U.S. patent application number 11/537537 was filed with the patent office on 2007-11-29 for cardiotrophin-1 compositions and methods for the treatment of tumor.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to David Botstein, Audrey Goddard, David A. Lawrence, Diane Pennica, Margaret Ann Roy, William I. Wood.
Application Number | 20070274995 11/537537 |
Document ID | / |
Family ID | 34199447 |
Filed Date | 2007-11-29 |
United States Patent
Application |
20070274995 |
Kind Code |
A1 |
Botstein; David ; et
al. |
November 29, 2007 |
CARDIOTROPHIN-1 COMPOSITIONS AND METHODS FOR THE TREATMENT OF
TUMOR
Abstract
The invention concerns compositions and methods for the
diagnosis and treatment of neoplastic cell growth and proliferation
in mammals, including humans. The invention is based on the
identification of cardiotrophin-1 gene amplified in the genome of
tumor cells. Such gene amplification is expected to be associated
with the overexpression of the gene product and contribute to
tumorigenesis. Accordingly, the cardiotrophin-1 polypeptide encoded
by the amplified gene is believed to be a useful target for the
diagnosis and/or treatment (including prevention) of certain
cancers, and may act as a predictor of the prognosis of tumor
treatment.
Inventors: |
Botstein; David; (Belmont,
CA) ; Goddard; Audrey; (San Francisco, CA) ;
Lawrence; David A.; (San Francisco, CA) ; Pennica;
Diane; (Burlingame, CA) ; Roy; Margaret Ann;
(Mountain View, CA) ; Wood; William I.;
(Cupertino, CA) |
Correspondence
Address: |
GENENTECH, INC.
1 DNA WAY
SOUTH SAN FRANCISCO
CA
94080
US
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
34199447 |
Appl. No.: |
11/537537 |
Filed: |
September 29, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10895545 |
Jul 21, 2004 |
7258983 |
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11537537 |
Sep 29, 2006 |
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09723703 |
Nov 28, 2000 |
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10895545 |
Jul 21, 2004 |
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09648183 |
Aug 25, 2000 |
6472585 |
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09723703 |
Nov 28, 2000 |
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09234730 |
Jan 21, 1999 |
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09648183 |
Aug 25, 2000 |
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09033114 |
Mar 2, 1998 |
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09234730 |
Jan 21, 1999 |
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08733850 |
Oct 18, 1996 |
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09033114 |
Mar 2, 1998 |
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08443129 |
May 17, 1995 |
5627073 |
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08733850 |
Oct 18, 1996 |
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08286304 |
Aug 5, 1994 |
5571893 |
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08443129 |
May 17, 1995 |
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08233609 |
Apr 25, 1994 |
5534615 |
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08286304 |
Aug 5, 1994 |
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60113296 |
Dec 22, 1998 |
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Current U.S.
Class: |
424/155.1 ;
435/252.33; 435/254.2; 435/320.1; 435/325; 435/358; 435/6.16;
435/69.1; 435/7.23; 514/16.4; 514/19.3; 514/9.6; 530/350;
530/388.8; 536/23.5 |
Current CPC
Class: |
C07K 14/4703 20130101;
C07K 16/18 20130101; C12Q 2600/136 20130101; C12Q 1/6886 20130101;
C07K 16/24 20130101; C07K 2319/02 20130101; G01N 2500/00 20130101;
C07K 2317/24 20130101; C07K 14/52 20130101; C07K 2319/00 20130101;
G01N 2333/52 20130101; A01K 2217/05 20130101; C07K 14/47 20130101;
A61K 38/00 20130101; G01N 33/57484 20130101 |
Class at
Publication: |
424/155.1 ;
435/006; 435/007.23; 514/012; 435/069.1; 435/320.1; 435/325;
530/350; 530/388.8; 536/023.5; 435/358; 435/252.33; 435/254.2 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12Q 1/68 20060101 C12Q001/68; G01N 33/574 20060101
G01N033/574; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; C12N 1/21 20060101 C12N001/21; C12N 1/18 20060101
C12N001/18; C12N 5/06 20060101 C12N005/06; C07K 16/30 20060101
C07K016/30; C07K 14/82 20060101 C07K014/82 |
Claims
1. A method of diagnosing tumor in a mammal, comprising detecting
the level of expression of a gene encoding a cardiotrophin-1 (CT-1)
polypeptide (a) in a test sample of tissue cells obtained from the
mammal, and (b) in a control sample of known normal tissue cells of
the same cell type, wherein a higher expression level in the test
sample indicates the presence of tumor in the mammal from which the
test tissue cells were obtained.
2. A method of diagnosing tumor in a mammal, comprising (a)
contacting an anti-CT-1 antibody with a test sample of tissue cells
obtained from the mammal, and (b) detecting the formation of a
complex between the anti-CT-1 antibody and the CT-1 polypeptide in
the test sample.
3. The method of claim 2 wherein said test sample is obtained from
an individual suspected to have neoplastic cell growth or
proliferation.
4. A cancer diagnostic kit, comprising an anti-CT-1 antibody and a
carrier in suitable packaging.
5. The kit of claim 4 further comprising instructions for using
said antibody to detect the CT-1 polypeptide.
6. A method for inhibiting the growth of tumor cells comprising
exposing a cell which overexpresses a CT-1 polypeptide to an
effective amount of an agent inhibiting the expression and/or
activity of the CT-1 polypeptide.
7. The method of claim 6 wherein said agent is an anti-CT-1
antibody.
8. The method of claim 7 wherein said tumor cells are further
exposed to radiation treatment or a cytotoxic or chemotherapeutic
agent.
9. An article of manufacture, comprising: a container; a label on
the container; and a composition comprising an active agent
contained within the container; wherein the composition is
effective for inhibiting the growth of tumor cells, the label on
the container indicates that the composition can be used for
treating conditions characterized by overexpression of a CT-1
polypeptide, and the active agent in the composition is an agent
inhibiting the expression and/or activity of the CT-1
polypeptide.
10. The article of manufacture of claim 9 wherein said active agent
is an anti-CT-1 antibody.
11. A method for identifying a compound capable of inhibiting the
expression or activity of a CT-1 polypeptide, comprising contacting
a candidate compound with a CT-1 polypeptide under conditions and
for a time sufficient to allow these two components to
interact.
12. The method of claim 11 wherein said candidate compound or said
CT-1 polypeptide is immobilized on a solid support.
13. The method of claim 12 wherein the non-immobilized component
carries a detectable label.
14. An isolated nucleic acid molecule comprising DNA having at
least an 80% sequence identity to (a) a DNA molecule encoding a
cardiotrophin-1 (CT-1) polypeptide having the sequence of amino
acid residues of FIG. 1A (SEQ ID NO:3), or (b) the complement of
the DNA molecule of (a).
15. The isolated nucleic acid molecule of claim 14 comprising the
sequence of FIGS. 1A and 1B (SEQ ID NO:1).
16. The isolated nucleic acid molecule of claim 14 comprising the
sequence of FIGS. 1A and 1B (SEQ ID NO:2).
17. An isolated nucleic acid molecule encoding a CT-1 polypeptide,
comprising DNA hybridizing to the complement of the nucleic acid
having the sequence of FIGS. 1A and 1B (SEQ ID NO:1) or (SEQ ID
NO:2).
18. A vector comprising the nucleic acid of claim 14.
19. The vector of claim 18 operably linked to control sequences
recognized by a host cell transformed with the vector.
20. A host cell comprising the vector of claim 18.
21. The host cell of claim 20, wherein said cell is a CHO cell.
22. The host cell of claim 20, wherein said cell is an E. coli.
23. The host cell of claim 20, wherein said cell is a yeast cell.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation application claiming priority to U.S.
application Ser. No. 10/895,545, filed Jul. 21, 2004, which is a
continuation application of U.S. application Ser. No. 09/723,703,
filed Nov. 28, 2000, which is a continuation application of U.S.
application Ser. No. 09/648,183, filed Aug. 25, 2000, issued Oct.
29, 2002 as U.S. Pat. No. 6,472,585, which is a continuation of
U.S. application Ser. No. 09/234,730, filed Jan. 21, 1999, which is
a continuation-in-part application of U.S. application Ser. No.
09/033,114, filed Mar. 2, 1998, which is a continuation of U.S.
application Ser. No. 08/733,850, filed Oct. 18, 1996, now
abandoned, which is a continuation of U.S. application Ser. No.
08/443,129 filed May 17, 1995, issued May 6, 1997 as U.S. Pat. No.
5,627,073, which is a divisional of U.S. application Ser. No.
08/286,304 filed Aug. 5, 1994, issued Nov. 5, 1996 as U.S. Pat. No.
5,571,893, which is a continuation-in-part of U.S. application Ser.
No. 08/233,609 field Apr. 25, 1994, issued Jul. 9, 1996 as U.S.
Pat. No. 5,534,615, and to U.S. Provisional Application Ser. No.
60/113,296, filed Dec. 22, 1998, which applications are herein
incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions and methods
for the diagnosis and treatment of tumor.
BACKGROUND OF THE INVENTION
[0003] Malignant tumors (cancers) are the second leading cause of
death in the United States, after heart disease (Boring et al., CA
Cancel J. Clin. 43, 7, [1993]).
[0004] Cancer is characterized by the increase in the number of
abnormal, or neoplastic, cells derived from a normal tissue which
proliferate to form a tumor mass, the invasion of adjacent tissues
by these neoplastic tumor cells, and the generation of malignant
cells which eventually spread via the blood or lymphatic system to
regional lymph nodes and to distant sites (metastasis). In a
cancerous state a cell proliferates under conditions in which
normal cells would not grow. Cancer manifests itself in a wide
variety of forms, characterized by different degrees of
invasiveness and aggressiveness.
[0005] Alteration of gene expression is intimately related to the
uncontrolled cell growth and de-differentiation which are a common
feature of all cancers. The genomes of certain well studied tumors
have been found to show decreased expression of recessive genes,
usually referred to as tumor suppression genes, which would
normally function to prevent malignant cell growth, and/or
overexpression of certain dominant genes, such as oncogenes, that
act to promote malignant growth. Each of these genetic changes
appears to be responsible for importing some of the traits that, in
aggregate, represent the full neoplastic phenotype (Hunter, Cell
64, 1129 [1991]; Bishop, Cell 64, 235-248 [1991]).
[0006] A well known mechanism of gene (e.g. oncogene)
overexpression in cancer cells is gene amplification. This is a
process where in the chromosome of the ancestral cell multiple
copies of a particular gene are produced. The process involves
unscheduled replication of the region of chromosome comprising the
gene, followed by recombination of the replicated segments back
into the chromosome (Alitalo et al., Adv. Cancer Res. 47, 235-281
[1986]). It is believed that the overexpression of the gene
parallels gene amplification, i.e. is proportionate to the number
of copies made.
[0007] Proto-oncogenes that encode growth factors and growth factor
receptors have been identified to play important roles in the
pathogenesis of various human malignancies, including breast
cancer. For example, it has been found that the human ErbB2 gene
(erbB2, also known as her2, or c-erbB-2), which encodes a 185-kd
transmembrane glycoprotein receptor (p185.sup.HER2; HER2) related
to the epidermal growth factor receptor (EGFR), is overexpressed in
about 25% to 30% of human breast cancer (Slamon et al., Science
235:177-182 [1987]; Slamon et al., Science 244:707-712 [1989]).
[0008] It has been reported that gene amplification of a
protooncogen is an event typically involved in the more malignant
forms of cancer, and could act as a predictor of clinical outcome
(Schwab et al., Genes Chromosomes Cancer 1, 181-193 [1990]; Alitalo
et al., supra). Thus, erbB2 overexpression is commonly regarded as
a predictor of a poor prognosis, especially in patients with
primary disease that involves axillary lymph nodes (Slamon et al.,
[1987] and [1989], supra; Ravdin and Chamness, Gene 159:19-27
[1995]; and Hynes and Stern, Biochim Biophys Acta 1198:165-184
[1994]), and has been linked to sensitivity and/or resistance to
hormone therapy and chemotherapeutic regimens, including CMF
(cyclophosphamide, methotrexate, and fluoruracil) and
anthracyclines (Baselga et al., Oncology 11(3 Suppl 1):43-48
[1997]). However, despite the association of erbB2 overexpression
with poor prognosis, the odds of HER2-positive patients responding
clinically to treatment with taxanes were greater than three times
those of HER2-negative patients (Ibid). A recombinant humanized
anti-ErbB2 (anti-HER2) monoclonal antibody (a humanized version of
the murine anti-ErbB2 antibody 4D5, referred to as rhuMAb HER2 or
Herceptin.RTM.) has been clinically active in patients with
ErbB2-overexpressing metastatic breast cancers that had received
extensive prior anticancer therapy. (Baselga et al., J. Clin.
Oncol. 14:737-744 [1996]).
SUMMARY OF THE INVENTION
[0009] The present invention concerns compositions and methods for
the diagnosis and treatment of neoplastic cell growth and
proliferation in mammals, including humans. The present invention
is based on the identification of a gene that are amplified in the
genome of tumor cells. Such gene amplification is expected to be
associated with the overexpression of the gene product and
contribute to tumorigenesis. Accordingly, the protein encoded by
the amplified gene is believed to be a useful target for the
diagnosis and/or treatment (including prevention) of certain
cancers, and may act of predictors of the prognosis of tumor
treatment.
[0010] A gene product, CT-1, is useful in the treatment of heart
failure and/or neurological disorders such as peripheral neuropathy
was disclosed in U.S. Pat. No. 5,571,675 (herein incorporated by
reference in its entirety). The surprising discovery that CT-1 is
amplified in tumor cells, such as lung and colon tumor cells, is
disclosed herein. Applicant's discovery that CT-1 is amplified in
tumor cells led to the additional discoveries of compositions for
treatment of tumor cells and methods of carrying out such
treatment.
[0011] In one embodiment, the present invention concerns an
isolated antibody which binds a CT-1 polypeptide. In one aspect,
the antibody induces death of a cell overexpressing a CT-1
polypeptide. In another aspect, the antibody is a monoclonal
antibody, which preferably has nonhuman complementarity determining
region (CDR) residues and human framework region (FR) residues. The
antibody may be labeled and may be immobilized on a solid support.
In a further aspect, the antibody is an antibody fragment, a
single-chain antibody, or an anti-idiotypic antibody.
[0012] In another embodiment, the invention concerns a composition
comprising an antibody which binds a CT-1 polypeptide in admixture
with a pharmaceutically acceptable carrier. In one aspect, the
composition comprises a therapeutically effective amount of the
antibody. In another aspect, the composition comprises a further
active ingredient, which may, for example, be a further antibody or
a cytotoxic or chemotherapeutic agent. Preferably, the composition
is sterile.
[0013] In a further embodiment, the invention concerns nucleic acid
encoding an anti-CT-1 antibody, and vectors and recombinant host
cells comprising such nucleic acid.
[0014] In a still further embodiment, the invention concerns a
method for producing an anti-CT-1 antibody by culturing a host cell
transformed with nucleic acid encoding the antibody under
conditions such that the antibody is expressed, and recovering the
antibody from the cell culture.
[0015] The invention further concerns antagonists and agonists of a
CT-1 polypeptide that inhibit one or more of the functions or
activities of the CT-1 polypeptide.
[0016] In another embodiment, the invention concerns a method for
determining the presence of a CT-1 polypeptide comprising exposing
a cell suspected of containing the CT-1 polypeptide to an anti-CT-1
antibody and determining binding of the antibody to the cell.
[0017] In yet another embodiment, the present invention concerns a
method of diagnosing tumor in a mammal, is comprising detecting the
level of expression of a gene encoding a CT-1 polypeptide (a) in a
test sample of tissue cells obtained from the mammal, and (b) in a
control sample of known normal tissue cells of the same cell type,
wherein a higher expression level in the test sample indicates the
presence of tumor in the mammal from which the test tissue cells
were obtained.
[0018] In another embodiment, the present invention concerns a
method of diagnosing tumor in a mammal, comprising (a) contacting
an anti-CT-1 antibody with a test sample of tissue cells obtained
from the mammal, and (b) detecting the formation of a complex
between the anti-CT-1 antibody and the CT-1 polypeptide in the test
sample. The detection may be qualitative or quantitative, and may
be performed in comparison with monitoring the complex formation in
a control sample of known normal tissue cells of the same cell
type. A larger quantity of complexes formed in the test sample
indicates the presence of tumor in the mammal from which the test
tissue cells were obtained. The antibody preferably carries a
detectable label. Complex formation can be monitored, for example,
by light microscopy, flow cytometry, fluorimetry, or other
techniques known in the art.
[0019] The test sample is usually obtained from an individual
suspected to have neoplastic cell growth or proliferation (e.g.
cancerous cells).
[0020] In another embodiment, the present invention concerns a
cancer diagnostic kit, comprising an anti-CT-1 antibody and a
carrier (e.g. a buffer) in suitable packaging. The kit preferably
contains instructions for using the antibody to detect the CT-1
polypeptide.
[0021] In yet another embodiment, the invention concerns a method
for inhibiting the growth of tumor cells comprising exposing a cell
which overexpresses a CT-1 polypeptide to an effective amount of an
agent inhibiting the expression and/or activity of the CT-1
polypeptide. The agent preferably is an anti-CT-1 antibody, a small
organic and inorganic molecule, peptide, phosphopeptide, antisense
or ribozyme molecule, or a triple helix molecule. In a specific
aspect, the agent, e.g. anti-CT-1 antibody induces cell death. In a
further aspect, the tumor cells are further exposed to radiation
treatment and/or a cytotoxic or chemotherapeutic agent.
[0022] In a further embodiment, the invention concerns an article
of manufacture, comprising:
[0023] a container;
[0024] a label on the container; and
[0025] a composition comprising an active agent contained within
the container; wherein the composition is effective for inhibiting
the growth of tumor cells, the label on the container indicates
that the composition can be used for treating conditions
characterized by overexpression of a CT-1 polypeptide, and the
active agent in the composition is an agent inhibiting the
expression and/or activity of the CT-1 polypeptide. In a preferred
aspect, the active agent is an anti-CT-1 antibody.
[0026] A method for identifying a compound capable of inhibiting
the expression and/or activity of a CT-1 polypeptide, comprising
contacting a candidate compound with a CT-1 polypeptide under
conditions and for a time sufficient to allow these two components
to interact. In a specific aspect, either the candidate compound or
the CT-1 polypeptide is immobilized on a solid support. In another
aspect, the non-immobilized component carries a detectable
label.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 (SEQ ID NO: 1 and 2) shows the nucleotide sequence of
DNA58125 that is a cDNA encoding a native sequence cardiotrophin-1
(CT-1). SEQ ID NO:1 is the coding strand of DNA58125 and SEQ ID
NO:2 is the complementary strand of DNA58125. SEQ ID NO:3 is the
derived amino acid sequence of a native sequence cardiotrophin-1
(CT-1).
[0028] FIG. 2 is a diagram of human chromosome 16 indicating the
regions of the chromosome at which DNA58125 and various marker
probes hybridize. The marker probes (P7, P55, P99, P154, and P208)
are located approximately every 20 Megabases along chromosome 16
and are used as controls for measurement of genetic amplification.
DNA58125 hybridizes to a region on the long arm between the
centromere and marker probe P99.
[0029] FIG. 3 is a three-dimensional representation of the results
of a framework analysis of DNA58125 (cardiotrophin-1) on lung tumor
Panel 1. The primary lung tumors tested are shown along the x-axis;
the marker probes and DNA58125 are shown along the z-axis; and the
relative genetic amplification in the area of each of the marker
probes is shown as bars on the y-axis. Bars project above the zero
plane for genetic regions amplified relative to DNA58125 in healthy
tissue, or below the zero plane indicating reduced genetic
quantitation in that region.
[0030] FIG. 4 is a three-dimensional representation of the results
of a framework analysis of DNA58125 (cardiotrophin-1) on lung tumor
Panel 2. The bar graph is arranged as generally described in FIG.
3.
[0031] FIG. 5 is a two-dimensional bar graph summary of the results
for DNA58125 from FIGS. 3 and 4. The mean .DELTA.Ct values
determined for each of the lung tumors lines tested are shown.
[0032] FIG. 6 is a three-dimensional representation of the results
of a framework analysis of DNA58125 (cardiotrophin-1) on colon
tumor Panel 1. The primary colon tumors tested are shown along the
x-axis; the marker probes and DNA58125 are shown along the z-axis;
and the relative genetic amplification in the area of each of the
marker probes is shown as bars on the y-axis. Bars project above
the zero plane for genetic regions amplified relative to DNA58125
in healthy tissue, or below the zero plane indicating reduced
genetic quantitation in that region.
[0033] FIG. 7 is a three-dimensional representation of the results
of a framework analysis of DNA58125 (cardiotrophin-1) on colon
tumor Panel 2. The bar graph is arranged as generally described in
FIG. 6.
[0034] FIG. 8 is a two-dimensional bar graph summary of the results
for DNA58125 from FIGS. 6 and 7. The mean .DELTA.Ct values
determined for each of the colon tumors tested are shown.
[0035] FIG. 9 is a three-dimensional representation of the results
of an epicenter analysis of DNA58125 (cardiotrophin-1) on lung
tumor Panel 1. The bar graph is arranged as generally described in
FIG. 3.
[0036] FIG. 10 is a three dimensional representation of the results
of an epicenter analysis of DNA58125 (CT-1) on lung tumor Panel 2.
The bar graph is arranged as generally described in FIG. 3.
[0037] FIG. 11 is a three dimensional representation of the results
of an epicenter analysis of DNA58125 (CT-1) on colon tumor Panel 1.
The bar graph is arranged as generally described in FIG. 6.
[0038] FIG. 12 is a three dimensional representation of the results
of an epicenter analysis of DNA58125 (CT-1) on colon tumor Panel 2.
The bar graph is arranged as generally described in FIG. 6.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0039] The phrases "gene amplification" and "gene duplication" are
used interchangeably and refer to a process by which multiple
copies of a gene or gene fragment are formed in a particular cell
or cell line. The duplicated region (a stretch of amplified DNA) is
often referred to as "amplicon." Usually, the amount of the
messenger RNA (mRNA) produced, i.e. the level of gene expression,
also increases in the proportion of the number of copies made of
the particular gene expressed.
[0040] "Tumor", as used herein, refers to all neoplastic cell
growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues.
[0041] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Examples of cancer include but are not
limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
More particular examples of such cancers include breast cancer,
prostate cancer, colon cancer, squamous cell cancer, small-cell
lung cancer, non-small cell lung cancer, gastrointestinal cancer,
pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer,
liver cancer, bladder cancer, hepatoma, colorectal cancer,
endometrial carcinoma, salivary gland carcinoma, kidney cancer,
liver cancer, vulval cancer, thyroid cancer, hepatic carcinoma and
various types of head and neck cancer.
[0042] "Treatment" is an intervention performed with the intention
of preventing the development or altering the pathology of a
disorder. Accordingly, "treatment" refers to both therapeutic
treatment and prophylactic or preventative measures. Those in need
of treatment include those already with the disorder as well as
those in which the disorder is to be prevented. In tumor (e.g.
cancer) treatment, a therapeutic agent may directly decrease the
pathology of tumor cells, or render the tumor cells more
susceptible to treatment by other therapeutic agents, e.g.
radiation and/or chemotherapy.
[0043] The "pathology" of cancer includes all phenomena that
compromise the well-being of the patient. This includes, without
limitation, abnormal or uncontrollable cell growth, metastasis,
interference with the normal functioning of neighboring cells,
release of cytokines or other secretory products at abnormal
levels, suppression or aggravation of inflammatory or immunological
response, etc.
[0044] "Mammal" for purposes of treatment refers to any animal
classified as a mammal, including humans, domestic and farm
animals, and zoo, sports, or pet animals, such as dogs, horses,
cats, cows, etc. Preferably, the mammal is human.
[0045] "Carriers" as used herein include pharmaceutically
acceptable carriers, excipients, or stabilizers which are nontoxic
to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often the physiologically acceptable
carrier is an aqueous pH buffered solution. Examples of
physiologically acceptable carriers include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid; low molecular weight (less than about 10 residues)
polypeptide; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants such as
TWEEN.TM., polyethylene glycol (PEG), and PLURONICS.TM..
[0046] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0047] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g. I.sup.131, I.sup.125, Y.sup.90 and
Re.sup.186), chemotherapeutic agents, and toxins such as
enzymatically active toxins of bacterial, fungal, plant or animal
origin, or fragments thereof.
[0048] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include adriamycin, doxorubicin, epirubicin, 5-fluorouracil,
cytosine arabinoside ("Ara-C"), cyclophosphamide, thiotepa,
busulfan, cytoxin, taxoids, e.g. paclitaxel (Taxol, Bristol-Myers
Squibb Oncology, Princeton, N.J.), and doxetaxel (Taxotere,
Rhone-Poulenc Rorer, Antony, Rnace), toxotere, methotrexate,
cisplatin, melphalan, vinblastine, bleomycin, etoposide,
ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine,
carboplatin, teniposide, daunomycin, caminomycin, aminopterin,
dactinomycin, mitomycins, esperamicins (see U.S. Pat. No.
4,675,187), melphalan and other related nitrogen mustards. Also
included in this definition are hormonal agents that act to
regulate or inhibit hormone action on tumors such as tamoxifen and
onapristone.
[0049] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell, especially
cancer cell overexpressing any of the genes identified herein,
either in vitro or in vivo. Thus, the growth inhibitory agent is
one which significantly reduces the percentage of cells
overexpressing such genes in S phase of the cell cycle. Examples of
growth inhibitory agents include agents that block cell cycle
progression (at a place other than S phase), such as agents that
induce G1 arrest and M-phase arrest. Classical M-phase blockers
include the vincas (vincristine and vinblastine), taxol, and topo
II inhibitors such as doxorubicin, epirubicin, daunorubicin,
etoposide, and bleomycin. Those agents that arrest G1 also spill
over into S-phase arrest, for example, DNA alkylating agents such
as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin,
methotrexate, 5-fluorouracil, and ara-C. Further information can be
found in The MolecularBasis of Cancer, Mendelsohn and Israel, eds.,
Chapter 1, entitled "Cell cycle regulation, oncogens, and
antineoplastic drugs" by Murakami et al. (W B Saunders:
Philadelphia, 1995), especially p. 13.
[0050] "Doxorubicin" is an athracycline antibiotic. The full
chemical name of doxorubicin is
(8S-cis)-10-[(3-amino-2,3,6-trideoxy-.alpha.-L-lyxo-hexapyranosyl)oxy]-7,-
8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-napht-
hacenedione.
[0051] The term "cytokine" is a generic term for proteins released
by one cell population which act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytokines
are growth hormone such as human growth hormone, N-methionyl human
growth hormone, and bovine growth hormone; parathyroid hormone;
thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein
hormones such as follicle stimulating hormone (FSH), thyroid
stimulating hormone (TSH), and luteinizing hormone (LH); hepatic
growth factor; fibroblast growth factor; prolactin; placental
lactogen; tumor necrosis factor-.alpha. and -.beta.;
mullerian-inhibiting substance; mouse gonadotropin-associated
peptide; inhibin; activin; vascular endothelial growth factor;
integrin; thrombopoietin (TPO); nerve growth factors such as
NGF-.beta.; platelet-growth factor; transforming growth factors
(TGFs) such as TGF-.alpha. and TGF-.beta.; insulin-like growth
factor-I and -II; erythropoietin (EPO); osteoinductive factors;
interferons such as interferon-.alpha., -.beta., and -.gamma.;
colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);
interleukins (ILs) such as IL-1, IL-1.alpha., IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor
such as TNF-.alpha. or TNF-.beta.; and other polypeptide factors
including LIF and kit ligand (KL). As used herein, the term
cytokine includes proteins from natural sources or from recombinant
cell culture and biologically active equivalents of the native
sequence cytokines.
[0052] As used herein, the terms a "CT-1" polypeptide is used to
refer to a polypeptide comprising a native sequence polypeptide
having the same amino acid sequence as a corresponding CT-1
polypeptide derived from nature, and fragments of such native
sequence polypeptides. Such native sequence CT-1 polypeptides can
be isolated from nature or, along with the respective fragments,
can be produced by recombinant and/or synthetic means. The term
specifically encompasses naturally-occurring truncated or secreted
forms (e.g., an extracellular domain sequence), naturally-occurring
variant forms (e.g., alternatively spliced forms) and
naturally-occurring allelic variants of the CT-1 polypeptide. In
one embodiment of the invention, the native sequence CT-1 is a
full-length native presequence or a mature form of a CT-1
polypeptide shown in FIG. 1 (SEQ ID NO:3). Fragments of the
respective native polypeptides herein include, but are not limited
to, polypeptide variants from which the native N-terminal signal
sequence has been fully or partially deleted or replaced by another
sequence, and extracellular domains of the respective native
sequences, regardless whether such truncated (secreted) forms occur
in nature.
[0053] An "isolated" nucleic acid molecule encoding a CT-1
polypeptide is a nucleic acid molecule that is identified and
separated from at least one contaminant nucleic acid molecule with
which it is ordinarily associated in the natural source of the
CT-1-encoding nucleic acid. An isolated CT-1-encoding nucleic acid
molecule is other than in the form or setting in which it is found
in nature. Isolated nucleic acid molecules therefore are
distinguished from the CT-1-encoding nucleic acid molecule as it
exists in natural cells. However, an isolated nucleic acid molecule
encoding a CT-1 polypeptide includes nucleic acid molecules
contained in cells that ordinarily express CT-1, where, for
example, the nucleic acid molecule is in a chromosomal location
different from that of natural cells.
[0054] 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.
[0055] 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.
[0056] "Stringency" of hybridization reactions is readily
determinable by one of ordinary skill in the art, and generally is
an empirical calculation dependent upon probe length, washing
temperature, and salt concentration. In general, longer probes
require higher temperatures for proper annealing, while shorter
probes need lower temperatures. Hybridization generally depends on
the ability of denatured DNA to reanneal when complementary strands
are present in an environment below their melting temperature. The
higher the degree of desired homology between the probe and
hybridizable sequence, the higher the relative temperature which
can be used. As a result, it follows that higher relative
temperatures would tend to make the reaction conditions more
stringent, while lower temperatures less so. For additional details
and explanation of stringency of hybridization reactions, see
Ausubel et al., Current Protocols in Molecular Biology, Wiley
Interscience Publishers, (1995).
[0057] "Stringent conditions" or "high stringency conditions", as
defined herein, may be identified by those that: (1) employ low
ionic strength and high temperature for washing, for example 0.015
M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl
sulfate at 50.degree. C.; (2) employ during hybridization a
denaturing agent, such as formamide, for example, 50% (v/v)
formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%
polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with
750 mM sodium chloride, 75 mM sodium citrate at 42.degree. C.; or
(3) employ 50% formamide, 5.times.SSC (0.75 M NaCl, 0.075 M sodium
citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium
pyrophosphate, 5.times.Denhardt's solution, sonicated salmon sperm
DNA (50 .mu.g/ml), 0.1% SDS, and 10% dextran sulfate at 42.degree.
C., with washes at 42.degree. C. in 0.2.times.SSC (sodium
chloride/sodium citrate) and 50% formamide at 55.degree. C.,
followed by a high-stringency wash consisting of 0.1.times.SSC
containing EDTA at 55.degree. C.
[0058] "Moderately stringent conditions" may be identified as
described by Sambrook et al., Molecular Cloning: A Laboratory
Manual, New York: Cold Spring Harbor Press, 1989, and include the
use of washing solution and hybridization conditions (e.g.,
temperature, ionic strength and % SDS) less stringent than those
described above. An example of moderately stringent conditions is
overnight incubation at 37.degree. C. in a solution comprising: 20%
formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times.Denhardt's solution, 10%
dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA,
followed by washing the filters in 1.times.SSC at about
37-50.degree. C. The skilled artisan will recognize how to adjust
the temperature, ionic strength, etc. as necessary to accommodate
factors such as probe length and the like.
[0059] The term "epitope tagged" when used herein refers to a
chimeric polypeptide comprising a CT-1 polypeptide fused to a "tag
polypeptide". The tag polypeptide has enough residues to provide an
epitope against which an antibody can be made, yet is short enough
such that it does not interfere with activity of the polypeptide to
which it is fused. The tag polypeptide preferably also is fairly
unique so that the antibody does not substantially cross-react with
other epitopes. Suitable tag polypeptides generally have at least
six amino acid residues and usually between about 8 and 50 amino
acid residues (preferably, between about 10 and 20 amino acid
residues).
[0060] "Active" or "activity" in the context of molecules
identified based upon the CT-1 polypeptides (or their coding
sequences) refers to polypeptides (e.g. antibodies) or organic or
inorganic small molecules, peptides, etc. which retain the
biological and/or immunological activities/properties of a native
or naturally-occurring CT-1.
[0061] "Biological activity" in the context of an antibody or
another molecule that can be identified by the screening assays
disclosed herein (e.g. an organic or inorganic small molecule,
peptide, etc.) is used to refer to the ability of such molecules to
bind or complex with the polypeptides encoded by the amplified
genes identified herein, or otherwise interfere with the
interaction of the encoded polypeptides with other cellular
proteins. A preferred biological activity is growth inhibition of a
target tumor cell. Another preferred biological activity is
cytotoxic activity resulting in the death of the target tumor
cell.
[0062] The phrase "immunological property" means immunological
cross-reactivity with at least one epitope of a CT-1
polypeptide.
[0063] "Immunological cross-reactivity" as used herein means that
the candidate polypeptide is capable of competitively inhibiting
the qualitative biological activity of a CT-1 polypeptide having
this activity with polyclonal antisera raised against the known
active CT-1 polypeptide. Such antisera are prepared in conventional
fashion by injecting goats or rabbits, for example, subcutaneously
with the known active analogue in complete Freund's adjuvant,
followed by booster intraperitoneal or subcutaneous injection in
incomplete Freunds. The immunological cross-reactivity preferably
is "specific", which means that the binding affinity of the
immunologically cross-reactive molecule (e.g. antibody) identified,
to the corresponding CT-1 polypeptide is significantly higher
(preferably at least about 2-times, more preferably at least about
4-times, even more preferably at least about 8-times, most
preferably at least about 8-times higher) than the binding affinity
of that molecule to any other known native polypeptide.
[0064] The term "antagonist" is used in the broadest sense, and
includes any molecule that partially or fully blocks, inhibits, or
neutralizes a biological activity of a native CT-1 polypeptide
disclosed herein. In a similar manner, the term "agonist" is used
in the broadest sense and includes any molecule that mimics a
biological activity of a native CT-1 polypeptide disclosed herein.
Suitable agonist or antagonist molecules specifically include
agonist or antagonist antibodies or antibody fragments, fragments
or amino acid sequence variants of native polypeptides, peptides,
small organic molecules, etc.
[0065] A "small molecule" is defined herein to have a molecular
weight below about 500 daltons.
[0066] "Antibodies" (Abs) and "immunoglobulins" (Igs) are
glycoproteins having the same structural characteristics. While
antibodies exhibit binding specificity to a specific antigen,
immunoglobulins include both antibodies and other antibody-like
molecules which lack antigen specificity. Polypeptides of the
latter kind are, for example, produced at low levels by the lymph
system and at increased levels by myelomas. The term "antibody" is
used in the broadest sense and specifically covers, without
limitation, intact monoclonal antibodies, polyclonal antibodies,
multispecific antibodies (e.g. bispecific antibodies) formed from
at least two intact antibodies, and antibody fragments so long as
they exhibit the desired biological activity.
[0067] "Native antibodies" and "native immunoglobulins" are usually
heterotetrameric glycoproteins of about 150,000 daltons, composed
of two identical light (L) chains and two identical heavy (H)
chains. Each light chain is linked to a heavy chain by one covalent
disulfide bond, while the number of disulfide linkages varies among
the heavy chains of different immunoglobulin isotypes. Each heavy
and light chain also has regularly spaced intrachain disulfide
bridges. Each heavy chain has at one end a variable domain
(V.sub.H) followed by a number of constant domains. Each light
chain has a variable domain at one end (V.sub.L) and a constant
domain at its other end; the constant domain of the light chain is
aligned with the first constant domain of the heavy chain, and the
light-chain variable domain is aligned with the variable domain of
the heavy chain. Particular amino acid residues are believed to
form an interface between the light- and heavy-chain variable
domains.
[0068] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
complementarity-determining regions (CDRs) or hypervariable regions
both in the light-chain and the heavy-chain variable domains. The
more highly conserved portions of variable domains are called the
framework (FR). The variable domains of native heavy and light
chains each comprise four FR regions, largely adopting a
.beta.-sheet configuration, connected by three CDRs, which form
loops connecting, and in some cases forming part of, the
.beta.-sheet structure. The CDRs in each chain are held together in
close proximity by the FR regions and, with the CDRs from the other
chain, contribute to the formation of the antigen-binding site of
antibodies (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pages
647-669 (1991)). The constant domains are not involved directly in
binding an antibody to an antigen, but exhibit various effector
functions, such as participation of the antibody in
antibody-dependent cellular toxicity.
[0069] "Antibody fragments" comprise a portion of an intact
antibody, preferably the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; linear antibodies
(Zapata et al., Protein Eng. 8(10):1057-1062 [1995]); single-chain
antibody molecules; and multispecific antibodies formed from
antibody fragments.
[0070] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen-combining
sites and is still capable of cross-linking antigen.
[0071] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and -binding site. This region
consists of a dimer of one heavy- and one light-chain variable
domain in tight, non-covalent association. It is in this
configuration that the three CDRs of each variable domain interact
to define an antigen-binding site on the surface of the
V.sub.H-V.sub.L dimer. Collectively, the six CDRs confer
antigen-binding specificity to the antibody. However, even a single
variable domain (or half of an Fv comprising only three CDRs
specific for an antigen) has the ability to recognize and bind
antigen, although at a lower affinity than the entire binding
site.
[0072] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CH1) of the heavy chain.
Fab fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0073] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa (.kappa.) and lambda (.lamda.), based on the
amino acid sequences of their constant domains.
[0074] Depending on the amino acid sequence of the constant domain
of their heavy chains, immunoglobulins can be assigned to different
classes. There are five major classes of immunoglobulins: IgA, IgD,
IgE, IgG, and IgM, and several of these may be further divided into
subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
The heavy-chain constant domains that correspond to the different
classes of immunoglobulins are called .alpha., .delta., .xi.,
.gamma., and .mu., respectively. The subunit structures and
three-dimensional configurations of different classes of
immunoglobulins are well known.
[0075] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations which typically include
different antibodies directed against different determinants
(epitopes), each monoclonal antibody is directed against a single
determinant on the antigen. In addition to their specificity, the
monoclonal antibodies are advantageous in that they are synthesized
by the hybridoma culture, uncontaminated by other immunoglobulins.
The modifier "monoclonal" indicates the character of the antibody
as being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of
the antibody by any particular method. For example, the monoclonal
antibodies to be used in accordance with the present invention may
be made by the hybridoma method first described by Kohler et al.,
Nature, 256:495 [1975], or may be made by recombinant DNA methods
(see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies"
may also be isolated from phage antibody libraries using the
techniques described in Clackson et al., Nature, 352:624-628 [1991]
and Marks et al., J. Mol. Biol., 222:581-597 (1991), for
example.
[0076] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins) in which a portion of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired biological activity (U.S. Pat. No. 4,816,567; Morrison et
al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 [1984]).
[0077] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric 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. For the most part,
humanized antibodies are human immunoglobulins (recipient antibody)
in which residues from a 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 FR residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Furthermore, humanized antibodies may comprise residues which are
found neither in the recipient antibody nor in the imported CDR or
framework sequences. These modifications are made to further refine
and maximize antibody performance. 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 sequence. The humanized antibody
optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature,
321:522-525 (1986); Reichmann et al., Nature, 332:323-329 [1988];
and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992). The
humanized antibody includes a PRIMATIZED.TM. antibody wherein the
antigen-binding region of the antibody is derived from an antibody
produced by immunizing macaque monkeys with the antigen of
interest.
[0078] "Single-chain Fv" or "sFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Preferably, the Fv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the sFv to form the
desired structure for antigen binding. For a review of sFv see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994).
[0079] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy-chain
variable domain (V.sub.H) connected to a light-chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L).
By using a linker that is too short to allow pairing between the
two domains on the same chain, the domains are forced to pair with
the complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, for
example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.
Acad. Sci. USA, 90:6444-6448 (1993).
[0080] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the antibody will be purified (1) to greater than 95%
by weight of antibody as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated antibody
includes the antibody in situ within recombinant cells since at
least one component of the antibody's natural environment will not
be present. Ordinarily, however, isolated antibody will be prepared
by at least one purification step.
[0081] The word "label" when used herein refers to a detectable
compound or composition which is conjugated directly or indirectly
to the antibody so as to generate a "labeled" antibody. The label
may be detectable by itself (e.g. radioisotope labels or
fluorescent labels) or, in the case of an enzymatic label, may
catalyze chemical alteration of a substrate compound or composition
which is detectable.
[0082] By "solid phase" is meant a non-aqueous matrix to which the
antibody of the present invention can adhere. Examples of solid
phases encompassed herein include those formed partially or
entirely of glass (e.g., controlled pore glass), polysaccharides
(e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol
and silicones. In certain embodiments, depending on the context,
the solid phase can comprise the well of an assay plate; in others
it is a purification column (e.g., an affinity chromatography
column). This term also includes a discontinuous solid phase of
discrete particles, such as those described in U.S. Pat. No.
4,275,149.
[0083] A "liposome" is a small vesicle composed of various types of
lipids, phospholipids and/or surfactant which is useful for
delivery of a drug (such as an CT-1 polypeptide or an antibody
thereto and, optionally, a chemotherapeutic agent) to a mammal. The
components of the liposome are commonly arranged in a bilayer
formation, similar to the lipid arrangement of biological
membranes.
[0084] As used herein, the term "immunoadhesin" designates
antibody-like molecules which combine the binding specificity of a
heterologous protein (an "adhesin") with the effector functions of
immunoglobulin constant domains. Structurally, the immunoadhesins
comprise a fusion of an amino acid sequence with the desired
binding specificity which is other than the antigen recognition and
binding site of an antibody (i.e., is "heterologous"), and an
immunoglobulin constant domain sequence. The adhesin part of an
immunoadhesin molecule typically is a contiguous amino acid
sequence comprising at least the binding site of a receptor or a
ligand. The immunoglobulin constant domain sequence in the
immunoadhesin may be obtained from any immunoglobulin, such as
IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and
IgA-2), IgE, IgD or IgM.
II. Compositions and Methods of the Invention
[0085] 1. Preparation of the CT-1 Polypeptides
[0086] The present invention provides methods for using DNA58125
encoding CT-1 for the production of compounds inhibiting neoplastic
growth as well as for the preparation of screening methods for
identifying growth inhibitory compounds (e.g. tumor compounds). In
particular, cDNAs encoding certain CT-1 polypeptides. For the sake
of simplicity, in the present specification the proteins encoded by
nucleic acid referred to as "DNA58125", as well as all further
native homologues and variants included in the foregoing definition
of CT-1 polypeptide, will be referred to as "CT-1" polypeptide,
regardless of their origin or mode of expression.
[0087] The description below relates primarily to production of
CT-1 polypeptides by culturing cells transformed or transfected
with a vector containing CT-1-encoding nucleic acid. It is, of
course, contemplated that alternative methods, which are well known
in the art, may be employed to prepare CT-1 polypeptides. For
instance, the CT-1 polypeptide sequence, or portions thereof, may
be produced by direct peptide synthesis using solid-phase
techniques [see, e.g., Stewart et al., Solid-Phase Peptide
Synthesis, W.H. Freeman Co., San Francisco, Calif. (1969);
Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)]. In vitro
protein synthesis may be performed using manual techniques or by
automation. Automated synthesis may be accomplished, for instance,
using an Applied Biosystems Peptide Synthesizer (Foster City,
Calif.) using manufacturer's instructions. Various portions of the
CT-1 polypeptide may be chemically synthesized separately and
combined using chemical or enzymatic methods to produce the
full-length CT-1.
[0088] i. Synthesis or Isolation of DNA Encoding a CT-1
Polypeptide.
[0089] DNA encoding CT-1, homologues, variants, or portions
thereof, may be produced by direct DNA synthesis using standard
nucleic acid synthetic techniques [see, e.g., Gait, M. J.,
Oligonucleotide Synthesis, IRL Press, Oxford, 1984]. In vitro DNA
synthesis may be performed using manual techniques or by
automation. Automated oliogonucleotide synthesis may be
accomplished, for instance, using standard techniques. Various
portions of the CT-1-encoding nucleic acid sequence may be
chemically synthesized separately and combined using chemical or
enzymatic methods to produce the full-length CT-1-encoding
sequence.
[0090] Alternatively, DNA encoding CT-1 may be obtained from a cDNA
library prepared from tissue believed to possess the CT-1 mRNA and
to express it at a detectable level. Accordingly, human CT-1 DNA
can be conveniently obtained from a cDNA library prepared from
human tissue, such as described in the Examples. The CT-1-encoding
gene may also be obtained from a genomic library or by
oligonucleotide synthesis.
[0091] Libraries can be screened with probes (such as antibodies to
the CT-1 polypeptide, or oligonucleotides of at least about 20-80
bases) designed to identify the gene of interest or the protein
encoded by it. Screening the cDNA or genomic library with the
selected probe may be conducted using standard procedures, such as
described in Sambrook et al., Molecular Cloning: A Laboratory
Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An
alternative means to isolate the gene encoding CT-1 is to use PCR
methodology [Sambrook et al., supra; Dieffenbach et al., PCR
Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press,
1995)].
[0092] The Examples below describe techniques for screening a cDNA
library. The oligonucleotide sequences selected as probes should be
of sufficient length and sufficiently unambiguous that false
positives are minimized. The oligonucleotide is preferably labeled
such that it can be detected upon hybridization to DNA in the
library being screened. Methods of labeling are well known in the
art, and include the use of radiolabels like .sup.32P-labeled ATP,
biotinylation or enzyme labeling. Hybridization conditions,
including moderate stringency and high stringency, are provided in
Sambrook et al., supra.
[0093] Sequences identified in such library screening methods can
be compared and aligned to other known sequences deposited and
available in public databases such as GenBank or other private
sequence databases. Sequence identity (at either the amino acid or
nucleotide level) within defined regions of the molecule or across
the full-length sequence can be determined through sequence
alignment using computer software programs such as ALIGN, DNAstar,
and INHERIT which employ various algorithms to measure
homology.
[0094] Nucleic acid having protein coding sequence may be obtained
by screening selected cDNA or genomic libraries using the deduced
amino acid sequence disclosed herein for the first time, and, if
necessary, using conventional primer extension procedures as
described in Sambrook et al., supra, to detect precursors and
processing intermediates of mRNA that may not have been
reverse-transcribed into cDNA.
[0095] ii. Selection and Transformation of Host Cells
[0096] Host cells are transfected or transformed with expression or
cloning vectors described herein for CT-1 production and cultured
in conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes
encoding the desired sequences. The culture conditions, such as
media, temperature, pH and the like, can be selected by the skilled
artisan without undue experimentation. In general, principles,
protocols, and practical techniques for maximizing the productivity
of cell cultures can be found in Mammalian Cell Biotechnology: a
Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook
et al., supra.
[0097] Methods of transfection are known to the ordinarily skilled
artisan, for example, CaPO.sub.4 and electroporation. Depending on
the host cell used, transformation is performed using standard
techniques appropriate to such cells. The calcium treatment
employing calcium chloride, as described in Sambrook et al., supra,
or electroporation is generally used for prokaryotes or other cells
that contain substantial cell-wall barriers. Infection with
Agrobacterium tumefaciens is used for transformation of certain
plant cells, as described by Shaw et al., Gene, 23:315 (1983) and
WO 89/05859 published 29 Jun. 1989. For mammalian cells without
such cell walls, the calcium phosphate precipitation method of
Graham and van der Eb, Virology, 52:456-457 (1978) can be employed.
General aspects of mammalian cell host system transformations have
been described in U.S. Pat. No. 4,399,216. Transformations into
yeast are typically carried out according to the method of Van
Solingen et al., J. Bact., 130:946 (1977) and Hsiao et al., Proc.
Natl. Acad. Sci. (USA), 76:3829 (1979). However, other methods for
introducing DNA into cells, such as by nuclear microinjection,
electroporation, bacterial protoplast fusion with intact cells, or
polycations, e.g., polybrene, polyornithine, may also be used. For
various techniques for transforming mammalian cells, see Keown et
al., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,
Nature, 336:348-352 (1988).
[0098] Suitable host cells for cloning or expressing the DNA in the
vectors herein include prokaryote, yeast, or higher eukaryote
cells. Suitable prokaryotes include, but are not limited to,
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, Enterobacteriaceae such as E. coli. Various E. coli
strains are publicly available, such as E. coli K12 strain MM294
(ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110
(ATCC 27,325) and K5 772 (ATCC 53,635).
[0099] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for CT-1-encoding vectors. Saccharomyces cerevisiae is a commonly
used lower eukaryotic host microorganism.
[0100] Suitable host cells for the expression of glycosylated CT-1
are derived from multicellular organisms. Examples of invertebrate
cells include insect cells such as Drosophila S2 and Spodoptera
Sf9, as well as plant cells. Examples of useful mammalian host cell
lines include Chinese hamster ovary (CHO) and COS cells. More
specific examples include monkey kidney CV1 line transformed by
SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or
293 cells subcloned for growth in suspension culture, Graham et
al., J. Gen Virol., 36:59 (1977)); Chinese hamster ovary
cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,
77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.,
23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human
liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562,
ATCC CCL51). The selection of the appropriate host cell is deemed
to be within the skill in the art.
[0101] iii. Selection and Use of a Replicable Vector
[0102] The nucleic acid (e.g., cDNA or genomic DNA) encoding CT-1
may be inserted into a replicable vector for cloning (amplification
of the DNA) or for expression. Various vectors are publicly
available. The vector may, for example, be in the form of a
plasmid, cosmid, viral particle, or phage. The appropriate nucleic
acid sequence may be inserted into the vector by a variety of
procedures. In general, DNA is inserted into an appropriate
restriction endonuclease site(s) using techniques known in the art.
Vector components generally include, but are not limited to, one or
more of a signal sequence, an origin of replication, one or more
marker genes, an enhancer element, a promoter, and a transcription
termination sequence. Construction of suitable vectors containing
one or more of these components employs standard ligation
techniques which are known to the skilled artisan.
[0103] The CT-1 polypeptide may be produced recombinantly not only
directly, but also as a fusion polypeptide with a heterologous
polypeptide, which may be a signal sequence or other polypeptide
having a specific cleavage site at the N-terminus of the mature
protein or polypeptide. In general, the signal sequence may be a
component of the vector, or it may be a part of the CT-1-encoding
DNA that is inserted into the vector. The signal sequence may be a
prokaryotic signal sequence selected, for example, from the group
of the alkaline phosphatase, penicillinase, lpp, or heat-stable
enterotoxin II leaders. For yeast secretion the signal sequence may
be, e.g., the yeast invertase leader, alpha factor leader
(including Saccharomyces and Kluyveromyces .alpha.-factor leaders,
the latter described in U.S. Pat. No. 5,010,182), or acid
phosphatase leader, the C. albicans glucoamylase leader (EP 362,179
published 4 Apr. 1990), or the signal described in WO 90/13646
published 15 Nov. 1990. In mammalian cell expression, mammalian
signal sequences may be used to direct secretion of the protein,
such as signal sequences from secreted polypeptides of the same or
related species, as well as viral secretory leaders.
[0104] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Such sequences are well known for a variety of
bacteria, yeast, and viruses. The origin of replication from the
plasmid pBR322 is suitable for most Gram-negative bacteria, the
2.mu. plasmid origin is suitable for yeast, and various viral
origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for
cloning vectors in mammalian cells.
[0105] Expression and cloning vectors will typically contain a
selection gene, also termed a selectable marker. Typical selection
genes encode proteins that (a) confer resistance to antibiotics or
other toxins, e.g., ampicillin, neomycin, methotrexate, or
tetracycline, (b) complement auxotrophic deficiencies, or (c)
supply critical nutrients not available from complex media, e.g.,
the gene encoding D-alanine racemase for Bacilli.
[0106] An example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the CT-1-encoding nucleic acid, such as DHFR or
thymidine kinase. An appropriate host cell when wild-type DHFR is
employed is the CHO cell line deficient in DHFR activity, prepared
and propagated as described by Urlaub et al., Proc. Natl. Acad.
Sci. USA, 77:4216 (1980). A suitable selection gene for use in
yeast is the trp1 gene present in the yeast plasmid YRp7
[Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene,
7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1 gene
provides a selection marker for a mutant strain of yeast lacking
the ability to grow in tryptophan, for example, ATCC No. 44076 or
PEP4-1 [Jones, Genetics, 85:12 (1977)].
[0107] Expression and cloning vectors usually contain a promoter
operably linked to the CT-1-encoding nucleic acid sequence to
direct mRNA synthesis. Promoters recognized by a variety of
potential host cells are well known. Promoters suitable for use
with prokaryotic hosts include the .beta.-lactamase and lactose
promoter systems [Chang et al., Nature, 275:615 (1978); Goeddel et
al., Nature, 281:544 (1979)], alkaline phosphatase, a tryptophan
(trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980);
EP 36,776], and hybrid promoters such as the tac promoter [deBoer
et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)]. Promoters for
use in bacterial systems also will contain a Shine-Dalgarno (S.D.)
sequence operably linked to the DNA encoding CT-1.
[0108] Examples of suitable promoting sequences for use with yeast
hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman
et al., J. Biol. Chem., 255:2073 (1980)] or other glycolytic
enzymes [Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland,
Biochemistry, 17:4900 (1978)], such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase.
[0109] Other yeast promoters, which are inducible promoters having
the additional advantage of transcription controlled by growth
conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible
for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in EP
73,657.
[0110] CT-1 transcription from vectors in mammalian host cells is
controlled, for example, by promoters obtained from the genomes of
viruses such as polyoma virus, fowlpox virus (UK 2,211,504
published 5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine
papilloma virus, avian sarcoma virus, cytomegalovirus, a
retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from
heterologous mammalian promoters, e.g., the actin promoter or an
immunoglobulin promoter, and from heat-shock promoters, provided
such promoters are compatible with the host cell systems.
[0111] Transcription of a DNA encoding a CT-1 polypeptide by higher
eukaryotes may be increased by inserting an enhancer sequence into
the vector. Enhancers are cis-acting elements of DNA, usually about
from 10 to 300 bp, that act on a promoter to increase its
transcription. Many enhancer sequences are now known from mammalian
genes (globin, elastase, albumin, .alpha.-fetoprotein, and
insulin). Typically, however, one will use an enhancer from a
eukaryotic cell virus. Examples include the SV40 enhancer on the
late side of the replication origin (bp 100-270), the
cytomegalovirus early promoter enhancer, the polyoma enhancer on
the late side of the replication origin, and adenovirus enhancers.
The enhancer may be spliced into the vector at a position 5' or 3'
to the CT-1 coding sequence, but is preferably located at a site 5'
from the promoter.
[0112] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human, or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from the 5' and, occasionally 3',
untranslated regions of eukaryotic or viral DNAs or cDNAs. These
regions contain nucleotide segments transcribed as polyadenylated
fragments in the untranslated portion of the mRNA encoding
CT-1.
[0113] Still other methods, vectors, and host cells suitable for
adaptation to the synthesis of CT-1 in recombinant vertebrate cell
culture are described in Gething et al., Nature, 293:620-625
(1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP
117,058.
[0114] iv. Detecting Gene Amplification/Expression
[0115] Gene amplification and/or expression may be measured in a
sample directly, for example, by conventional Southern blotting,
Northern blotting to quantitate the transcription of mRNA [Thomas,
Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA
analysis), or in situ hybridization, using an appropriately labeled
probe, based on the sequences provided herein. Alternatively,
antibodies may be employed that can recognize specific duplexes,
including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes
or DNA-protein duplexes. The antibodies in turn may be labeled and
the assay may be carried out where the duplex is bound to a
surface, so that upon the formation of duplex on the surface, the
presence of antibody bound to the duplex can be detected.
[0116] Gene expression, alternatively, may be measured by
immunological methods, such as immunohistochemical staining of
cells or tissue sections and assay of cell culture or body fluids,
to quantitate directly the expression of gene product. Antibodies
useful for immunohistochemical staining and/or assay of sample
fluids may be either monoclonal or polyclonal, and may be prepared
in any mammal. Conveniently, the antibodies may be prepared against
a native sequence CT-1 polypeptide or against a synthetic peptide
based on the DNA sequences provided herein or against exogenous
sequence fused to CT-1 DNA and encoding a specific antibody
epitope.
[0117] v. Purification of Polypeptide
[0118] Forms of CT-1 polypeptides may be recovered from culture
medium or from host cell lysates. If membrane-bound, it can be
released from the membrane using a suitable detergent solution
(e.g. Triton-X 100) or by enzymatic cleavage. Cells employed in
expression of CT-1 can be disrupted by various physical or chemical
means, such as freeze-thaw cycling, sonication, mechanical
disruption, or cell lysing agents.
[0119] It may be desired to purify CT-1 from recombinant cell
proteins or polypeptides. The following procedures are exemplary of
suitable purification procedures: by fractionation on an
ion-exchange column; ethanol precipitation; reverse phase HPLC;
chromatography on silica or on a cation-exchange resin such as
DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation;
gel filtration using, for example, Sephadex G-75; protein A
Sepharose columns to remove contaminants such as IgG; and metal
chelating columns to bind epitope-tagged forms of the CT-1
polypeptides. Various methods of protein purification may be
employed and such methods are known in the art and described for
example in Deutscher, Methods in Enzymology, 182 (1990); Scopes,
Protein Purification: Principles and Practice, Springer-Verlag, New
York (1982). The purification step(s) selected will depend, for
example, on the nature of the production process used and the
particular CT-1 polypeptide produced.
[0120] 2. Amplification of Genes Encoding the CT-1 Polypeptides in
Tumor Tissues and Cell Lines
[0121] The present invention is based on the identification and
characterization of genes which are amplified in certain cancer
cells.
[0122] The genome of prokaryotic and eukaryotic organisms is
subjected to two seemingly conflicting requirements. One is the
preservation and propagation of DNA as the genetic information in
its original form, to guarantee stable inheritance through multiple
generations. On the other hand, cells or organisms must be able to
adapt to lasting environmental changes. The adaptive mechanisms can
include qualitative or quantitative modifications of the genetic
material. Qualitative modifications include DNA mutations, in which
coding sequences are altered resulting in a structurally and/or
functionally different protein. Gene amplification is a
quantitative modification, whereby the actual number of complete
coding sequence, i.e. a gene, increases, leading to an increased
number of available templates for transcription, an increased
number of translatable transcripts, and, ultimately, to an
increased abundance of the protein encoded by the amplified
gene.
[0123] The phenomenon of gene amplification and its underlying
mechanisms have been investigated in vitro in several prokaryotic
and eukaryotic culture systems. The best-characterized example of
gene amplification involves the culture of eukaryotic cells in
medium containing variable concentrations of the cytotoxic drug
methotrexate (MTX). MTX is a folic acid analogue and interferes
with DNA synthesis by blocking the enzyme dihydrofolate reductase
(DHFR). During the initial exposure to low concentrations of MTX
most cells (>99.9%) will die. A small number of cells survive,
and are capable of growing in increasing concentrations of MTX by
producing large amounts of DHFR-RNA and protein. The basis of this
overproduction is the amplification of the single DHFR gene. The
additional copies of the gene are found as extrachromosomal copies
in the form of small, supernumerary chromosomes (double minutes) or
as integrated chromosomal copies.
[0124] Gene amplification is most commonly encountered in the
development of resistance to cytotoxic drugs (antibiotics for
bacteria and chemotherapeutic agents for eukaryotic cells) and
neoplastic transformation. Transformation of a eukaryotic cell as a
spontaneous event or due to a viral or chemical/environmental
insult is typically associated with changes in the genetic material
of that cell. One of the most common genetic changes observed in
human malignancies are mutations of the p53 protein. p53 controls
the transition of cells from the stationary (G1) to the replicative
(S) phase of the cell cycle and prevents this transition in the
presence of DNA damage. In other words, one of the main
consequences of disabling p53 mutations is the accumulation and
propagation of DNA damage, i.e. genetic changes. Common types of
genetic changes in neoplastic cells are, in addition to point
mutations, amplifications and gross, structural alterations, such
as translocations.
[0125] The amplification of DNA sequences may indicate specific
functional requirement as illustrated in the DHFR experimental
system. Therefore, the amplification of certain oncogenes in
malignancies points toward a causative role of these genes in the
process of malignant transformation and maintenance of the
transformed phenotype. This hypothesis has gained support in recent
studies. For example, the bcl-2 protein was found to be amplified
in certain types of non-Hodgkin's lymphoma. This protein inhibits
apoptosis and leads to the progressive accumulation of neoplastic
cells. Members of the gene family of growth factor receptors have
been found to be amplified in various types of cancers suggesting
that overexpression of these receptors may make neoplastic cells
less susceptible to limiting amounts of available growth factor.
Examples include the amplification of the androgen receptor in
recurrent prostate cancer during androgen deprivation therapy and
the amplification of the growth factor receptor homologue ERB2 in
breast cancer. Lastly, genes involved in intracellular signaling
and control of cell cycle progression can undergo amplification
during malignant transformation. This is illustrated by the
amplification of the bcl-I and ras genes in various epithelial and
lymphoid neoplasms.
[0126] These earlier studies illustrate the feasibility of
identifying amplified DNA sequences in neoplasms, because this
approach can identify genes important for malignant transformation.
The case of ERB2 also demonstrates the feasibility from a
therapeutic standpoint, since transforming proteins may represent
novel and specific targets for tumor therapy.
[0127] Several different techniques can be used to demonstrate
amplified genomic sequences. Classical cytogenetic analysis of
chromosome spreads prepared from cancer cells is adequate to
identify gross structural alterations, such as translocations,
deletions and inversions. Amplified genomic regions can only be
visualized, if they involve large regions with high copy numbers or
are present as extrachromosomal material. While cytogenetics was
the first technique to demonstrate the consistent association of
specific chromosomal changes with particular neoplasms, it is
inadequate for the identification and isolation of manageable DNA
sequences. The more recently developed technique of comparative
genomic hybridization (CGH) has illustrated the widespread
phenomenon of genomic amplification in neoplasms. Tumor and normal
DNA are hybridized simultaneously onto metaphases of normal cells
and the entire genome can be screened by image analysis for DNA
sequences that are present in the tumor at an increased frequency.
(WO 93/18,186; Gray et al., Radiation Res. 137, 275-289 [1994]). As
a screening method, this type of analysis has revealed a large
number of recurring amplicons (a stretch of amplified DNA) in a
variety of human neoplasms. Although CGH is more sensitive than
classical cytogenetic analysis in identifying amplified stretches
of DNA, it does not allow a rapid identification and isolation of
coding sequences within the amplicon by standard molecular genetic
techniques.
[0128] The most sensitive methods to detect gene amplification are
polymerase chain reaction (PCR)-based assays. These assays utilize
very small amount of tumor DNA as starting material, are
exquisitely sensitive, provide DNA that is amenable to further
analysis, such as sequencing and are suitable for high-volume
throughput analysis.
[0129] The above-mentioned assays are not mutually exclusive, but
are frequently used in combination to identify amplifications in
neoplasms. While cytogenetic analysis and CGH represent screening
methods to survey the entire genome for amplified regions,
PCR-based assays are most suitable for the final identification of
coding sequences, i.e. genes in amplified regions.
[0130] According to the present invention, such genes have been
identified by quantitative PCR(S. Gelmini et al., Clin. Chem. 43,
752 [1997]), by comparing DNA from a variety of primary tumors,
including breast, lung, colon, prostate, brain, liver, kidney,
pancreas, spleen, thymus, testis, ovary, uterus, etc. tumor, or
tumor cell lines, with pooled DNA from healthy donors. Quantitative
PCR was performed using a TaqMan instrument (ABI). Gene-specific
primers and fluorogenic probes were designed based upon the coding
sequences of the DNAs.
[0131] Human lung carcinoma cell lines include A549 (SRCC768),
Calu-1 (SRCC769), Calu-6 (SRCC770), H157 (SRCC771), H441 (SRCC772),
H460 (SRCC773), SKMES-1 (SRCC774) and SW900 (SRCC775), all
available from ATCC. Primary human lung tumor cells usually derive
from adenocarcinomas, squamous cell carcinomas, large cell
carcinomas, non-small cell carcinomas, small cell carcinomas, and
broncho alveolar carcinomas, and include, for example, SRCC724
(squamous cell carcinoma abbreviated as "SqCCa"), SRCC725
(non-small cell carcinoma, abbreviated as "NSCCa"), SRCC726
(adenocarcinoma, abbreviated as "AdenoCa"), SRCC727
(adenocarcinoma), SRCC728 (squamous cell carcinoma), SRCC729
(adenocarcinoma), SRCC730 (adeno/squamous cell carcinoma), SRCC731
(adenocarcinoma), SRCC732 (squamous cell carcinoma), SRCC733
(adenocarcinoma), SRCC734 (adenocarcinoma), SRCC735 (broncho
alveolar carcinoma, abbreviated as "BAC"), SRCC736 (squamous cell
carcinoma), SRCC738 (squamous cell carcinoma), SRCC739 (squamous
cell carcinoma), SRCC740 (squamous cell carcinoma), SRCC740 (lung
cell carcinoma, abbreviated as "LCCa").
[0132] Colon cancer cell lines include, for example, ATCC cell
lines SW480 (adenocarcinoma, SRCC776), SW620 (lymph node metastasis
of colon adenocarcinoma, SRCC777), COL0320 (adenocarcinoma,
SRCC778), HT29 (adenocarcinoma, SRCC779), HM7 (carcinoma, SRCC780),
CaWiDr (adenocarcinoma, srcc781), HCT116 (carcinoma, SRCC782),
SKCO1 (adenocarcinoma, SRCC783), SW403 (adenocarcinoma, SRCC784),
LS174T (carcinoma, SRCC785), and HM7 (a high mucin producing
variant of ATCC colon adenocarcinoma cell line LS174T, obtained
from Dr. Robert Warren, UCSF). Primary colon tumors include colon
adenoocarcinomas designated ColT2 (SRCC742), ColT3 (SRCC743), ColT8
(SRCC744), ColT10 (SRCC745), ColT12 (SRCC746), ColT14 (SRCC747),
ColT15 (SRCC748), ColT17 (SRCC750), ColT1 (SRCC751), ColT4
(SRCC752), ColT5 (SRCC753), ColT6 (SRCC754), ColT7 (SRCC755), ColT9
(SRCC756), ColT11 (SRCC757), ColT18 (SRCC758), and DcR3, BACrev,
BACfwd, T160, and T159.
[0133] Human breast carcinoma cell lines include, for example,
HBL100 (SRCC759), MB435s (SRCC760), T47D (SRCC761), MB468(SRCC762),
MB175 (SRCC763), MB361 (SRCC764), BT20 (SRCC765), MCF7 (SRCC766),
SKBR3 (SRCC767).
[0134] 3. Tissue Distribution
[0135] The results of the gene amplification assays herein can be
verified by further studies, such as, by determining mRNA
expression in various human tissues.
[0136] As noted before, gene amplification and/or gene expression
in various tissues may be measured by conventional Southern
blotting, Northern blotting to quantitate the transcription of mRNA
(Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 [1980]), dot
blotting (DNA analysis), or in situ hybridization, using an
appropriately labeled probe, based on the sequences provided
herein. Alternatively, antibodies may be employed that can
recognize specific duplexes, including DNA duplexes, RNA duplexes,
and DNA-RNA hybrid duplexes or DNA-protein duplexes.
[0137] Gene expression in various tissues, alternatively, may be
measured by immunological methods, such as immunohistochemical
staining of tissue sections and assay of cell culture or body
fluids, to quantitate directly the expression of gene product.
Antibodies useful for immunohistochemical staining and/or assay of
sample fluids may be either monoclonal or polyclonal, and may be
prepared in any mammal. Conveniently, the antibodies may be
prepared against a native sequence CT-1 polypeptide or against a
synthetic peptide based on the DNA sequences provided herein or
against exogenous sequence fused to CT-1 DNA and encoding a
specific antibody epitope. General techniques for generating
antibodies, and special protocols for Northern blotting and in situ
hybridization are provided hereinbelow.
[0138] 4. Chromosome Mapping
[0139] If the amplification of a given gene is functionally
relevant, then that gene should be amplified more than neighboring
genomic regions which are not important for tumor survival. To test
this, the gene can be mapped to a particular chromosome, e.g. by
radiation-hybrid analysis. The amplification level is then
determined at the location identified, and at neighboring genomic
region. Selective or preferential amplification at the genomic
region to which to gene has been mapped is consistent with the
possibility that the gene amplification observed promotes tumor
growth or survival. Chromosome mapping includes both framework and
epicenter mapping. For further details see e.g., Stewart et al.,
Genome Research 7, 422-433 (1997).
[0140] 5. Antibody Binding Studies
[0141] The results of the gene amplification study can be further
verified by antibody binding studies, in which the ability of
anti-CT-1 antibodies to inhibit the effect of the CT-1 polypeptides
on tumor (cancer) cells is tested. Exemplary antibodies include
polyclonal, monoclonal, humanized, bispecific, and heteroconjugate
antibodies, the preparation of which will be described
hereinbelow.
[0142] Antibody binding studies may be carried out in any known
assay method, such as competitive binding assays, direct and
indirect sandwich assays, and immunoprecipitation assays. Zola,
Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC
Press, Inc., 1987).
[0143] Competitive binding assays rely on the ability of a labeled
standard to compete with the test sample analyte for binding with a
limited amount of antibody. The amount of target protein (encoded
by a gene amplified in a tumor cell) in the test sample is
inversely proportional to the amount of standard that becomes bound
to the antibodies. To facilitate determining the amount of standard
that becomes bound, the antibodies preferably are insolubilized
before or after the competition, so that the standard and analyte
that are bound to the antibodies may conveniently be separated from
the standard and analyte which remain unbound.
[0144] Sandwich assays involve the use of two antibodies, each
capable of binding to a different immunogenic portion, or epitope,
of the protein to be detected. In a sandwich assay, the test sample
analyte is bound by a first antibody which is immobilized on a
solid support, and thereafter a second antibody binds to the
analyte, thus forming an insoluble three-part complex. See, e.g.,
U.S. Pat. No. 4,376,110. The second antibody may itself be labeled
with a detectable moiety (direct sandwich assays) or may be
measured using an anti-immunoglobulin antibody that is labeled with
a detectable moiety (indirect sandwich assay). For example, one
type of sandwich assay is an ELISA assay, in which case the
detectable moiety is an enzyme.
[0145] For immunohistochemistry, the tumor sample may be fresh or
frozen or may be embedded in paraffin and fixed with a preservative
such as formalin, for example.
[0146] 6. Cell-Based Tumor Assays
[0147] Cell-based assays and animal models for tumors (e.g.
cancers) can be used to verify the findings of the gene
amplification assay, and further understand the relationship
between the genes identified herein and the development and
pathogenesis of neoplastic cell growth. The role of gene products
identified herein in the development and pathology of tumor or
cancer can be tested by using primary tumor cells or cells lines
that have been identified to amplify the genes herein. Such cells
include, for example, the breast, colon and lung cancer cells and
cell lines listed above.
[0148] In a different approach, cells of a cell type known to be
involved in a particular tumor are transfected with the cDNAs
herein, and the ability of these cDNAs to induce excessive growth
is analyzed. Suitable cells include, for example, stable tumor
cells lines such as, the B104-1-1 cell line (stable NIH-3T3 cell
line transfected with the neu protooncogene) and ras-transfected
NIH-3T3 cells, which can be transfected with the desired gene, and
monitored for tumorogenic growth. Such transfected cell lines can
then be used to test the ability of poly- or monoclonal antibodies
or antibody compositions to inhibit tumorogenic cell growth by
exerting cytostatic or cytotoxic activity on the growth of the
transformed cells, or by mediating antibody-dependent cellular
cytotoxicity (ADCC). Cells transfected with the coding sequences of
the genes identified herein can further be used to identify drug
candidates for the treatment of cancer.
[0149] In addition, primary cultures derived from tumors in
transgenic animals (as described below) can be used in the
cell-based assays herein, although stable cell lines are preferred.
Techniques to derive continuous cell lines from transgenic animals
are well known in the art (see, e.g. Small et al., Mol. Cell. Biol.
5, 642-648 [1985]).
[0150] 7. Animal Models
[0151] A variety of well known animal models can be used to further
understand the role of the genes identified herein in the
development and pathogenesis of tumors, and to test the efficacy of
candidate therapeutic agents, including antibodies, and other
antagonists of the native polypeptides, including small molecule
antagonists. The in vivo nature of such models makes them
particularly predictive of responses in human patients. Animal
models of tumors and cancers (e.g. breast cancer, colon cancer,
prostate cancer, lung cancer, etc.) include both non-recombinant
and recombinant (transgenic) animals. Non-recombinant animal models
include, for example, rodent, e.g., murine models. Such models can
be generated by introducing tumor cells into syngeneic mice using
standard techniques, e.g. subcutaneous injection, tail vein
injection, spleen implantation, intraperitoneal implantation,
implantation under the renal capsule, or orthopin implantation,
e.g. colon cancer cells implanted incolonic tissue. (See, e.g. PCT
publication No. WO 97/33551, published Sep. 18, 1997).
[0152] Probably the most often used animal species in oncological
studies are immunodeficient mice and, in particular, nude mice. The
observation that the nude mouse with hypo/aplasia could
successfully act as a host for human tumor xenografts has lead to
its widespread use for this purpose. The autosomal recessive nu
gene has been introduced into a very large number of distinct
congenic strains of nude mouse, including, for example, ASW, A/He,
AKR, BALB/c, B10.LP, C17, C3H, C57BL, C57, CBA, DBA, DDD, I/st, NC,
NFR, NFS, NFS/N, NZB, NZC, NZW, P, RIII and SJL. In addition, a
wide variety of other animals with inherited immunological defects
other than the nude mouse have been bred and used as recipients of
tumor xenografts. For further details see, e.g. The Nude Mouse in
Oncology Research, E. Boven and B. Winograd, eds., CRC Press, Inc.,
1991.
[0153] The cells introduced into such animals can be derived from
known tumor/cancer cell lines, such as, any of the above-listed
tumor cell lines, and, for example, the B104-1-1 cell line (stable
NIH-3T3 cell line transfected with the neu protooncogene);
ras-transfected NIH-3T3 cells; Caco-2 (ATCC HTB-37); a moderately
well-differentiated grade II human colon adenocarcinoma cell line,
HT-29 (ATCC HTB-38), or from tumors and cancers. Samples of tumor
or cancer cells can be obtained from patients undergoing surgery,
using standard conditions, involving freezing and storing in liquid
nitrogen (Karmali et al., Br. J. Cancer 48, 689-696 [1983]). Tumor
cells can be introduced into animals, such as nude mice, by a
variety of procedures. The subcutaneous (s.c.) space in mice is
very suitable for tumor implantation. Tumors can be transplanted
s.c. as solid blocks, as needle biopsies by use of a trochar, or as
cell suspensions. For solid block or trochar implantation, tumor
tissue fragments of suitable size are introduced into the s.c.
space. Cell suspensions are freshly prepared from primary tumors or
stable tumor cell lines, and injected subcutaneously. Tumor cells
can also be injected as subdermal implants. In this location, the
inoculum is deposited between the lower part of the dermal
connective tissue and the s.c. tissue. Boven and Winograd (1991),
supra.
[0154] Animal models of breast cancer can be generated, for
example, by implanting rat neuroblastoma cells (from which the neu
oncogen was initially isolated), or neu-transformed NIH-3T3 cells
into nude mice, essentially as described by Drebin et al. PNAS USA
83, 9129-9133 (1986).
[0155] Similarly, animal models of colon cancer can be generated by
passaging colon cancer cells in animals, e.g. nude mice, leading to
the appearance of tumors in these animals. An orthotopic transplant
model of human colon cancer in nude mice has been described, for
example, by Wang et al., Cancer Research 54, 4726-4728 (1994) and
Too et al., Cancer Research 55, 681-684 (1995). This model is based
on the so-called "METAMOUSE.TM." sold by AntiCancer, Inc. (San
Diego, Calif.).
[0156] Tumors that arise in animals can be removed and cultured in
vitro. Cells from the in vitro cultures can then be passaged to
animals. Such tumors can serve as targets for further testing or
drug screening. Alternatively, the tumors resulting from the
passage can be isolated and RNA from pre-passage cells and cells
isolated after one or more rounds of passage analyzed for
differential expression of genes of interest. Such passaging
techniques can be performed with any known tumor or cancer cell
lines.
[0157] For example, Meth A, CMS4, CMS5, CMS21, and WEHI-164 are
chemically induced fibrosarcomas of BALB/c female mice (DeLeo et
al., J. Exp. Med. 146,720 [1977]), which provide a highly
controllable model system for studying the anti-tumor activities of
various agents (Palladino et al., J. Immunol. 138, 4023-4032
[1987]). Briefly, tumor cells are propagated in vitro in cell
culture. Prior to injection into the animals, the cell lines are
washed and suspended in buffer, at a cell density of about
10.times.10.sup.6 to 10.times.10.sup.7 cells/ml. The animals are
then infected subcutaneously with 10 to 100 .mu.l of the cell
suspension, allowing one to three weeks for a tumor to appear.
[0158] In addition, the Lewis lung (3LL) carcinoma of mice, which
is one of the most thoroughly studied experimental tumors, can be
used as an investigational tumor model. Efficacy in this tumor
model has been correlated with beneficial effects in the treatment
of human patients diagnosed with small cell carcinoma of the lung
(SCCL). This tumor can be introduced in normal mice upon injection
of tumor fragments from an affected mouse or of cells maintained in
culture (Zupi et al., Br. J. Cancer 41, suppl. 4, 309 [1980]), and
evidence indicates that tumors can be started from injection of
even a single cell and that a very high proportion of infected
tumor cells survive. For further information about this tumor model
see Zacharski, Haemostasis 16, 300-320 [1986]).
[0159] One way of evaluating the efficacy of a test compound in an
animal model is implanted tumor is to measure the size of the tumor
before and after treatment. Traditionally, the size of implanted
tumors has been measured with a slide caliper in two or three
dimensions. The measure limited to two dimensions does not
accurately reflect the size of the tumor, therefore, it is usually
converted into the corresponding volume by using a mathematical
formula. However, the measurement of tumor size is very inaccurate.
The therapeutic effects of a drug candidate can be better described
as treatment-induced growth delay and specific growth delay.
Another important variable in the description of tumor growth is
the tumor volume doubling time. Computer programs for the
calculation and description of tumor growth are also available,
such as the program reported by Rygaard and Spang-Thomsen, Proc.
6th Int. Workshop on Immune-Deficient Animals, Wu and Sheng eds.,
Basel, 1989, 301. It is noted, however, that necrosis and
inflammatory responses following treatment may actually result in
an increase in tumor size, at least initially. Therefore, these
changes need to be carefully monitored, by a combination of a
morphometric method and flow cytometric analysis.
[0160] Recombinant (transgenic) animal models can be engineered by
introducing the coding portion of the genes identified herein into
the genome of animals of interest, using standard techniques for
producing transgenic animals. Animals that can serve as a target
for transgenic manipulation include, without limitation, mice,
rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human
primates, e.g. baboons, chimpanzees and monkeys. Techniques known
in the art to introduce a transgene into such animals include
pronucleic microinjection (Hoppe and Wanger, U.S. Pat. No.
4,873,191); retrovirus-mediated gene transfer into germ lines
(e.g., Van der Putten et al., Proc. Natl. Acad. Sci. USA 82,
6148-615 [1985]); gene targeting in embryonic stem cells (Thompson
et al., Cell 56, 313-321 [1989]); electroporation of embryos (Lo,
Mol. Cel. Biol 3, 1803-1814 [1983]); sperm-mediated gene transfer
(Lavitrano et al., Cell 57, 717-73 [1989]). For review, see, for
example, U.S. Pat. No. 4,736,866.
[0161] For the purpose of the present invention, transgenic animals
include those that carry the transgene only in part of their cells
("mosaic animals"). The transgene can be integrated either as a
single transgene, or in concatamers, e.g., head-to-head or
head-to-tail tandems. Selective introduction of a transgene into a
particular cell type is also possible by following, for example,
the technique of Lasko et al., Proc. Natl. Acad. Sci. USA 89,
6232-636 (1992).
[0162] The expression of the transgene in transgenic animals can be
monitored by standard techniques. For example, Southern blot
analysis or PCR amplification can be used to verify the integration
of the transgene. The level of mRNA expression can then be analyzed
using techniques such as in situ hybridization, Northern blot
analysis, PCR, or immunocytochemistry. The animals are further
examined for signs of tumor or cancer development.
[0163] Alternatively, "knock out" animals can be constructed which
have a defective or altered gene encoding a CT-1 polypeptide
identified herein, as a result of homologous recombination between
the endogenous gene encoding the polypeptide and altered genomic
DNA encoding the same polypeptide introduced into an embryonic cell
of the animal. For example, cDNA encoding a particular CT-1
polypeptide can be used to clone genomic DNA encoding that
polypeptide in accordance with established techniques. A portion of
the genomic DNA encoding a particular CT-1 polypeptide can be
deleted or replaced with another gene, such as a gene encoding a
selectable marker which can be used to monitor integration.
Typically, several kilobases of unaltered flanking DNA (both at the
5' and 3' ends) are included in the vector [see e.g., Thomas and
Capecchi, Cell, 51:503 (1987) for a description of homologous
recombination vectors]. The vector is introduced into an embryonic
stem cell line (e.g., by electroporation) and cells in which the
introduced DNA has homologously recombined with the endogenous DNA
are selected [see e.g., Li et al., Cell, 69:915 (1992)]. The
selected cells are then injected into a blastocyst of an animal
(e.g., a mouse or rat) to form aggregation chimeras [see e.g.,
Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. A
chimeric embryo can then be implanted into a suitable
pseudopregnant female foster animal and the embryo brought to term
to create a "knock out" animal. Progeny harboring the homologously
recombined DNA in their germ cells can be identified by standard
techniques and used to breed animals in which all cells of the
animal contain the homologously recombined DNA. Knockout animals
can be characterized for instance, by their ability to defend
against certain pathological conditions and by their development of
pathological conditions due to absence of the CT-1 polypeptide.
[0164] The efficacy of antibodies specifically binding the
polypeptides identified herein and other drug candidates, can be
tested also in the treatment of spontaneous animal tumors. A
suitable target for such studies is the feline oral squamous cell
carcinoma (SCC). Feline oral SCC is a highly invasive, malignant
tumor that is the most common oral malignancy of cats, accounting
for over 60% of the oral tumors reported in this species. It rarely
metastasizes to distant sites, although this low incidence of
metastasis may merely be a reflection of the short survival times
for cats with this tumor. These tumors are usually not amenable to
surgery, primarily because of the anatomy of the feline oral
cavity. At present, there is no effective treatment for this tumor.
Prior to entry into the study, each cat undergoes complete clinical
examination, biopsy, and is scanned by computed tomography. Cats
diagnosed with sublingual oral squamous cell tumors are excluded
from the study. The tongue can become paralyzed as a result of such
tumor, and even if the treatment kills the tumor, the animals may
not be able to feed themselves. Each cat is treated repeatedly,
over a longer period of time. Photographs of the tumors will be
taken daily during the treatment period, and at each subsequent
recheck. After treatment, each cat undergoes another computed
tomography scan. Computed tomography scans and thoracic radiograms
are evaluated every 8 weeks thereafter. The data are evaluated for
differences in survival, response and toxicity as compared to
control groups. Positive response may require evidence of tumor
regression, preferably with improvement of quality of life and/or
increased life span.
[0165] In addition, other spontaneous animal tumors, such as
fibrosarcoma, adenocarcinoma, lymphoma, chrondroma, leiomyosarcoma
of dogs, cats, and baboons can also be tested. Of these mammary
adenocarcinoma in dogs and cats is a preferred model as its
appearance and behavior are very similar to those in humans.
However, the use of this model is limited by the rare occurrence of
this type of tumor in animals.
[0166] 8. Screening Assays for Drug Candidates
[0167] Screening assays for drug candidates are designed to
identify compounds that bind or complex with the polypeptides
encoded by the genes identified herein, or otherwise interfere with
the interaction of the encoded polypeptides with other cellular
proteins. Such screening assays will include assays amenable to
high-throughput screening of chemical libraries, making them
particularly suitable for identifying small molecule drug
candidates. Small molecules contemplated include synthetic organic
or inorganic compounds, including peptides, preferably soluble
peptides, (poly)peptide-immunoglobulin fusions, and, in particular,
antibodies including, without limitation, poly- and monoclonal
antibodies and antibody fragments, single-chain antibodies,
anti-idiotypic antibodies, and chimeric or humanized versions of
such antibodies or fragments, as well as human antibodies and
antibody fragments. The assays can be performed in a variety of
formats, including protein-protein binding assays, biochemical
screening assays, immunoassays and cell based assays, which are
well characterized in the art.
[0168] All assays are common in that they call for contacting the
drug candidate with a polypeptide encoded by a nucleic acid
identified herein under conditions and for a time sufficient to
allow these two components to interact.
[0169] In binding assays, the interaction is binding and the
complex formed can be isolated or detected in the reaction mixture.
In a particular embodiment, the polypeptide encoded by the gene
identified herein or the drug candidate is immobilized on a solid
phase, e.g. on a microtiter plate, by covalent or non-covalent
attachments. Non-covalent attachment generally is accomplished by
coating the solid surface with a solution of the polypeptide and
drying. Alternatively, an immobilized antibody, e.g. a monoclonal
antibody, specific for the polypeptide to be immobilized can be
used to anchor it to a solid surface. The assay is performed by
adding the non-immobilized component, which may be labeled by a
detectable label, to the immobilized component, e.g. the coated
surface containing the anchored component. When the reaction is
complete, the non-reacted components are removed, e.g. by washing,
and complexes anchored on the solid surface are detected. When the
originally non-immobilized component carries a detectable label,
the detection of label immobilized on the surface indicates that
complexing occurred. Where the originally non-immobilized component
does not carry a label, complexing can be detected, for example, by
using a labeled antibody specifically binding the immobilized
complex.
[0170] If the candidate compound interacts with but does not bind
to a particular CT-1 polypeptide encoded by a nucleic acid sequence
described herein, its interaction with that polypeptide can be
assayed by methods well known for detecting protein-protein
interactions. Such assays include traditional approaches, such as,
cross-linking, co-immunoprecipitation, and co-purification through
gradients or chromatographic columns. In addition, protein-protein
interactions can be monitored by using a yeast-based genetic system
described by Fields and co-workers [Fields and Song, Nature
(London) 340, 245-246 (1989); Chien et al., Proc. Natl. Acad. Sci.
USA 88, 9578-9582 (1991)] as disclosed by Chevray and Nathans
[Proc. Natl. Acad. Sci. USA 89, 5789-5793 (1991)]. Many
transcriptional activators, such as yeast GAL4, consist of two
physically discrete modular domains, one acting as the DNA-binding
domain, while the other one functioning as the transcription
activation domain. The yeast expression system described in the
foregoing publications (generally referred to as the "two-hybrid
system") takes advantage of this property, and employs two hybrid
proteins, one in which the target protein is fused to the
DNA-binding domain of GAL4, and another, in which candidate
activating proteins are fused to the activation domain. The
expression of a GAL1-lacZ reporter gene under control of a
GAL4-activated promoter depends on reconstitution of GAL4 activity
via protein-protein interaction. Colonies containing interacting
polypeptides are detected with a chromogenic substrate for
.beta.-galactosidase. A complete kit (MATCHMAKER.TM.) for
identifying protein-protein interactions between two specific
proteins using the two-hybrid technique is commercially available
from Clontech. This system can also be extended to map protein
domains involved in specific protein interactions as well as to
pinpoint amino acid residues that are crucial for these
interactions.
[0171] Compounds that interfere with the interaction of a
CT-1-encoding gene identified herein and other intra- or
extracellular components can be tested as follows: usually a
reaction mixture is prepared containing the product of the
amplified gene and the intra- or extracellular component under
conditions and for a time allowing for the interaction and binding
of the two products. To test the ability of a test compound to
inhibit binding, the reaction is run in the absence and in the
presence of the test compound. In addition, a placebo may be added
to a third reaction mixture, to serve as positive control. The
binding (complex formation) between the test compound and the
intra- or extracellular component present in the mixture is
monitored as described hereinabove. The formation of a complex in
the control reaction(s) but not in the reaction mixture containing
the test compound indicates that the test compound interferes with
the interaction of the test compound and its reaction partner.
[0172] 9. Compositions and Methods for the Treatment of Tumors
[0173] The compositions useful in the treatment of tumors
associated with the amplification of the genes identified herein
include, without limitation, antibodies, small organic and
inorganic molecules, peptides, phosphopeptides, antisense and
ribozyme molecules, triple helix molecules, etc. that inhibit the
expression and/or activity of the target gene product.
[0174] For example, antisense RNA and RNA molecule act to directly
block the translation of mRNA by hybridizing to targeted mRNA and
preventing protein translation. When antisense DNA is used,
oligodeoxyribonucleotides derived from the translation initiation
site, e.g. between about -10 and +10 positions of the target gene
nucleotide sequence, are preferred.
[0175] Ribozymes are enzymatic RNA molecules capable of catalyzing
the specific cleavage of RNA. Ribozymes act by sequence-specific
hybridization to the complementary target RNA, followed by
endonucleolytic cleavage. Specific ribozyme cleavage sites within a
potential RNA target can be identified by known techniques. For
further details see, e.g. Rossi, Current Biology 4, 469-471 (1994),
and PCT publication No. WO 97/33551 (published Sep. 18, 1997).
[0176] Nucleic acid molecules in triple helix formation used to
inhibit transcription should be single-stranded and composed of
deoxynucleotides. The base composition of these oligonucleotides is
designed such that it promotes triple helix formation via Hoogsteen
base pairing rules, which generally require sizeable stretches of
purines or pyrimidines on one strand of a duplex. For further
details see, e.g. PCT publication No. WO 97/33551, supra.
[0177] These molecules can be identified by any or any combination
of the screening assays discussed hereinabove and/or by any other
screening techniques well known for those skilled in the art.
[0178] 9.1 Antibodies
[0179] Some of the most promising drug candidates according to the
present invention are antibodies and antibody fragments which may
inhibit the production or the gene product of the amplified genes
identified herein and/or reduce the activity of the gene
products.
[0180] i. Polyclonal Antibodies
[0181] 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
CT-1 polypeptide 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.
[0182] ii. Monoclonal Antibodies
[0183] The anti-CT-1 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.
[0184] The immunizing agent will typically include the CT-1
polypeptide, including fragments, or a fusion protein of such
protein or a fragment 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.
[0185] Preferred immortalized cell lines are those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More preferred immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif. and
the American Type Culture Collection (ATCC), Manassas, Va. Human
myeloma and mouse-human heteromyeloma cell lines also have been
described for the production of human monoclonal antibodies
[Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal
Antibody Production Techniques and Applications, Marcel Dekker,
Inc., New York, (1987) pp. 51-63].
[0186] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against CT-1. Preferably, the binding specificity of
monoclonal antibodies produced by the hybridoma cells is determined
by immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay
(ELISA). Such techniques and assays are known in the art. The
binding affinity of the monoclonal antibody can, for example, be
determined by the Scatchard analysis of Munson and Pollard, Anal.
Biochem., 107:220 (1980).
[0187] After the desired hybridoma cells are identified, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods [Goding, supra]. Suitable culture media for this
purpose include, for example, Dulbecco's Modified Eagle's Medium
and RPMI-1640 medium. Alternatively, the hybridoma cells may be
grown in vivo as ascites in a mammal.
[0188] The monoclonal antibodies secreted by the subclones may be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0189] The monoclonal antibodies may also be made by recombinant
DNA methods, such as those described in U.S. Pat. No. 4,816,567.
DNA encoding the monoclonal antibodies of the invention can be
readily isolated and sequenced using conventional procedures (e.g.,
by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells of the invention serve as a
preferred source of such DNA. Once isolated, the DNA may be placed
into expression vectors, which are then transfected into host cells
such as simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. The DNA also may be modified, for example, by
substituting the coding sequence for human heavy and light chain
constant domains in place of the homologous murine sequences [U.S.
Pat. No. 4,816,567; Morrison et al., supra] or by covalently
joining to the immunoglobulin coding sequence all or part of the
coding sequence for a non-immunoglobulin polypeptide. Such a
non-immunoglobulin polypeptide can be substituted for the constant
domains of an antibody of the invention, or can be substituted for
the variable domains of one antigen-combining site of an antibody
of the invention to create a chimeric bivalent antibody.
[0190] The antibodies may be monovalent antibodies. Methods for
preparing monovalent antibodies are well known in the art. For
example, one method involves recombinant expression of
immunoglobulin light chain and modified heavy chain. The heavy
chain is truncated generally at any point in the Fc region so as to
prevent heavy chain crosslinking. Alternatively, the relevant
cysteine residues are substituted with another amino acid residue
or are deleted so as to prevent crosslinking.
[0191] In vitro methods are also suitable for preparing monovalent
antibodies. Digestion of antibodies to produce fragments thereof,
particularly, Fab fragments, can be accomplished using routine
techniques known in the art.
[0192] iii. Human and Humanized Antibodies
[0193] The anti-CT-1 antibodies may further comprise humanized
antibodies or human antibodies. Humanized forms of non-human (e.g.,
murine) antibodies are chimeric 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 from 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)].
[0194] 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.
[0195] 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).
[0196] iv. Bispecific Antibodies
[0197] Bispecific antibodies are monoclonal, preferably human or
humanized, antibodies that have binding specificities for at least
two different antigens. In the present case, one of the binding
specificities is for a CT-1 polypeptide, the other one is for any
other antigen, and preferably for a cell-surface protein or
receptor or receptor subunit.
[0198] Methods for making bispecific antibodies are known in the
art. Traditionally, the recombinant production of bispecific
antibodies is based on the co-expression of two immunoglobulin
heavy-chain/light-chain pairs, where the two heavy chains have
different specificities (Milstein and Cuello, Nature, 305:537-539
[1983]). Because of the random assortment of immunoglobulin heavy
and light chains, these hybridomas (quadromas) produce a potential
mixture of ten different antibody molecules, of which only one has
the correct bispecific structure. The purification of the correct
molecule is usually accomplished by affinity chromatography steps.
Similar procedures are disclosed in WO 93/08829, published 13 May
1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
[0199] Antibody variable domains with the desired binding
specificities (antibody-antigen combining sites) can be fused to
immunoglobulin constant domain sequences. The fusion preferably is
with an immunoglobulin heavy-chain constant domain, comprising at
least part of the hinge, CH2, and CH3 regions. It is preferred to
have the first heavy-chain constant region (CH1) containing the
site necessary for light-chain binding present in at least one of
the fusions. DNAs encoding the immunoglobulin heavy-chain fusions
and, if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable
host organism. For further details of generating bispecific
antibodies see, for example, Suresh et al., Methods in Enzymology,
121:210 (1986).
[0200] v. Heteroconjugate Antibodies
[0201] Heteroconjugate antibodies are composed of two covalently
joined antibodies. Such antibodies have, for example, been proposed
to target immune system cells to unwanted cells [U.S. Pat. No.
4,676,980], and for treatment of HIV infection [WO 91/00360; WO
92/200373; EP 03089]. It is contemplated that the antibodies may be
prepared in vitro using known methods in synthetic protein
chemistry, including those involving crosslinking agents. For
example, immunotoxins may be constructed using a disulfide exchange
reaction or by forming a thioether bond. Examples of suitable
reagents for this purpose include iminothiolate and
methyl-4-mercaptobutyrimidate and those disclosed, for example, in
U.S. Pat. No. 4,676,980.
[0202] vi. Effector Function Engineering
[0203] It may be desirable to modify the antibody of the invention
with respect to effector function, so as to enhance the
effectiveness of the antibody in treating cancer, for example. For
example cysteine residue(s) may be introduced in the Fc region,
thereby allowing interchain disulfide bond formation in this
region. The homodimeric antibody thus generated may have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B.
J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with
enhanced anti-tumor activity may also be prepared using
heterobifunctional cross-linkers as described in Wolff et al.
Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can
be engineered which has dual Fc regions and may thereby have
enhanced complement lysis and ADCC capabilities. See Stevenson et
al., Anti-Cancer Drug Design 3:219-230 (1989).
[0204] vii. Immunoconjugates
[0205] The invention also pertains to immunoconjugates comprising
an antibody conjugated to a cytotoxic agent such as a
chemotherapeutic agent, toxin (e.g. an enzymatically active toxin
of bacterial, fungal, plant or animal origin, or fragments
thereof), or a radioactive isotope (i.e., a radioconjugate).
[0206] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above. Enzymatically active
toxins and fragments thereof which can be used include diphtheria A
chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin and the tricothecenes. A variety of
radionuclides are available for the production of radioconjugated
antibodies. Examples include .sup.212Bi, .sup.131I, .sup.131In,
.sup.90Y and .sup.186Re.
[0207] Conjugates of the antibody and cytotoxic agent are made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026.
[0208] In another embodiment, the antibody may be conjugated to a
"receptor" (such streptavidin) for utilization in tumor
pretargeting wherein the antibody-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g. avidin) which is conjugated to a
cytotoxic agent (e.g. a radionucleotide).
[0209] viii. Immunoliposomes
[0210] The antibodies disclosed herein may also be formulated as
immunoliposomes. Liposomes containing the antibody are prepared by
methods known in the art, such as described in Epstein et al.,
Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc.
Natl. Acad. Sci. USA, 77:4030 (1980); and U.S. Pat. Nos. 4,485,045
and 4,544,545. Liposomes with enhanced circulation time are
disclosed in U.S. Pat. No. 5,013,556.
[0211] Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al.,
J. Biol. Chem. 257: 286-288 (1982) via a disulfide interchange
reaction. A chemotherapeutic agent (such as Doxorubicin) is
optionally contained within the liposome. See Gabizon et al., J.
National Cancer Inst. 81(19)1484 (1989).
[0212] 10. Pharmaceutical Compositions
[0213] Antibodies specifically binding the product of an amplified
gene identified herein, as well as other molecules identified by
the screening assays disclosed hereinbefore, can be administered
for the treatment of tumors, including cancers, in the form of
pharmaceutical compositions.
[0214] If the protein encoded by the amplified gene is
intracellular and whole antibodies are used as inhibitors,
internalizing antibodies are preferred. However, lipofections or
liposomes can also be used to deliver the antibody, or an antibody
fragment, into cells. Where antibody fragments are used, the
smallest inhibitory fragment which specifically binds to the
binding domain of the target protein is preferred. For example,
based upon the variable region sequences of an antibody, peptide
molecules can be designed which retain the ability to bind the
target protein sequence. Such peptides can be synthesized
chemically and/or produced by recombinant DNA technology (see, e.g.
Marasco et al., Proc. Natl. Acad. Sci. USA 90, 7889-7893
[1993]).
[0215] Therapeutic formulations of the antibody are prepared for
storage by mixing the antibody having the desired degree of purity
with optional pharmaceutically acceptable carriers, excipients or
stabilizers (Remington's Pharmaceutical Sciences 16th edition,
Osol, A. Ed. [1980]), in the form of lyophilized formulations or
aqueous solutions. Acceptable carriers, excipients, or stabilizers
are nontoxic to recipients at the dosages and concentrations
employed, and include buffers such as phosphate, citrate, and other
organic acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0216] Non-antibody compounds identified by the screening assays of
the present invention can be formulated in an analogous manner,
using standard techniques well known in the art.
[0217] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. Alternatively, or in addition, the
composition may comprise a cytotoxic agent, cytokine or growth
inhibitory agent. Such molecules are suitably present in
combination in amounts that are effective for the purpose
intended.
[0218] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington's Pharmaceutical Sciences
16th edition, Osol, A. Ed. (1980).
[0219] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0220] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated antibodies remain in
the body for a long time, they may denature or aggregate as a
result of exposure to moisture at 37.degree. C., resulting in a
loss of biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on
the mechanism involved. For example, if the aggregation mechanism
is discovered to be intermolecular S--S bond formation through
thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
[0221] 11. Methods of Treatment
[0222] It is contemplated that the antibodies and other anti-tumor
compounds of the present invention may be used to treat various
conditions, including those characterized by overexpression and/or
activation of the amplified genes identified herein. Exemplary
conditions or disorders to be treated with such antibodies and
other compounds, including, but not limited to, small organic and
inorganic molecules, peptides, antisense molecules, etc. include
benign or malignant tumors (e.g. renal, liver, kidney, bladder,
breast, gastric, ovarian, colorectal, prostate, pancreatic, ling,
vulval, thyroid, hepatic carcinomas; sarcomas; glioblastomas; and
various head and neck tumors); leukemias and lymphoid malignancies;
other disorders such as neuronal, glial, astrocytal, hypothalamic
and other glandular, macrophagal, epithelial, stromal and
blastocoelic disorders; and inflammatory, angiogenic and
immunologic disorders.
[0223] The anti-tumor agents of the present invention, e.g.
antibodies, are administered to a mammal, preferably a human, in
accord with known methods, such as intravenous administration as a
bolus or by continuous infusion over a period of time, by
intramuscular, intraperitoneal, intracerobrospinal, subcutaneous,
intra-articular, intrasynovial, intrathecal, oral, topical, or
inhalation routes. Intravenous administration of the antibody is
preferred.
[0224] Other therapeutic regimens may be combined with the
administration of the anti-cancer agents, e.g. antibodies of the
instant invention. For example, the patient to be treated with such
anti-cancer agents may also receive radiation therapy.
Alternatively, or in addition, a chemotherapeutic agent may be
administered to the patient. Preparation and dosing schedules for
such chemotherapeutic agents may be used according to
manufacturers' instructions or as determined empirically by the
skilled practitioner. Preparation and dosing schedules for such
chemotherapy are also described in Chemotherapy Service Ed., M. C.
Perry, Williams & Wilkins, Baltimore, Md. (1992). The
chemotherapeutic agent may precede, or follow administration of the
anti-tumor agent, e.g. antibody, or may be given simultaneously
therewith. The antibody may be combined with an anti-oestrogen
compound such as tamoxifen or an anti-progesterone such as
onapristone (see, EP 616812) in dosages known for such
molecules.
[0225] It may be desirable to also administer antibodies against
other tumor associated antigens, such as antibodies which bind to
the ErbB2, EGFR, ErbB3, ErbB4, or vascular endothelial factor
(VEGF). Alternatively, or in addition, two or more antibodies
binding the same or two or more different antigens disclosed herein
may be co-administered to the patient. Sometimes, it may be
beneficial to also administer one or more cytokines to the patient.
In a preferred embodiment, the antibodies herein are
co-administered with a growth inhibitory agent. For example, the
growth inhibitory agent may be administered first, followed by an
antibody of the present invention. However, simultaneous
administration or administration of the antibody of the present
invention first is also contemplated. Suitable dosages for the
growth inhibitory agent are those presently used and may be lowered
due to the combined action (synergy) of the growth inhibitory agent
and the antibody herein.
[0226] For the prevention or treatment of disease, the appropriate
dosage of an anti-tumor agent, e.g. an antibody herein will depend
on the type of disease to be treated, as defined above, the
severity and course of the disease, whether the agent is
administered for preventive or therapeutic purposes, previous
therapy, the patient's clinical history and response to the agent,
and the discretion of the attending physician. The agent is
suitably administered to the patient at one time or over a series
of treatments.
[0227] For example, depending on the type and severity of the
disease, about 1 .mu.g/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of
antibody is an initial candidate dosage for administration to the
patient, whether, for example, by one or more separate
administrations, or by continuous infusion. A typical daily dosage
might range from about 1 .mu.g/kg to 100 mg/kg or more, depending
on the factors mentioned above. For repeated administrations over
several days or longer, depending on the condition, the treatment
is sustained until a desired suppression of disease symptoms
occurs. However, other dosage regimens may be useful. The progress
of this therapy is easily monitored by conventional techniques and
assays.
[0228] 12. Articles of Manufacture
[0229] In another embodiment of the invention, an article of
manufacture containing materials useful for the diagnosis or
treatment of the disorders described above is provided. The article
of manufacture comprises a container and a label. Suitable
containers include, for example, bottles, vials, syringes, and test
tubes. The containers may be formed from a variety of materials
such as glass or plastic. The container holds a composition which
is effective for diagnosing or treating the condition and may have
a sterile access port (for example the container may be an
intravenous solution bag or a vial having a stopper pierceable by a
hypodermic injection needle). The active agent in the composition
is usually an anti-tumor agent capable of interfering with the
activity of a gene product identified herein, e.g. an antibody. The
label on, or associated with, the container indicates that the
composition is used for diagnosing or treating the condition of
choice. The article of manufacture may further comprise a second
container comprising a pharmaceutically-acceptable buffer, such as
phosphate-buffered saline, Ringer's solution and dextrose solution.
It may further include other materials desirable from a commercial
and user standpoint, including other buffers, diluents, filters,
needles, syringes, and package inserts with instructions for
use.
[0230] 13. Diagnosis and Prognosis of Tumors
[0231] While cell surface proteins, such as growth receptors
overexpressed in certain tumors are excellent targets for drug
candidates or tumor (e.g. cancer) treatment, the same proteins
along with secreted proteins encoded by the genes amplified in
tumor cells find additional use in the diagnosis and prognosis of
tumors. For example, antibodies directed against the proteins
products of genes amplified in tumor cells can be used as tumor
diagnostics or prognostics.
[0232] For example, antibodies, including antibody fragments, can
be used to qualitatively or quantitatively detect the expression of
proteins encoded by the amplified genes ("marker gene products").
The antibody preferably is equipped with a detectable, e.g.
fluorescent label, and binding can be monitored by light
microscopy, flow cytometry, fluorimetry, or other techniques known
in the art. These techniques are particularly suitable, if the
amplified gene encodes a cell surface protein, e.g. a growth
factor. Such binding assays are performed essentially as described
in section 5 above.
[0233] In situ detection of antibody binding to the marker gene
products can be performed, for example, by immunofluorescence or
immunoelectron microscopy. For this purpose, a histological
specimen is removed from the patient, and a labeled antibody is
applied to it, preferably by overlaying the antibody on a
biological sample. This procedure also allows for determining the
distribution of the marker gene product in the tissue examined. It
will be apparent for those skilled in the art that a wide variety
of histological methods are readily available for in situ
detection.
[0234] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way.
[0235] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
EXAMPLES
[0236] Commercially available reagents referred to in the examples
were used according to manufacturer's instructions unless otherwise
indicated. The source of those cells identified in the following
examples, and throughout the specification, by ATCC accession
numbers is the American Type Culture Collection, Manassas, Va.
Unless otherwise noted, the present invention uses standard
procedures of recombinant DNA technology, such as those described
hereinabove and in the following textbooks: Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press
N.Y., 1989; Ausubel et al., Current Protocols in Molecular Biology,
Green Publishing Associates and Wiley Interscience, N.Y., 1989;
Innis et al., PCR Protocols: A Guide to Methods and Applications,
Academic Press, inc., N.Y., 1990; Harlow et al., Antibodies: A
Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor,
1988; Gait, M. J., Oligonucleotide Synthesis, IRL Press, Oxford,
1984; R. I. Freshney, Animal Cell Culture, 1987; Coligan et al.,
Current Protocols in Immunology, 1991.
Example 1
Gene Amplification
[0237] This example shows that the CT-1-encoding gene is amplified
in the genome of certain human lung and colon cancer cell lines.
Amplification is associated with overexpression of the gene
product, indicating that the CT-1 proteins are useful targets for
therapeutic intervention in certain cancers such as colon, lung,
breast and other cancers. Therapeutic agent may take the form of
antagonists of CT-1-encoding gene products, for example,
murine-human chimeric, humanized or human antibodies against a CT-1
(CT-1) polypeptide.
[0238] The starting material for the screen was genomic DNA
isolated from a variety of cancers. The DNA is quantitated
precisely, e.g. fluorometrically. As a negative control, DNA was
isolated from the cells of ten normal healthy individuals which was
pooled and used as assay controls for the gene copy in healthy
individuals (not shown). The 5' nuclease assay (for example,
TaqMan.TM.) and real-time quantitative PCR (for example, ABI Prizm
7700 Sequence Detection System.TM. (Perkin Elmer, Applied
Biosystems Division, Foster City, Calif.)), were used to find genes
potentially amplified in certain cancers. The results were used to
determine whether the DNA encoding CT-1 is over-represented in any
of the primary lung or colon cancers or cancer cell lines that were
screened. The primary lung cancers were obtained from individuals
with tumors of the type and stage as indicated in Table 1. An
explanation of the abbreviations used for the designation of the
primary tumors listed in Table 1 and the primary tumors and cell
lines referred to throughout this example has been given
hereinbefore. The results of the Taqman.TM. are reported in delta
(.DELTA.) Ct units. One unit corresponds to one PCR cycle or
approximately a 2-fold amplification relative to normal, two units
corresponds to 4-fold, 3 units to 8-fold amplification and so on.
Quantitation was obtained using primers and a Taqman.TM.
fluorescent prove derived from the CT-1-encoding gene. Regions of
CT-1 which are most likely to contain unique nucleic acid sequences
and which are least likely to have spliced out introns are
preferred for the primer and probe derivation, e.g. a
3'-untranslated region. The sequences for the primers and probes
(forward, reverse and probe) used for the CT-1 gene amplification
were as follows: TABLE-US-00001 CT-1 (DNA58125): 58125.tm.fl
5'-TTCCCAGCCTCTCTTTGCTTT-3' (SEQ ID NO:4) 58125.tm.rl
5'-TCAGACGGAGTTACCATGCAGA-3' (SEQ ID NO:5) 58125.tm.pl
5'-TGCCCCGTTCTCTTAACTCTTGGACCC-3' (SEQ ID NO:6)
[0239] The 5' nuclease assay reaction is a fluorescent PCR-based
technique which makes use of the 5' exonuclease activity of Taq DNA
polymerase enzyme to monitor amplification in real time. Two
oligonucleotide primers are used to generate an amplicon typical of
a PCR reaction. A third oligonucleotide, or probe, is designed to
detect nucleotide sequence located between the two PCR primers. The
probe is non-extendible by Taq DNA polymerase enzyme, and is
labeled with a reporter fluorescent dye and a quencher fluorescent
dye. Any laser-induced emission from the reporter dye is quenched
by the quenching dye when the two dyes are located close together
as they are on the probe. During the amplification reaction, the
Taq DNA polymerase enzyme cleaves the probe in a template-dependent
manner. The resultant probe fragments disassociate in solution, and
signal from the released reporter dye is free from the quenching
effect of the second fluorophore. One molecule of reporter dye is
liberated for each new molecule synthesized, and detection of the
unquenched reporter dye provides the basis for quantitative
interpretation of the data.
[0240] The 5' nuclease procedure is run on a real-time quantitative
PCR device such as the ABI Prism 7700.TM. Sequence Detection. The
system consists of a thermocycler, laser, charge-coupled device
(CCD) camera and computer. The system amplifies samples in a
96-well format on a thermocycler. During amplification,
laser-induced fluorescent signal is collected in real-time through
fiber optics cables for all 96 wells, and detected at the CCD. The
system includes software for running the instrument and for
analyzing the data.
[0241] 5' Nuclease assay data are initially expressed as Ct, or the
threshold cycle. This is defined as the cycle at which the reporter
signal accumulates above the background level of fluorescence. The
.DELTA.Ct values are used as quantitative measurement of the
relative number of starting copies of a particular target sequence
in a nucleic acid sample when comparing cancer DNA results to
normal human DNA results.
[0242] Table 1 describes the stage, T stage and N stage, of various
primary tumors which were used to screen the CT-1 compounds of the
invention. TABLE-US-00002 TABLE 1 Primary Lung and Colon Tumor
Profiles Dukes T N Primary Tumor Stage Other Stage Stage Stage
Stage Human lung tumor SqCCA (SRCC724) [LT1] IB -- -- T1 N1 Human
lung tumor NSCCa (SRCC725) [LT1a] IA -- -- T3 N0 Human lung tumor
AdenoCa (SRCC726) [LT2] IB -- -- T2 N0 Human lung tumor AdenoCa
(SRCC727) [LT3] IB -- -- T1 N2 Human lung tumor SqCCq (SRCC728)
[LT4] IIB -- -- T2 N0 Human lung tumor AdenoCa (SRCC729) [LT6] IV
-- -- T1 N0 Human lung tumor Aden/SqCCa (SRCC730) [LT7] IB -- -- T1
N0 Human lung tumor AdenoCa (SRCC731) [LT9] IIB -- -- T2 N0 Human
lung tumor SqCCa (SRCC732) [LT10] IA -- -- T2 N1 Human lung tumor
AdenoCa (SRCC733) [LT11] IB -- -- T1 N1 Human lung tumor AdenoCa
(SRCC734) [LT12] IIA -- -- T2 N0 Human lung tumor BAC (SRCC735)
[LT13] IB -- -- T2 N0 Human lung tumor SqCCa (SRCC736) [LT15] IB --
-- T2 N0 Human lung tumor SqCCa (SRCC737) [LT16] IB -- -- T2 N0
Human lung tumor SqCCa (SRCC738) [LT17] IIB -- -- T2 N1 Human lung
tumor SqCCa (SRCC739) [LT18] IB -- -- T2 N0 Human lung tumor SqCCa
(SRCC740) [LT19] IB -- -- T2 N0 Human lung tumor LCCa (SRCC741)
[LT21] IIB -- -- T3 N1 Human colon AdenoCa (SRCC742) [ColT2] -- M1
D pT4 N0 Human colon AdenoCa (SRCC743) [ColT3] -- B pT3 N0 Human
colon AdenoCa (SRCC 744) [ColT8] B T3 N0 Human colon AdenoCa
(SRCC745) [ColT10] A pT2 N0 Human colon AdenoCa (SRCC746) [ColT12]
MO, R1 B T3 N0 Human colon AdenoCa (SRCC747) [ColT14] pMO, RO B pT3
pN0 Human colon AdenoCa (SRCC748) [ColT15] M1, R2 D T4 N2 Human
colon AdenoCa (SRCC749) [ColT16] pMO B pT3 pN0 Human colon AdenoCa
(SRCC750) [ColT17] C1 pT3 pN1 Human colon AdenoCa (SRCC751) [ColT1]
MO, R1 B pT3 N0 Human colon AdenoCa (SRCC752) [ColT4] B pT3 M0
Human colon AdenoCa (SRCC753) [ColT5] G2 C1 pT3 pN0 Human colon
AdenoCa (SRCC754) [ColT6] pMO, RO B pT3 pN0 Human colon AdenoCa
(SRCC755) [ColT7] G1 A pT2 pN0 Human colon AdenoCa (SRCC756)
[ColT9] G3 D pT4 pN2 Human colon AdenoCa (SRCC757) [ColT11] B T3 N0
Human colon AdenoCa (SRCC758) [ColT18] MO, RO B pT3 pN0
DNA Preparation:
[0243] DNA was prepared from cultured cell lines, primary tumors,
normal human blood. The isolation was performed using purification
kit, buffer set and protease and all from Quiagen, according to the
manufacturer's instructions and the description below.
[0244] Cell Culture Lysis:
[0245] Cells were washed and trypsinized at a concentration of
7.5.times.10.sup.8 per tip and pelleted by centrifuging at 1000 rpm
for 5 minutes at 4.degree. C., followed by washing again with 1/2
volume of PBS recentrifugation. The pellets were washed a third
time, the suspended cells collected and washed 2.times. with PBS.
The cells were then suspended into 10 mL PBS. Buffer C1 was
equilibrated at 4.degree. C. Quiagen protease #19155 was diluted
into 6.25 ml cold ddH.sub.2O to a final concentration of 20 mg/ml
and equilibrated at 4.degree. C. 10 mL of G2 Buffer was prepared by
diluting Quiagen RNAse A stock (100 mg/ml) to a final concentration
of 200 .mu.g/ml.
[0246] Buffer C1 (10 mL, 4.degree. C.) and ddH2O (40 mL, 4.degree.
C.) were then added to the 10 mL of cell suspension, mixed by
inverting and incubated on ice for 10 minutes. The cell nuclei were
pelleted by centrifuging in a Beckman swinging bucket rotor at 2500
rpm at 4.degree. C. for 15 minutes. The supernatant was discarded
and the nuclei were suspended with a vortex into 2 mL Buffer C1 (at
4.degree. C.) and 6 mL ddH.sub.2O, followed by a second 4.degree.
C. centrifugation at 2500 rpm for 15 minutes. The nuclei were then
resuspended into the residual buffer using 200 .mu.l per tip. G2
buffer (10 ml) was added to the suspended nuclei while gentle
vortexing was applied. Upon completion of buffer addition, vigorous
vortexing was applied for 30 seconds. Quiagen protease (200 .mu.L,
prepared as indicated above) was added and incubated at 50.degree.
C. for 60 minutes. The incubation and centrifugation was repeated
until the lysates were clear (e.g., incubating additional 30-60
minutes, pelleting at 3000.times.g for 10 min., 4.degree. C.).
[0247] Solid Human Tumor Sample Preparation and Lysis:
[0248] Tumor samples were weighed and placed into 50 ml conical
tubes and held on ice. Processing was limited to no more than 250
mg tissue per preparation (1 tip/preparation). The protease
solution was freshly prepared by diluting into 6.25 ml cold
ddH.sub.2O to a final concentration of 20 mg/ml and stored at
4.degree. C. G2 buffer (20 ml) was prepared by diluting DNase A to
a final concentration of 200 mg/ml (from 100 mg/ml stock). The
tumor tissue was homogenated in 19 ml G2 buffer for 60 seconds
using the large tip of the polytron in a laminar-flow TC hood to
order to avoid inhalation of aerosols, and held at room
temperature. Between samples, the polytron was cleaned by spinning
at 2.times.30 seconds each in 2 L ddH.sub.2O, followed by G2 buffer
(50 ml). If tissue was still present on the generator tip, the
apparatus was disassembled and cleaned.
[0249] Quiagen protease (prepared as indicated above, 1.0 ml) was
added, followed by vortexing and incubation at 50.degree. C. for 3
hours. The incubation and centrifugation was repeated until the
lysates were clear (e.g., incubating additional 30-60 minutes,
pelleting at 3000.times.g for 10 min., 4.degree. C.).
[0250] Human Blood Preparation and Lysis:
[0251] Blood was drawn from healthy volunteers using standard
infectious agent protocols and titrated into 10 ml samples per tip.
Quiagen protease was freshly prepared by dilution into 6.25 ml cold
ddH.sub.2O to a final concentration of 20 mg/ml and stored at
4.degree. C. G2 buffer was prepared by diluting RNase A to a final
concentration of 200 .mu.g/ml from 100 mg/ml stock. The blood (10
ml) was placed into a 50 ml conical tube and 10 ml C1 buffer and 30
ml ddH.sub.2O (both previously equilibrated to 4.degree. C.) were
added, and the components mixed by inverting and held on ice for 10
minutes. The nuclei were pelleted with a Beckman swinging bucket
rotor at 2500 rpm, 4.degree. C. for 15 minutes and the supernatant
discarded. With a vortex, the nuclei were suspended into 2 ml C1
buffer (4.degree. C.) and 6 ml ddH.sub.2O (4.degree. C.). Vortexing
was repeated until the pellet was white. The nuclei were then
suspended into the residual buffer using a 200 .mu.l tip. G2 buffer
(10 ml) were added to the suspended nuclei while gently vortexing,
followed by vigorous vortexing for 30 seconds. Quiagen protease was
added (200 .mu.l) and incubated at 50.degree. C. for 60 minutes.
The incubation and centrifugation was repeated until the lysates
were clear (e.g., incubating additional 30-60 minutes, pelleting at
3000.times.g for 10 min., 4.degree. C.).
[0252] Purification of Cleared Lysates:
[0253] (1) Isolation of Genomic DNA:
[0254] Genomic DNA was equilibrated (1 sample per maxi tip
preparation) with 10 ml QBT buffer. QF elution buffer was
equilibrated at 50.degree. C. The samples were vortexed for 30
seconds, then loaded onto equilibrated tips and drained by gravity.
The tips were washed with 2.times.15 ml QC buffer. The DNA was
eluted into 30 ml silanized, autoclaved 30 ml Corex tubes with 15
ml QF buffer (50.degree. C.). Isopropanol (10.5 ml) was added to
each sample, the tubes covered with paraffin and mixed by repeated
inversion until the DNA precipitated. Samples were pelleted by
centrifugation in the SS-34 rotor at 15,000 rpm for 10 minutes at
4.degree. C. The pellet location was marked, the supernatant
discarded, and 10 ml 70% ethanol (4.degree. C.) was added. Samples
were pelleted again by centrifugation on the SS-34 rotor at 10,000
rpm for 10 minutes at 4.degree. C. The pellet location was marked
and the supernatant discarded. The tubes were then placed on their
side in a drying rack and dried 10 minutes at 37.degree. C., taking
care not to over dry the samples.
[0255] After drying, the pellets were dissolved into 1.0 ml TE (pH
8.5) and placed at 50.degree. C. for 1-2 hours. Samples were held
overnight at 4.degree. C. as dissolution continued. The DNA
solution was then transferred to 1.5 ml tubes with a 26 gauge
needle on a tuberculin syringe. The transfer was repeated 5.times.
in order to shear the DNA. Samples were then placed at 50.degree.
C. for 1-2 hours.
[0256] Quantitation of Genomic DNA and Preparation for Gene
Amplification Assay:
[0257] The DNA levels in each tube were quantified by standard
A.sub.260, A.sub.280 spectrophotometry on a 1:20 dilution (5 .mu.l
DNA+95 .mu.l ddH.sub.2O) using the 0.1 ml quartz cuvetts in the
Beckman DU640 spectrophotometer. A.sub.260/A.sub.280 ratios were in
the range of 1.8-1.9. Each DNA samples was then diluted further to
approximately 200 ng/ml in TE (pH 8.5). If the original material
was highly concentrated (about 700 ng/.mu.l), the material was
placed at 50.degree. C. for several hours until resuspended.
[0258] Fluorometric DNA quantitation was then performed on the
diluted material (20-600 ng/ml) using the manufacturer's guidelines
as modified below. This was accomplished by allowing a Hoeffer DyNA
Quant 200 fluorometer to warm-up for about 15 minutes. The Hoechst
dye working solution (#H33258, 10 .mu.l, prepared within 12 hours
of use) was diluted into 100 ml 1.times.TNE buffer. A 2 ml cuvette
was filled with the fluorometer solution, placed into the machine,
and the machine was zeroed. pGEM 3Zf(+) (2 .mu.l, lot #360851026)
was added to 2 ml of fluorometer solution and calibrated at 200
units. An additional 2 .mu.l of pGEM 3Zf(+) DNA was then tested and
the reading confirmed at 400.+-.10 units. Each sample was then read
at least in triplicate. When 3 samples were found to be within 10%
of each other, their average was taken and this value was used as
the quantification value.
[0259] The fluorometricly determined concentration was then used to
dilute each sample to 10 ng/.mu.l in ddH.sub.2O. This was done
simultaneously on all template samples for a single TaqMan.TM.
plate assay, and with enough material to run 500-1000 assays. The
samples were tested in triplicate with Taqman.TM. primers and probe
both B-actin and GAPDH on a single plate with normal human DNA and
no-template controls. The diluted samples were used provided that
the Ct value of normal human DNA subtracted from test DNA was .+-.1
Ct. The diluted, lot-qualified genomic DNA was stored in 1.0 ml
aliquots at -80.degree. C. Aliquots which were subsequently to be
used in the gene amplification assay were stored at 4.degree. C.
Each 1 ml aliquot is enough for 8-9 plates or 64 tests.
[0260] Gene Amplification Assay:
[0261] The CT-1 (cardiotrophin-1) compounds of the invention were
screened in the following primary tumors and the resulting
.DELTA.Ct values are reported in Table 2. TABLE-US-00003 TABLE 2
Screening of DNA58125 .DELTA.Ct values in lung and colon primary
tumor models Lung Lung Colon Colon Tumor Tumor Tumor Tumor Panel 1
.DELTA.Ct Panel 2 .DELTA.Ct Panel 1 .DELTA.Ct Panel 2 .DELTA.Ct
LT1.1 -0.07 LT11 0.91 ColT2 2.19 ColT1 1.17 LT1a 0.79 LT12 1.05
ColT3 1.65 ColT4 1.10 LT2 0.25 LT13 1.36 ColT8 1.11 ColT5 2.03 LT3
0.92 LT15 2.20 ColT10 1.65 ColT6 0.92 LT4 0.56 LT16 0.75 ColT12
1.06 ColT7 0.28 LT6 0.45 LT17 1.31 ColT14 1.63 ColT9 0.72 LT7 0.61
LT18 1.12 ColT15 1.26 ColT11 2.13 LT9 0.59 LT22 0.29 ColT16 1.30
ColT18 0.77 LT10 0.81 -- -- ColT17 0.89 -- -- S.D. 0.02 -- 0.13 --
0.13 -- 0.17
CT-1:
[0262] CT-1 (cardiotrophin-1,DNA58125) was reexamined with both
framework and epicenter mapping using tumors selected from the
above initial screen. FIGS. 3-8 and Tables 3-5 provide the results
of chromosome 16 mapping of the framework markers in lung and colon
tumors. The framework markers are located approximately every 20
megabases and were used as controls for determining amplification.
Tables 6-8 and FIGS. 9-12 show the results of chromosome 16 mapping
of the epicenter markers near DNA58125. TABLE-US-00004 TABLE 3
Framework Markers Map Position Stanford Human Genome Center Marker
Name P7 SHGC-2835 P55 SHGC-9643 P99 GATA7B02 P154 SHGC-33727 P208
SHGC-13574
[0263] The .DELTA.Ct values of the above described framework
markers along Chromosome 16 relative to CT-1 are indicated for
selected lung and colon tumors in Tables 4 and 5, respectively.
TABLE-US-00005 TABLE 4 Amplification of framework markers relative
to DNA58125 in Lung Tumor Lung Framework Markers (.DELTA.Ct) Tumor
P7 P55 P99 P154 P208 DNA58125 Panel 1 LT1.1 -3.62 -0.07 0.03 -0.22
-0.06 0.18 LT1a -1.90 -0.13 0.10 0.45 0.28 0.75 LT2 -0.41 -0.05
0.07 -0.07 0.41 0.36 LT3 0.18 -0.37 -0.17 -0.18 0.19 1.02 LT4 -3.58
-0.25 -0.13 -0.05 0.04 0.65 LT6 -0.57 -0.26 0.05 -0.23 0.09 0.34
LT7 -1.60 -0.46 1.14 0.25 -0.54 0.43 LT9 -0.77 -0.14 0.33 -0.18
0.43 0.36 LT10 -2.60 -0.28 0.20 -0.02 0.39 0.50 S.D. 0.36 0.11 0.01
0.04 0.21 0.01 Panel 2 LT11 -0.64 -0.15 -0.02 -0.08 -0.55 0.86 LT12
-1.19 -0.11 -0.50 -0.74 -0.97 1.00 LT13 -0.31 -0.27 0.02 -0.38
-0.40 1.33 LT15 -0.90 -1.90 -0.07 -0.18 -0.39 1.83 LT16 -1.29 -0.92
-0.68 -0.43 -0.90 0.97 LT17 -0.13 -0.15 0.02 -0.15 -0.52 1.03 LT18
-1.24 -0.43 -0.04 -0.13 -0.45 1.08 LT22 -1.86 -0.29 -0.09 -0.12
-0.26 0.05 -- -- -- -- -- -- -- S.D. 0.30 0.04 0.09 0.07 0.28
0.10
[0264] FIGS. 3 and 4 provide a three-dimensional graphical
representation of the data in Table 4, Panels 1 and 2 respectively.
The lung tumors are plotted along the x-axis, the markers and
DNA58125 are plotted along the z-axis, and the relative
amplification of chromosome 16 in the region of the marker is
indicated along the y-axis by the height of the bar. FIG. 5 is a
two-dimensional bar graph summarizing the data in Table 4 for
DNA58125 and showing that the chromosomal DNA encoding CT-1 is
amplified in some of the lung tumors (mean .DELTA.Ct values above
1.0 are single underlined and values above 2.0 are double
underlined). TABLE-US-00006 TABLE 5 Amplification of framework
markers relative to DNA58125 in Colon Tumors Colon Framework
Markers (.DELTA.Ct) Tumor P7 P55 P99 P154 P208 DNA58125 Panel 1
ColT2 2.72 0.93 0.72 0.48 -0.13 2.27 ColT3 0.01 0.07 0.53 -0.27
-0.52 1.34 ColT8 -1.01 1.05 0.69 0.60 0.04 1.23 ColT10 0.95 0.84
0.75 -0.17 -0.57 1.74 ColT12 -0.73 0.49 0.71 0.60 -0.88 1.13 ColT14
-0.16 1.49 0.83 0.33 -0.38 1.74 ColT15 -1.23 0.72 0.60 -0.29 -0.70
1.30 ColT16 0.05 1.07 0.59 -0.13 -0.66 0.93 ColT17 0.27 1.06 0.83
-0.15 -0.77 0.91 S.D. 0.15 0.67 0.88 0.57 0.49 0.04 Panel 2 ColT1
-0.73 0.35 -0.09 0.05 -0.03 1.08 ColT4 -0.99 -0.07 -0.61 -0.43
-0.09 1.13 ColT5 0.09 0.34 -0.04 -0.19 -0.01 2.17 ColT6 -1.36 -0.29
-0.03 -0.16 0.27 1.41 ColT7 -1.36 0.09 -0.18 -0.17 -0.13 0.24 ColT9
1.73 0.29 0.08 0.22 0.13 0.95 ColT11 1.03 0.51 -0.08 0.61 0.16 2.24
ColT18 0.32 0.81 0.74 0.55 0.36 1.04 S.D. 0.23 0.03 0.27 0.23 0.26
0.04
[0265] FIGS. 6 and 7 provide a three-dimensional graphical
representation of the data in Table 5, Panels 1 and 2 respectively.
The colon tumors are plotted along the x-axis, the markers and
DNA58125 are plotted along the z-axis, and the relative
amplification of chromosome 16 in the region of the marker is
indicated along the y-axis by the height of the bar. FIG. 8 is a
two-dimensional bar graph summarizing the data in Table 5 for
DNA58125 and showing that the chromosomal DNA encoding CT-1 is
amplified in several of the colon tumors (mean .DELTA.Ct values
above 1.0 are single underlined and values above 2.0 are double
underlined).
[0266] Table 6 describes the epicenter markers that were employed
in association with CT-1 (DNA58125). These markers are located in
close proximity to DNA58125 and are used to assess the
amplification status of the region of chromosome 16 in which
DNA58125 is located. The distance between individual markers is
measured in centirays, which is a radiation breakage unit
approximately equal to a 1% chance of a breakage between two
markers. One cR is very roughly equivalent to 20 kilobases. The
marker SHGC-36123 is the marker found to be the closest to the
location on chromosome 16 where DNA58125 most closely maps.
However, the Taqman.TM. primers and probes for SHGC-2726 failed in
our assay due to technical difficulties related to PCR.
TABLE-US-00007 TABLE 6 Epicenter Markers Distance to Map Position
Stanford Human Genome Center Next Marker on Chromosome 16 Marker
Name (cR.sup.1) P89 SHGC-11302 27 P90 EST00087 8 P92 SHGC-2726 23
DNA58125 -- -- P93 SHGC-36123.sup.2 42 P94 SHGC-35326 23 P95 IB391
-- .sup.1cR = Centiray. Distance between markers is measured in cR,
which is a radiation breadage unit approximately equal to a one
perent chance of a breakage between two markers. One cR corresponds
roughly to 20 kilobases. SHGC-36123 is the marker to which DNA58125
most closely maps.
[0267] Table 7 indicates the .DELTA.Ct values for results of
epicenter mapping relative to DNA58125 in lung tumors, indicating
the relative amplification in the region more immediate to the
actual location of DNA58125 along chromosome 16. TABLE-US-00008
TABLE 7 Amplification of Epicenter Markers Relative to DNA58125 in
Lung Tumors P89 P90 P92 P93 P94 P95 DNA58125 Panel 1 LT1.1 -0.11
0.00 -0.10 -0.52 -0.01 -0.13 -0.02 LT1a.1 -0.03 0.00 0.06 0.19
-0.33 -0.25 0.65 LT2.2 0.02 0.00 0.17 -0.32 0.11 -0.13 0.38 LT3.1
-0.15 0.00 0.05 0.10 0.13 0.04 0.77 LT4.2 0.08 0.00 0.02 -0.72 0.15
-0.43 0.36 LT6.1 -0.82 0.00 -0.40 -1.18 0.09 0.23 0.07 LT7.1 0.09
0.00 -0.04 0.03 0.29 0.32 0.41 LT9.1 -0.09 0.00 0.12 0.04 0.18 0.09
0.40 LT10.1 -1.65 0.00 -0.79 0.78 0.00 -0.93 -0.43 S.D. 0.29 Failed
0.25 0.88 0.04 0.18 0.11 Panel 2 LT11.1 0.15 0.00 0.17 0.10 0.23
0.31 0.91 LT12.1 -1.03 0.00 -0.07 -0.30 0.29 0.27 1.02 LT13.1 0.42
0.00 0.44 -0.12 0.23 0.27 1.52 LT15.1 0.48 0.00 0.35 0.37 0.00 0.22
2.04 LT16.2 -0.09 0.00 -0.47 -0.62 0.32 0.54 1.09 LT17.2 0.81 0.00
0.46 0.72 0.46 0.45 1.32 LT18.2 -0.10 0.00 -0.35 -0.56 0.33 -0.53
0.56 LT22.1 0.75 0.00 0.67 0.14 0.13 -0.16 0.22 S.D. 0.17 Failed
0.03 0.06 0.18 0.13 0.17
[0268] Table 8 indicates the .DELTA.Ct values for results of
epicenter mapping relative to DNA58125 in lung tumors, indicating
the relative amplification in the region more immediate to the
actual location of DNA58125 along chromosome 16. TABLE-US-00009
TABLE 8 Amplification of Epicenter Markers Relative to DNA58125 in
Colon Tumors P89 P90 P92 P93 P94 P95 DNA58125 Panel 1 ColT2 0.17
0.00 0.18 0.41 0.17 0.05 1.07 ColT3 -0.73 0.00 -0.50 -1.04 0.21
-0.61 0.66 ColT8 0.54 0.00 0.59 0.76 0.46 0.52 2.27 ColT10 0.46
0.00 0.29 0.32 0.46 0.12 1.50 ColT12 0.09 0.00 -0.15 0.05 0.57 0.01
0.81 ColT14 0.37 0.00 0.22 -0.84 0.50 0.43 0.47 ColT16 0.50 0.00
0.14 0.15 0.64 0.08 2.24 ColT17 0.15 0.00 0.26 -0.42 0.07 -0.02
0.82 S.D. 0.01 Failed 0.06 0.02 0.06 0.12 0.04 Panel 2 ColT2 0.40
0.00 0.22 0.33 0.21 0.68 2.29 ColT4 -0.20 0.00 -0.21 0.81 0.13
-0.07 1.49 ColT5 0.25 0.00 0.17 -0.30 0.14 -0.12 0.71 ColT6 0.38
0.00 0.39 0.31 0.21 0.01 1.83 ColT7 0.37 0.00 0.19 0.44 0.27 -0.12
1.20 ColT9 0.53 0.00 0.47 0.52 0.20 0.20 1.67 ColT11 0.10 0.00 0.09
0.18 0.05 -0.08 1.02 ColT18 0.02 0.00 0.12 0.21 0.05 -0.07 0.78
S.D. 0.01 Failed 0.08 0.25 0.06 0.02 0.10
DISCUSSION
[0269] The .DELTA.Ct values for DNA58125 (CT-1) in a variety of
lung and colon tumors are reported in Tables 2 (initial screen), 4
and 5 (showing amplification by framework analysis relative to
markers elsewhere on Chromosome 16), 7 and 8 (showing amplification
by epicenter analysis relative to markers in the chromosomal area
that DNA58125 is located), as well as in FIGS. 3-12. A .DELTA.Ct
value>1 (values with a single underline) was typically used as
the threshold value for amplification scoring, as this represents a
doubling of the gene copy. Table 4 indicates that significant
amplification of DNA58125 occurred in primary lung tumors LT3,
LT12, LT13, LT15, LT17, and LT18. The average .DELTA.Ct values were
1.02, 1.00, 1.33, 1.83, 1.03, 1.08, respectively, for the lung
tumors. This represents approximately a 2.0, 2.0, 2.5, 3.6, 2.0,
and 2.1 fold increase, respectively, in gene copy for the lung
tumors relative to normal tissue.
[0270] Table 5 indicates that significant amplification of DNA58125
occurred in primary colon tumors ColT2, ColT3, ColT8, ColT10,
ColT12, ColT14, ColT15, ColT1, ColT4, ColT5, ColT6, ColT11 and
ColT18. The average .DELTA.Ct values were 2.27, 1.34, 1.23, 1.74,
1.13, 1.74, 1.30, 1.08, 1.13, 2.17, 1.41, 2.24 and 1.04,
respectively for the colon tumors. This represents approximately a
4.8, 2.5, 2.3, 3.3, 2.2, 3.3, 2.5, 2.1, 2.2, 4.5, 2.6, 4.7 and 2.0
fold increase in gene copy, respectively, for the colon tumors
relative to normal tissue.
[0271] In contrast, the amplification of the closest known markers
(Tables 7 and 8) are not amplified to a greater extent than
DNA58125. Amplification of the closest markers to DNA58125 does not
occur to a greater extent than that of DNA58125. This strongly
suggests that DNA58125 is the gene that is the cause for the
amplification of the particular region on Chromosome 16.
[0272] Because amplification of DNA58125 (CT-1) occurs in various
tumors, it is likely to play a significant role in tumor formation
or growth. As a result, antagonists (e.g., antibodies) directed
against the protein encoded by DNA58125 (CT-1) would be expected to
be useful in cancer therapy.
Example 2
In situ Hybridization
[0273] In situ hybridization is a powerful and versatile technique
for the detection and localization of nucleic acid sequences within
cell or tissue preparations. It may be useful, for example, to
identify sites of gene expression, analyze the tissue distribution
of transcription, identify and localize viral infection, follow
changes in specific mRNA synthesis and aid in chromosome
mapping.
[0274] Studies of tissue distribution of cardiotrophin-1 in human
tissue was assessed in related U.S. application Ser. No. 08/286,304
filed Aug. 5, 1994, now U.S. Pat. No. 5,571,893 issued Nov. 5,
1996, herein incorporated by reference in its entirety. Such
studies are also described by Pennica, D. et al in Cytokine
8(3):183-9 (1996), herein incorporated by reference in its
entirety. Poly(A)RNA from several adult human tissues was screened
using a probe from mouse CT-1 cDNA clones. Blot hybridization with
a 180 bp mouse CT-1 probe (extending from 19 bp 5' of the
initiating ATG through amino acid 50) in 20% formamide, 5.times.SSC
at 42.degree. C. with a final wash at 0.25.times.SSC at 52.degree.
C. A 1.7 kb CT-1 mRNA was shown to be expressed in adult human
heart, skeletal muscle, ovary, colon, prostate and testis and in
fetal kidney and lung.
[0275] In situ hybridization may also be performed following an
optimized version of the protocol by Lu and Gillett, Cell Vision 1:
169-176 (1994), using PCR-generated .sup.33P-labeled riboprobes.
Briefly, formalin-fixed, paraffin-embedded human tissues are
sectioned, deparaffinized, deproteinated in proteinase K (20 g/ml)
for 15 minutes at 37.degree. C., and further processed for in situ
hybridization as described by Lu and Gillett, supra. A [.sup.33-p]
UTP-labeled antisense riboprobe is generated from a PCR product and
hybridized at 55.degree. C. overnight. The slides are dipped in
Kodak NTB2 nuclear track emulsion and exposed for 4 weeks.
[0276] .sup.33P-Riboprobe Synthesis [0277] 6.0 .mu.l (125 mCi) of
.sup.33P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) were speed vac
dried. To each tube containing dried .sup.33P-UTP, the following
ingredients were added: [0278] 2.0 .mu.l 5.times. transcription
buffer [0279] 1.0 .mu.l DTT (100 mM) [0280] 2.0 .mu.l NTP mix (2.5
mM: 10 .mu.l; each of 10 mM GTP, CTP & ATP+10 .mu.l H.sub.2O)
[0281] 1.0 .mu.l UTP (50 .mu.M) [0282] 1.0 .mu.l Rnasin [0283] 1.0
.mu.l DNA template (1 .mu.g) [0284] 1.0 .mu.l H.sub.2O [0285] 1.0
.mu.l RNA polymerase (for PCR products T3=AS, T7=S, usually)
[0286] The tubes were incubated at 37.degree. C. for one hour. 1.0
.mu.l RQ1 DNase were added, followed by incubation at 37.degree. C.
for 15 minutes. 90 .mu.l TE (10 mM Tris pH 7.6/1 mM EDTA pH 8.0)
were added, and the mixture was pipetted onto DE81 paper. The
remaining solution was loaded in a Microcon-50 ultrafiltration
unit, and spun using program 10 (6 minutes). The filtration unit
was inverted over a second tube and spun using program 2 (3
minutes). After the final recovery spin, 100 .mu.l TE were added. 1
.mu.l of the final product was pipetted on DE81 paper and counted
in 6 ml of Biofluor II.
[0287] The probe was run on a TBE/urea gel. 1-3 .mu.l of the probe
or 5 .mu.l of RNA Mrk III were added to 3 .mu.l of loading buffer.
After heating on a 95.degree. C. heat block for three minutes, the
gel was immediately placed on ice. The wells of gel were flushed,
the sample loaded, and run at 180-250 volts for 45 minutes. The gel
was wrapped in saran wrap and exposed to XAR film with an
intensifying screen in -70.degree. C. freezer one hour to
overnight.
[0288] .sup.33P-Hybridization
[0289] Pretreatment of frozen sections The slides were removed from
the freezer, placed on aluminum trays and thawed at room
temperature for 5 minutes. The trays were placed in 55.degree. C.
incubator for five minutes to reduce condensation. The slides were
fixed for 10 minutes in 4% paraformaldehyde on ice in the fume
hood, and washed in 0.5.times.SSC for 5 minutes, at room
temperature (25 ml 20.times.SSC+975 ml SQ H.sub.2O). After
deproteination in 0.5 .mu.g/ml proteinase K for 10 minutes at
37.degree. C. (12.5 .mu.l of 10 mg/ml stock in 250 ml prewarmed
RNase-free RNAse buffer), the sections were washed in 0.5.times.SSC
for 10 minutes at room temperature. The sections were dehydrated in
70%, 95%, 100% ethanol, 2 minutes each.
[0290] Pretreatment of paraffin-embedded sections The slides were
deparaffinized, placed in SQ H.sub.2O, and rinsed twice in
2.times.SSC at room temperature, for 5 minutes each time. The
sections were deproteinated in 20 .mu.g/ml proteinase K (500 .mu.l
of 10 mg/ml in 250 ml RNase-free RNase buffer; 37.degree. C., 15
minutes)-human embryo, or 8.times. proteinase K (100 .mu.l in 250
ml Rnase buffer, 37.degree. C., 30 minutes)-formalin tissues.
Subsequent rinsing in 0.5.times.SSC and dehydration were performed
as described above.
[0291] Prehybridization The slides were laid out in plastic box
lined with Box buffer (4.times.SSC, 50% formamide)-saturated filter
paper. The tissue was covered with 50 .mu.l of hybridization buffer
(3.75 g Dextran Sulfate+6 ml SQ H.sub.2O), vortexed and heated in
the microwave for 2 minutes with the cap loosened. After cooling on
ice, 18.75 ml formamide, 3.75 ml 20.times.SSC and 9 ml SQ H.sub.2O
were added, the tissue was vortexed well, and incubated at
42.degree. C. for 1-4 hours.
[0292] Hybridization 1.0.times.10.sup.6 cpm probe and 1.0 .mu.l
tRNA (50 mg/ml stock) per slide were heated at 95.degree. C. for 3
minutes. The slides were cooled on ice, and 48 .mu.l hybridization
buffer were added per slide. After vortexing, 50 .mu.l .sup.33P mix
were added to 50 .mu.l prehybridization on slide. The slides were
incubated overnight at 55.degree. C.
[0293] Washes Washing was done 2.times.10 minutes with 2.times.SSC,
EDTA at room temperature (400 ml 20.times.SSC+16 ml 0.25M EDTA,
V.sub.f=4 L), followed by RNaseA treatment at 37.degree. C. for 30
minutes (500 .mu.l of 10 mg/ml in 250 ml Rnase buffer=20 .mu.g/ml),
The slides were washed 2.times.10 minutes with 2.times.SSC, EDTA at
room temperature. The stringency wash conditions were as follows: 2
hours at 55.degree. C., 0.1.times.SSC, EDTA (20 ml 20.times.SSC+16
ml EDTA, V.sub.f=4 L).
Example 3
Use of CT-1 as a Hybridization Probe
[0294] The following method describes use of a nucleotide sequence
encoding a CT-1 polypeptide as a hybridization probe.
[0295] DNA comprising the coding sequence of full-length or mature
CT-1 (as shown in FIG. 1, SEQ ID NO:1 and 2) is employed as a probe
to screen for homologous DNAs (such as those encoding
naturally-occurring variants of CT-1) in human tissue cDNA
libraries or human tissue genomic libraries.
[0296] Hybridization and washing of filters containing either
library DNAs is performed under the following high stringency
conditions. Hybridization of radiolabeled CT-1-derived probe to the
filters is performed in a solution of 50% formamide, 5.times.SSC,
0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH
6.8, 2.times.Denhardt's solution, and 10% dextran sulfate at
42.degree. C. for 20 hours. Washing of the filters is performed in
an aqueous solution of 0.1.times.SSC and 0.1% SDS at 42.degree.
C.
[0297] DNAs having a desired sequence identity with the DNA
encoding full-length native sequence CT-1 can then be identified
using standard techniques known in the art.
Example 4
Expression of CT-1 in E. coli
[0298] This example illustrates preparation of an unglycosylated
form of CT-1 by recombinant expression in E. coli.
[0299] The DNA sequence encoding CT-1 (SEQ ID NO:1) is initially
amplified using selected PCR primers. The primers should contain
restriction enzyme sites which correspond to the restriction enzyme
sites on the selected expression vector. A variety of expression
vectors may be employed. An example of a suitable vector is pBR322
(derived from E. coli; see Bolivar et al., Gene, 2:95 (1977)) which
contains genes for ampicillin and tetracycline resistance. The
vector is digested with restriction enzyme and dephosphorylated.
The PCR amplified sequences are then ligated into the vector. The
vector will preferably include sequences which encode for an
antibiotic resistance gene, a trp promoter, a polyhis leader
(including the first six STII codons, polyhis sequence, and
enterokinase cleavage site), the CT-1 coding region, lambda
transcriptional terminator, and an argU gene.
[0300] The ligation mixture is then used to transform a selected E.
coli strain using the methods described in Sambrook et al., supra.
Transformants are identified by their ability to grow on LB plates
and antibiotic resistant colonies are then selected. Plasmid DNA
can be isolated and confirmed by restriction analysis and DNA
sequencing.
[0301] Selected clones can be grown overnight in liquid culture
medium such as LB broth supplemented with antibiotics. The
overnight culture may subsequently be used to inoculate a larger
scale culture. The cells are then grown to a desired optical
density, during which the expression promoter is turned on.
[0302] After culturing the cells for several more hours, the cells
can be harvested by centrifugation. The cell pellet obtained by the
centrifugation can be solubilized using various agents known in the
art, and the solubilized CT-1 protein can then be purified using a
metal chelating column under conditions that allow tight binding of
the protein.
[0303] CT-1 is expressed in E. coli in a poly-His tagged form,
using the following procedure. The DNA encoding CT-1 is initially
amplified using selected PCR primers. The primers contain
restriction enzyme sites which correspond to the restriction enzyme
sites on the selected expression vector, and other useful sequences
providing for efficient and reliable translation initiation, rapid
purification on a metal chelation column, and proteolytic removal
with enterokinase. The PCR-amplified, poly-His tagged sequences are
then ligated into an expression vector, which is used to transform
an E. coli host based on strain 52 (W3110 fuhA(tonA) lon galE
rpoHts (htpRts) clpP(lacIq). Transformants are first grown in LB
containing 50 mg/ml carbenicillin at 30.degree. C. with shaking
until an O.D.600 of 3-5 is reached. Cultures were then diluted
50-100 fold into CRAP media (prepared by mixing 3.57 g
(NH.sub.4).sub.2SO.sub.4, 0.71 g sodium citrate-2H2O, 1.07 g KCl,
5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 mL
water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM
MgSO.sub.4) and grown for approximately 20-30 hours at 30.degree.
C. with shaking. Samples are removed to verify expression by
SDS-PAGE analysis, and the bulk culture is centrifuged to pellet
the cells. Cell pellets are frozen until purification and
refolding.
[0304] E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets)
is resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH
8 buffer. Solid sodium sulfite and sodium tetrathionate is added to
make final concentrations of 0.1M and 0.02 M, respectively, and the
solution was stirred overnight at 4.degree. C. This step results in
a denatured protein with all cysteine residues blocked by
sulfitolization. The solution is centrifuged at 40,000 rpm in a
Beckman Ultracentifuge for 30 min. The supernatant is diluted with
3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM
Tris, pH 7.4) and filtered through 0.22 micron filters to clarify.
Depending the clarified extract is loaded onto a 5 ml Qiagen Ni-NTA
metal chelate column equilibrated in the metal chelate column
buffer. The column is washed with additional buffer containing 50
mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein is
eluted with buffer containing 250 mM imidazole. Fractions
containing the desired protein are pooled and stored at 4.degree.
C. Protein concentration is estimated by its absorbance at 280 nm
using the calculated extinction coefficient based on its amino acid
sequence.
[0305] The proteins are refolded by diluting sample slowly into
freshly prepared refolding buffer consisting of: 20 mM Tris, pH
8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM
EDTA. Refolding volumes are chosen so that the final protein
concentration is between 50 to 100 micrograms/ml. The refolding
solution is stirred gently at 4.degree. C. for 12-36 hours. The
refolding reaction is quenched by the addition of TFA to a final
concentration of 0.4% (pH of approximately 3). Before further
purification of the protein, the solution is filtered through a
0.22 micron filter and acetonitrile is added to 2-10% final
concentration. The refolded protein is chromatographed on a Poros
R1/H reversed phase column using a mobile buffer of 0.1% TFA with
elution with a gradient of acetonitrile from 10 to 80%. Aliquots of
fractions with A.sub.280 absorbance are analyzed on SDS
polyacrylamide gels and fractions containing homogeneous refolded
protein are pooled. Generally, the properly refolded species of
most proteins are eluted at the lowest concentrations of
acetonitrile since those species are the most compact with their
hydrophobic interiors shielded from interaction with the reversed
phase resin. Aggregated species are usually eluted at higher
acetonitrile concentrations. In addition to resolving misfolded
forms of proteins from the desired form, the reversed phase step
also removes endotoxin from the samples.
[0306] Fractions containing the desired folded CT-1 proteins,
respectively, are pooled and the acetonitrile removed using a
gentle stream of nitrogen directed at the solution. Proteins are
formulated into 20 mM Hepes, pH 6.8 with 0.14 M sodium chloride and
4% mannitol by dialysis or by gel filtration using G25 Superfine
(Pharmacia) resins equilibrated in the formulation buffer and
sterile filtered.
Example 5
Expression of CT-1 in Mammalian Cells
[0307] This example illustrates preparation of a glycosylated form
of CT-1 by recombinant expression in mammalian cells.
[0308] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989),
is employed as the expression vector. Optionally, the CT-1 DNA is
ligated into pRK5 with selected restriction enzymes to allow
insertion of the CT-1 DNA using ligation methods such as described
in Sambrook et al., supra. The resulting vector is called
pRK5-CT-1.
[0309] In one embodiment, the selected host cells may be 293 cells.
Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue
culture plates in medium such as DMEM supplemented with fetal calf
serum and optionally, nutrient components and/or antibiotics. About
10 .mu.g pRK5-CT-1 DNA is mixed with about 1 .mu.g DNA encoding the
VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] and dissolved
in 500 .mu.l of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl.sub.2. To
this mixture is added, dropwise, 500 .mu.l of 50 mM HEPES (pH
7.35), 280 mM NaCl, 1.5 mM NaPO.sub.4, and a precipitate is allowed
to form for 10 minutes at 25.degree. C. The precipitate is
suspended and added to the 293 cells and allowed to settle for
about four hours at 37.degree. C. The culture medium is aspirated
off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The
293 cells are then washed with serum free medium, fresh medium is
added and the cells are incubated for about 5 days.
[0310] Approximately 24 hours after the transfections, the culture
medium is removed and replaced with culture medium (alone) or
culture medium containing 200 .mu.Ci/ml .sup.35S-cysteine and 200
.mu.Ci/ml .sup.35S-methionine. After a 12 hour incubation, the
conditioned medium is collected, concentrated on a spin filter, and
loaded onto a 15% SDS gel. The processed gel may be dried and
exposed to film for a selected period of time to reveal the
presence of CT-1 polypeptide. The cultures containing transfected
cells may undergo further incubation (in serum free medium) and the
medium is tested in selected bioassays.
[0311] In an alternative technique, CT-1 DNA may be introduced into
293 cells transiently using the dextran sulfate method described by
Somparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981). 293
cells are grown to maximal density in a spinner flask and 700 .mu.g
pRK5-CT-1 DNA is added. The cells are first concentrated from the
spinner flask by centrifugation and washed with PBS. The
DNA-dextran precipitate is incubated on the cell pellet for four
hours. The cells are treated with 20% glycerol for 90 seconds,
washed with tissue culture medium, and re-introduced into the
spinner flask containing tissue culture medium, 5 .mu.g/ml bovine
insulin and 0.1 .mu.g/ml bovine transferrin. After about four days,
the conditioned media is centrifuged and filtered to remove cells
and debris. The sample containing expressed CT-1 can then be
concentrated and purified by any selected method, such as dialysis
and/or column chromatography.
[0312] In another embodiment, CT-1 can be expressed in CHO cells.
The pRK5-CT-1 vector can be transfected into CHO cells using known
reagents such as CaPO.sub.4 or DEAE-dextran. As described above,
the cell cultures can be incubated, and the medium replaced with
culture medium (alone) or medium containing a radiolabel such as
.sup.35S-methionine. After determining the presence of CT-1
polypeptide, the culture medium may be replaced with serum free
medium. Preferably, the cultures are incubated for about 6 days,
and then the conditioned medium is harvested. The medium containing
the expressed CT-1 can then be concentrated and purified by any
selected method.
[0313] Epitope-tagged CT-12 may also be expressed in host CHO
cells. The CT-1 may be subcloned out of the pRK5 vector. The
subclone insert can undergo PCR to fuse in frame with a selected
epitope tag such as a poly-His tag into a Baculovirus expression
vector. The poly-His tagged CT-1 insert can then be subcloned into
a SV40 driven vector containing a selection marker such as DHFR for
selection of stable clones. Finally, the CHO cells can be
transfected (as described above) with the SV40 driven vector.
Labeling may be performed, as described above, to verify
expression. The culture medium containing the expressed poly-His
tagged CT-1 can then be concentrated and purified by any selected
method, such as by Ni.sup.2+-chelate affinity chromatography.
[0314] CT-1 was expressed in CHO cells by both a transient and a
stable expression procedure. Stable expression in CHO cells was
performed using the following procedure. The proteins were
expressed as an IgG construct (immunoadhesin), in which the coding
sequences for the soluble forms (e.g. extracellular domains) of the
respective proteins were fused to an IgG1 constant region sequence
containing the hinge, CH2 and CH2 domains and/or is a poly-His
tagged form.
[0315] Following PCR amplification, the DNA58125 is subcloned in a
CHO expression vector using standard techniques as described in
Ausubel et al., Current Protocols of Molecular Biology, Unit 3.16,
John Wiley and Sons (1997). CHO expression vectors are constructed
to have compatible restriction sites 5' and 3' of the DNA of
interest to allow the convenient shuttling of cDNA's. The vector
uses expression in CHO cells is as described in Lucas et al., Nucl.
Acids Res. 24: 9 (1774-1779 (1996), and uses the SV40 early
promoter/enhancer to drive expression of the cDNA of interest and
dihydrofolate reductase (DHFR). DHFR expression permits selection
for stable maintenance of the plasmid following transfection.
[0316] Twelve micrograms of the desired plasmid DNA were introduced
into approximately 10 million CHO cells using commercially
available transfection reagents Superfect.RTM. (Quiagen),
Dosper.RTM. or Fugene.RTM. (Boehringer Mannheim). The cells were
grown and described in Lucas et al., supra. Approximately
3.times.10.sup.-7 cells are frozen in an ampule for further growth
and production as described below.
[0317] The ampules containing the plasmid DNA were thawed by
placement into a water bath and mixed by vortexing. The contents
were pipetted into a centrifuge tube containing 10 mLs of media and
centrifuged at 1000 rpm for 5 minutes. The supernatant was
aspirated and the cells were resuspended in 10 mL of selective
media (0.2 .mu.m filtered PS20 with 5% 0.2 .mu.m diafiltered fetal
bovine serum). The cells were then aliquoted into a 100 mL spinner
containing 90 mL of selective media. After 1-2 days, the cells were
transferred into a 250 mL spinner filled with 150 mL selective
growth medium and incubated at 37.degree. C. After another 2-3
days, a 250 mL, 500 mL and 2000 mL spinners were seeded with
3.times.10.sup.5 cells/mL. The cell media was exchanged with fresh
media by centrifugation and resuspension in production medium.
Although any suitable CHO media may be employed, a production
medium described in U.S. Pat. No. 5,122,469, issued Jun. 16, 1992
was actually used. 3 L production spinner is seeded at
1.2.times.10.sup.6 cells/mL. On day 0, the cell number pH were
determined. On day 1, the spinner was sampled and sparging with
filtered air was commenced. On day 2, the spinner was sampled, the
temperature shifted to 33.degree. C., and 30 mL of 500 g/L glucose
and 0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane
emulsion, Dow Corning 365 Medical Grade Emulsion). Throughout the
production, pH was adjusted as necessary to maintain a pH of about
7.2. After 10 days, or until viability dropped below 70%, the cell
culture was harvested by centrifugation and filtering through a
0.22 .mu.m filter. The filtrate was either stored at 4.degree. C.
or immediately loaded onto columns for purification.
[0318] For the poly-His tagged constructs, the proteins were
purified using a Ni-NTA column (Qiagen). Before purification,
imidazole was added to the conditioned media to a concentration of
5 mM. The conditioned media was pumped onto a 6 ml Ni-NTA column
equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl
and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4.degree. C.
After loading, the column was washed with additional equilibration
buffer and the protein eluted with equilibration buffer containing
0.25 M imidazole. The highly purified protein was subsequently
desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl
and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia)
column and stored at -80.degree. C.
[0319] CT-1 may be produced by transient expression in COS cells,
as well, using standard techniques.
Example 6
Expression of CT-1 in Yeast
[0320] The following method describes recombinant expression of
CT-1 in yeast.
[0321] First, yeast expression vectors are constructed for
intracellular production or secretion of CT-1 from the ADH2/GAPDH
promoter. DNA58125 encoding CT-1 and the promoter is inserted into
suitable restriction enzyme sites in the selected plasmid to direct
intracellular expression of CT-1. For secretion, DNA encoding CT-1
can be cloned into the selected plasmid, together with DNA encoding
the ADH2/GAPDH promoter, a native CT-1 signal peptide or other
mammalian signal peptide, or, for example, a yeast alpha-factor or
invertase secretory signal/leader sequence, and linker sequences
(if needed) for expression of CT-1.
[0322] Yeast cells, such as yeast strain AB110, can then be
transformed with the expression plasmids described above and
cultured in selected fermentation media. The transformed yeast
supernatants can be analyzed by precipitation with 10%
trichloroacetic acid and separation by SDS-PAGE, followed by
staining of the gels with Coomassie Blue stain.
[0323] Recombinant CT-1 can subsequently be isolated and purified
by removing the yeast cells from the fermentation medium by
centrifugation and then concentrating the medium using selected
cartridge filters. The concentrate containing CT-1 may further be
purified using selected column chromatography resins.
Example 7
Expression of CT-1 in Baculovirus-Infected Insect Cells
[0324] The following method describes recombinant CT-1 expression
in Baculovirus-infected insect cells.
[0325] The sequence coding for CT-1 is fused upstream of an epitope
tag contained within a baculovirus expression vector. Such epitope
tags include poly-His tags and immunoglobulin tags (like Fc regions
of IgG). A variety of plasmids may be employed, including plasmids
derived from commercially available plasmids such as pVL1393
(Novagen). Briefly, the sequence encoding CT-1 or the desired
portion of the coding sequence of CT-1 (such as the sequence
encoding the extracellular domain of a transmembrane protein or the
sequence encoding the mature protein if the protein is
extracellular) is amplified by PCR with primers complementary to
the 5' and 3' regions. The 5' primer may incorporate flanking
(selected) restriction enzyme sites. The product is then digested
with those selected restriction enzymes and subcloned into the
expression vector.
[0326] Recombinant baculovirus is generated by co-transfecting the
above plasmid and BaculoGold.TM. virus DNA (Pharmingen) into
Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using
lipofectin (commercially available from GIBCO-BRL). After 4-5 days
of incubation at 28.degree. C., the released viruses are harvested
and used for further amplifications. Viral infection and protein
expression are performed as described by O'Reilley et al.,
Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford
University Press (1994).
[0327] Expressed poly-His tagged CT-1 can then be purified, for
example, by Ni.sup.2+-chelate affinity chromatography as follows.
Extracts are prepared from recombinant virus-infected Sf9 cells as
described by Rupert et al., Nature, 362:175-179 (1993). Briefly,
Sf9 cells are washed, resuspended in sonication buffer (25 mL
Hepes, pH 7.9; 12.5 mM MgCl.sub.2; 0.1 mM EDTA; 10% glycerol; 0.1%
NP-40; 0.4 M KCl), and sonicated twice for 20 seconds on ice. The
sonicates are cleared by centrifugation, and the supernatant is
diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl,
10% glycerol, pH 7.8) and filtered through a 0.45 .mu.m filter. A
Ni.sup.2+-NTA agarose column (commercially available from Qiagen)
is prepared with a bed volume of 5 mL, washed with 25 mL of water
and equilibrated with 25 mL of loading buffer. The filtered cell
extract is loaded onto the column 0.5 mL per minute. The column is
washed to baseline A.sub.280 with loading buffer, at which point
fraction collection is started. Next, the column is washed with a
secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol,
pH 6.0), which elutes nonspecifically bound protein. After reaching
A.sub.280 baseline again, the column is developed with a 0 to 500
mM imidazole gradient in the secondary wash buffer. One mL
fractions are collected and analyzed by SDS-PAGE and silver
staining or Western blot with Ni.sup.2+-NTA-conjugated to alkaline
phosphatase (Qiagen). Fractions containing the eluted
His.sub.10-tagged CT-1 are pooled and dialyzed against loading
buffer. Alternatively, purification of the IgG tagged (or Fc
tagged) CT-1 can be performed using known chromatography
techniques, including for instance, Protein A or Protein G column
chromatography.
[0328] While the CT-1 expression is performed in a 0.5-2 L scale,
it can be readily scaled up for larger (e.g. 8 L) preparations.
CT-1 is also expressed as an IgG construct (immunoadhesin), in
which the protein extracellular region is fused to an IgG1 constant
region sequence containing the hinge, CH2 and CH3 domains and/or in
poly-His tagged forms.
[0329] Following PCR amplification, the coding sequence is
subcloned into a baculovirus expression vector (pb.PH.IgG for IgG
fusions and pb.PH.His.c for poly-His tagged proteins), and the
vector and Baculogold.RTM. baculovirus DNA (Pharmingen) is
co-transfected into 105 Spondoptera frugiperda ("Sf9") cells (ATCC
CRL 1711), using Lipofectin (Gibco BRL). pb.PH.IgG and pb.PH.His
are modifications of the commercially available baculovirus
expression vector pVL1393 (Pharmingen), with modified polylinker
regions to include the His or Fc tag sequences. The cells are grown
in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone). Cells
are incubated for 5 days at 28.degree. C. The supernatant is
harvested and subsequently used for the first viral amplification
by infecting Sf9 cells in Hink's TNM-FH medium supplemented with
10% FBS at an approximate multiplicity of infection (MOI) of 10.
Cells are incubated for 3 days at 28.degree. C. The supernatant is
harvested and the expression of the constructs in the baculovirus
expression vector is determined by batch binding of 1 mL of
supernatant to 25 mL of NI-NTA beads (Qiagen) for histidine tagged
proteins or Protein-A Sepharose CL-4B beads (Pharmacia) for IgG
tagged proteins followed by SDS-PAGE analysis comparing to a known
concentration of protein standard by Coomassie blue staining.
[0330] The first viral amplification supernatant is used to infect
a spinner culture (500 mL) of Sf9 cells grown in ESF-921 medium
(Expression Systems LLC) at an approximate MOI of 0.1. Cells are
incubated for 3 days at 28.degree. C. The supernatant is harvested
and filtered. Batch binding and SDS-PAGE analysis is repeated, as
necessary, until expression of the spinner culture is
confirmed.
[0331] The conditioned medium from the transfected cells (0.5 to 3
L) is harvested by centrifugation to remove the cells and filtered
through 0.22 micron filters. For the poly-His tagged constructs,
the protein construct are purified using a Ni-NTA column (Qiagen).
Before purification, imidazole is added to the conditioned media to
a concentration of 5 mM. The conditioned media are pumped onto a 6
mL Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer
containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5
mL/min. at 4.degree. C. After loading, the column is washed with
additional equilibration buffer and the protein eluted with
equilibration buffer containing 0.25 M imidazole. The highly
purified protein is subsequently desalted into a storage buffer
containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a
25 mL G25 Superfine (Pharmacia) column and stored at -80.degree.
C.
[0332] Immunoadhesin (Fc containing) constructs of proteins are
purified from the conditioned medium as follows. The conditioned
medium is pumped onto a 5 mL Protein A column (Pharmacia) which had
been equilibrated in 20 mM sodium phosphate buffer, pH 6.8. After
loading, the column is washed extensively with equilibration buffer
before elution with 100 mM citric acid, pH 3.5. The eluted protein
is immediately neutralized by collecting 1 mL fractions into tubes
containing 275 of 1 M Tris buffer, pH 9. The highly purified
protein is subsequently desalted into storage buffer as described
above for the poly-His tagged proteins. The homogeneity of the
proteins is verified by SDS-PAGE and N-terminal amino acid
sequencing by Edman degradation.
Example 8
Preparation of Antibodies that Bind CT-1
[0333] This example illustrates preparation of monoclonal
antibodies which can specifically bind CT-1.
[0334] Techniques for producing the monoclonal antibodies are known
in the art and are described, for instance, in Goding, supra.
Immunogens that may be employed include purified CT-,1 fusion
proteins containing CT-1, and cells expressing recombinant CT-1 on
the cell surface. Selection of the immunogen can be made by the
skilled artisan without undue experimentation.
[0335] Mice, such as BAlb/c, are immunized with the CT-1 immunogen
emulsified in complete Freund's adjuvant and injected
subcutaneously or intraperitoneally in an amount from 1-100
micrograms. Alternatively, the immunogen is emulsified in MPL-TDM
adjuvant (Ribi Immunochemical Research, Hamilton, Mont.) and
injected into the animal's hind foot pads. The immunized mice are
then boosted 10 to 12 days later with additional immunogen
emulsified in the selected adjuvant. Thereafter, for several weeks,
the mice may also be boosted with additional immunization
injections. Serum samples may be periodically obtained from the
mice by retro-orbital bleeding for testing in ELISA assays to
detect anti-CT-1 antibodies.
[0336] After a suitable antibody titer has been detected, the
animals "positive" for antibodies can be injected with a final
intravenous injection of CT-1. Three to four days later, the mice
are sacrificed and the spleen cells are harvested. The spleen cells
are then fused (using 35% polyethylene glycol) to a selected murine
myeloma cell line such as P3X63AgU.1, available from ATCC, No. CRL
1597. The fusions generate hybridoma cells which can then be plated
in 96 well tissue culture plates containing HAT (hypoxanthine,
aminopterin, and thymidine) medium to inhibit proliferation of
non-fused cells, myeloma hybrids, and spleen cell hybrids.
[0337] The hybridoma cells are screened in an ELISA for reactivity
against CT-1. Determination of "positive" hybridoma cells secreting
the desired monoclonal antibodies against CT-1 is within the skill
in the art.
[0338] The positive hybridoma cells can be injected
intraperitoneally into syngeneic Balb/c mice to produce ascites
containing the anti-CT-1 monoclonal antibodies. Alternatively, the
hybridoma cells can be grown in tissue culture flasks or roller
bottles. Purification of the monoclonal antibodies produced in the
ascites can be accomplished using ammonium sulfate precipitation,
followed by gel exclusion chromatography. Alternatively, affinity
chromatography based upon binding of antibody to Protein A or
Protein G can be employed.
Deposit of Material
[0339] The following material, a plasmid encoding CT-1 (disclosed
in U.S. Ser. No. 08,286,304 filed Aug. 5, 1994, now U.S. Pat. No.
5,571,893, issued Nov. 5, 1996), has been deposited with the
American Type Culture Collection, 10801 University Blvd., Manassas,
Va. 20110-2209, USA (ATCC): TABLE-US-00010 Material ATCC Dep. No.
Deposit Date pBSSK + .huCT1.h5 74,841 Jul. 26, 1994
[0340] This deposit was made under the provisions of the Budapest
Treaty on the International Recognition of the Deposit of
Microorganisms for the purpose of Patent Procedure and the
Regulations thereunder (Budapest Treaty). This assures maintenance
of a viable culture of the deposit for 30 years from the date of
deposit. The deposit will be made available by ATCC under the terms
of the Budapest Treaty, and subject to an agreement between
Genentech, Inc. and ATCC, which assures permanent and unrestricted
availability of the progeny of the culture of the deposit to the
public upon issuance of the pertinent U.S. patent or upon laying
open to the public of any U.S. or foreign patent application,
whichever comes first, and assures availability of the progeny to
one determined by the U.S. Commissioner of Patents and Trademarks
to be entitled thereto according to 35 U.S.C. .sctn. 122 and the
Commissioner's rules pursuant thereto (including 37 CFR .sctn. 1.14
with particular reference to 8860G 638).
[0341] The assignee of the present application has agreed that if a
culture of the materials on deposit should die or be lost or
destroyed when cultivated under suitable conditions, the materials
will be promptly replaced on notification with another of the same.
Availability of the deposited material is not to be construed as a
license to practice the invention in contravention of the rights
granted under the authority of any government in accordance with
its patent laws.
[0342] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
the construct deposited, since the deposited embodiment is intended
as a single illustration of certain aspects of the invention and
any constructs that are functionally equivalent are within the
scope of the this invention. The deposit of material herein does
not constitute an admission that the written description herein
contained is inadequate to enable the practice of any aspect of the
invention, including the best mode thereof, nor is it to be
construed as limiting the scope of the claims to the specific
illustrations that it represents. Indeed, various modifications of
the invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
Sequence CWU 1
1
6 1 1539 DNA Homo sapiens 1 gtgaagggag ccgggatcag ccaggggcca
gcatgagccg gagggaggga 50 agtctggaag acccccagac tgattcctca
gtctcacttc ttccccactt 100 ggaggccaag atccgtcaga cacacagcct
tgcgcacctc ctcaccaaat 150 acgctgagca gctgctccag gaatatgtgc
agctccaggg agaccccttc 200 gggctgccca gcttctcgcc gccgcggctg
ccggtggccg gcctgagcgc 250 cccggctccg agccacgcgg ggctgccagt
gcacgagcgg ctgcggctgg 300 acgcggcggc gctggccgcg ctgcccccgc
tgctggacgc agtgtgtcgc 350 cgccaggccg agctgaaccc gcgcgcgccg
cgcctgctgc gccgcctgga 400 ggacgcggcg cgccaggccc gggccctggg
cgccgccgtg gaggccttgc 450 tggccgcgct gggcgccgcc aaccgcgggc
cccgggccga gccccccgcc 500 gccaccgcct cagccgcctc cgccaccggg
gtcttccccg ccaaggtgct 550 ggggctccgc gtttgcggcc tctaccgcga
gtggctgagc cgcaccgagg 600 gcgacctggg ccagctgctg cccgggggct
cggcctgagc gccgcggggc 650 agctcgcccc gcctcctccc gctgggttcc
gtctctcctt ccgcttcttt 700 gtctttctct gccgctgtcg gtgtctgtct
gtctgctctt agctgtctcc 750 attgcctcgg ccttctttgc tttttgtggg
ggagagggga ggggacgggc 800 agggtctctg tcgcccaggc tggggtgcag
tggcgcgatc ccagcactgc 850 agcctcaacc tcctgggctc aagccatcct
tccgcctcag cttccccagc 900 agctgggact acaggcacgc gccaccacag
ccggctaatt ttttatttaa 950 ttttttgtag agacgaggtt tcgccatgtt
gcccaggctg gtcttgaact 1000 ccggggctca agcgatcctc ccgcttcagc
ctccctaagt gctgggattg 1050 caggcgtgag ccactttccc agcctctctt
tgctttgcct gccccgttct 1100 cttaactctt ggaccctcct cgtctgcatg
gtaactccgt ctgagtctac 1150 cattttcttg ctctccctcc ttccttgggc
ctgcctcagt tccctttggc 1200 ctcccccttt acccagctct tggggtgtct
ctgttttttc catccccact 1250 tcctgccttc tcgtggccct gtggtagcac
atgtgtacat ctcagcctta 1300 tctcaaggag gtgacacctt ctctccttgt
ccccatctgg ccgtctctct 1350 gtgcttccct ggccaggggc gtgcctgctg
gtcctatggg gggaaggcta 1400 ctccgcatct cagccacctt cctcaggctc
actccaccta catccccagt 1450 ctgccacacc ccatcccttt gggcctcagc
cctgtccctt tgatgtcctc 1500 ctttccttca gcccctctgc cctgtccctg
cacacctcc 1539 2 1539 DNA Homo sapiens 2 ggaggtgtgc agggacaggg
cagaggggct gaaggaaagg aggacatcaa 50 agggacaggg ctgaggccca
aagggatggg gtgtggcaga ctggggatgt 100 aggtggagtg agcctgagga
aggtggctga gatgcggagt agccttcccc 150 ccataggacc agcaggcacg
cccctggcca gggaagcaca gagagacggc 200 cagatgggga caaggagaga
aggtgtcacc tccttgagat aaggctgaga 250 tgtacacatg tgctaccaca
gggccacgag aaggcaggaa gtggggatgg 300 aaaaaacaga gacaccccaa
gagctgggta aagggggagg ccaaagggaa 350 ctgaggcagg cccaaggaag
gagggagagc aagaaaatgg tagactcaga 400 cggagttacc atgcagacga
ggagggtcca agagttaaga gaacggggca 450 ggcaaagcaa agagaggctg
ggaaagtggc tcacgcctgc aatcccagca 500 cttagggagg ctgaagcggg
aggatcgctt gagccccgga gttcaagacc 550 agcctgggca acatggcgaa
acctcgtctc tacaaaaaat taaataaaaa 600 attagccggc tgtggtggcg
cgtgcctgta gtcccagctg ctggggaagc 650 tgaggcggaa ggatggcttg
agcccaggag gttgaggctg cagtgctggg 700 atcgcgccac tgcaccccag
cctgggcgac agagaccctg cccgtcccct 750 cccctctccc ccacaaaaag
caaagaaggc cgaggcaatg gagacagcta 800 agagcagaca gacagacacc
gacagcggca gagaaagaca aagaagcgga 850 aggagagacg gaacccagcg
ggaggaggcg gggcgagctg ccccgcggcg 900 ctcaggccga gcccccgggc
agcagctggc ccaggtcgcc ctcggtgcgg 950 ctcagccact cgcggtagag
gccgcaaacg cggagcccca gcaccttggc 1000 ggggaagacc ccggtggcgg
aggcggctga ggcggtggcg gcggggggct 1050 cggcccgggg cccgcggttg
gcggcgccca gcgcggccag caaggcctcc 1100 acggcggcgc ccagggcccg
ggcctggcgc gccgcgtcct ccaggcggcg 1150 cagcaggcgc ggcgcgcgcg
ggttcagctc ggcctggcgg cgacacactg 1200 cgtccagcag cgggggcagc
gcggccagcg ccgccgcgtc cagccgcagc 1250 cgctcgtgca ctggcagccc
cgcgtggctc ggagccgggg cgctcaggcc 1300 ggccaccggc agccgcggcg
gcgagaagct gggcagcccg aaggggtctc 1350 cctggagctg cacatattcc
tggagcagct gctcagcgta tttggtgagg 1400 aggtgcgcaa ggctgtgtgt
ctgacggatc ttggcctcca agtggggaag 1450 aagtgagact gaggaatcag
tctgggggtc ttccagactt ccctccctcc 1500 ggctcatgct ggcccctggc
tgatcccggc tcccttcac 1539 3 201 PRT Homo sapiens 3 Met Ser Arg Arg
Glu Gly Ser Leu Glu Asp Pro Gln Thr Asp Ser 1 5 10 15 Ser Val Ser
Leu Leu Pro His Leu Glu Ala Lys Ile Arg Gln Thr 20 25 30 His Ser
Leu Ala His Leu Leu Thr Lys Tyr Ala Glu Gln Leu Leu 35 40 45 Gln
Glu Tyr Val Gln Leu Gln Gly Asp Pro Phe Gly Leu Pro Ser 50 55 60
Phe Ser Pro Pro Arg Leu Pro Val Ala Gly Leu Ser Ala Pro Ala 65 70
75 Pro Ser His Ala Gly Leu Pro Val His Glu Arg Leu Arg Leu Asp 80
85 90 Ala Ala Ala Leu Ala Ala Leu Pro Pro Leu Leu Asp Ala Val Cys
95 100 105 Arg Arg Gln Ala Glu Leu Asn Pro Arg Ala Pro Arg Leu Leu
Arg 110 115 120 Arg Leu Glu Asp Ala Ala Arg Gln Ala Arg Ala Leu Gly
Ala Ala 125 130 135 Val Glu Ala Leu Leu Ala Ala Leu Gly Ala Ala Asn
Arg Gly Pro 140 145 150 Arg Ala Glu Pro Pro Ala Ala Thr Ala Ser Ala
Ala Ser Ala Thr 155 160 165 Gly Val Phe Pro Ala Lys Val Leu Gly Leu
Arg Val Cys Gly Leu 170 175 180 Tyr Arg Glu Trp Leu Ser Arg Thr Glu
Gly Asp Leu Gly Gln Leu 185 190 195 Leu Pro Gly Gly Ser Ala 200 4
21 DNA Homo sapiens 4 ttcccagcct ctctttgctt t 21 5 22 DNA Homo
sapiens 5 tcagacggag ttaccatgca ga 22 6 27 DNA Homo sapiens 6
tgccccgttc tcttaactct tggaccc 27
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