024] FIG. 2 shows an embodiment of an amino acid sequence of
CJA8. A putative transmembrane region is shaded.
[0025] FIG. 3 shows the relative amount of expression of CJA8 in
various samples of colorectal cancer tissue (dark bars) and many
normal tissue types (light bars).
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention provides novel methods for diagnosis
and prognosis evaluation for colorectal cancer, as well as methods
for screening for compositions which modulate colorectal cancer and
compositions which bind to modulators of colorectal cancer. 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 colorectal cancer tissue, and within
colorectal cancer tissue, different prognosis states (good or poor
long term survival prospects, for example) may be determined. By
comparing expression profiles of colorectal cancer tissue in
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 colorectal cancer tissue 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 colorectal cancer 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 colorectal
cancer 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 colorectal cancer proteins can be
evaluated for diagnostic and prognostic purposes or to screen
candidate agents. In addition, the colorectal cancer nucleic acid
sequences can be administered for gene therapy purposes, including
the administration of antisense nucleic acids, or the colorectal
cancer proteins (including antibodies and other modulators thereof)
administered as therapeutic drugs.
[0027] Thus the present invention provides nucleic acid and protein
sequences that are differentially expressed in colorectal cancer
when compared to normal tissue. The differentially expressed
sequences provided herein are termed "colorectal cancer sequences".
As outlined below, colorectal cancer sequences include those that
are up-regulated (i.e. expressed at a higher level) in colorectal
cancer, as well as those that are down-regulated (i.e. expressed at
a lower level) in colorectal cancer. In a preferred embodiment, the
colorectal cancer sequences are from humans; however, as will be
appreciated by those in the art, colorectal cancer sequences from
other organisms may be useful in animal models of disease and drug
evaluation; thus, other colorectal cancer 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). Colorectal cancer sequences
from other organisms may be obtained using the techniques outlined
below.
[0028] In a preferred embodiment, the colorectal cancer sequences
are those of nucleic acids encoding CJA8 or fragments thereof.
Preferably, the colorectal cancer sequences are those depicted in
FIG. 1, or fragments thereof. Preferably, the colorectal cancer
sequences encode a protein having the amino acid sequence depicted
in FIG. 2, or a fragment thereof. In a preferred embodiment, CJA8
has the sequence of a human type II membrane serine protease.
[0029] Colorectal cancer sequences can include both nucleic acid
and amino acid sequences. In a preferred embodiment, the colorectal
cancer 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.
[0030] 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 all 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
colorectal cancer 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 not normally found in
nature, as in the addition of an epitope tag or amino acid
substitutions, insertions and deletions, as discussed below.
[0031] In a preferred embodiment, the colorectal cancer sequences
are nucleic acids. As will be appreciated by those in the art and
is more fully outlined below, colorectal cancer 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 colorectal cancer 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 Scripta 26:141 91986)),
phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437 (1991);
and U.S. Pat. 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. Pat. 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. Pat.
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) pp
169-176). Several nucleic acid analogs are described in Rawls, C
& E News Jun. 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.
[0032] 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.
[0033] 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.degree. C. drop in Tm for an internal mismatch. With the
non-ionic PNA backbone, the drop is closer to 7-9.degree. 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.
[0034] 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.
[0035] A colorectal cancer sequence can be initially identified by
substantial nucleic acid and/or amino acid sequence homology to the
colorectal cancer 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.
[0036] The colorectal cancer 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 commercially
available, for example from Affymetrix. Gene expression profiles as
described herein are generated, and the data analyzed.
[0037] 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, colorectal, kidney, muscle, prostate,
small intestine, large intestine, spleen, bone, and placenta. In a
preferred embodiment, those genes identified during the colorectal
cancer 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.
[0038] In a preferred embodiment, colorectal cancer sequences are
those that are up-regulated in colorectal cancer; 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 50% increase, preferably a two-fold change,
more 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 are found to
be expressed in a limited amount or not at all in bladder, bone
marrow, brain, breast, fibroblasts, heart, kidney, liver, lung,
muscle, pancreas, prostate, skin, small intestine, spleen, stomach
and testes.
[0039] In a preferred embodiment, the gene for CJA8 is upregulated
in colon cancer tissue as compared with normal colon tissue.
[0040] In another embodiment, colorectal cancer sequences are those
that are down-regulated in colorectal cancer; that is, the
expression of these genes is lower in, for example, 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.
[0041] Colorectal cancer proteins of the present invention may be
classified as secreted proteins, transmembrane proteins or
intracellular proteins. In a preferred embodiment the colorectal
cancer protein is an intracellular protein. Intracellular proteins
may be found in the cytoplasm and/or 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, polymerase 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.
[0042] 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 and/or
molecules with which the protein may associate.
[0043] In a preferred embodiment, the colorectal cancer 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.
[0044] 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.
[0045] 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 sequence of a particular protein, the localization and
number of transmembrane domains within the protein may be
predicted.
[0046] The extracellular 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.
Immunoglobulin-like domains are highly conserved. Mucin-like
domains may be involved in cell adhesion and leucine-rich repeats
participate in protein-protein interactions.
[0047] 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.
[0048] Colorectal cancer 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.
[0049] 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.
[0050] In a preferred embodiment, the colorectal cancer 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 endocrine manner (acting on cells
at a distance). Thus secreted molecules find use in modulating or
altering numerous aspects of physiology. Colorectal cancer 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.
[0051] A colorectal cancer sequence is initially identified by
substantial nucleic acid and/or amino acid sequence homology to the
colorectal cancer 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.
[0052] As used herein, a nucleic acid is a "colorectal cancer
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 most 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 & Waterman, 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, Wis.), 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.
[0053] 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 shown in FIG.
1 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.
[0054] 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 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.
[0055] Another example of a useful algorithm is the BLAST
algorithm, described in Altschul et al., J. Mol. Biol. 215,
403-410, (1990) and Karlin 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/b- last/READ.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).
[0056] 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 FIG. 1.
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.
[0057] 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.
[0058] 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 colorectal cancer 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.
[0059] Ausubel, et al., 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.degree. 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.degree. C. for short probes (e.g.
10 to 50 nucleotides) and at least about 60.degree. 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.
[0060] 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.
[0061] In addition, the colorectal cancer 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 colorectal
cancer 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.
[0062] Once the colorectal cancer nucleic acid is identified, it
can be cloned and, if necessary, its constituent parts recombined
to form the entire colorectal cancer 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 colorectal cancer nucleic acid can be further-used as a
probe to identify and isolate other colorectal cancer nucleic
acids, for example additional coding regions. It can also be used
as a "precursor" nucleic acid to make modified or variant
colorectal cancer nucleic acids and proteins.
[0063] The colorectal cancer nucleic acids of the present invention
are used in several ways. In a first embodiment, nucleic acid
probes to the colorectal cancer 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 therapy
and/or antisense applications. Alternatively, the colorectal cancer
nucleic acids that include coding regions of colorectal cancer
proteins can be put into expression vectors for the expression of
colorectal cancer proteins, again either for screening purposes or
for administration to a patient.
[0064] In a preferred embodiment, nucleic acid probes to colorectal
cancer 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 colorectal cancer
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.
[0065] 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.
[0066] 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.
[0067] 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 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.
[0068] 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.
[0069] 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, TeflonJ, 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 fluorescese. A preferred substrate is
described in copending application entitled Reusable Low
Fluorescent Plastic Biochip filed Mar. 15, 1999, herein
incorporated by reference in its entirety.
[0070] 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.
[0071] 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 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.
[0072] 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 may be attached to the solid support, or
attachment may be via an internal nucleoside.
[0073] 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.
[0074] 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. Pat. 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 GeneChip.TM.
technology.
[0075] In a preferred embodiment, colorectal cancer nucleic acids
encoding colorectal cancer proteins are used to make a variety of
expression vectors to express colorectal cancer 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 colorectal cancer 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.
[0076] 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 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 colorectal cancer protein; for
example, transcriptional and translational regulatory nucleic acid
sequences from Bacillus are preferably used to express the
colorectal cancer protein in Bacillus. Numerous types of
appropriate expression vectors, and suitable regulatory sequences
are known in the art for a variety of host cells.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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.
[0081] The colorectal cancer proteins of the present invention are
produced by culturing a host cell transformed with an expression
vector containing nucleic acid encoding a colorectal cancer
protein, under the appropriate conditions to induce or cause
expression of the colorectal cancer protein. The conditions
appropriate for colorectal cancer 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.
[0082] 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.
[0083] In a preferred embodiment, the colorectal cancer 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/US97/01019 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.
[0084] 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.
[0085] In a preferred embodiment, colorectal cancer 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 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 colorectal cancer 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.
[0086] 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.
[0087] In one embodiment, colorectal cancer 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.
[0088] In a preferred embodiment, colorectal cancer 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.
[0089] The colorectal cancer 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 colorectal cancer protein may be fused to a carrier
protein to form an immunogen. Alternatively, the colorectal cancer
protein may be made as a fusion protein to increase expression, or
for other reasons. For example, when the colorectal cancer protein
is a colorectal cancer peptide, the nucleic acid encoding the
peptide may be linked to other nucleic acid for expression
purposes.
[0090] In one embodiment, the colorectal cancer 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 antigens; and c) colored or
fluorescent dyes. The labels may be incorporated into the
colorectal cancer 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 .sup.3H, .sup.14C, .sup.32P,
.sup.35S, or .sup.125I, 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).
[0091] Accordingly, the present invention also provides colorectal
cancer protein sequences. A colorectal cancer 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 colorectal cancer 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 0.85
default. This results in the generation of a putative protein
sequence.
[0092] Also included within one embodiment of colorectal cancer
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.
[0093] Colorectal cancer 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
colorectal cancer proteins are portions or fragments of the wild
type sequences, herein. In addition, as outlined above, the
colorectal cancer nucleic acids of the invention may be used to
obtain additional coding regions, and thus additional protein
sequence, using techniques known in the art.
[0094] In a preferred embodiment, the colorectal cancer proteins
are derivative or variant colorectal cancer proteins as compared to
the wild-type sequence. That is, as outlined more fully below, the
derivative colorectal cancer 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 colorectal cancer peptide.
[0095] Also included in an embodiment of colorectal cancer 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 colorectal cancer 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
colorectal cancer 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 colorectal cancer 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.
[0096] 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 colorectal cancer
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 colorectal cancer protein activities.
[0097] 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.
[0098] 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 molecule. However, larger changes may be
tolerated in certain circumstances. When small alterations in the
characteristics of the colorectal cancer protein are desired,
substitutions are generally made in accordance with the following
chart:
1 CHART I Original Residue Exemplary Substitutions Ala Ser Arg Lys
Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly 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
[0099] 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.
[0100] 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 colorectal cancer proteins as
needed. Alternatively, the variant may be designed such that the
biological activity of the colorectal cancer protein is altered.
For example, glycosylation sites may be altered or removed.
[0101] Covalent modifications of colorectal cancer polypeptides are
included within the scope of this invention. One type of covalent
modification includes reacting targeted amino acid residues of a
colorectal cancer polypeptide with an organic derivatizing agent
that is capable of reacting with selected side chains or the N-or
C-terminal residues of a colorectal cancer polypeptide.
Derivatization with bifunctional agents is useful, for instance,
for crosslinking colorectal cancer to a water-insoluble support
matrix or surface for use in the method for purifying
anti-colorectal cancer 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-hydroxysuccinimide 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]propioimidate.
[0102] 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 .alpha.-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.
[0103] Another type of covalent modification of the colorectal
cancer 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 colorectal cancer
polypeptide, and/or adding one or more glycosylation sites that are
not present in the native sequence colorectal cancer
polypeptide.
[0104] Addition of glycosylation sites to colorectal cancer
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 colorectal cancer polypeptide (for
O-linked glycosylation sites). The colorectal cancer amino acid
sequence may optionally be altered through changes at the DNA
level, particularly by mutating the DNA encoding the colorectal
cancer polypeptide at preselected bases such that codons are
generated that will translate into the desired amino acids.
[0105] Another means of increasing the number of carbohydrate
moieties on the colorectal cancer polypeptide is by chemical or
enzymatic coupling of glycosides to the polypeptide. Such methods
are described in the art, e.g., in WO 87/05330 published Sep. 11,
1987, and in Aplin and Wriston, colorectal cancer Crit. Rev.
Biochem., pp. 259-306 (1981).
[0106] Removal of carbohydrate moieties present on the colorectal
cancer 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).
[0107] Another type of covalent modification of colorectal cancer
protein comprises linking the colorectal cancer 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. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;
4,670,417; 4,791,192 or 4,179,337.
[0108] Colorectal cancer polypeptides of the present invention may
also be modified in a way to form chimeric molecules comprising a
colorectal cancer polypeptide fused to another, heterologous
polypeptide or amino acid sequence. In one embodiment, such a
chimeric molecule comprises a fusion of a colorectal cancer
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
colorectal cancer polypeptide. The presence of such epitope-tagged
forms of a colorectal cancer polypeptide can be detected using an
antibody against the tag polypeptide. Also, provision of the
epitope tag enables the colorectal cancer 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 colorectal cancer polypeptide with an immunoglobulin or
a particular region of an immunoglobulin. For a bivalent form of
the chimeric molecule, such a fusion could be to the Fc region of
an IgG molecule.
[0109] 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 [Hopp 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)].
[0110] Also included with the definition of colorectal cancer
protein in one embodiment are other colorectal cancer proteins of
the colorectal cancer family, and colorectal cancer 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 colorectal cancer
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 colorectal cancer 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.
[0111] In addition, as is outlined herein, colorectal cancer
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.
[0112] Colorectal cancer proteins may also be identified as being
encoded by colorectal cancer nucleic acids.
[0113] Thus, colorectal cancer proteins are encoded by nucleic
acids that will hybridize to the sequences of the sequence
listings, or their complements, as outlined herein.
[0114] In a preferred embodiment, when the colorectal cancer
protein is to be used to generate antibodies, for example for
immunotherapy, the colorectal cancer 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 colorectal cancer 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.
[0115] In one embodiment, the term "antibody" includes antibody
fragments, as are known in the art, including Fab, Fab.sub.2,
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.
[0116] 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
CJA8 or 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.
[0117] 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 CJA8 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 [Goding, 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.
[0118] 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 the CJA8 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.
[0119] In a preferred embodiment, the antibodies to colorectal
cancer are capable of reducing or eliminating the biological
function of colorectal cancer, as is described below. That is, the
addition of anti-colorectal cancer antibodies (either polyclonal or
preferably monoclonal) to colorectal cancer (or cells containing
colorectal cancer) may reduce or eliminate the colorectal cancer
activity. 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.
[0120] In a preferred embodiment the antibodies to the colorectal
cancer proteins are humanized antibodies.
[0121] 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').sub.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 will 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)].
[0122] 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. Pat. 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.
[0123] 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., 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. Pat. 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/Technology 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).
[0124] By immunotherapy is meant treatment of colorectal cancer
with an antibody raised against colorectal cancer 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.
[0125] In a preferred embodiment the colorectal cancer 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 colorectal cancer
protein.
[0126] In another preferred embodiment, the colorectal cancer
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 colorectal cancer 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 colorectal cancer 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
colorectal cancer protein. The antibody is also an antagonist of
the colorectal cancer protein. Further, the antibody prevents
activation of the transmembrane colorectal cancer protein. In one
aspect, when the antibody prevents the binding of other molecules
to the colorectal cancer protein, the antibody prevents growth of
the cell. The antibody also sensitizes the cell to cytotoxic
agents, including, but not limited to TNF-.alpha., TNF-.beta.,
IL-1, INF-.gamma. and IL-2, or chemotherapeutic agents including
5FU, 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, colorectal cancer is
treated by administering to a patient antibodies directed against
the transmembrane colorectal cancer protein.
[0127] 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 colorectal cancer
protein.
[0128] In another aspect the therapeutic moiety modulates the
activity of molecules associated with or in close proximity to the
colorectal cancer protein. The therapeutic moiety may inhibit
enzymatic activity such as protease or protein kinase activity
associated with colorectal cancer.
[0129] In 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
colorectal cancer. 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
colorectal cancer proteins, or binding of a radionuclide to a
chelating agent that has been covalently attached to the antibody.
Targeting the therapeutic moiety to transmembrane colorectal cancer
proteins not only serves to increase the local concentration of
therapeutic moiety in the colorectal cancer afflicted area, but
also serves to reduce deleterious side effects that may be
associated with the therapeutic moiety.
[0130] In another preferred embodiment, the PC 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 PC 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.
[0131] The colorectal cancer antibodies of the invention
specifically bind to colorectal cancer 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.sup.=4-10.sup.-6
M.sup.-1, with a preferred range being 10.sup.-7-10.sup.-9
M.sup.-1.
[0132] In a preferred embodiment, the colorectal cancer protein is
purified or isolated after expression. Colorectal cancer 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 colorectal cancer protein may be purified using a
standard anti-colorectal cancer 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 colorectal cancer protein. In
some instances no purification will be necessary.
[0133] Once expressed and purified if necessary, the colorectal
cancer proteins and nucleic acids are useful in a number of
applications.
[0134] In one aspect, the expression levels of genes are determined
for different cellular states in the colorectal cancer phenotype;
that is, the expression levels of genes in normal colon tissue and
in colorectal cancer tissue (and in some cases, for varying
severities of colorectal cancer 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 colorectal cancer tissue.
[0135] "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 colorectal cancer gene
can qualitatively have its expression altered, including an
activation or inactivation, in, for example, normal versus
colorectal cancer 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
characterization techniques as outlined below, such as by use of
Affymetrix GeneChip.TM. 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.
[0136] 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
colorectal cancer protein and standard immunoassays (ELISAs, etc.)
or other techniques, including mass spectroscopy assays, 2D gel
electrophoresis assays, etc. Thus, the proteins corresponding to
colorectal cancer genes, i.e. those identified as being important
in a colorectal cancer phenotype, can be evaluated in a colorectal
cancer diagnostic test.
[0137] 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.
[0138] In this embodiment, the colorectal cancer nucleic acid
probes are attached to biochips as outlined herein for the
detection and quantification of colorectal cancer sequences in a
particular cell. The assays are further described below in the
example.
[0139] In a preferred embodiment nucleic acids encoding the
colorectal cancer protein are detected. Although DNA or RNA
encoding the colorectal cancer protein may be detected, of
particular interest are methods wherein the mRNA encoding a
colorectal cancer protein is detected. The presence of mRNA in a
sample is an indication that the colorectal cancer 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 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 colorectal cancer 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.
[0140] 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
colorectal cancer proteins, antibodies, nucleic acids, modified
proteins and cells containing colorectal cancer 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.
[0141] As described and defined herein, colorectal cancer proteins,
including intracellular, transmembrane or secreted proteins, find
use as markers of colorectal cancer. Detection of these proteins in
putative colorectal cancer tissue of patients allows for a
determination or diagnosis of colorectal cancer. Numerous methods
known to those of ordinary skill in the art find use in detecting
colorectal cancer.
[0142] In one embodiment, antibodies are used to detect colorectal
cancer 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 colorectal cancer protein is detected
by immunoblotting with antibodies raised against the colorectal
cancer protein. Methods of immunoblotting are well known to those
of ordinary skill in the art.
[0143] In another preferred method, antibodies to the colorectal
cancer protein find use in in situ imaging techniques. In this
method cells are contacted with from one to many antibodies to the
colorectal cancer 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 colorectal
cancer protein(s) 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 pluralilty of colorectal cancer
proteins. As will be appreciated by one of ordinary skill in the
art, numerous other histological imaging techniques are useful in
the invention.
[0144] 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.
[0145] In another preferred embodiment, antibodies find use in
diagnosing colorectal cancer from blood samples. As previously
described, certain colorectal cancer proteins are
secreted/circulating molecules. Blood samples, therefore, are
useful as samples to be probed or tested for the presence of
secreted colorectal cancer proteins. Antibodies can be used to
detect the colorectal cancer 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.
[0146] In a preferred embodiment, in situ hybridization of labeled
colorectal cancer nucleic acid probes to tissue arrays is done. For
example, arrays of tissue samples, including colorectal cancer
tissue and/or normal tissue, are made. In situ hybridization as is
known in the art can then be done.
[0147] 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.
[0148] In a preferred embodiment, the colorectal cancer proteins,
antibodies, nucleic acids, modified proteins and cells containing
colorectal cancer sequences are used in prognosis assays. As above,
gene expression profiles can be generated that correlate to
colorectal cancer 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 colorectal cancer probes are
attached to biochips for the detection and quantification of
colorectal cancer sequences in a tissue or patient. The assays
proceed as outlined for diagnosis.
[0149] In a preferred embodiment, any of the three classes of
proteins as described herein are used in drug screening assays. The
colorectal cancer proteins, antibodies, nucleic acids, modified
proteins and cells containing colorectal cancer 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.
[0150] In a preferred embodiment, the colorectal cancer proteins,
antibodies, nucleic acids, modified proteins and cells containing
the native or modified colorectal cancer proteins are used in
screening assays. That is, the present invention provides novel
methods for screening for compositions which modulate the
colorectal cancer 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.
[0151] Having identified the colorectal cancer 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 colorectal
cancer, 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.
[0152] 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 colorectal cancer
protein and standard immunoassays.
[0153] 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.
[0154] In this embodiment, the colorectal cancer nucleic acid
probes are attached to biochips as outlined herein for the
detection and quantification of colorectal cancer sequences in a
particular cell. The assays are further described below.
[0155] 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 colorectal cancer proteins,
binds to a colorectal cancer protein, or interferes between the
binding of a colorectal cancer protein and an antibody.
[0156] The term "candidate bioactive agent" or "drug candidate" or
grammatical equivalents 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 the
colorectal cancer phenotype or the expression of a colorectal
cancer 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 colorectal cancer phenotype, for
example to a normal colon tissue fingerprint. Similarly, the
candidate agent preferably suppresses a severe colorectal cancer
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.
[0157] 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.
[0158] 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.
[0159] 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 chemical modifications, such as acylation,
alkylation, esterification, amidification to produce structural
analogs.
[0160] 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.
[0161] In 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.
[0162] 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.
[0163] 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 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.
[0164] In a preferred embodiment, the candidate bioactive agents
are nucleic acids, as defined above.
[0165] 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.
[0166] In a preferred embodiment, the candidate bioactive agents
are organic chemical moieties, a wide variety of which are
available in the literature.
[0167] 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
and/or 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.
[0168] 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.
[0169] 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. Pat. 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, 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.
[0170] 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.
[0171] These parameters may also be used to control non-specific
binding, as is generally outlined in U.S. Pat. No. 5,681,697. Thus
it may be desirable to perform certain steps at higher stringency
conditions to reduce non-specific binding.
[0172] 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.
[0173] 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.
[0174] The screens are done to identify drugs or bioactive agents
that modulate the colorectal cancer 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 colorectal cancer
similar to the expression profile of normal colon tissue is
expected to result in a suppression of the colorectal cancer
phenotype. Thus, in this embodiment, mimicking an expression
profile, or changing one profile to another, is the goal.
[0175] In a preferred embodiment, as for the diagnosis and
prognosis applications, having identified the colorectal cancer
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.
[0176] In a preferred embodiment, screening is done to alter the
biological function of the expression product of the colorectal
cancer gene. Again, having identified the importance of a gene in a
particular state, screening for agents that bind and/or modulate
the biological activity of the gene product can be run as is more
fully outlined below.
[0177] Thus, screening of candidate agents that modulate the
colorectal cancer phenotype either at the gene expression level or
the protein level can be done.
[0178] In addition screens can be done for novel genes that are
induced in response to a candidate agent.
[0179] After identifying a candidate agent based upon its ability
to suppress a colorectal cancer expression pattern leading to a
normal expression pattern, or modulate a single colorectal cancer
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 colorectal cancer tissue reveals genes that are not
expressed in normal colon tissue or colorectal cancer 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 colorectal cancer 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 colorectal cancer tissue
sample.
[0180] Thus, in one embodiment, a candidate agent is administered
to a population of colorectal cancer cells, that thus has an
associated colorectal cancer 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.
[0181] 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.
[0182] Thus, for example, colorectal cancer tissue may be screened
for agents that reduce or suppress the colorectal cancer phenotype.
A change in at least one gene of the expression profile indicates
that the agent has an effect on colorectal cancer activity. By
defining such a signature for the colorectal cancer 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.
[0183] In a preferred embodiment, as outlined above, screens may be
done on individual genes and gene products (proteins). That is,
having identified a particular colorectal cancer 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 colorectal cancer genes are sometimes referred to
herein as "colorectal cancer proteins" or "colorectal cancer
modulating proteins" or "BCMP". Additionally, "modulator" and
"modulating" proteins are sometimes used interchangeably herein. In
one embodiment, the colorectal cancer protein is termed CJA8. CJA8
sequences can be identified as described herein for colorectal
cancer sequences. In one embodiment, CJA8 protein sequences are as
depicted in FIG. 2. The colorectal cancer protein may be a
fragment, or alternatively, be the full length protein to the
fragment shown herein. Preferably, the colorectal cancer protein is
a fragment. In a preferred embodiment, the amino acid sequence
which is used to determine sequence identity or similarity is that
depicted in FIG. 2. In another embodiment, the sequences are
naturally occurring allelic variants of a protein having the
sequence depicted in FIG. 2. In another embodiment, the sequences
are sequence variants as further described herein.
[0184] Preferably, the colorectal cancer protein is a fragment of
approximately 14 to 24 amino acids long. More preferably the
fragment is a soluble fragment. Preferably, the fragment includes a
non-transmembrane region. In a preferred embodiment, the fragment
has an N-terminal Cys to aid in solubility. 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.
Preferably, the fragment of approximately 14 to 24 amino acids
long. More preferably the fragment is a soluble fragment. In
another embodiment, a CJA8 fragment has at least one CJA8
bioactivity as defined below.
[0185] In one embodiment the colorectal cancer proteins are
conjugated to an immunogenic agent as discussed herein. In one
embodiment the colorectal cancer 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.
[0186] In a preferred embodiment, screens are designed to first
find candidate agents that can bind to colorectal cancer proteins,
and then these agents may be used in assays that evaluate the
ability of the candidate agent to modulate colorectal cancer
activity.
[0187] 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.
[0188] 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 colorectal cancer 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 colorectal cancer proteins can
be used in the assays.
[0189] Thus, in a preferred embodiment, the methods comprise
combining a colorectal cancer protein and a candidate bioactive
agent, and determining the binding of the candidate agent to the
colorectal cancer protein. Preferred embodiments utilize the human
colorectal cancer 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 colorectal cancer proteins may be used.
[0190] Generally, in a preferred embodiment of the methods herein,
the colorectal cancer 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.). It is
understood that alternatively, soluble assays known in the art may
be performed. 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, teflon.TM., 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 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.
[0191] In a preferred embodiment, the colorectal cancer 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 colorectal cancer 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 like.
[0192] The determination of the binding of the candidate bioactive
agent to the colorectal cancer protein may be done in a number of
ways. In a preferred embodiment, the candidate bioactive agent is
labelled, and binding determined directly. For example, this may be
done by attaching all or a portion of the colorectal cancer protein
to a solid support, adding a labelled 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.
[0193] 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.
[0194] 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 .sup.125I, or with
fluorophores. Alternatively, more than one component may be labeled
with different labels; using .sup.125I for the proteins, for
example, and a fluorophor for the candidate agents.
[0195] 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. colorectal
cancer), 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.
[0196] 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.degree. 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.
[0197] 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 colorectal cancer protein and thus is capable of
binding to, and potentially modulating, the activity of the
colorectal cancer 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.
[0198] In 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 colorectal cancer 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 colorectal cancer protein.
[0199] In a preferred embodiment, the methods comprise differential
screening to identity bioactive agents that are capable of
modulating the activity of the colorectal cancer proteins. In this
embodiment, the methods comprise combining a colorectal cancer
protein and a competitor in a first sample. A second sample
comprises a candidate bioactive agent, a colorectal cancer 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 colorectal cancer 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 colorectal cancer protein.
[0200] Alternatively, a preferred embodiment utilizes differential
screening to identify drug candidates that bind to the native
colorectal cancer protein, but cannot bind to modified colorectal
cancer proteins. The structure of the colorectal cancer protein may
be modeled, and used in rational drug design to synthesize agents
that interact with that site. Drug candidates that affect
colorectal cancer bioactivity are also identified by screening
drugs for the ability to either enhance or reduce the activity of
the protein.
[0201] 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.
[0202] 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.
[0203] Screening for agents that modulate the activity of
colorectal cancer proteins may also be done. In a preferred
embodiment, methods for screening for a bioactive agent capable of
modulating the activity of colorectal cancer proteins comprise the
steps of adding a candidate bioactive agent to a sample of
colorectal cancer proteins, as above, and determining an alteration
in the biological activity of colorectal cancer proteins.
"Modulating the activity" of colorectal cancer 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 colorectal cancer 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 colorectal cancer proteins.
[0204] Thus, in this embodiment, the methods comprise combining a
colorectal cancer sample and a candidate bioactive agent, and
evaluating the effect on colorectal cancer activity. By "colorectal
cancer activity" or grammatical equivalents herein is meant at
least one of colorectal cancer's biological activities, including,
but not limited to, cell division, preferably in colon tissue, cell
proliferation, tumor growth, transformation of cells and serine
protease activity. In one embodiment, colorectal cancer activity
includes activation of CJA8 or a substrate thereof by CJA8. An
inhibitor of colorectal cancer activity is an agent which inhibits
any one or more colorectal cancer activities.
[0205] In a preferred embodiment, the activity of the colorectal
cancer protein is increased; in another preferred embodiment, the
activity of the colorectal cancer 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.
[0206] In a preferred embodiment, the invention provides methods
for screening for bioactive agents capable of modulating the
activity of a colorectal cancer protein. The methods comprise
adding a candidate bioactive agent, as defined above, to a cell
comprising colorectal cancer proteins. Preferred cell types include
almost any cell. The cells contain a recombinant nucleic acid that
encodes a colorectal cancer protein. In a preferred embodiment, a
library of candidate agents are tested on a plurality of cells.
[0207] 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.
[0208] In this way, bioactive agents are identified. Compounds with
pharmacological activity are able to enhance or interfere with the
activity of the colorectal cancer protein. In one embodiment,
"colorectal cancer protein activity" as used herein includes at
least one of the following: colorectal cancer activity, binding to
CJA8, activation of CJA8 or activation of substrates of CJA8 by
CJA8. An inhibitor of CJA8 inhibits at least one of CJA8's
bioactivities.
[0209] In one embodiment, a method of inhibiting colorectal cancer
cell division is provided. The method comprises administration of a
colorectal cancer inhibitor.
[0210] In another embodiment, a method of inhibiting colorectal
tumor growth is provided. The method comprises administration of a
colorectal cancer inhibitor. In a preferred embodiment, the
inhibitor is an inhibitor of CJA8.
[0211] In a further embodiment, methods of treating cells or
individuals with colorectal cancer are provided. The method
comprises administration of a colorectal cancer inhibitor. In a
preferred embodiment, the inhibitor is an inhibitor of CJA8.
[0212] 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
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. (BioTechniques 6:958,
1988).
[0213] 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.
[0214] 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 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.
[0215] 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 containing the
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.
[0216] Without being bound by theory, it appears that the various
colorectal cancer sequences are important in colorectal cancer.
Accordingly, disorders based on mutant or variant colorectal cancer
genes may be determined. In one embodiment, the invention provides
methods for identifying cells containing variant colorectal cancer
genes comprising determining all or part of the sequence of at
least one endogeneous colorectal cancer gene 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 colorectal cancer genotype of
an individual comprising determining all or part of the sequence of
at least one colorectal cancer 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 gene to a known gene, i.e. a wild-type gene.
[0217] The sequence of all or part of the colorectal cancer gene
can then be compared to the sequence of a known colorectal cancer
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 difference in the sequence
between the colorectal cancer gene of the patient and the known
colorectal cancer gene is indicative of a disease state or a
propensity for a disease state, as outlined herein.
[0218] In a preferred embodiment, the colorectal cancer genes are
used as probes to determine the number of copies of the colorectal
cancer gene in the genome.
[0219] In another preferred embodiment colorectal cancer genes are
used as probed to determine the chromosomal localization of the
colorectal cancer genes. Information such as chromosomal
localization finds use in providing a diagnosis or prognosis in
particular when chromosomal abnormalities such as translocations,
and the like are identified in colorectal cancer gene loci.
[0220] Thus, in one embodiment, methods of modulating colorectal
cancer in cells or organisms are provided. In one embodiment, the
methods comprise administering to a cell an antibody that reduces
or eliminates the biological activity of an endogenous colorectal
cancer protein. Alternatively, the methods comprise administering
to a cell or organism a recombinant nucleic acid encoding a
colorectal cancer 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 colorectal cancer sequence is
down-regulated in colorectal cancer, the activity of the colorectal
cancer gene is increased by increasing the amount in the cell, for
example by overexpressing the endogenous protein or by
administering a gene encoding the sequence, using known
gene-therapy techniques, for example. In a preferred embodiment,
the gene therapy techniques include the incorporation of the
exogenous 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
colorectal cancer sequence is up-regulated in colorectal cancer,
the activity of the endogeneous gene is decreased, for example by
the administration of an inhibitor of colorectal cancer, such as an
antisense nucleic acid.
[0221] In one embodiment, the colorectal cancer proteins of the
present invention may be used to generate polyclonal and monoclonal
antibodies to colorectal cancer proteins, which are useful as
described herein. Similarly, the colorectal cancer proteins can be
coupled, using standard technology, to affinity chromatography
columns. These columns may then be used to purify colorectal cancer
antibodies. In a preferred embodiment, the antibodies are generated
to epitopes unique to a colorectal cancer 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 colorectal cancer antibodies may be coupled to standard
affinity chromatography columns and used to purify colorectal
cancer proteins. The antibodies may also be used as blocking
polypeptides, as outlined above, since they will specifically bind
to the colorectal cancer protein.
[0222] In one embodiment, a therapeutically effective dose of a
colorectal cancer 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 will 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 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.
[0223] 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.
[0224] The administration of the colorectal cancer 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 colorectal cancer
proteins and modulators may be directly applied as a solution or
spray.
[0225] The pharmaceutical compositions of the present invention
comprise a colorectal cancer 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, maleic 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.
[0226] 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.
[0227] In a preferred embodiment, colorectal cancer proteins and
modulators are administered as therapeutic agents, and can be
formulated as outlined above. Similarly, colorectal cancer genes
(including both the full-length sequence, partial sequences, or
regulatory sequences of the colorectal cancer coding regions) can
be administered in gene therapy applications, as is known in the
art. These colorectal cancer 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.
[0228] In a preferred embodiment, colorectal cancer genes are
administered as DNA vaccines, either single genes or combinations
of colorectal cancer genes. Naked DNA vaccines are generally known
in the art. Brower, Nature Biotechnology, 16:1304-1305 (1998).
[0229] In one embodiment, colorectal cancer 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 colorectal cancer gene or portion of a
colorectal cancer gene under the control of a promoter for
expression in a patient with colorectal cancer. The colorectal
cancer gene used for DNA vaccines can encode full-length colorectal
cancer proteins, but more preferably encodes portions of the
colorectal cancer proteins including peptides derived from the
colorectal cancer protein. In a preferred embodiment a patient is
immunized with a DNA vaccine comprising a plurality of nucleotide
sequences derived from a colorectal cancer gene. Similarly, it is
possible to immunize a patient with a plurality of colorectal
cancer 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
colorectal cancer proteins.
[0230] 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 colorectal cancer 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.
[0231] In another preferred embodiment colorectal cancer genes find
use in generating animal models of colorectal cancer. For example,
as is appreciated by one of ordinary skill in the art, when the
colorectal cancer gene identified is repressed or diminished in
colorectal cancer tissue, gene therapy technology wherein antisense
RNA directed to the colorectal cancer gene will also diminish or
repress expression of the gene. An animal generated as such serves
as an animal model of colorectal cancer 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 colorectal
cancer protein. When desired, tissue-specific expression or
knockout of the colorectal cancer protein may be necessary.
[0232] It is also possible that the colorectal cancer protein is
overexpressed in colorectal cancer. As such, transgenic animals can
be generated that overexpress the colorectal cancer protein.
Depending on the desired expression level, promoters 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 colorectal cancer and are additionally useful in
screening for bioactive molecules to treat disorders related to the
colorectal cancer protein.
[0233] 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
[0234] Tissue Preparation, Labeling Chips, and Fingerprints
[0235] Purify Total RNA from Tissue Using TRIzol Reagent
[0236] Estimate tissue weight. Homogenize tissue samples in 1 ml of
TRIzol per 50 mg 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 20 mm generator for
tissue weighing more than 0.6 g. If the working volume is greater
than 2 ml, then homogenize tissue in a 15 ml polypropylene tube
(Falcon 2059). Fill tube no greater than 10 ml.
[0237] Homogenization
[0238] Before using generator, it should have been cleaned after
last usage by running it through soapy H.sub.2O and rinsing
thoroughly. Run through with EtOH to sterilize. Keep tissue frozen
until ready. Add TRIzol directly to frozen tissue then
homogenize.
[0239] Following homogenization, remove insoluble material from the
homogenate by centrifugation at 7500.times.g for 15 min. in a
Sorvall superspeed or 12,000.times.g for 10 min. in an Eppendorf
centrifuge at 4.degree. C. Transfer the cleared homogenate to a new
tube(s). The samples may be frozen now at -60 to -70.degree. C.
(and kept for at least one month) or you may continue with the
purification.
[0240] Phase Separation
[0241] Incubate the homogenized samples for 5 minutes at room
temperature. Add 0.2 ml of chloroform per 1 ml of TRIzol reagent
used in the original homogenization. Cap tubes securely and shake
tubes vigorously by hand (do not vortex) for 15 seconds. Incubate
samples at room temp. for 2-3 minutes. Centrifuge samples at 6500
rpm in a Sorvall superspeed for 30 min. at 4.degree. C. (You may
spin at up to 12,000.times.g for 10 min. but you risk breaking your
tubes in the centrifuge.)
[0242] RNA Precipitation
[0243] Transfer the aqueous phase to a fresh tube. Save the organic
phase if isolation of DNA or protein is desired. Add 0.5 ml of
isopropyl alcohol per 1 ml of TRIzol reagent used in the original
homogenization. Cap tubes securely and invert to mix. Incubate
samples at room temp. for 10 minutes. Centrifuge samples at 6500
rpm in Sorvall for 20 min. at 4.degree. C.
[0244] RNA Wash
[0245] Pour off the supernate. Wash pellet with cold 75% ethanol.
Use 1 ml of 75% ethanol per 1 ml of TRIzol reagent used in the
initial homogenization. Cap tubes securely and invert several times
to loosen pellet. (Do not vortex). Centrifuge at <800 rpm
(<7500.times.g) for 5 minutes at 4.degree. 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 tube(s) you want to precipitate the RNA in.
When I tried leaving the RNA in the large 15 ml 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 H.sub.2O. Try for 2-5 ug/ul. Take
absorbance readings.
[0246] Purify poly A+ mRNA from Total RNA or Clean Up Total RNA
with Qiagen's RNeasy Kit
[0247] Purification of poly A.sup.+ mRNA from total RNA. Heat
oligotex suspension to 37.degree. C. and mix immediately before
adding to RNA. Incubate Elution Buffer at 70.degree. C. Warm up
2.times.Binding Buffer at 65.degree. C. if there is precipitate in
the buffer. Mix total RNA with DEPC-treated water, 2.times.Binding
Buffer, and Oligotex according to Table 2 on page 16 of the
Oligotex Handbook. Incubate for 3 minutes at 65.degree. C. Incubate
for 10 minutes at room temperature.
[0248] 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.sup.+ mRNA has
occurred.
[0249] 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.
[0250] Transfer spin column to a new collection tube and gently
resuspend in Wash Buffer OW2 and centrifuge as describe herein.
[0251] Transfer spin column to a new tube and elute with 20 to 100
ul of preheated (70.degree. 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.
[0252] Read absorbance, using diluted Elution Buffer as the
blank.
[0253] 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.
[0254] Ethanol Precipitation
[0255] Add 0.4 vol. of 7.5 M NH.sub.4OAc+2.5 vol. of cold 100%
ethanol. Precipitate at -20.degree. C. 1 hour to overnight (or
20-30 min. at -70.degree. C.). Centrifuge at 14,000-16,000.times.g
for 30 minutes at 4.degree. C. Wash pellet with 0.5 ml of
80%ethanol (-20.degree. C.) then centrifuge at
14,000-16,000.times.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 H.sub.2O at 1
ug/ul concentration.
[0256] Clean up total RNA using Qiagen's RNeasy Kit
[0257] Add no more than 100 ug to an RNeasy column. Adjust sample
to a volume of 100 ul with RNase-free water. Add 350 ul Buffer RLT
then 250 ul 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 >10,000 rpm. If concerned about yield,
re-apply flowthrough to column and centrifuge again.
[0258] Transfer column to a new 2-ml collection tube. Add 500 ul
Buffer RPE and centrifuge for 15 sec at >10,000 rpm. Discard
flowthrough. Add 500 ul Buffer RPE and centrifuge for 15 sec at
>10,000 rpm. 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 30-50 ul of RNase-free water
directly onto column membrane. Centrifuge 1 min at >10,000 rpm.
Repeat elution. Take absorbance reading. If necessary, ethanol
precipitate with ammonium acetate and 2.5.times.volume 100%
ethanol.
[0259] Make cDNA using Gibco's "SuperScript Choice System for cDNA
Synthesis" Kit
[0260] First Strand cDNA Synthesis
[0261] Use 5 ug of total RNA or lug of polyA+ mRNA as starting
material. For total RNA, use 2 ul of SuperScript RT. For
polyA+mRNA, use 1 ul of SuperScript RT. Final volume of first
strand synthesis mix is 20 ul. RNA must be in a volume no greater
than 10 ul. Incubate RNA with 1 ul of 100 pmol T7-T24 oligo for 10
min at 70C. On ice, add 7 ul of: 4 ul 5.times.1.sup.st Strand
Buffer, 2 ul of 0.1M DTT, and 1 ul of 10 mM dNTP mix. Incubate at
37C for 2 min then add SuperScript RT Incubate at 37C for 1
hour.
[0262] Second Strand Synthesis
[0263] Place 1.sup.st strand reactions on ice.
[0264] Add:
[0265] 91 ul DEPC H20
[0266] 30 ul 5.times.2.sup.nd Strand Buffer
[0267] 3 ul 10 mM dNTP mix
[0268] 1 ul 10U/ul E. coli DNA Ligase
[0269] 4 ul 10U/ul E. coli DNA Polymerase
[0270] 1 ul 2U/ul RNase H
[0271] Make the above into a mix if there are more than 2 samples.
Mix and incubate 2 hours at 16C. Add 2 ul T4 DNA Polymerase.
Incubate 5 min at 16C. Add 10 ul of 0.5M EDTA
[0272] Clean Up cDNA
[0273] Phenol:Chloroform:Isoamyl 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
7.5.times.5M NH40ac and 2.5.times.volume of 100% ethanol.
Centrifuge immediately at room temp. for 20 min, maximum speed.
Remove sup then wash pellet 2.times.with cold 80% ethanol. Remove
as much ethanol wash as possible then let pellet air dry. Resuspend
pellet in 3 ul RNase-free water.
[0274] In vitro Transcription (IVT) and Labeling with Biotin
[0275] Pipet 1.5 ul of cDNA into a thin-wall PCR tube.
[0276] Make NTP Labeling Mix:
[0277] Combine at room temperature:
[0278] 2 ul T7 10.times.ATP (75 mM) (Ambion)
[0279] 2 ul T7 10.times.GTP (75 mM) (Ambion)
[0280] 1.5 ul T7 10.times.CTP (75 mM) (Ambion)
[0281] 1.5 ul T7 10.times.UTP (75 mM) (Ambion)
[0282] 3.75 ul 10 mM Bio-11-UTP (Boehringer-Mannheim/Roche or
Enzo)
[0283] 3.75 ul 10 mM Bio-16-CTP (Enzo)
[0284] 2 ul 10.times.T7 transcription buffer (Ambion)
[0285] 2 ul 10.times.T7 enzyme mix (Ambion)
[0286] Final volume of total reaction is 20 ul. Incubate 6 hours at
37C in a PCR machine.
[0287] RNeasy Clean-Up of IVT Product
[0288] Follow previous instructions for RNeasy columns or refer to
Qiagen's RNeasy protocol handbook.
[0289] cRNA will most likely need to be ethanol precipitated.
Resuspend in a volume compatible with the fragmentation step.
[0290] Fragmentation
[0291] 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.times.Fragmentation buffer.
[0292] 5.times.Fragmentation Buffer:
[0293] 200 mM Tris-acetate, pH 8.1
[0294] 500 mM KOAc
[0295] 150 mM MgOAc
[0296] 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
[0297] Hybridization
[0298] 200 ul (10 ug 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.
[0299] Hybrization Mix: fragment labeled RNA (50 ng/ul final
conc.)
[0300] 50 pM 948-b control oligo
[0301] 1.5 pM BioB
[0302] 5 pM BioC
[0303] 25 pM BioD
[0304] 100 pM CRE
[0305] 0.1 mg/ml herring sperm DNA
[0306] 0.5 mg/ml acetylated BSA
[0307] to 300 ul with 1.times.MES hyb. buffer
[0308] The instruction manuals for the products used herein are
incorporated herein in their entirety.
2 Labeling 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 .mu.g: .mu.l
Random Hexamers (1 .mu.g/.mu.l): 4 .mu.l H.sub.2O: .mu.l 14 .mu.l
Incubate 70.degree. C., 10 min. Put on ice. Reverse transcription:
5X First Strand (BRL) buffer: 6 .mu.l 0.1 M DTT: 3 .mu.l 50X dNTP
mix: 0.6 .mu.l H2O: 2.4 .mu.l Cy3 or Cy5 dUTP (1 mM): 3 .mu.l SS RT
II (BRL): 1 .mu.l 16 .mu.l
[0309] Add to hybridization reaction.
[0310] Incubate 30 min., 42.degree. C.
[0311] Add 1 .mu.l SSII and let go for another hour.
[0312] Put on ice.
[0313] 50.times.dNTP mix (25 mM of cold dATP, dCTP, and dGTP, 10 mM
of dTTP: 25 .mu.l each of 100 mM dATP, dCTP, and dGTP; 10 .mu.l, of
100 mM dTTP to 15 .mu.l, H.sub.2O. dNTPs from Pharmacia)
3 RNA degradation: 86 .mu.l H.sub.2O Add 1.5 .mu.l 1 M NaOH/2 mM
EDTA, 10 .mu.l 10 N NaOH incubate at 65.degree. C., 10 min. 4 .mu.l
50 mM EDTA
[0314] U-Con 30
[0315] 500 .mu.l TE/sample spin at 7000 g for 10 min, save flow
through for purification
[0316] Qiagen Purification:
[0317] suspend u-con recovered material in 500 .mu.l buffer PB
[0318] proceed w/normal Qiagen protocol
[0319] DNAse Digest:
[0320] Add 1 .mu.l of {fraction (1/100)} dil of DNAse/30 .mu.l Rx
and incubate at 37.degree. C. for 15 min.
[0321] 5 min 95.degree. C. to denature enzyme
4 Sample preparation: Add: Cot-1 DNA: 10 .mu.l 50X dNTPs: 1 .mu.l
20X SSC: 2.3 .mu.l Na pyro phosphate: 7.5 .mu.l 10 mg/ml Herring
sperm DNA 1 ul of 1/10 dilution 21.8 final vol.
[0322] Dry down in speed vac.
[0323] Resuspend in 15 .mu.l H.sub.2O.
[0324] Add 0.38 .mu.l 10% SDS.
[0325] Heat 95.degree. C., 2 min.
[0326] Slow cool at room temp. for 20 min.
[0327] Put on slide and hybridize overnight at 64.degree. C.
5 Washing after the hybridization: 3X SSC/0.03% SDS: 2 min. 37.5
mls 20X SSC + 0.75 mls 10% SDS in 250 mls H.sub.2O 1X SSC: 5 min.
12.5 mls 20X SSC in 250 mls H.sub.2O 0.2X SSC: 5 min. 2.5 mls 20X
SSC in 250 mls H.sub.2O
[0328] Dry slides in centrifuge, 1000 RPM, 1 min.
[0329] Scan at appropriate PMT's and channels.
Example 2
[0330] Expression studies were performed herein. As indicated in
FIG. 3, CJA8 is upregulated in colorectal cancer tissue. CJA8 is
located on chromosome 11.
* * * * *
References