U.S. patent application number 15/786960 was filed with the patent office on 2018-06-07 for cancer targets and uses thereof.
The applicant listed for this patent is CELERA CORPORATION. Invention is credited to Sudeepta AGGARWAL, Dong FANG, Paul MOORE, Steve RUBEN.
Application Number | 20180155421 15/786960 |
Document ID | / |
Family ID | 45990807 |
Filed Date | 2018-06-07 |
United States Patent
Application |
20180155421 |
Kind Code |
A1 |
FANG; Dong ; et al. |
June 7, 2018 |
CANCER TARGETS AND USES THEREOF
Abstract
Methods and compositions are provided for assessing, treating,
and preventing diseases, especially cancer, using cancer-associated
targets ("CAT"). Methods and compositions are also provided for
determining or predicting the effectiveness of a treatment for
these diseases or for selecting a treatment, using CAT. Methods and
compositions are further provided for modulating cell function
using CAT. Also provided are compositions that modulate CAT (e.g.,
antagonists or agonists), such as antibodies, proteins, small
molecule compounds, and nucleic acid agents (e.g., RNAi and
antisense agents), as well as pharmaceutical compositions thereof.
Further provided are methods of screening for agents that modulate
CAT, and agents identified by these screening methods.
Inventors: |
FANG; Dong; (North Potomac,
MD) ; MOORE; Paul; (North Bethesda, MD) ;
RUBEN; Steve; (Brookeville, MD) ; AGGARWAL;
Sudeepta; (North Potomac, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CELERA CORPORATION |
San Clemente |
CA |
US |
|
|
Family ID: |
45990807 |
Appl. No.: |
15/786960 |
Filed: |
October 18, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14960642 |
Dec 7, 2015 |
|
|
|
15786960 |
|
|
|
|
13959893 |
Aug 6, 2013 |
|
|
|
14960642 |
|
|
|
|
13436411 |
Mar 30, 2012 |
8524238 |
|
|
13959893 |
|
|
|
|
12275779 |
Nov 21, 2008 |
8168586 |
|
|
13436411 |
|
|
|
|
61003930 |
Nov 21, 2007 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/574 20130101;
G01N 33/57484 20130101; C07K 16/18 20130101; C12N 15/113 20130101;
A61K 2039/505 20130101; C07K 2317/732 20130101; C12Q 2600/158
20130101; G01N 33/5011 20130101; C12Q 1/6886 20130101; C12Q
2600/118 20130101; C12Q 2600/106 20130101; C12N 2310/14 20130101;
C07K 16/30 20130101; C07K 14/47 20130101; A61K 39/39533 20130101;
C07K 2317/622 20130101; C07K 14/57 20130101; C07K 2317/734
20130101; C07K 2317/54 20130101; C07K 16/249 20130101; C07K 2317/55
20130101; C07K 2317/21 20130101; C07K 2317/24 20130101; G01N
2500/10 20130101 |
International
Class: |
C07K 16/24 20060101
C07K016/24; A61K 39/395 20060101 A61K039/395; C07K 14/47 20060101
C07K014/47; C07K 14/57 20060101 C07K014/57; C07K 16/18 20060101
C07K016/18; G01N 33/50 20060101 G01N033/50; G01N 33/574 20060101
G01N033/574; C12N 15/113 20100101 C12N015/113; C12Q 1/6886 20180101
C12Q001/6886; C07K 16/30 20060101 C07K016/30 |
Claims
1. An isolated protein comprising an amino acid sequence selected
from the group consisting of SEQ ID NOS:1-722, 1975, and 1977 and
1282-1974.
2. (canceled)
3. An isolated nucleic acid molecule comprising a nucleotide
sequence selected from the group consisting of: a) SEQ ID
NOS:723-1281, 1976, and 1978; b) nucleotide sequences that encode a
protein comprising an amino acid sequence selected from the group
consisting of SEQ ID NOS:1-722, 1975, and 1977 and 1282-1974; and
c) nucleotide sequences that are completely complementary to the
nucleotide sequences of a) or b).
4. An isolated RNAi or antisense nucleic acid molecule that
selectively binds to the nucleic acid molecule of claim 3.
5. An isolated antibody that selectively binds to the protein of
claim 1.
6. The antibody of claim 5, wherein the antibody is at least one of
a monoclonal, polyclonal, fully human, humanized, chimeric,
single-chain, or anti-idiotypic antibody.
7. (canceled)
8. The antibody of claim 5, wherein the antibody is coupled to a
composition selected from the group consisting of detectable
substances and therapeutic agents.
9. A composition comprising the antibody of claim 5 and a
pharmaceutically acceptable carrier.
10. An isolated antibody fragment of the antibody of claim 5,
wherein the antibody fragment comprises a fragment selected from
the group consisting of: a) an Fab fragment; b) an F(ab').sub.2
fragment; and c) an Fv fragment.
11. A method of modulating cell proliferation or apoptosis, the
method comprising contacting a cell with the antibody of claim
5.
12. The method of claim 11, wherein the method comprises either
inhibiting proliferation of cancer cells or stimulating apoptosis
of cancer cells.
13. A method of modulating cell proliferation or apoptosis, the
method comprising contacting a cell with the RNAi or antisense
nucleic acid molecule of claim 4.
14. A method of detecting the protein of claim 1 in a sample, the
method comprising contacting the sample with an isolated antibody
that selectively binds to the protein and determining whether the
antibody binds to the protein.
15. A method of detecting the nucleic acid molecule of claim 3 in a
sample, the method comprising contacting the sample with an
oligonucleotide that specifically hybridizes to the nucleic acid
molecule and determining whether the oligonucleotide binds to the
nucleic acid molecule.
16. A method of diagnosing, prognosing, or determining risk of
cancer in a subject, the method comprising detecting at least one
molecule in a sample, wherein the presence or abundance of the
molecule is indicative of cancer, and wherein the molecule is
selected from the group consisting of: a) proteins comprising an
amino acid sequence selected from the group consisting of SEQ ID
NOS:1-722, 1975, and 1977 and 1282-1974; b) antibodies that
selectively bind to the protein of a); c) nucleic acid molecules
comprising a nucleotide sequence selected from the group consisting
of SEQ ID NOS:723-1281, 1976, and 1978 and nucleotide sequences
that encode the protein of a); and d) nucleic acid molecules
comprising a nucleotide sequence that is completely complementary
to the nucleic acid molecule of c).
17. A method of treating cancer, the method comprising
administering a therapeutically effective amount of the antibody of
claim 5 to a subject.
18-20. (canceled)
21. A method of detecting or isolating a cancer stem cell, wherein
the method comprises contacting the cancer stem cell with the
antibody of claim 5.
22. A method of detecting or isolating a protein expressed by a
cancer stem cell, wherein the method comprises contacting the
cancer stem cell with the antibody of claim 5.
23. A method of selectively targeting a cancer stem cell, wherein
the method comprises contacting a heterogenous population of cancer
cells with the antibody of claim 5, wherein the antibody
selectively binds to a cancer stem cell.
24. The method of claim 23, wherein the method modulates
proliferation or apoptosis of the cancer stem cell.
25. The method of claim 24, wherein the method inhibits
proliferation or stimulates apoptosis of the cancer stem cell.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
non-provisional application Ser. No. 14/960,642, filed Dec. 7,
2015, which is a continuation application of U.S. non-provisional
application Ser. No. 13/959,893, filed Aug. 6, 2013, which is a
divisional application of U.S. non-provisional application Ser. No.
13/436,411, filed Mar. 30, 2012, which is a divisional application
of U.S. non-provisional application Ser. No. 12/275,779, filed Nov.
21, 2008 (and issued as U.S. Pat. No. 8,168,586 on May 1, 2012),
which claims priority to U.S. provisional application Ser. No.
61/003,930, filed on Nov. 21, 2007, the contents of each of which
are hereby incorporated by reference in their entirety into this
application.
FIELD OF THE INVENTION
[0002] This invention relates to the field of disease assessment
and therapy. The invention provides compositions and methods for
assessing and treating diseases, especially cancer.
BACKGROUND OF THE INVENTION
[0003] Cancer
[0004] Cancer is one of the leading causes of death worldwide, and
cancer is difficult to diagnose and treat effectively. Accordingly,
there is a need in the art for new compositions and methods for
assessing and treating various cancers.
[0005] Cancer Stem Cells (CSCs)
[0006] Cancer stem cells (CSCs), which may also be referred to as
tumor stem cells, are cancer cells that are operationally similar
to normal stem cells and that typically have the ability to grow
(or re-grow) a tumor. For example, like normal stem cells, CSCs
typically possess the properties of self-renewal ability, extensive
proliferation potential, and the potential to differentiate into
multiple phenotypically different cell types. CSCs can potentially
arise from, for example, mutation of a normal adult stem cell or
from mutations that impart stem cell-like properties to another
type of cell. Therefore, CSCs may or may not originate from their
normal stem cell counterpart. However, regardless of whether or not
the cell of origin of a CSC is in fact a true stem cell, the term
CSC is used in the art on the basis of the cancer cell having
properties that are similar to normal stem cells.
[0007] For example, recent studies have demonstrated that in many
types of cancer, only a small subset of cancer cells within a tumor
have the ability to proliferate extensively, to initiate the growth
of new tumors (including metastatic growth), and to differentiate
into the various different cell types that make up a typical,
complex heterogeneous tumor. These cells are the CSCs, and these
properties are typical of CSCs. Some CSCs have previously been
isolated (e.g., from leukemia, as well as brain, breast, and lung
cancers) and, when transplanted into an animal model (or even
serially passaged into multiple animals), have been shown to be
necessary and sufficient to initiate the growth of new tumors,
whereas the vast majority of other cells within a tumor do not have
the capability to initiate the growth of new tumors.
[0008] However, current cancer therapies (including, for example,
chemotherapy and radiation therapy) generally attempt to
non-discriminately kill proliferating cells, and potential
therapeutic agents are commonly selected based on their ability to
reduce tumor size. Furthermore, clinical trials of anti-cancer
agents are commonly designed with the objective of demonstrating a
reduction in tumor size. However, because cancer stem cells
typically make up only a small proportion of a tumor, reduction in
tumor size may not be indicative of any reduction in cancer stem
cell populations, and therefore reduction in tumor size may not
accurately reflect longer-term cancer prognosis. Because CSCs
typically represent only a minor portion of a tumor and can be
quiescent (non-proliferating), cancer therapies may primarily kill
other cells that make up the majority of the tumor, thereby often
leading to tumor shrinkage and possibly temporary cancer remission
or other clinical improvement. However, because the CSCs have not
been directly targeted and killed, CSCs may survive the cancer
therapy and eventually initiate re-growth of the cancer. Thus, to
permanently eradicate a cancer and to prevent metastasis, it would
be desirable to specifically kill the CSCs. Similarly, to gain a
more comprehensive diagnosis of a cancer, such as in predicting the
reoccurrence of a cancer following treatment or the threat of
metastasis, it would be desirable to specifically assess the
CSCs.
[0009] Therefore, there is a need in the art to identify cancer
stem cell markers (e.g., proteins expressed by cancer stem cells,
and the encoding nucleic acid molecules), as well as agents (e.g.,
antibodies) that target these cancer stem cell markers, so that
more effective cancer therapies and diagnostics can be implemented
that specifically target the small population of tumor cells within
a tumor that are cancer stem cells and which can be most harmful to
a patient with regards to tumor growth, metastasis and initiation
of new tumors, and re-growth of tumors following treatment.
[0010] For a further review of CSCs, see the following (each of
these references is incorporated herein by reference): Fang et al.,
"A tumorigenic subpopulation with stem cell properties in
melanomas", Cancer Res. 2005 Oct. 15; 65(20):9328-37; Lee et al.,
"Tumor stem cells derived from glioblastomas cultured in bFGF and
EGF more closely mirror the phenotype and genotype of primary
tumors than do serum-cultured cell lines", Cancer Cell. 2006 May;
9(5):391-403; Nishizuka, "Profiling cancer stem cells using protein
array technology", Eur J Cancer. 2006 June; 42(9):1273-1282; Reya
et al., "Stem cells, cancer, and cancer stem cells", Nature 2001
Nov. 1; 414(6859):105-11; Huff et al., "The paradox of response and
survival in cancer therapeutics", Blood 2006 Jan. 15; 107(2):431-4;
Feinberg et al., "The epigenetic progenitor origin of human
cancer", Nat Rev Genet 2006 January; 7(1):21-33; Al-Hajj et al.,
"Therapeutic implications of cancer stem cells", Curr Opin Genet
Dev. 2004 Feburary; 14(1):43-7; Soltysova et al., "Cancer stem
cells", Neoplasma 2005; 52(6):435-40; Wang et al., "Cancer stem
cells: lessons from leukemia", Trends Cell Biol. 2005 September;
15(9):494-501; Jordan, "Cancer stem cell biology: from leukemia to
solid tumors", Curr Opin Cell Biol. 2004 December; 16(6):708-12;
Liu et al., "Adult stem cells and cancer stem cells: tie in or tear
apart?", J Cancer Res Clin Oncol. 2005 October; 131(10):631-8;
Bjerkvig et al., "Opinion: the origin of the cancer stem cell:
current controversies and new insights", Nat Rev Cancer 2005
November; 5(11):899-904; Brabletz et al., "Opinion: migrating
cancer stem cells--an integrated concept of malignant tumour
progression", Nat Rev Cancer 2005 September; 5(9):744-9; Jones et
al., "Cancer stem cells: are we missing the target?", J Natl Cancer
Inst. 2004 Apr. 21; 96(8):583-5; Al-Hajj et al., "Self-renewal and
solid tumor stem cells", Oncogene 2004 Sep. 20; 23(43):7274-82;
Kopper et al., "Tumor stem cells", Pathol Oncol Res. 2004;
10(2):69-73; Cheng, "Cell cycle inhibitors in normal and tumor stem
cells", Oncogene 2004 Sep. 20; 23(43):7256-66; and U.S. Pat. No.
6,984,522 ("Isolation and Use of Solid Tumor Stem Cells").
[0011] Description of the Files Contained on the CD-R Named
CL001733CDR
[0012] The CD-R named CL001733CDR contains the following three text
(ASCII) files:
[0013] 1) File SEQLIST_1733ORD.txt provides the Sequence Listing.
The Sequence Listing provides exemplary protein sequences (SEQ ID
NOS:1-722, 1975, and 1977), transcript sequences (SEQ ID
NOS:723-1281, 1976, and 1978), and peptide sequences (SEQ ID
NOS:1282-1974) as shown in Table 1. File SEQLIST_1733ORD.txt is
5,905 KB in size and was created on Nov. 20, 2008.
[0014] 2) File TABLE1_1733.txt provides Table 1, which is 1,018 KB
in size and was created on Nov. 20, 2007.
[0015] 3) File TABLE2_1733.txt provides Table 2, which is 14 KB in
size and was created on Nov. 20, 2007.
[0016] The material contained on the CD-R named CL001733CDR is
hereby incorporated by reference pursuant to 37 CFR 1.77(b)(4).
[0017] Description of Table 1
[0018] Table 1 (provided on the CD-R) discloses cancer-associated
target ("CAT") proteins, transcripts, and peptides (each protein,
transcript, and peptide is represented by a SEQ ID NO in Table 1,
and the corresponding sequence is provided in the Sequence Listing;
the range of numbers in parentheses following each peptide SEQ ID
NO represents the amino acid residues of the location of the
peptide within its corresponding protein), the disease (e.g.,
cancer) cell lines or tissues, the expression ratio ("ratio") of
the peptide in the disease sample compared to a control sample, and
the type of cancer (or, if "adipose" is indicated, adipose disease
such as diabetes or obesity) in which differential expression of
the target was observed ("disease").
[0019] The expression ratio is based on measuring the expression
level of the peptides, which are fragments of each full-length
protein. Thus, the expression level of the peptides is indicative
of the expression level of the corresponding protein of which the
peptide is a fragment. Numerical representation of over-expression
is indicated by 2.0 or more, whereas numerical representation of
under-expression is indicated by 0.5 or less. Over-expressed
singleton indicates that a peptide peak was detected in a disease
(e.g., cancer) sample but no peak was detected in a control sample.
Under-expressed singleton indicates that a peptide peak was
detected in a control sample but no peak was detected in a disease
sample.
[0020] The protein/gene/transcript information provided for each
target includes any or all of the information selected from the
following: [0021] a protein SEQ ID NO [0022] an internal
identification number for the protein (hCP and/or UID) [0023] a
public protein accession number (Genbank, e.g., RefSeq NP number,
Swiss-prot, or Derwent) for the protein [0024] a protein name
recognized in the art [0025] an internal identification number for
the gene (hCG and/or UID) [0026] an art-recognized gene symbol
[0027] OMIM ("Online Mendelian Inheritance in Man" database; Johns
Hopkins University/NCBI) gene/protein name(s) and/or symbol(s)
[0028] a transcript SEQ ID NO [0029] an internal identification
number for the transcript (hCT and/or UID) [0030] a public
transcript accession number (Genbank, e.g., RefSeq NM number, or
Derwent)
[0031] Description of Table 2
[0032] Table 2 discloses tumor staging information for tumor tissue
samples listed in Table 1, as follows (all tumor staging
designations in Table 2 are conventional in the art):
[0033] Sample ID Number (corresponding to a tumor tissue sample
listed in Table 1), sample type (labeled "Sample"), cancer type
(labeled "Type"), Lymph Nodes (the "N" in the TNM staging system,
for Node, which designates the spread of a tumor to the lymph
nodes), Distant Metastasis (the "M" in the TNM staging system, for
Metastasis, which designates the extent of tumor metastasis),
Extent of Invasion (the "T" in the TNM staging system, for Tumor,
which designates the size and location of a primary tumor), and
AJCC Stage (which is assigned based on a combination of the T, N,
and M classifications).
[0034] Description of Table 3
[0035] Table 3 summarizes results of expression and functional
validation analyses for the cancer-associated targets ("CAT")
provided herein.
[0036] The column labeled "Target Symbol" provides the target
symbol identifying the target, which can be used to cross-reference
targets between Table 3 and Table 1.
[0037] The column labeled "IHC" provides results of
immunohistochemistry analysis of target protein expression in tumor
tissue samples. For IHC studies, the expression of each target was
typically evaluated in 10 tumor specimen for 10-14 cancer
indications and 2 normal specimens of the same tissue type. A
pathology score was given to each specimen by a certified
pathologist. The pathology scores were based on: (i) staining
intensity that ranged from 0-4, 0 being the weakest and 4 being the
strongest staining, and (ii) the number of cells staining positive;
for a specimen to be considered positively stained, >25% percent
of cells needed to have a staining score of 1 or above. Once the
scores are assigned for each specimen, the percent of tumors that
have 2 or more pathology scores above the highest normal is
calculated. These percentages are listed in Table 3 (in the column
labeled "IHC").
[0038] The columns labeled "Apoptosis" and "Proliferation" provide
results of apoptosis and cell proliferation functional assays,
respectively, in cancer cell lines in which each target was knocked
down by RNAi. In the proliferation or apoptosis columns, a "+"
indicates that the target has been functionally validated based on
an RNAi screen. Experimentally, a pool of four RNAi duplexes
against a target was transfected in one or more cell lines for each
indication. If the transfection results in >35% decrease in
proliferation or >2-fold increase in apoptosis in a cell line,
the results were considered significant and the target was assigned
a "+" notation (which is indicated in Table 3).
[0039] Once a cell line has passed a screen, further validation
steps were undertaken. First, the screen positive cell line is
transfected with maximum concentration (100 nM) of each of four
individual duplexes that were included in the pool in the above
description. Typically, 1-2 duplexes out of the pool of 4 will show
similar effects on proliferation and/or apoptosis as the pool
transfection. Effect of mRNA knockdown by individual duplexes was
also measured at this stage. Next, decreasing concentrations of the
selected duplex (100 nM down to 1 nM) were transfected into the
cell line to confirm specificity of the selected duplex. If
decreasing the duplex concentration resulted in decreased effect on
proliferation and apoptosis of the cell line, the target was
considered to be functionally validated and was therefore assigned
a "++" notation (which is indicated in Table 3).
[0040] For selected targets, if an antibody is commercially
available or has been custom generated, the effect of inhibition of
the cell line proliferation was measured in the presence of
different concentrations of the antibody and its isotype control.
If the presence of an antibody against a target showed inhibition
of proliferation in one or more cell lines, the target was
considered functionally validated using an antibody and was
therefore assigned a "+++" notation (which is indicated in Table 3)
(a target with a "+++" notation was not necessarily also "++"
validated).
[0041] The column labeled "Matrix" refers to the experiments where
the comparisons were made between "case" and "control" to identify
the over-expressed protein/peptides. Each matrix entry is
identified by a prefix that defines the cell line and a suffix that
defines the comparison or culture condition used to generate the
cell line. As examples,
[0042] 1. H1299_Stemcell_Cys_only:
[0043] Spheroids (stem cells) generated from lung H1299 cell line
were compared to adherent cells obtained either from (i) primary
culture or (ii) spheroid population; only cystine containing
peptides were analyzed in this experiment.
[0044] 2. H1299_Stemcell_Glyco:
[0045] Spheroids (stem cells) generated from lung H1299 cell line
were compared to adherent cells obtained either from (i) primary
culture or (ii) spheroid population; only peptides captured based
on glycosylated residues were analyzed in this experiment.
[0046] 3. CBT026_HES_Primary
[0047] Spheroids (stem cells) generated from colon tumor tissue
CBT026T were compared to the primary processed tumor tissue.
[0048] 4. HT29_Stemcell:
[0049] Spheroids (stem cells) generated from colorectal cell line
HT29 were compared to adherent cells obtained from primary
culture.
[0050] 5. CBT026_T:
[0051] Spheroids (stem cells) generated from colon tumor tissue
CBT026T were compared to the adherent cells in culture obtained
from the same tumor tissue.
[0052] 6. CBT026_N:
[0053] Spheroids (stem cells) generated from normal colon tissue
CBT026N were compared to the adherent cells in culture obtained
from the same tissue.
[0054] 7. RKO Sphere:
[0055] Spheroids (stem cells) generated from colorectal cell line
RKO were compared to adherent cells obtained from primary
culture.
[0056] 8. Melanoma Stemcell_EGT003D:
[0057] Spheroids (stem cells) generated from melanoma tumor tissue
EGT003D were compared to the adherent cells in culture obtained
from the same tissue.
[0058] 9. LS123_Stemcell:
[0059] Spheroids (stem cells) generated from colorectal cell line
LS123 were compared to adherent cells obtained from primary
culture.
[0060] 10. CBT026_12Maps:
[0061] Spheroids (stem cells) generated from colon tumor tissue
CBT026T and normal tissue CBT026N were compared with adherent cells
obtained from culturing these tissues.
[0062] 11. CBT026_Five Sets with N Primary:
[0063] Spheroids (stem cells) generated from colon tumor tissue
CBT026T or adherent cells were compared to spheroids and adherent
cells from normal tissue.
Further information about cell lines: [0064] Lung metastatic
non-small cell lung cancer cell line H1299 (catalog no. CRL-5803)
was purchased through ATCC and cultured according to the vendor
recommendations. [0065] Colorectal cancer cell lines HT-29 (Catalog
no. HTB-39), LS123 (catalog number: CCL-255), and RKO (catalog no.
CRL-2577) were also obtained from ATCC and cultured according to
the vendor recommendations. [0066] Spheroid cells (stem cells) were
generated from colorectal tumor tissue CBT026T (from 62 yr old
male; grade: G2; stage: IIIC; TNM Status: T4N2MX) and its adjacent
normal colorectal tissue CBT026N. The spheroid cell line generated
from colorectal tumor tissue CBT026T (which was used in comparisons
that are designated in the "Matrix" column of Table 3 as "CBT026_T"
and "CBT026_HES_Primary") has been deposited with the American Type
Culture Collection (ATCC), 10801 University Boulevard, Manassas,
Va. 20110-2209, USA, under the terms of the Budapest Treaty on the
International Recognition of the Deposit of Microorganisms for the
Purpose of Patent Procedure on Jul. 21, 2006 and bears the ATCC
accession number PTA-7742. [0067] Spheroid cells (stem cells) were
generated from melanoma tumor tissue EGT003D (identified in the
"Matrix" column of Table 3 as "Melanoma StemCell_EGT003D").
[0068] The column labeled "Subcellular Location" indicates the
cellular location of the target (either cell surface or
secreted).
DESCRIPTION OF THE FIGURES
[0069] FIGS. 1-4. Results of experimental validation (TaqMan, RNAi,
and, for ANGPTL4, Flow Cytometry) for Angiopoietin-like 4 protein
(ANGPTL4) (FIG. 1), Tweety Homolog 3 (TTYH3) (FIG. 2), CEACAM6
(FIG. 3), and Protein FLJ11273 (FIG. 4).
[0070] In the figures, with respect to RNAi validation results,
"Apo." refers to an apoptosis assay, and "Prol." refers to a cell
proliferation assay. "+", "++", and "-" indicate relative degree of
RNAi-mediated effect observed in the assay (either inducement of
apoptosis or inhibition of cell proliferation) in various cancer
cell lines (as indicated), with "+" indicating that an
RNAi-mediated effect was observed, "++" indicating that a strong
RNAi-mediated effect was observed, and "-" indicating that an
RNAi-mediated effect was not observed.
[0071] H1299 is a lung cancer cell line, CBT026T is a colon cancer
cell line, and EGT003D is a melanoma cell line.
[0072] Cell lines labeled "DMEM" refer to differentiated (adherent)
cell lines (DMEM indicates a type of culture medium suitable for
the growth of differentiated cells). Cell lines labeled "HES" and
"HES-S" refer to cancer stem cell lines (HES and HES-S, which are
used herein interchangeably, indicate a type of culture media
suitable for the growth of human embryonic stem, or "HES", cells
and cancer stem cells). Cell lines labeled "H/D" refer to
differentiated cell populations derived from cancer stem cells.
Cell lines labeled "MBM4" refer to adherent melanoma cells
generated in vitro from a primary tumor by culturing in
serum-containing melanoma cell-derived MBM4 medium.
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS OF THE INVENTION
[0073] The invention will best be understood by reference to the
following detailed description of the exemplary embodiments, taken
in conjunction with the accompanying table(s) and/or figure(s). The
discussion below is exemplary and is not to be taken as limiting
the scope defined by the claims.
[0074] The invention generally relates to molecules that have been
identified using proteomic analysis techniques such as
MALDI-TOF/TOF LC/MS-based protein expression analysis to determine
the expression levels of proteins in disease tissues and/or disease
cell lines (tissues and cell lines may be collectively referred to
as "samples") and in normal tissues and/or normal cell lines, such
that proteins that are differentially expressed (e.g., over- or
under-expressed) in disease samples compared with normal samples
are identified.
[0075] Exemplary embodiments of the invention provide the targets
shown in Tables 1 and 3 and methods of using these targets. Four of
these targets (ANGPTL4, TTYH3, CEACAM6, and Protein FLJ11273) are
also shown in the figures and described in section 13 of the
Examples section (which is entitled "Specific Examples of Results
from Experimental Validation"). All of these targets (i.e., the
targets provided in Table 1, Table 3, and/or the figures) are
collectively referred to herein as "CAT" ("cancer-associated
targets"). In particular, exemplary embodiments of the invention
provide methods of using these targets for assessing (e.g.,
diagnosing, prognosing, or determining future susceptibility),
treating, and preventing cancer. Each of these targets is
associated with specific types of cancers in particular, as shown
in Tables 1 and 3, the figures, and/or described in section 13 of
the Examples section. These targets also are expressed by cancer
stem cells and therefore these targets have utilities with respect
to cancer stem cells, in addition to their other cancer-related
uses, such as for specifically targeting cancer stems cells (e.g.,
for therapeutic or diagnostic purposes) or for detecting,
isolating, and/or labeling cancer stem cells.
[0076] Based on, for example, the finding that certain proteins,
referred to herein as cancer-associated target ("CAT") proteins,
are differentially expressed in cancer samples in comparison with
normal samples, exemplary embodiments of the invention provide
methods and compositions for assessing, treating, and preventing
diseases, especially cancer, particularly the cancers identified in
Table 1 (as indicated for each peptide) and/or Table 3, using CAT.
Furthermore, the compositions and methods of the invention may be
suitable for other types of cancer, particularly other epithelial
cell-related cancers and solid tumors.
[0077] Cancer-associated target ("CAT") proteins and fragments
thereof (e.g., CAT peptides), and CAT nucleic acid molecules and
fragments thereof encoding CAT proteins and peptides, are
collectively referred to as CAT or "targets" (which may be
interchangeably referred to as "markers" or "biomarkers").
Exemplary CAT proteins are provided as SEQ ID NOS:1-722, 1975, and
1977, exemplary fragments of these CAT proteins are provided as the
peptide sequences of SEQ ID NOS:1282-1974, and exemplary CAT
transcript sequences (which encode the CAT proteins of SEQ ID
NOS:1-722, 1975, and 1977) are provided as SEQ ID NOS:723-1281,
1976, and 1978. These targets can be, for example, cell surface
proteins, cytosolic proteins, or secreted proteins, as well as
nucleic acid molecules that encode these proteins.
[0078] The terms "protein" and "polypeptide" are used herein
interchangeably. Exemplary CAT proteins/polypeptides are provided
as SEQ ID NOS:1-722, 1975, and 1977. A "peptide" typically refers
to a fragment of a protein/polypeptide. Thus, peptides are
interchangeably referred to as fragments. Exemplary CAT peptides
are provided as SEQ ID NOS:1282-1974. SEQ ID NOS:1282-1974 are
fragments of SEQ ID NOS:1-722, 1975, and 1977 (in Table 1, the
range of numbers in parentheses following each peptide SEQ ID NO
represents the amino acid residues of the location of the peptide
within its corresponding protein).
[0079] References herein to proteins, peptides, nucleic acid
molecules, and antibodies typically are not limited to the
full-size or full-length molecule, but also can encompass fragments
of these molecules (unless a particular sequence or structure is
explicitly stated).
[0080] Exemplary embodiments of the invention, which are discussed
in greater detail below, provide antibodies, proteins, immunogenic
peptides (e.g., peptides which induce a T-cell response), or other
biomolecules, as well as small molecules, nucleic acid agents
(e.g., RNAi and antisense nucleic acid agents), and other
compositions that modulate the targets (e.g., agonists and
antagonists), such as by binding to or otherwise interacting with
or affecting the targets. These compositions can be used for
assessing, treating, and preventing diseases, especially cancer,
particularly the cancers identified in Table 1 (as indicated for
each peptide) and/or Table 3, as well as other uses. Moreover, the
invention provides methods for assessing, treating, and preventing
diseases such as these based on CAT, such as by using these
compositions. Further provided are methods of screening for agents
that modulate CAT, such as by affecting the function, activity,
and/or expression level ("expression level" may be interchangeably
referred to as "abundance level" or "level") of CAT, and agents
identified by these screening methods.
[0081] Exemplary embodiments of the invention also provide methods
of modulating cell function. In particular, the invention provides
methods of modulating cell proliferation and/or apoptosis. For
example, for cancer/tumor cells, the invention provides methods of
inhibiting cell proliferation and/or stimulating apoptosis. Such
methods can be applied to the treatment of diseases, especially
cancer.
[0082] Exemplary embodiments of the invention further provide
methods of determing or predicting effectiveness or response to a
particular treatment, and methods of selecting a treatment for an
individual. For example, targets that are differentially expressed
by cells that are more or less responsive or resistant to a
particular treatment, such as a cancer treatment, are useful for
determing or predicting effectiveness or response to the treatment
or for selecting a treatment for an individual. Exemplary
embodiments of the invention also provide methods of selecting
individuals for a clinical trial of a therapeutic agent. For
example, the targets can be used to identify individuals for
inclusion in a clinical trial who are more likely to respond to a
particular treatment. Alternatively, the targets can be used to
exclude individuals from a clinical trial who are less likely to
respond to a particular treatment or who are more likely to
experience toxic or other undesirable side effects from a
particular treatment.
[0083] Further exemplary embodiments of the invention are described
in greater detail below.
[0084] Cancer-Associated Targets (CAT) as Cancer Stem Cell (CSC)
Targets
[0085] The CAT proteins and encoding nucleic acid molecules
provided in Table 1 and/or Table 3 are expressed by cancer stem
cells ("CSCs") and therefore are useful as CSC targets (as
exemplified in the figures for certain targets). Thus, the
cancer-associated targets ("CAT") provided herein may be
interchangeably referred to herein as CSC targets. Because the CAT
provided herein are expressed by cancer stem cells, the targets
provided herein are particularly useful for cancer stem
cell-related purposes, such as for specifically targeting cancer
stems cells (e.g., for therapeutic or diagnostic purposes) or for
detecting, isolating, and/or labeling cancer stem cells. For
example, the targets provided herein are particularly useful as
therapeutic targets for cancer. A therapeutic agent can
specifically target a CAT protein, such as in a heterogenous tumor
cell population, in order to more effectively and efficiently
(e.g., by enabling the use of a lower dose of a therapeutic agent,
thereby minimizing side effects such as toxicity) treat a cancer by
targeting a cancer stem cell.
[0086] CSCs may be cultured under conditions (e.g., using a
serum-free culture medium such as media designated for human
embryonic stem cells) in which the CSCs proliferate as non-adherent
3-dimensional ("3D") spheres, closely mimicking in vivo tumor
growth. Hence, CSCs may be interchangeably referred to herein as
"cancer spheroid cells", "spheroids", or "tumorospheres". These 3D
cultures of CSCs, which can be stably maintained in vitro, are
useful for therapeutic target discovery and drug testing, among
other uses.
[0087] Typical cancer cells or heterogeneous populations of cancer
cells that are not CSCs (i.e., non-stem cell-like cancer cells) may
be interchangeably referred to herein as "differentiated",
"adherent", or "parental" cells (although "parental" cells
typically more specifically refer to differentiated/adherent cells
from which cancer stem cells are derived from), or as
"counterparts" to cancer stem cells.
[0088] In certain exemplary embodiments of the invention, the CAT
proteins or encoding nucleic acid molecules provided in Table 1
and/or Table 3 are used for selectively targeting CSCs (such as in
a tumor or other heterogenous population of cancer cells), such as
for therapeutic, preventative, diagnostic, or prognostic purposes.
For example, by selectively targeting a CSC target provided herein
(e.g., with an antibody or small molecule compound that selectively
binds to a protein provided herein, or a nucleic acid agent such as
an RNAi or antisense molecule that hybridizes to a nucleic acid
molecule that encodes a protein provided herein), proliferation or
apoptosis of CSCs can be modulated, such as to inhibit
proliferation or stimulate apoptosis of CSCs (such as for the
purpose of killing CSCs).
[0089] Exemplary embodiments of the invention provide agents, such
as antibodies, antibody-drug conjugates, detectably labeled
antibodies, small molecule compounds, and nucleic acid agents,
which selectively target the CSC targets provided herein, as well
as methods of using these agents. Exemplary methods of using these
agents include, but are not limited to, methods for modulating CSC
proliferation or apoptosis (e.g., inhibiting CSC proliferation or
stimulating CSC apoptosis), and methods for the treatment,
prevention, or diagnosis of cancer (such as may be achieved by
selectively targeting CSCs).
[0090] The CSC targets provided herein are also useful to screen
drug candidates for agents that selectively bind, interact with, or
otherwise target CSCs. Accordingly, certain exemplary embodiments
of the invention provide screening methods that utilize the CSC
targets provided herein, as well as agents identified using these
CSC-specific drug screening methods and methods of using these
agents.
[0091] The CSC targets provided herein are also useful in methods
of eliciting a CSC-specific immune response. For example, the CSC
targets or fragments thereof can be used in the preparation of
immunogens or cancer vaccines that utilize CSC antigens, either for
prophylactic or therapeutic purposes. Thus, certain exemplary
embodiments of the invention provide immunogens or cancer vaccines
that comprise one or more CSC targets provided herein, or fragments
thereof, and methods of producing and using such immunogens or
cancer vaccines.
[0092] With respect to methods of using the CSC targets provided
herein, examples of various embodiments of the invention include,
but are not limited to, methods for distinguishing functionally
different populations of cancer cells; methods for diagnosing an
individual with cancer; methods for predicting an individual's
likelihood of developing cancer in the future; methods for
determining the severity of cancer or predicting cancer progression
(e.g., likelihood and extent of metastasis, or rapidity and extent
of tumor growth); methods for determining the effects of
therapeutic agents (e.g., antibodies, small molecules, proteins,
nucleic acid compositions such as antisense or RNAi nucleic acids,
etc.) on tumors or the interaction of these agents with CSCs;
methods for selecting a therapeutic strategy (e.g., based on
predicted response of the cancer to a particular therapeutic
agent); methods for screening, identifying, and testing therapeutic
agents that target cancer stem cells; etc.
[0093] Thus, the CSC targets are useful in the diagnosis,
prognosis, treatment, or prevention of cancer, particularly by
selectively targeting CSCs, or for screening for therapeutic or
diagnostic agents for cancer (e.g., small molecule compounds or
antibodies that selectively bind to the CSC targets), particularly
therapeutic or diagnostic agents that target CSCs. Agents that
target CSC targets, such as antibodies, antibody-drug conjugates,
and small molecule compounds, can be used to inhibit proliferation
or stimulate apoptosis of CSCs. Accordingly, agents such as these
that target CSC targets can be used to treat cancer. Furthermore,
agents that target (e.g., selectively bind to) CSC targets can be
used for such purposes as, for example, diagnostic or prognostic
purposes, for selecting and/or prescribing a therapeutic agent to
be administered to an individual or selecting a treatment regimen
for an individual, for monitoring or predicting an individual's
response to a particular treatment, for monitoring cancer
progression or remission, for determining or predicting cancer
recurrence, or for determining a specific stage, sub-type, or other
classification or characteristic of a cancer. Uses such as these
can be achieved, for example, by using an agent (such as an
antibody, for example, which may optionally be coupled to a
detectable label) that selectively binds to a CSC target provided
herein in order to, for example, determine the presence or
abundance of CSCs or CSC targets in a tumor or to determine the
proportion of CSCs relative to other (non-stem cell-like) cancer
cells in a tumor. The CSC targets provided herein are useful for,
for example, isolating or differentiating CSCs (e.g., isolating
CSCs from tumor tissue samples).
[0094] CSCs can typically be isolated or cultured using a
serum-free culture medium such as media designated for human
embryonic stem cells. For example, CSC populations can be derived
from their adherent parental cells by culturing the adherent
parental cells in a serum-free culture medium such as that
designated for human embryonic stem cells. Furthermore, CSC
populations can typically be differentiated back to adherent cells
upon exposure of the CSC populations to serum.
[0095] CSCs may demonstrate higher tumorigenic capacity or retain
tumorigenicity (as assayed by an anchorage-independent assay)
compared with their adherent cell counterparts. Furthermore, CSCs
may demonstrate increased resistance relative to their adherent
counterparts to chemotherapeutic drugs (e.g., irinotecan, a first
line treatment for colon cancer), whereas their adherent
counterparts undergo apoptosis and/or demonstrate growth inhibition
when exposed to the same drug.
[0096] 1. CAT Proteins
[0097] Exemplary embodiments of the invention provide isolated CAT
proteins that consist of, consist essentially of, or comprise the
amino acid sequences of SEQ ID NOS:1-722, 1975, and 1977 (which are
encoded by the nucleotide sequences of SEQ ID NOS:723-1281, 1976,
and 1978, respectively), as well as all obvious variants of these
proteins and nucleic acid molecules that are within the art to make
and use. Examples of such obvious variants include, but are not
limited to, naturally-occurring allelic variants, pre-processed or
mature processed forms of a protein, non-naturally occurring
recombinantly-derived variants, orthologs, and paralogs. Such
variants can readily be generated using art-known techniques in the
fields of recombinant nucleic acid technology and protein
biochemistry.
[0098] A protein is said to be "isolated" or "purified" when it is
substantially free of cellular material or free of chemical
precursors or other chemicals. CAT proteins can be purified to
homogeneity or other degrees of purity. The level of purification
can be based on the intended use. The primary consideration is that
the preparation allows for the desired function of the protein,
even if in the presence of considerable amounts of other
components.
[0099] In some uses, "substantially free of cellular material"
includes preparations of a protein having less than about 30% (by
dry weight) other proteins (i.e., contaminating protein), less than
about 20% other proteins, less than about 10% other proteins, or
less than about 5% other proteins. When the protein is
recombinantly produced, it can also be substantially free of
culture medium, i.e., culture medium represents less than about 20%
of the volume of the protein preparation.
[0100] The language "substantially free of chemical precursors or
other chemicals" includes preparations of a protein in which the
protein is separated from chemical precursors or other chemicals
that are involved in the protein's synthesis. In one embodiment,
the language "substantially free of chemical precursors or other
chemicals" includes preparations of a CAT protein having less than
about 30% (by dry weight) chemical precursors or other chemicals,
less than about 20% chemical precursors or other chemicals, less
than about 10% chemical precursors or other chemicals, or less than
about 5% chemical precursors or other chemicals.
[0101] Isolated CAT proteins can be purified from cells that
naturally express it, purified from cells that have been altered to
express it (recombinant), or synthesized using known protein
synthesis methods (e.g., Sambrook et al., Molecular Cloning: A
Laboratory Manual. 3rd. ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., (2001)). For example, a nucleic acid
molecule encoding a CAT protein can be cloned into an expression
vector, the expression vector introduced into a host cell, and the
protein expressed in the host cell. The protein can then be
isolated from the cells by an appropriate purification scheme using
standard protein purification techniques.
[0102] A CAT protein or fragment thereof can be attached to
heterologous sequences to form chimeric or fusion proteins. Such
chimeric and fusion proteins comprise a protein operatively linked
to a heterologous protein having an amino acid sequence not
substantially homologous to the protein. "Operatively linked"
indicates that the protein and the heterologous protein are fused
in-frame. The heterologous protein can be fused to the N-terminus
or C-terminus of the protein.
[0103] In some uses, the fusion protein does not affect the
activity of the protein per se. For example, the fusion protein can
include, but is not limited to, beta-galactosidase fusions, yeast
two-hybrid GAL fusions, poly-His fusions, MYC-tagged, HI-tagged,
and Ig fusions. Such fusion proteins, particularly poly-His
fusions, can facilitate the purification of recombinant CAT
proteins. In certain host cells (e.g., mammalian host cells),
expression and/or secretion of a protein can be increased by using
a heterologous signal sequence.
[0104] A chimeric or fusion CAT protein can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for
different protein sequences can be ligated together in-frame in
accordance with conventional techniques. In another embodiment, a
fusion gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers that give
rise to complementary overhangs between two consecutive gene
fragments that can subsequently be annealed and re-amplified to
generate a chimeric gene sequence (Ausubel et al., Current
Protocols in Molecular Biology, 1992-2006). Moreover, many
expression vectors are commercially available that already encode a
fusion moiety (e.g., a GST protein). A CAT-encoding nucleic acid
can be cloned into such an expression vector such that the fusion
moiety is linked in-frame to the CAT protein.
[0105] To determine the percent identity of two amino acid
sequences or two nucleic acid sequences, the sequences can be
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). In an
exemplary embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90%
or more of the length of a reference sequence can be aligned for
comparison purposes. The amino acid residues or nucleotides at
corresponding amino acid positions or nucleotide positions can then
be compared. When a position in the first sequence is occupied by
the same amino acid residue or nucleotide as the corresponding
position in the second sequence, then the molecules are identical
at that position (as used herein, amino acid or nucleic acid
"identity" is equivalent to amino acid or nucleic acid "homology").
The percent identity between the two sequences is a function of the
number of identical positions shared by the sequences, taking into
account the number of gaps, and the length of each gap, that are
introduced for optimal alignment of the two sequences.
[0106] The comparison of sequences and determination of percent
identity and similarity between two sequences can be accomplished
using a mathematical algorithm. (Computational Molecular Biology,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds., Stockton Press, New York, 1991). In an
exemplary embodiment, the percent identity between two amino acid
sequences is determined using the Needleman and Wunsch (J. Mol.
Biol. (48):444-453 (1970)) algorithm which has been incorporated
into the GAP program in the GCG software package, using either a
Blossom 62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12,
10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6. In
another exemplary embodiment, the percent identity between two
nucleotide sequences can be determined using the GAP program in the
GCG software package (Devereux et al., Nucleic Acids Res. 12(1):387
(1984)) using a NWSgapdna.CMP matrix and a gap weight of 40, 50,
60, 70, or 80, and a length weight of 1, 2, 3, 4, 5, or 6. In
another exemplary embodiment, the percent identity between two
amino acid or nucleotide sequences is determined using the
algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which
has been incorporated into the ALIGN program (version 2.0), using a
PAM120 weight residue table, a gap length penalty of 12, and a gap
penalty of 4.
[0107] The sequences of the proteins and nucleic acid molecules of
the invention can further be used as a "query sequence" to perform
a search against sequence databases to, for example, identify other
protein family members or related sequences. Such searches can be
performed using the NBLAST and XBLAST programs (version 2.0) of
Altschul et al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide
searches can be performed with the NBLAST program, score=100,
wordlength=12, to obtain nucleotide sequences homologous to the
query nucleic acid molecule. BLAST protein searches can be
performed with the XBLAST program, score=50, wordlength=3, to
obtain amino acid sequences homologous to the query proteins. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al. (Nucleic Acids Res.
25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used.
[0108] As used herein, two proteins (or a region or domain of the
proteins) have significant homology/identity (also referred to as
substantial homology/identity) when the amino acid sequences are
typically at least about 70-80%, 80-90%, 90-95%, 96%, 97%, 98%, or
99% identical A significantly homologous amino acid sequence can be
encoded by a nucleic acid molecule that hybridizes to a CAT
protein-encoding nucleic acid molecule under stringent conditions,
as more fully described below.
[0109] Orthologs of a CAT protein typically have some degree of
significant sequence homology to at least a portion of a CAT
protein and are encoded by a gene from another organism. Preferred
orthologs are isolated from mammals, preferably non-human primates,
for the development of human therapeutic targets and agents. Such
orthologs can be encoded by a nucleic acid molecule that hybridizes
to a CAT protein-encoding nucleic acid molecule under moderate to
stringent conditions, as more fully described below, depending on
the degree of relatedness of the two organisms yielding the
proteins.
[0110] Non-naturally occurring variants of the CAT proteins can
readily be generated using recombinant techniques. Such variants
include, but are not limited to, deletions, additions, and
substitutions in the amino acid sequence of the CAT protein. For
example, one class of substitutions is conserved amino acid
substitutions. Such substitutions are those that substitute a given
amino acid in a CAT protein by another amino acid of like
characteristics. Typically seen as conservative substitutions are
the replacements, one for another, among the aliphatic amino acids
Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser
and Thr; exchange of the acidic residues Asp and Glu; substitution
between the amide residues Asn and Gln; exchange of the basic
residues Lys and Arg; and replacements among the aromatic residues
Phe and Tyr. Guidance concerning which amino acid changes are
likely to be phenotypically silent are found in Bowie et al.,
Science 247:1306-1310 (1990).
[0111] Variant CAT proteins can be fully functional or can lack
function in one or more activities, e.g., ability to bind
substrate, ability to phosphorylate substrate, ability to mediate
signaling, etc. Fully functional variants typically contain only
conservative variations or variation in non-critical residues or in
non-critical regions.
[0112] Non-functional variants typically contain one or more
non-conservative amino acid substitutions, deletions, insertions,
inversions, or truncations, or a substitution, insertion,
inversion, or deletion in a critical residue or critical
region.
[0113] Amino acids that are essential for function can be
identified by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham et al.,
Science 244:1081-1085 (1989)). The latter procedure introduces
single alanine mutations at every residue in the molecule. The
resulting mutant molecules are then tested for biological activity
or in assays such as in vitro proliferative activity. Sites that
are critical for binding partner/substrate binding can also be
determined by structural analysis such as crystallization, nuclear
magnetic resonance, or photoaffinity labeling (Smith et al., J.
Mol. Biol. 224:899-904 (1992); de Vos et al., Science 255:306-312
(1992)).
[0114] Exemplary embodiments of the invention provide fragments of
a CAT, and peptides that comprise and consist of such fragments. An
exemplary fragment typically comprises at least about 5, 6, 8, 10,
12, 14, 16, 18, 20 or more contiguous amino acid residues of a CAT
protein. Such fragments can be chosen based on the ability to
retain one or more of the biological activities of CAT or can be
chosen for the ability to perform a function, e.g., bind a
substrate or act as an immunogen. Particularly important fragments
are biologically active fragments, such as peptides that are, for
example, about 8 or more amino acids in length. Such fragments can
include a domain or motif of a CAT, e.g., an active site, a
transmembrane domain, or a binding domain. Further, possible
fragments include, but are not limited to, soluble peptide
fragments and fragments containing immunogenic structures. Domains
and functional sites can readily be identified, for example, by
computer programs well known and readily available to those of
skill in the art (e.g., PROSITE analysis).
[0115] Proteins can contain amino acids other than the 20 amino
acids commonly referred to as the 20 naturally-occurring amino
acids. Further, many amino acids, including the terminal amino
acids, can be modified by natural processes, such as processing and
other post-translational modifications, or by chemical modification
techniques well known in the art. Common modifications that occur
naturally in proteins are well known to those of skill in the
art.
[0116] Known modifications include, but are not limited to,
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent cros
slinks, formation of cystine, formation of pyroglutamate,
formylation, gamma carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, tRNA-mediated addition of
amino acids to proteins such as arginylation, and
ubiquitination.
[0117] Such modifications are well known to those of skill in the
art and have been described in the scientific literature. Several
particularly common modifications, glycosylation, lipid attachment,
sulfation, gamma-carboxylation of glutamic acid residues,
hydroxylation and ADP-ribosylation, for instance, are described in
most basic texts, such as Proteins-Structure and Molecular
Properties, 2nd Ed., T.E. Creighton, W. H. Freeman and Company, New
York (1993). Many detailed reviews are available on this subject,
such as by Wold (Posttranslational Covalent Modification of
Proteins, B. C. Johnson, Ed., Academic Press, New York 1-12
(1983)); Seifter et al. (Meth. Enzymol. 182: 626-646 (1990)); and
Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
[0118] Accordingly, exemplary CAT proteins and fragments thereof of
the invention can also encompasses derivatives or analogs in which,
for example, a substituted amino acid residue is not one encoded by
the genetic code, in which a substituent group is included, in
which a mature CAT is fused with another composition, such as a
composition to increase the half-life of a CAT (e.g., polyethylene
glycol or albumin), or in which additional amino acids are fused to
a mature CAT, such as a leader or secretory sequence or a sequence
for purification of a mature CAT or a pro-protein sequence.
[0119] 2. Antibodies to CAT Proteins
[0120] Exemplary embodiments of the invention provide antibodies to
CAT proteins, including, for example, monoclonal and polyclonal
antibodies; chimeric, humanized, and fully human antibodies; and
antigen-binding fragments and variants thereof, as well as other
embodiments.
[0121] Antibodies that selectively bind to a CAT protein can be
made using standard procedures known to those of ordinary skills in
the art. The term "antibody" is used in the broadest sense, and
specifically covers, for example, monoclonal antibodies, polyclonal
antibodies, multispecific antibodies (e.g., bispecific antibodies),
chimeric antibodies, humanized antibodies, fully human antibodies,
and antibody fragments (e.g., Fab, F(ab').sub.2, Fv and
Fv-containing binding proteins), so long as they exhibit the
desired biological activity. Antibodies (Ab's) and immunoglobulins
(Ig's) are glycoproteins typically having the same structural
characteristics. While antibodies exhibit binding specificity to a
specific antigen, immunoglobulins include both antibodies and other
antibody-like molecules that lack antigen specificity. Antibodies
can be of the IgG, IgE, IgM, IgD, and IgA class or subclass thereof
(e.g., IgG1, IgG2, IgG3, IgG4, IgAl and IgA2). Antibodies may be
interchangeably referred to as "antigen-binding molecules".
[0122] 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 substantially identical except for possible
naturally occurring mutations that may be present in minor amounts.
Monoclonal antibodies are highly specific and are typically
directed against a single antigenic site. Furthermore, in contrast
to polyclonal antibody preparations, which typically include
different antibodies directed against different determinants
(epitopes), each monoclonal antibody is typically directed against
a single determinant on an antigen. In addition to their
specificity, monoclonal antibodies are advantageous in that
substantially homogenous antibodies can be produced by a hybridoma
culture which is uncontaminated by other immunoglobulins or
antibodies. The modifier "monoclonal" antibody 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, monoclonal antibodies can be made by hybridoma methods
such as described by Kohler and Milstein, Nature 256: 495-497
(1975), by recombinant methods (e.g., as described in U.S. Pat. No.
4,816,567), or can be isolated from phage antibody libraries such
as by using the techniques described in Clackson et al., Nature
352: 624-628 (1991) or Marks et al., J. Mol. Biol. 222: 581-597
(1991).
[0123] "Humanized" forms of non-human (e.g., murine or rabbit)
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. Typically,
humanized antibodies are human immunoglobulins (a recipient
antibody) in which residues from a complementarity determining
regions ("CDR") of the recipient are replaced by residues from a
CDR of a non-human species (a 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.
Furthermore, a humanized antibody may comprise residues which are
found neither in the recipient antibody nor in the imported CDR or
framework region (FR) sequences. These modifications can be made to
further refine and optimize antibody performance. In general, a
humanized antibody can comprise substantially all of at least one,
and typically two, variable domains, in which all or substantially
all of the CDRs correspond to those of a non-human immunoglobulin
and all or substantially all of the FRs are those of a human
immunoglobulin consensus sequence. A humanized antibody can also
comprise at least a portion of an immunoglobulin constant region
(Fc), typically that of a human immunoglobulin. For further details
concerning humanized antibodies, see: Jones et al., Nature
321:522-525 (1986); Reichmann et al., Nature 332:323-327 (1988);
Presta, Curr. Op. Struct. Biol. 2:593-596 (1992); Queen et al.,
U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370; and
Winter, U.S. Pat. No. 5,225,539.
[0124] Antibodies, as used herein, include antibody fragments,
particularly antigen-binding fragments, as well as other modified
antibody structures and antigen-binding scaffolds (such as modified
antibody structures that are smaller or have less than all domains
or chains compared with a typical naturally occurring, full-size
human antibody). Examples of antibody fragments and other modified
antibody structures and antigen-binding scaffolds are known in the
art by such terms as minibodies (e.g., U.S. Pat. No. 5,837,821),
Nanobodies (llama heavy chain antibodies; Ablynx, Ghent, Belgium),
Adnectins (fibronectin domains; Adnexus Therapeutics, Waltham,
Mass.), Affibodies (protein-binding domain of Staphylococcus aureus
protein A; Affibody, Stockholm, Sweden), peptide aptamers
(synthetic peptides; Aptanomics, Lyon, France), Avimers (A-domains
derived from cell surface receptors; Avidia, Mountain View, Calif.
(acquired by Amgen)), Transbodies (transferrin; BioRexis
Pharmaceuticals, King of Prussia, PA (acquired by Pfizer)),
trimerized tetranectin domains (Borean Pharma, Aarhus, Denmark),
Domain antibodies (heavy or light chain antibodies; Domantis,
Cambridge, UK (acquired by GlaxoSmithKline)), Evibodies (derived
from V-like domains of T-cell receptors CTLA-4, CD28 and inducible
T-cell costimulator; EvoGenix Therapeutics, Sydney, Australia),
scFV fragments (stable single chain antibody fragments; ESBATech,
Zurich, Switzerland), Unibodies (monovalent IgG4 mAbs fragments;
Genmab, Copenhagen, Denmark), BiTEs (bispecific, T-cell activating
single-chain antibody fragments; Micromet, Munich, Germany),
DARPins (designed ankyrin repeat proteins; Molecular Partners,
Zurich, Switzerland), Anticalins (derived from lipocalins; Pieris,
Freising-Weihenstephan, Germany), Affilins (derived from human lens
protein gamma crystalline; Scil Proteins, Halle, Germany), and
SMIPs (small modular immunopharmaceuticals; Trubion
Pharmaceuticals, Seattle, WA) (Sheridan, Nature Biotechnology, 2007
April; 25(4):365-6).
[0125] An "isolated" or "purified" antibody is one that has been
identified and separated and/or recovered from a component of the
environment in which it is produced. Contaminant components of its
production environment are materials that would interfere with
diagnostic or therapeutic uses for the antibody, and may include
enzymes, hormones, and other proteinaceous or nonproteinaceous
solutes. In exemplary embodiments, the antibody can be purified as
measurable by any of at least three different methods: 1) to
greater than 95% by weight of antibody as determined by the Lowry
method, 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 non-reducing conditions
using Coomasie blue or silver stain. Isolated antibody can include
an antibody in situ within recombinant cells since at least one
component of the antibody's natural environment will not be
present. Ordinarily, however, an isolated antibody can be be
prepared by at least one purification step.
[0126] An "antigenic region", "antigenic determinant", or "epitope"
includes any protein determinant capable of specific binding to an
antibody. This is the site on an antigen to which each distinct
antibody molecule binds. Epitopic determinants can be active
surface groupings of molecules such as amino acids or sugar side
chains and may have specific three-dimensional structural
characteristics or charge characteristics.
[0127] "Antibody specificity" refers to an antibody that has a
stronger binding affinity for an antigen from a first subject
species than it has for a homologue of that antigen from a second
subject species. Typically, an antibody "binds specifically" to a
human antigen (e.g., has a binding affinity (Kd) value of no more
than about 1.times.10.sup.-7 M, preferably no more than about
1.times.10.sup.-8 M, and most preferably no more than about
1.times.10.sup.-9 M) but has a binding affinity for a homologue of
the antigen from a second subject species which is at least about
50-fold, or at least about 500-fold, or at least about 1000-fold,
weaker than its binding affinity for the human antigen. The
antibodies can be of any of the various types of antibodies as
described herein, such as humanized or fully human antibodies.
[0128] An antibody "selectively" or "specifically" binds a target
protein when the antibody binds the target protein and does not
significantly bind to unrelated proteins. An antibody can still be
considered to selectively or specifically bind a target protein
even if it also binds to other proteins that are not substantially
homologous with the target protein as long as such proteins share
homology with a fragment or domain of the target protein. In this
case, it would be understood that antibody binding to the target
protein is still selective despite some degree of
cross-reactivity.
[0129] Exemplary embodiments of the invention provide an "antibody
variant", which refers to an amino acid sequence variant of an
antibody wherein one or more of the amino acid residues have been
modified. Such variants necessarily have less than 100% sequence
identity with the amino acid sequence of the antibody, and have at
least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid
sequence identity with the amino acid sequence of either the heavy
or light chain variable domain of the antibody.
[0130] The term "antibody fragment" refers to a portion of a
full-length antibody, including the antigen binding or variable
region or the antigen-binding portion thereof. Examples of antibody
fragments include Fab, Fab', F(ab').sub.2 and Fv fragments. Papain
digestion of antibodies typically produces two identical antigen
binding fragments, called the Fab fragment, each with a single
antigen binding site, and a residual "Fc" fragment. Pepsin
treatment typically yields an F(ab').sub.2 fragment that has two
antigen binding fragments which are capable of crosslinking
antigen, and a residual other fragment (which is termed pFc').
Examples of additional antigen-binding fragments can include
diabodies, triabodies, tetrabodies, single-chain Fv, single-chain
Fv-Fc, SMIPs, and multispecific antibodies formed from antibody
fragments. A "functional fragment", with respect to antibodies,
typically refers to an Fv, F(ab), F(ab').sub.2 or other
antigen-binding fragments comprising one or more CDRs that has
substantially the same antigen-binding specificity as an
antibody.
[0131] An "Fv" fragment is an example of an antibody fragment that
contains a complete antigen recognition and binding site. This
region typically consists of a dimer of one heavy and one light
chain variable domain in a tight, non-covalent association
(V.sub.H-V.sub.L dimer). 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.
[0132] An "Fab" fragment (also designated as "F(ab)") 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 carboxyl
terminus of the heavy chain CH1 domain, including one or more
cysteines from the antibody hinge region. Fab'-SH is the
designation for Fab' in which the cysteine residue(s) of the
constant domains have a free thiol group. F(ab') fragments are
produced by cleavage of the disulfide bond at the hinge cysteines
of the F(ab').sub.2 pepsin digestion product. Additional chemical
couplings of antibody fragments are known to those of ordinary
skill in the art.
[0133] A "single-chain Fv" or "scFv" antibody fragment contains
V.sub.H and V.sub.L domains, wherein these domains are present in a
single polypeptide chain. Typically, the Fv polypeptide further
comprises a polypeptide linker between the V.sub.H and V.sub.L
domains that enables the scFv to form the desired structure for
antigen binding. For a review of scFv, see Pliickthun in The
Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and
Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). A single
chain Fv-Fc is an scFv linked to a Fc region.
[0134] A "diabody" is a small antibody fragment with two
antigen-binding sites, which fragments comprise a variable heavy
domain (V.sub.H) connected to a variable light domain (V.sub.L) in
the same polypeptide chain. 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 0 404 097; WO 93/11161;
and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448
(1993). Triabodies, tetrabodies and other antigen-binding antibody
fragments have been described by Hollinger and Hudson, 2005, Nature
Biotechnology 23:1126.
[0135] A "small modular immunopharmaceutical" (or "SMIP") is a
single-chain polypeptide including a binding domain (e.g., an scFv
or an antigen binding portion of an antibody), a hinge region, and
an effector domain (e.g., an antibody Fc region or a portion
thereof). SMIPs are described in published U.S. Patent Application
No. 20050238646.
[0136] Many methods are known for generating and/or identifying
antibodies to a given target protein. Several such methods are
described by Kohler et al., 1975, Nature 256: 495-497; Lane, 1985,
J. Immunol. Meth. 81:223-228; Harlow et al., 1988, Antibodies: A
Laboratory Manual. Cold Spring Harbor Laboratory Press; Harlow et
al., 1998, Using Antibodies, Cold Spring Harbor Press; Zhong et
al., 1997, J. Indust. Microbiol. Biotech. 19(1):71-76; and Berry et
al., 2003, Hybridoma and Hybridomics 22(1): 23-31.
[0137] Polyclonal antibodies can be prepared by any known method or
modifications of these methods, including obtaining antibodies from
patients. In certain exemplary methods for generating antibodies
such as polyclonal antibodies, an isolated protein can be used as
an immunogen which is administered to a mammalian organism, such as
a rat, rabbit, or mouse. For example, a complex of an immunogen
such as a CAT protein (or fragment thereof) and a carrier protein
can be prepared and an animal immunized by the complex. Serum or
plasma containing antibodies against the protein can be recovered
from the immunized animal and the antibodies separated and purified
(in the same manner as for monoclonal antibodies, for example). The
gamma globulin fraction or the IgG antibodies can be obtained, for
example, by use of saturated ammonium sulfate or DEAE SEPHADEX, or
other techniques known to those skilled in the art. The antibody
titer in the antiserum can be measured in the same manner as in the
supernatant of a hybridoma culture.
[0138] A full-length CAT protein, an antigenic peptide fragment, or
a fusion protein thereof, can be used as an immunogen. A protein
used as an immunogen is not limited to any particular type of
immunogen. In one aspect, antibodies can be prepared from regions
or discrete fragments (e.g., functional domains, extracellular
domains, or portions thereof) of a CAT protein. Antibodies can be
prepared from any region of a protein as described herein. In
particular, the proteins can be selected from the group consisting
of SEQ ID NOS:1-722, 1975, and 1977 and fragments thereof. An
antigenic fragment can typically comprise at least 8, 10, 12, 14,
16, or more contiguous amino acid residues, for example. Such
fragments can be selected based on a physical property, such as
fragments that correspond to regions located on the surface of a
protein (e.g., hydrophilic regions) or can be selected based on
sequence uniqueness.
[0139] Antibodies can also be produced by inducing production in a
lymphocyte population or by screening antibody libraries or panels
of highly specific binding reagents, such as disclosed in Orlandi
et al. (Proc. Natl. Acad. Sci. 86:3833-3837 (1989)) or Winter et
al. (Nature 349:293-299 (1991)). A protein can be used in screening
assays of phagemid or B-lymphocyte immunoglobulin libraries to
identify antibodies having a desired specificity. Numerous
protocols for competitive binding or immunoassays using either
polyclonal or monoclonal antibodies with established specificities
are well known in the art (e.g., Smith, Curr. Opin. Biotechnol. 2:
668-673 (1991)).
[0140] Antibodies can also be generated using various phage display
methods known in the art. In representative phage display methods,
functional antibody domains are displayed on the surface of phage
particles which carry nucleic acid molecules that encode the
antibody domains. In particular, such phage can be utilized to
display antigen-binding domains expressed from a repertoire or
combinatorial antibody library (e.g., human or murine). Phage
expressing an antigen binding domain that binds an antigen of
interest can be selected or identified with the antigen, e.g.,
using labeled antigen or antigen bound or captured to a solid
surface or bead. Phage used in methods such as these can typically
be filamentous phage including fd and M13 binding domains expressed
from phage with Fab, Fv, or disulfide stabilized Fv antibody
domains recombinantly fused to either the phage gene III or gene
VIII protein. Examples of phage display methods that can be used to
make antibodies include methods described in Brinkman et al., J.
Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods
184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.
24:952-958 (1994); Persic et al., Gene 187:9-18 (1997); Burton et
al., Advances in Immunology 57:191-280 (1994); PCT application No.
PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO
92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and
U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717;
5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637;
5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is
incorporated herein by reference in its entirety.
[0141] Antibodies, antigen binding fragments, and/or antibody
variants can be produced by recombinant and genetic engineering
methods well known in the art. For example, methods of expressing
heavy and light chain genes in E. coli are described in PCT
publication numbers WO901443, WO901443, and WO9014424, and in Huse
et al., 1989 Science 246:1275-1281. When using recombinant
techniques, such as to produce an antibody variant, the antibody
variant can be produced intracellularly, in the periplasmic space,
or directly secreted into the medium. If an antibody variant is
produced intracellularly, as a first step, the particulate debris,
either host cells or lysed fragments, can be removed, for example,
by centrifugation or ultrafiltration. Carter et al. (Bio/Technology
10: 163-167 (1992)) describe a procedure for isolating antibodies
that are secreted to the periplasmic space of E. coli. Briefly,
cell paste can be thawed in the presence of sodium acetate (pH
3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30
minutes. Cell debris can be removed by centrifugation. Where an
antibody variant is secreted into the medium, supernatants from
such expression systems can first be concentrated using a
commercially available protein concentration filter (e.g., an
Amicon or Millipore PELLICON ultrafiltration unit). A protease
inhibitor such as PMSF can be included in any of the foregoing
steps to inhibit proteolysis, and antibiotics can be included to
prevent the growth of contaminating microorganisms.
[0142] An antibody composition prepared from cells can be purified
using, for example, affinity chromatography, hydroxylapatite
chromatography, gel electrophoresis, and/or dialysis. The
suitability of protein A as an affinity ligand typically depends on
the species and isotype of the immunoglobulin Fc domain of an
antibody. Protein A can be used to purify antibodies that are based
on human delta1, delta2, or delta4 heavy chains (Lindmark et al.,
J. Immunol Meth. 62: 1-13 (1983)). Protein G can be used for all
mouse isotypes and for human delta3 (Guss et al., EMBO J. 5:
1567-1575 (1986)). The matrix to which the affinity ligand is
attached can be, for example, agarose or mechanically stable
matrices such as controlled pore glass or
poly(styrenedivinyl)benzene. Where the antibody comprises a CH3
domain, the BAKERBOND ABX.TM. resin (J. T. Baker, Phillipsburg,
N.J.) can be used for purification. Other exemplary techniques for
antibody purification include, but are not limited to,
fractionation on an ion-exchange column, ethanol precipitation,
reverse phase HPLC, chromatography on silica, chromatography on
heparin hepharos, chromatography on an anion or cation exchange
resin (such as a polyaspartic acid column), chromatofocusing,
SDS-PAGE, and ammonium sulfate precipitation.
[0143] Following any preliminary purification step(s), contaminants
in a mixture containing an antibody of interest can be removed by
low pH hydrophobic interaction chromatography using an elution
buffer at a pH between about 2.5-4.5, preferably performed at low
salt concentrations (e.g., from about 0-0.25M salt).
[0144] Full-length antibodies, as well as antibody fragments, can
also be expressed and isolated from bacteria such as E. coli, such
as described in Mazor et al., "Isolation of engineered, full-length
antibodies from libraries expressed in Escherichia coli", Nat
Biotechnol. 2007 May; 25(5):563-5 and Sidhu, "Full-length
antibodies on display", Nat Biotechnol. 2007 May; 25(5):537-8.
[0145] Further details regarding antibodies are set forth in the
following U.S. Pat. No. 6,248,516 (Winter et al.); U.S. Pat. No.
6,291,158 (Winter et al.); U.S. Pat. No. 5,885,793 (Griffiths et
al.); U.S. Pat. No. 5,969,108 (McCafferty et al.); U.S. Pat. No.
5,939,598 (Kucherlapati et al.); U.S. Pat. No. 4,816,397 (Boss et
al.); U.S. Pat. No. 4,816,567 (Cabilly et al.); U.S. Pat. No.
6,331,415 (Cabilly et al.); U.S. Pat. No. 5,770,429 (Lonberg et
al.); U.S. Pat. No. 5,639,947 (Hiatt et al.); and U.S. Pat. No.
5,260,203 (Ladner et al.), each of which is incorporated herein by
reference, and in the following published U.S. patent applications:
US20040132101 (Lazar et al.), US20050064514 (Stavenhagen et al.),
US20040261148 (Dickey et al.), and US20050014934 (Hinton et al.),
each of which is incorporated herein by reference. Antibody
engineering is further described in Jain et al., "Engineering
antibodies for clinical applications", Trends Biotechnol. 2007
July; 25(7):307-16.
[0146] 3. Antibody-Drug Conjugates to CAT Proteins
[0147] An antibody against CAT can be coupled (e.g., covalently
bonded) to a suitable therapeutic agent (as further discussed
herein) either directly or indirectly (e.g., via a linker group). A
direct reaction between an antibody and a therapeutic agent is
possible when each possesses a substituent capable of reacting with
the other. For example, a nucleophilic group, such as an amino or
sulfhydryl group, on one molecule may be capable of reacting with a
carbonyl-containing group, such as an anhydride or an acid halide,
or with an alkyl group containing a good leaving group (e.g., a
halide) on the other molecule.
[0148] Alternatively, it may be desirable to couple a therapeutic
agent and an antibody via a linker group. A linker group can
function as a spacer to distance an antibody from an agent in order
to avoid interference with binding capabilities. A linker group can
also serve to increase the chemical reactivity of a substituent on
an agent or an antibody, and thus increase the coupling efficiency.
An increase in chemical reactivity may also facilitate the use of
agents, or functional groups on agents, which otherwise would not
be possible.
[0149] A variety of bifunctional or polyfunctional reagents, both
homo- and hetero-functional (such as those described in the catalog
of the Pierce Chemical Co., Rockford, Ill.), can be employed as the
linker group. Coupling can be effected, for example, through amino
groups, carboxyl groups, sulfhydryl groups, or oxidized
carbohydrate residues (e.g., U.S. Pat. No. 4,671,958).
[0150] Where a therapeutic agent is more potent when free from the
antibody portion of an immunoconjugate, it may be desirable to use
a linker group that is cleavable during or upon internalization
into a cell. A number of different cleavable linker groups have
been described. Mechanisms for the intracellular release of an
agent from these linker groups include cleavage by reduction of a
disulfide bond (e.g., U.S. Pat. No. 4,489,710), by irradiation of a
photolabile bond (e.g., U.S. Pat. No. 4,625,014), by hydrolysis of
derivatized amino acid side chains (e.g., U.S. Pat. No. 4,638,045),
by serum complement-mediated hydrolysis (e.g., U.S. Pat. No.
4,671,958), by protease cleavable linker (e.g., U.S. Pat. No.
6,214,345), and by acid-catalyzed hydrolysis (e.g., U.S. Pat. No.
4,569,789).
[0151] It may be desirable to couple more than one agent to an
antibody. Multiple molecules of an agent can be coupled to one
antibody molecule, and more than one type of agent can be coupled
to the same antibody. For example, about 1, 2, 4, 6, 8, 10, 12, 14,
16, 18, 20, or 22 (or any other number in-between) molecules of
therapeutic agents can be coupled to an antibody. The average
number or quantitative distribution of therapeutic agent molecules
per antibody molecule in a preparation of conjugation reactions can
be determined by conventional means such as mass spectroscopy,
ELISA, or HPLC. Separation, purification, and characterization of
homogeneous antibody-drug conjugates having a certain number of
therapeutic agents conjugated thereto can be achieved by means such
as reverse phase HPLC or electrophoresis (see, e.g., Hamblett et
al., Clinical Cancer Res. 10:7063-70 (2004).
[0152] Examples of suitable therapeutic agents that can be
conjugated to an antibody include, but are not limited to,
chemotherapeutic agents (e.g., cytotoxic or cytostatic agents or
immunomodulatory agents), radiotherapeutic agents, therapeutic
antibodies, small molecule drugs, peptide drugs, immunomodulatory
agents, differentiation inducers, and toxins.
[0153] Examples of useful classes of cytotoxic or immunomodulatory
agents include, but are not limited to, antitubulin agents,
auristatins, DNA minor groove binders, DNA replication inhibitors,
alkylating agents (e.g., platinum complexes such as cis-platin,
mono(platinum), bis(platinum) and tri-nuclear platinum complexes
and carboplatin), anthracyclines, antibiotics, antifolates,
antimetabolites, chemotherapy sensitizers, duocarmycins,
etoposides, fluorinated pyrimidines, ionophores, lexitropsins,
nitrosoureas, platinols, pre-forming compounds, purine
antimetabolites, puromycins, radiation sensitizers, steroids,
taxanes, topoisomerase inhibitors, vinca alkaloids, and the
like.
[0154] Examples of individual cytotoxic or immunomodulatory agents
include, but are not limited to, androgen, anthramycin (AMC),
asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan,
buthionine sulfoximine, calicheamicin or calicheamicin derivatives,
camptothecin or camptothecins derivatives, carboplatin, carmustine
(BSNU), CC-1065, chlorambucil, cisplatin, colchicine,
cyclophosphamide, cytidine arabinoside (cytarabine), cytochalasin
B, dacarbazine, dactinomycin (formerly actinomycin), daunorubicin,
decarbazine, docetaxel, doxorubicin, etoposide, estrogen,
5-fluordeoxyuridine, 5-fluorouracil, gemcitabine, gramicidin D,
hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU),
maytansine, mechlorethamine, melphalan, 6-mercaptopurine,
methotrexate, mithramycin, mitomycin C, mitoxantrone,
nitroimidazole, paclitaxel, palytoxin, plicamycin, procarbizine,
rhizoxin, streptozotocin, tenoposide, 6-thioguanine, thioTEPA,
topotecan, vinblastine, vincristine, vinorelbine, VP-16, and
VM-26.
[0155] Examples of other suitable cytotoxic agents include, but are
not limited to, DNA minor groove binders (e.g., enediynes and
lexitropsins, a CBI compound; see also U.S. Pat. No. 6,130,237),
duocarmycins, taxanes (e.g., paclitaxel and docetaxel), puromycins,
vinca alkaloids, CC-1065, SN-38, topotecan, morpholino-doxorubicin,
rhizoxin, cyanomorpholino-doxorubicin, echinomycin, combretastatin,
netropsin, epothilone A and B, estramustine, cryptophysins,
cemadotin, a maytansinoid, discodermolide, eleutherobin, and
mitoxantrone.
[0156] Examples of other suitable agents include, but are not
limited to, radionuclides, differentiation inducers, drugs, toxins,
and derivatives thereof. Exemplary radionuclides include .sup.90Y,
.sup.123I, .sup.125I, .sup.131I, .sup.186Re, .sup.188Re,
.sup.211At, and .sup.212Bi. Exemplary drugs include methotrexate,
and pyrimidine and purine analogs. Exemplary differentiation
inducers include phorbol esters and butyric acid. Exemplary toxins
include ricin, abrin, diptheria toxin, cholera toxin, gelonin,
Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral
protein.
[0157] In some embodiments, the therapeutic agent used in an
antibody-drug conjugate is an anti-tubulin agent. Examples of
anti-tubulin agents include, but are not limited to, taxanes (e.g.,
Taxol.RTM. (paclitaxel), Taxotere.RTM. (docetaxel)), T67 (Tularik)
and vinca alkyloids (e.g., vincristine, vinblastine, vindesine, and
vinorelbine). Other antitubulin agents include, for example,
baccatin derivatives, taxane analogs (e.g., epothilone A and B),
nocodazole, colchicine and colcimid, estramustine, cryptophysins,
cemadotin, maytansinoid, combretastatins, discodermolide, and
eleutherobin.
[0158] In certain embodiments, the cytotoxic agent is a
maytansinoid, another group of anti-tubulin agents. For example, in
specific embodiments, the maytansinoid is maytansine, DM-1
(ImmunoGen, Inc.; see also Chari et al., Cancer Res. 52:127-131
(1992)) or DM-4. In some embodiments, the therapeutic agent is an
auristatin, such as auristatin E (also known in the art as
dolastatin-10) or a derivative thereof. Typically, an auristatin E
derivative is, e.g., an ester formed between auristatin E and a
keto acid. For example, auristatin E can be reacted with paraacetyl
benzoic acid or benzoylvaleric acid to produce AEB and AEVB,
respectively. Other typical auristatin derivatives include AFP,
MMAF, and MMAE. The synthesis and structure of auristatin
derivatives are described in U.S. Patent Application Publication
Nos. 2003-0083263, 2005-0238649 and 2005-0009751; PCT Publication
Nos WO 04/010957 and WO 02/088172, and U.S. Pat. Nos. 6,323,315;
6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483;
5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024;
5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444;
and 4,486,414.
[0159] 4. CAT Nucleic Acid Molecules
[0160] Exemplary isolated CAT nucleic acid molecules of the
invention consist of, consist essentially of, or comprise a
nucleotide sequence that encodes a CAT protein of the invention, an
allelic variant thereof, or an ortholog or paralog thereof, for
example. As used herein, an "isolated" nucleic acid molecule is one
that is separated from other nucleic acid present in the natural
source of the nucleic acid. Preferably, an "isolated" nucleic acid
is free of sequences which naturally flank the nucleic acid (i.e.,
sequences located at the 5' and 3' ends of the nucleic acid) in the
genomic DNA of the organism from which the nucleic acid is derived.
However, there can be some flanking nucleotide sequences, for
example up to about 5 kilobases (KB), 4 KB, 3 KB, 2 KB, or 1 KB or
less, particularly contiguous protein-encoding sequences and
protein-encoding sequences within the same gene but separated by
introns in the genomic sequence, and flanking nucleotide sequences
that contain regulatory elements. The primary consideration is that
the nucleic acid is isolated from remote and unimportant flanking
sequences such that it can be subjected to the specific
manipulations described herein such as recombinant expression,
preparation of probes and primers, and other uses specific to the
nucleic acid molecules. Moreover, an "isolated" nucleic acid
molecule, such as a transcript/cDNA molecule, can be substantially
free of other cellular material, or culture medium when produced by
recombinant techniques, or chemical precursors or other chemicals
when chemically synthesized.
[0161] A nucleic acid molecule can be fused to other coding or
regulatory sequences and still be considered isolated. Isolated
nucleic acid molecules can include heterologous nucleotide
sequences, such as heterologous nucleotide sequences that are fused
to a nucleic acid molecule by recombinant techniques. For example,
recombinant DNA molecules contained in a vector are considered
isolated. Further examples of isolated DNA molecules include
recombinant DNA molecules maintained in heterologous host cells, or
purified (partially or substantially) DNA molecules in solution.
Isolated RNA molecules include in vivo or in vitro RNA transcripts
of isolated DNA molecules. Isolated nucleic acid molecules further
include such molecules produced synthetically.
[0162] Isolated nucleic acid molecules can encode a mature protein
plus additional amino or carboxyl-terminal amino acids, or amino
acids interior to the mature protein (when the mature form has more
than one peptide chain, for instance). Such sequences may play a
role in processing of a protein from precursor to a mature form,
facilitate protein trafficking, prolong or shorten protein
half-life, or facilitate manipulation of a protein for assay or
production, among other things. As generally is the case in situ,
additional amino acids may be processed away from the mature
protein by cellular enzymes.
[0163] Isolated nucleic acid molecules include, but are not limited
to, sequences encoding a CAT protein alone, sequences encoding a
mature protein with additional coding sequences (such as a leader
or secretory sequence (e.g., a pre-pro or pro-protein sequence)),
and sequences encoding a mature protein (with or without additional
coding sequences) plus additional non-coding sequences (e.g.,
introns and non-coding 5' and 3' sequences such as transcribed but
non-translated sequences that play a role in transcription, mRNA
processing (including splicing and polyadenylation signals),
ribosome binding, and/or stability of mRNA). In addition, nucleic
acid molecules can be fused to a marker sequence encoding, for
example, a peptide that facilitates purification.
[0164] Isolated nucleic acid molecules can be in the form of RNA,
such as mRNA, or in the form of DNA, including cDNA and genomic DNA
obtained by cloning or produced by chemical synthetic techniques or
by a combination thereof. Nucleic acid molecules, especially DNA,
can be double-stranded or single-stranded. Single-stranded nucleic
acid can be the coding strand (sense strand) or the non-coding
strand (anti-sense strand).
[0165] Exemplary embodiments of the invention further provide
isolated nucleic acid molecules that encode fragments of a CAT
protein as well as nucleic acid molecules that encode obvious
variants of a CAT protein. Such nucleic acid molecules may be
naturally occurring, such as allelic variants (same locus),
paralogs (different locus), and orthologs (different organism), or
can be constructed by recombinant DNA methods or by chemical
synthesis. Such non-naturally occurring variants can be made by
mutagenesis techniques, including those applied to nucleic acid
molecules, cells, or organisms. Accordingly, nucleic acid molecule
variants can contain nucleotide substitutions, deletions,
inversions, and/or insertions. Variations can occur in either or
both the coding and non-coding regions, and variations can produce
conservative and/or non-conservative amino acid substitutions.
[0166] A fragment of a nucleic acid molecule typically comprises a
contiguous nucleotide sequence at least 8, 10, 12, 15, 16, 18, 20,
22, 25, 30, 40, 50, 100, 150, 200, 250, 500 (or any other number
in-between) or more nucleotides in length. The length of a fragment
can be based on its intended use. For example, a fragment can
encode epitope bearing regions of a protein, or can be used as DNA
probes and primers. Isolated fragments can be produced by
synthesizing an oligonucleotide probe using known techniques, for
example, and can optionally be labeled and used to screen a cDNA
library, genomic DNA, or mRNA, for example. Primers can be used in
PCR reactions to clone specific regions of a gene.
[0167] A probe/primer typically comprises substantially a purified
oligonucleotide or oligonucleotide pair. An oligonucleotide
typically comprises a nucleotide sequence that hybridizes under
stringent conditions to at least about 8, 10, 12, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50 (or any other number
in-between) or more contiguous nucleotides.
[0168] Allelic variants, orthologs, and homologs can be identified
using methods well known in the art. These variants can comprise a
nucleotide sequence encoding a protein that is typically 60-70%,
70-80%, 80-90%, 90-95%, 96%, 97%, 98%, or 99% homologous to the
nucleotide sequence. Such nucleic acid molecules can readily be
identified as being able to hybridize under moderate to stringent
conditions, to a nucleotide sequence shown in the Sequence Listing
or a fragment thereof.
[0169] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences encoding a protein at
least 60-70% homologous to each other typically remain hybridized
to each other. The conditions can be such that sequences at least
about 60%, at least about 70%, or at least about 80% or more
homologous to each other typically remain hybridized to each other.
Such stringent conditions are known to those skilled in the art and
can be found in, for example, Current Protocols in Molecular
Biology, John Wiley & Sons, N.Y. (1989-2006), 6.3.1-6.3.6. One
example of stringent hybridization conditions is hybridization in
6.times. sodium chloride/sodium citrate (SSC) at about 45.degree.
C., followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
50-65.degree. C. Examples of moderate to low stringency
hybridization conditions are well known in the art.
[0170] Exemplary embodiments of the invention also include kits for
detecting the presence of CAT nucleic acid (e.g., DNA or mRNA) in a
biological sample. For example, a kit can comprise reagents such as
a labeled or labelable nucleic acid and/or other agents capable of
detecting CAT nucleic acid in a biological sample; means for
determining the amount of CAT nucleic acid in the sample; and means
for comparing the amount of CAT nucleic acid in the sample with a
standard. The nucleic acid and/or other agent can be packaged in
one or more suitable containers. The kit can further comprise
instructions for using the kit to detect CAT nucleic acid.
[0171] 5. Vectors and Host Cells
[0172] Exemplary embodiments of the invention also provide vectors
containing CAT nucleic acid molecules. The term "vector" refers to
a vehicle, such as a nucleic acid molecule, which can transport the
CAT nucleic acid molecules. When the vector is a nucleic acid
molecule, the CAT nucleic acid molecules are covalently linked to
the vector nucleic acid. A vector can be, for example, a plasmid,
single or double stranded phage, a single or double stranded RNA or
DNA viral vector, or artificial chromosome, such as a BAC, PAC,
YAC, OR MAC.
[0173] A vector can be maintained in a host cell as an
extrachromosomal element where it replicates and produces
additional copies of the CAT nucleic acid molecules. Alternatively,
a vector can integrate into a host cell genome and produce
additional copies of the CAT nucleic acid molecules when the host
cell replicates.
[0174] Exemplary embodiments of the invention provide vectors for
maintenance (cloning vectors) and vectors for expression
(expression vectors) of the nucleic acid molecules, for example.
Expression vectors can express a portion of, or all of, a protein
sequence. Vectors can function in prokaryotic or eukaryotic cells
or in both (shuttle vectors). Vectors also include insertion
vectors, which integrate a nucleic acid molecule into another
nucleic acid molecule, such as into the cellular genome (such as to
alter in situ expression of a gene and/or gene product). For
example, an endogenous protein-coding sequence can be entirely or
partially replaced via homologous recombination with a
protein-coding sequence containing one or more specifically
introduced mutations.
[0175] Expression vectors can contain cis-acting regulatory regions
that are operably-linked in the vector to the nucleic acid
molecules such that transcription of the nucleic acid molecules is
allowed in a host cell. The nucleic acid molecules can be
introduced into the host cell with a separate nucleic acid molecule
capable of affecting transcription. The separate nucleic acid
molecule may provide, for example, a trans-acting factor
interacting with the cis-regulatory control region to allow
transcription of the nucleic acid molecules from the vector.
Alternatively, a trans-acting factor may be supplied by a host
cell. Additionally, a trans-acting factor can be produced from a
vector itself. It is understood, however, that transcription and/or
translation of nucleic acid molecules can occur in cell-free
systems.
[0176] Regulatory sequences to which CAT nucleic acid molecules can
be operably linked include, for example, promoters for directing
mRNA transcription. These include, but are not limited to, the left
promoter from bacteriophage, the lac, TRP, and TAC promoters from
E. coli, the early and late promoters from SV40, the CMV immediate
early promoter, the adenovirus early and late promoters, and
retrovirus long-terminal repeats.
[0177] In addition to control regions that promote transcription,
expression vectors can also include regions that modulate
transcription, such as repressor binding sites and enhancers.
Examples include the SV40 enhancer, the cytomegalovirus immediate
early enhancer, polyoma enhancer, adenovirus enhancers, and
retrovirus LTR enhancers.
[0178] In addition to containing sites for transcription initiation
and control, expression vectors can also contain sequences
necessary for transcription termination and, in the transcribed
region, a ribosome binding site for translation. Other regulatory
control elements for expression include initiation and termination
codons as well as polyadenylation signals. Numerous regulatory
sequences useful in expression vectors are well known in the art
(e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual.
3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (2001)).
[0179] A variety of expression vectors can be used to express a
nucleic acid molecule. Such vectors include chromosomal, episomal,
and virus-derived vectors, for example vectors derived from
bacterial plasmids, from bacteriophage, from yeast episomes, from
yeast chromosomal elements, including yeast artificial chromosomes,
from viruses such as baculoviruses, papovaviruses such as SV40,
Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses,
and retroviruses. Vectors may also be derived from combinations of
these sources such as those derived from plasmid and bacteriophage
genetic elements, e.g. cosmids and phagemids. Appropriate cloning
and expression vectors for prokaryotic and eukaryotic hosts are
described in Sambrook et al., Molecular Cloning: A Laboratory
Manual. 3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (2001).
[0180] A regulatory sequence can provide constitutive expression in
one or more host cells (e.g., tissue specific) or can provide for
inducible expression in one or more cell types such as by
temperature, nutrient additive, or exogenous factors such as a
hormone or other ligand. A variety of vectors providing for
constitutive and inducible expression in prokaryotic and eukaryotic
hosts are well known in the art.
[0181] Nucleic acid molecules can be inserted into vector nucleic
acid by well-known methodology. For example, the DNA sequence that
will ultimately be expressed can be joined to an expression vector
by cleaving the DNA sequence and the expression vector with one or
more restriction enzymes and then ligating the fragments together.
Procedures for restriction enzyme digestion and ligation are well
known in the art.
[0182] A vector containing a nucleic acid molecule of interest can
be introduced into an appropriate host cell for propagation or
expression using well-known techniques. Bacterial cells include,
but are not limited to, E. coli, Streptomyces, and Salmonella
typhimurium. Eukaryotic cells include, but are not limited to,
yeast, insect cells such as Drosophila, animal cells such as COS
and CHO cells (e.g., DG44 or CHO-s), and plant cells.
[0183] As described herein, it may be desirable to express a
protein as a fusion protein. Accordingly, exemplary embodiments of
the invention provide fusion vectors that allow for the production
of fusion proteins. Fusion vectors can, for example, increase the
expression of a recombinant protein; increase the solubility of a
recombinant protein, and/or aid in the purification of a protein
such as by acting as a ligand for affinity purification. A
proteolytic cleavage site can be introduced at the junction of the
fusion moiety so that the desired protein can ultimately be
separated from the fusion moiety. Proteolytic enzymes include, but
are not limited to, factor Xa, thrombin, and enteroenzyme. Typical
fusion expression vectors include pGEX (Smith et al., Gene 67:31-40
(1988)), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5
(Pharmacia, Piscataway, N.J.), which fuse glutathione S-transferase
(GST), maltose E binding protein, or protein A, respectively, to a
target recombinant protein. Examples of suitable inducible
non-fusion E. coli expression vectors include pTrc (Amann et al.,
Gene 69:301-315 (1988)) and pET 11d (Studier et al., Gene
Expression Technology: Methods in Enzymology 185:60-89 (1990)).
[0184] Recombinant protein expression can be maximized in host
bacteria by providing a genetic background wherein the host cell
has an impaired capacity to proteolytically cleave the recombinant
protein (Gottesman, S., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990), pp.
119-128). Alternatively, the sequence of a nucleic acid molecule of
interest can be altered to provide preferential codon usage for a
specific host cell, such as E. coli (Wada et al., Nucleic Acids
Res. 20:2111-2118 (1992)).
[0185] CAT nucleic acid molecules can, for example, be expressed by
expression vectors in a yeast host. Examples of vectors for
expression in yeast (e.g., S. cerevisiae) include pYepSec1
(Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kurjan et al.,
Cell 30:933-943 (1982)), pJRY88 (Schultz et al., Gene 54:113-123
(1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).
Nucleic acid molecules can also be expressed in insect cells using,
for example, baculovirus expression vectors. Baculovirus vectors
available for expression of proteins in cultured insect cells
(e.g., Sf 9 cells) include the pAc series (Smith et al., Mol. Cell
Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow et al.,
Virology 170:31-39 (1989)). Nucleic acid molecules can also be
expressed in mammalian cells using mammalian expression vectors.
Examples of mammalian expression vectors include pCDM8 (Seed, B.
Nature 329:840 (1987)), pMT2PC (Kaufman et al., EMBO J. 6:187-195
(1987)), and CHEF (U.S. Pat. No. 5,888,809).
[0186] The expression vectors listed herein are provided by way of
example only of well-known vectors available to those of ordinary
skill in the art that would be useful to express CAT nucleic acid
molecules. The person of ordinary skill in the art would be aware
of other vectors suitable for maintenance, propagation, and/or
expression of CAT nucleic acid molecules (e.g., Sambrook et al.
Molecular Cloning: A Laboratory Manual. 3rd. ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001).
[0187] Exemplary embodiments of the invention also encompasses
vectors in which CAT nucleic acid molecules are cloned into a
vector in reverse orientation, but operably linked to a regulatory
sequence that permits transcription of antisense RNA. Thus, an
antisense transcript can be produced to all, or to a portion, of a
CAT nucleic acid molecule, including coding and non-coding regions.
Expression of this antisense RNA may be subject to each of the
parameters described above in relation to expression of the sense
RNA (e.g., regulatory sequences, constitutive or inducible
expression, tissue-specific expression).
[0188] Exemplary embodiments of the invention provide recombinant
host cells containing the vectors described herein. Host cells
include, for example, prokaryotic cells, lower eukaryotic cells
such as yeast, other eukaryotic cells such as insect cells, and
higher eukaryotic cells such as mammalian cells.
[0189] Recombinant host cells can be prepared by introducing vector
constructs, such as described herein, into cells by techniques
readily available to a person of ordinary skill in the art. These
techniques include, but are not limited to, calcium phosphate
transfection, DEAE-dextran-mediated transfection, cationic
lipid-mediated transfection, electroporation, transduction,
infection, lipofection, microinjection, and other techniques such
as those found in Sambrook, et al. (Molecular Cloning: A Laboratory
Manual. 3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (2001).
[0190] For example, using techniques such as these, a retroviral or
other viral vector can be introduced into mammalian cells. Examples
of mammalian cells into which a retroviral vector can be introduced
include, but are not limited to, primary mammalian cultures or
continuous mammalian cultures, COS cells, NIH3T3, 293 cells (ATCC
#CRL 1573), and dendritic cells.
[0191] Host cells can contain more than one vector. Thus, different
nucleotide sequences can be introduced on different vectors of the
same cell. Similarly, nucleic acid molecules of interest can be
introduced either alone or with other unrelated nucleic acid
molecules such as those providing trans-acting factors for
expression vectors. When more than one vector is introduced into a
cell, the vectors can be introduced independently, co-introduced,
or joined to the nucleic acid molecule vector.
[0192] Bacteriophage and viral vectors can be introduced into cells
as packaged or encapsulated virus by standard procedures for
infection and transduction. Viral vectors can be
replication-competent or replication-defective. If viral
replication is defective, replication can occur in host cells that
provide functions that complement the defects.
[0193] Vectors can include selectable markers that enable the
selection of a subpopulation of cells that contain the recombinant
vector constructs. Markers can be contained in the same vector that
contains the nucleic acid molecules of interest or can be on a
separate vector. Exemplary markers include tetracycline or
ampicillin-resistance genes for prokaryotic host cells, and
dihydrofolate reductase or neomycin resistance for eukaryotic host
cells. However, any marker that provides selection for a phenotypic
trait can be used.
[0194] While mature proteins can be produced in bacteria, yeast,
mammalian cells, and other cells under the control of appropriate
regulatory sequences, cell-free transcription and translation
systems can also be used to produce these proteins using RNA
derived from the DNA constructs described herein.
[0195] If secretion of a protein is desired, appropriate secretion
signals can be incorporated into a vector. The signal sequence can
be endogenous or heterologous to the protein.
[0196] If a protein is not secreted into a medium, the protein can
be isolated from a host cell by standard disruption procedures,
including freeze/thaw, sonication, mechanical disruption, use of
lysing agents, and the like. A protein can then be recovered and
purified by well-known purification methods including, for example,
ammonium sulfate precipitation, acid extraction, anion or cationic
exchange chromatography, phosphocellulose chromatography,
hydrophobic-interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, lectin chromatography, or high
performance liquid chromatography.
[0197] It is also understood that, depending upon the host cell
used in recombinant production of a protein, proteins can have
various glycosylation patterns or can be non-glycosylated, such as
when produced in bacteria. In addition, proteins can include an
initial modified methionine in some instances as a result of a
host-mediated process.
[0198] Recombinant host cells that express a CAT protein have a
variety of uses. For example, such host cells are useful for
producing CAT proteins, which can be further purified to produce
desired amounts of the protein or fragments thereof. Thus, host
cells containing expression vectors are useful for protein
production.
[0199] Host cells are also useful for conducting cell-based assays
involving a CAT protein or fragments thereof. For example, a
recombinant host cell expressing a CAT protein can be used to assay
compounds that stimulate or inhibit the protein's function.
[0200] Host cells are also useful for identifying mutant CAT
proteins in which the protein's function is affected. Host cells
expressing mutant proteins are useful for assaying compounds that
have a desired effect on the mutant proteins (e.g., stimulating or
inhibiting function), particularly if the mutant proteins naturally
occur and give rise to a pathology.
[0201] 6. Diagnosis and Treatment in General
[0202] The following terms, as used in the present specification
and claims, are intended to have the meaning as defined below,
unless indicated otherwise.
[0203] As used herein, a "biological sample" (or just "sample") can
comprise, for example, tissue, blood, sera, cells, cell lines, or
biological fluids such as plasma, interstitial fluid, urine,
cerebrospinal fluid, and the like. A biological sample is
typically, although not necessarily, obtained from an individual by
a medical practitioner.
[0204] As used herein, a "subject" can be a mammalian subject or
non-mammalian subject, preferably a mammalian subject. A mammalian
subject can be a human or non-human, preferably a human. The terms
"subject", "individual", and "patient" are used herein
interchangeably.
[0205] A "healthy" or "normal" subject or biological sample is a
subject or biological sample in which the disease of interest is
not detectable, as ascertained by using conventional diagnostic
methods (such a biological sample can interchangeably be referred
to as a "control" sample).
[0206] As used herein, "disease(s)" include cancer, particularly
the cancers identified in Table 1 (as indicated for each peptide)
and/or Table 3, and associated diseases and pathologies.
[0207] The terms "diagnose" (or "diagnosing", etc.) and "assess"
(or "assessing", etc.) are used herein interchangeably. Diagnosing
or assessing diseases can include, for example, initially detecting
the presence of a disease; determining a specific stage, sub-type,
or other classification or characteristic of a disease; prognosing
the future course of a disease; monitoring disease progression
(e.g., monitoring metastatic spread of a cancer) or remission;
determining or predicting response to a treatment; determining or
predicting recurrence of a disease; and/or determining the
likelihood of developing a disease in the future.
[0208] "Treat", "treating", or "treatment" of a disease includes:
(1) inhibiting the disease, i.e., arresting or reducing the
development of the disease or its clinical symptoms, or (2)
relieving the disease, i.e., causing regression of the disease or
its clinical symptom(s).
[0209] The term "prophylaxis" is used to distinguish from
"treatment," and to encompass both "preventing" and "suppressing."
It is not always possible to distinguish between "preventing" and
"suppressing," as the ultimate inductive event or events may be
unknown, latent, or the patient is not ascertained until well after
the occurrence of the event or events. Therefore, the term
"protection", as used herein, is meant to include
"prophylaxis."
[0210] A "therapeutically effective amount" means the amount of an
agent that, when administered to a subject for treating a disease,
is sufficient to effect such treatment for the disease. The
"therapeutically effective amount" can vary depending on such
factors as the agent, the disease and its severity, and the age,
weight, etc., of the subject to be treated.
[0211] A "differential level" is a level of a target (e.g., CAT
protein or nucleic acid) in a test sample (e.g., disease sample, or
drug resistant cells) either above or below the level of the same
target in a corresponding control or normal sample (e.g., a control
cell line or a biological sample from a healthy individual, or
cells responsive/sensitive to a drug).
[0212] Exemplary embodiments of the invention provide methods for
treating diseases, especially cancer, particularly the cancers
identified in Table 1 (as indicated for each peptide) and/or Table
3, comprising administering to a patient a therapeutically
effective amount of an antagonist, agonist, or a pharmaceutical
composition thereof. Exemplary embodiments of the invention further
provide agonists and antagonists to CAT proteins, as well as
pharmaceutical compositions that comprise an agonist or antagonist
with a suitable carrier such as a pharmaceutically acceptable
excipient.
[0213] Exemplary agonists or antagonists include antibodies that
specifically bind to a CAT protein. Antibodies can be used alone or
in combination with one or more other therapeutic agents (e.g., as
an antibody-drug conjugate or a combination therapy). Further
examples of molecules that can be used as antagonists include, but
are not limited to, small molecules that inhibit the function or
abundance level of CAT, and inhibitory nucleic acid molecules such
as RNAi or antisense nucleic acid molecules that specifically
hybridize to CAT nucleic acid.
[0214] Exemplary embodiments of the invention further encompass
novel agents identified by screening assays using CAT, such as the
screening assays described herein, as well as methods of using
these agents, such as for treatment or diagnostic purposes. For
example, an agent identified as described herein (e.g., a
CAT-modulating agent, a CAT-specific nucleic acid molecule such as
an RNAi or antisense molecule, a CAT-specific antibody, a
CAT-specific antibody-drug conjugate, or a CAT-binding partner) can
be used in an animal or other model, such as to determine efficacy,
toxicity, or side effects of treatment with the agent.
[0215] Modulators of CAT protein activity, such as modulators
identified according to the drug screening assays described herein,
can be used to treat a subject with a disorder mediated by a CAT,
e.g., by treating cells or tissues that express CAT at a
differential level. Methods of treatment can include the step of
administering a modulator of CAT activity in a pharmaceutical
composition to a subject in need of such treatment.
[0216] In certain exemplary embodiments, if decreased expression or
activity of a protein is desired, an antibody to the protein or an
inhibitor/antagonist and the like, or a pharmaceutical agent
containing one or more of these molecules, can be administered to
an individual. In other exemplary embodiments, if increased
expression or activity of a protein is desired, the protein itself
or an agonist/enhancer and the like, or a pharmaceutical agent
containing one or more of these molecules, can be administered.
Administration can be effected by methods well known in the art and
may include delivery by an antibody specifically targeted to the
protein. Neutralizing antibodies, which inhibit dimer formation,
can be used when decreased expression or activity of a protein is
desired.
[0217] Although modulating agents can be administered in a pure or
substantially pure form, modulating agents can also be administed
as pharmaceutical compositions, formulations, or preparations with
a carrier. Exemplary formulations of the invention, such as for
human or veterinary use, comprise a suitable active CAT-modulating
agent, together with one or more pharmaceutically acceptable
carriers and, optionally, other therapeutic ingredients. The
carrier(s) are "acceptable" in the sense of being compatible with
other ingredients of a formulation and not deleterious to the
recipient thereof. The formulations can be presented in unit dosage
form and can be prepared by any method known to the skilled
artisan.
[0218] Examples of suitable pharmaceutical carriers include
proteins such as albumins (e.g., U.S. Pat. No. 4,507,234), peptides
and polysaccharides such as aminodextran (e.g., U.S. Pat. No.
4,699,784), and water. A carrier can also bear an agent by
noncovalent bonding or by encapsulation, such as within a liposome
vesicle (e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088). Carriers
specific for radionuclide agents include radiohalogenated small
molecules and chelating compounds. For example, U.S. Pat. No.
4,735,792 discloses representative radiohalogenated small molecules
and their synthesis. A radionuclide chelate can be formed from
chelating compounds that include those containing nitrogen and
sulfur atoms as the donor atoms for binding the metal, metal oxide,
radionuclide. For example, U.S. Pat. No. 4,673,562 discloses
representative chelating compounds and their synthesis.
[0219] Methods of preparing pharmaceutical formulations typically
include the step of bringing into association the active ingredient
with the carrier, which constitutes one or more accessory
ingredients. Formulations can be prepared by uniformly and
intimately bringing into association the active ingredient with
liquid carriers or finely divided solid carriers, or both, and
then, if necessary, shaping the product into the desired
formulation.
[0220] Formulations suitable for intravenous, intramuscular,
subcutaneous, or intraperitoneal administration can comprise
sterile aqueous solutions of the active ingredient with solutions,
which can be isotonic with the blood of the recipient. Such
formulations can be prepared by dissolving solid active ingredient
in water containing physiologically compatible substances such as
sodium chloride (e.g., 0.1-2.0 M), glycine, and the like, and
having a buffered pH compatible with physiological conditions to
produce an aqueous solution, and rendering the solution sterile.
These may be present in unit or multi-dose containers, for example,
sealed ampoules or vials.
[0221] Exemplary formulations of the invention can incorporate a
stabilizer. Exemplary stabilizers include polyethylene glycol,
proteins, saccharides, amino acids, inorganic acids, detergents,
and organic acids, which can be used either alone or as admixtures.
These stabilizers can be incorporated in an amount of, for example,
0.11-10,000 parts by weight per part by weight of an agent. If two
or more stabilizers are to be used, their total amount can be
within the range specified above. These stabilizers can be used in
aqueous solutions at an appropriate concentration and pH. The
specific osmotic pressure of such aqueous solutions can be in the
range of 0.1-3.0 osmoles, preferably in the range of 0.8-1.2. The
pH of the aqueous solution can be adjusted to be within the range
of 5.0-9.0, preferably within the range of 6-8. In formulating an
antibody or antibody-drug conjugate, an anti-adsorption agent can
be used.
[0222] Additional pharmaceutical methods can be employed to control
duration of action. Controlled release can be achieved through the
use of polymer to complex or absorb the proteins or their
derivatives. Controlled delivery can be achieved by selecting
appropriate macromolecules (e.g., polyester, polyamino acids,
polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose,
carboxymethylcellulose, or protamine sulfate) and the concentration
of macromolecules as well as the methods of incorporation in order
to control release. Another possible method to control the duration
of action by controlled-release preparations is to incorporate an
anti-CAT antibody into particles of a polymeric material such as
polyesters, polyamino acids, hydrogels, poly(lactic acid) or
ethylene vinylacetate copolymers. Alternatively, instead of
incorporating these agents into polymeric particles, it is possible
to entrap these materials in microcapsules prepared, for example,
by coacervation techniques or by interfacial polymerization, for
example, hydroxymethylcellulose or gelatin-microcapsules and
poly(methylmethacylate) microcapsules, respectively, or in
colloidal drug delivery systems, for example, liposomes, albumin
microspheres, microemulsions, nanoparticles, and nanocapsules or in
macroemulsions.
[0223] When oral preparations are desired, the compositions can be
combined with typical carriers, such as lactose, sucrose, starch,
talc magnesium stearate, crystalline cellulose, methyl cellulose,
carboxymethyl cellulose, glycerin, sodium alginate or gum arabic,
among others.
[0224] Any of the therapeutic agents provided herein may be
administered in combination with other therapeutic agents.
Selection of agents for use in combination therapy can be made by
one of ordinary skill in the art according to conventional
pharmaceutical principles. A combination of therapeutic agents may
act synergistically to affect treatment of a particular disorder at
a lower dosage of each agent.
[0225] 7. Methods of Detection and Diagnosis Based on CAT
Proteins
[0226] CAT proteins are useful for diagnosing a disease, or
predisposition to a disease, particularly diseases in which the
protein is over- or under-expressed, especially cancer,
particularly the cancers identified in Table 1 (as indicated for
each peptide) and/or Table 3. The diagnostic methods may be further
suitable for monitoring disease progression in patients undergoing
treatment, or for testing for reoccurrence of disease in patients
who were previously treated for a disease, for example.
Accordingly, exemplary embodiments of the invention provide methods
for detecting the presence of, or abundance levels of, a CAT
protein in a biological sample.
[0227] In vitro techniques for detection of proteins include, but
are not limited to, enzyme linked immunosorbent assays (ELISAs),
Western blots, immunoprecipitations, and immunofluorescence using a
detection reagent, such as an antibody or protein binding agent.
Alternatively, a protein can be detected in vivo in a subject by
introducing into the subject a labeled antibody (or other types of
detection agent) specific for the protein target. For example, an
antibody can be labeled with a radioactive marker whose presence
and location in a subject can be detected by standard imaging
techniques. Particularly useful are methods that detect variants of
a protein (e.g., allelic variants or mutations) and methods that
detect fragments of a protein in a sample.
[0228] Proteins can be isolated from a biological sample (such as
from a patient having a disease) and assayed for the presence of a
mutation. A mutation can include, for example, one or more amino
acid substitutions, deletions, insertions, rearrangements (such as
from aberrant splicing events), or inappropriate post-translational
modifications. Examples of analytic methods useful for detecting
mutations in a protein include, but are not limited to, altered
electrophoretic mobility, altered tryptic peptide digest, altered
protein activity in cell-based or cell-free assays, alteration in
substrate or antibody-binding patterns, altered isoelectric point,
and direct amino acid sequencing.
[0229] Information obtained by detecting a protein can be used, for
example, to determine prognosis and appropriate course of treatment
for a disease. For example, individuals with a particular CAT
expression level or stage of disease may respond differently to a
given treatment that individuals lacking CAT expression, or
individuals over- or under-expressing CAT. Information obtained
from diagnostic methods of the invention can provide for the
personalization of diagnosis and treatment.
[0230] In exemplary embodiments, the invention provides methods for
diagnosing disease (including, for example, monitoring treatment
response or recurrence of disease following treatment) in a subject
comprising: determining the abundance level of CAT (e.g., CAT
protein or nucleic acid, or protein or nucleic acid fragments
thereof) in a test sample from the subject; wherein a difference in
the abundance level of CAT relative to the abundance level of CAT
in a test sample from a healthy subject, or the level established
for a healthy subject, is indicative of disease.
[0231] Exemplary embodiments of the invention provide methods for
diagnosing diseases having differential protein expression. For
example, normal, control, or standard values (e.g., that represent
typical expression levels of a protein in healthy subjects) can be
established, such as by combining body fluids, tissues, or cell
extracts taken from a normal healthy mammalian or human subject
with specific antibodies to a protein under conditions for complex
formation. Standard values for complex formation in normal and
disease tissues can be established by various methods, such as
photometric means. Complex formation, as it is expressed in a test
sample, can be compared with the standard values. Deviation from a
normal standard and toward a disease standard can provide
parameters for disease diagnosis or prognosis while deviation away
from a disease standard and toward a normal standard can be used to
evaluate treatment efficacy, for example.
[0232] Immunological methods for detecting and measuring complex
formation as a measure of protein expression using either specific
polyclonal or monoclonal antibodies are known in the art. Examples
of such techniques include ELISAs, radioimmunoassays (RIAs), flow
cytometry (also referred to as fluorescence-activated cell sorting,
or FACS), and antibody arrays. Such immunoassays typically involve
the measurement of complex formation between a protein and its
specific antibody. These assays and their quantitation against
purified, labeled standards are well known in the art (Ausubel,
supra, unit 10.1-10.6). For example, a two-site, monoclonal-based
immunoassay utilizing antibodies reactive to two non-interfering
epitopes can be utilized, and competitive binding assay can also be
utilized (Pound (1998) Immunochemical Protocols, Humana Press,
Totowa N.J.).
[0233] For diagnostic applications, an antibody can be labeled with
a detectable moiety (interchangeably referred to as a "label" or
"detectable substance"), such as to facilitate detection by various
imaging methods. Methods for detection of labels include, but are
not limited to, fluorescence, light, confocal, and electron
microscopy; magnetic resonance imaging and spectroscopy;
fluoroscopy, computed tomography and positron emission tomography.
Examples of suitable labels include, but are not limited to,
fluorescein, rhodamine, eosin and other fluorophores,
radioisotopes, gold, gadolinium and other lanthanides, paramagnetic
iron, fluorine-18 and other positron-emitting radionuclides.
Additionally, labels may be bi- or multi-functional and be
detectable by more than one of the methods listed. Antibodies may
be directly or indirectly labeled. Attachment of labels to
antibodies includes covalent attachment of a label, incorporation
of a label into an antibody, and covalent attachment of a chelating
compound for binding of a label, among others well known in the
art.
[0234] Numerous detectable moieties are available for labeling
antibodies, including, but not limited to, those in the following
categories:
[0235] (a) Radioisotopes, such as .sup.36S, .sup.14C, .sup.125I,
.sup.3H, and .sup.131I. An antibody can be labeled with a
radioisotope using the techniques described in Current Protocols in
Immunology, vol 1-2, Coligen et al., Ed., Wiley-Interscience, New
York, Pubs. (1991-2006), for example, and radioactivity can be
measured using scintillation counting.
[0236] (b) Fluorescent labels such as rare earth chelates (europium
chelates) or fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, Lissamine, phycoerythrin and Texas Red are
available. Fluorescent labels can be conjugated to an antibody
using the techniques disclosed in Current Protocols in Immunology,
supra, for example. Fluorescence can be quantified using a
fluorometer.
[0237] (c) Various enzyme-substrate labels are available (e.g.,
U.S. Pat. Nos. 4,275,149 and 4,318,980). An enzyme generally
catalyzes a chemical alteration of a chromogenic substrate which
can be measured using various techniques. For example, an enzyme
may catalyze a color change in a substrate, which can be measured
spectrophotometrically. Alternatively, an enzyme may alter the
fluorescence or chemiluminescence of a substrate. Techniques for
quantifying a change in fluorescence are described herein and well
known in the art A chemiluminescent substrate becomes
electronically excited by a chemical reaction and may then emit
light which can be measured (using a chemiluminometer, for example)
or donates energy to a fluorescent acceptor. Examples of enzymatic
labels include luciferases (e.g., firefly luciferase and bacterial
luciferase; U.S. Pat. No. 4,737,456), luciferin,
2,3-dihydrophthalazinediones, malate dehydrogenase, urease,
peroxidase such as horseradish peroxidase (HRPO), alkaline
phosphatase, .beta.-galactosidase, glucoamylase, lysozyme,
saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and
glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as
uricase and xanthine oxidase), lactoperoxidase, microperoxidase,
and the like. Techniques for conjugating enzymes to antibodies are
described in O'Sullivan et al., Methods for the Preparation of
Enzyme-Antibody Conjugates for Use in Enzyme Immunoassay, in
Methods in Enzyme. (Ed. J. Langone & H. Van Vunakis), Academic
press, New York, 73: 147-166 (1981).
[0238] A label can be indirectly conjugated with an antibody. The
skilled artisan will be aware of various techniques for achieving
this. For example, an antibody can be conjugated with biotin and
any of the three broad categories of labels mentioned above can be
conjugated with avidin, or vice versa. Biotin binds selectively to
avidin and thus, the label can be conjugated with the antibody in
this indirect manner. Alternatively, to achieve indirect
conjugation of a label with an antibody, an antibody can be
conjugated with a small hapten (e.g., digoxin) and one of the
different types of labels mentioned above can be conjugated with an
anti-hapten antibody (e.g., anti-digoxin antibody). Thus, indirect
conjugation of a label with an antibody can be achieved.
[0239] Antibodies can be used to isolate CAT proteins by standard
techniques, such as affinity chromatography or immunoprecipitation,
and antibodies can facilitate the purification of the natural
protein from cells and recombinantly-produced protein expressed in
host cells. Biological samples can be tested directly for the
presence of a CAT protein by assays (e.g., ELISA or
radioimmunoassay) and format (e.g., microwells, dipstick, etc., as
described in International Patent Publication WO 93/03367).
Alternatively, proteins in a sample can be size separated (e.g., by
polyacrylamide gel electrophoresis (PAGE)), in the presence or
absence of sodium dodecyl sulfate (SDS), and the presence of a CAT
detected by immunoblotting (e.g., Western blotting).
[0240] Antibody binding can also be detected by "sandwich"
immunoassays, immunoradiometric assays, gel diffusion precipitation
reactions, immunodiffusion assays, in situ immunoassays (e.g.,
using colloidal gold, enzyme or radioisotope labels, for example),
precipitation reactions, agglutination assays (e.g., gel
agglutination assays, hemagglutination assays, etc.), complement
fixation assays, immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc.
[0241] In certain exemplary embodiments, antibody binding can be
detected by detecting a label on the primary antibody. In other
exemplary embodiments, a primary antibody can be detected by
detecting binding of a secondary antibody or reagent to the primary
antibody. In further exemplary embodiments, the secondary antibody
is labeled. Numerous means are known in the art for detecting
binding in an immunoassay and are within the scope of the
invention. In some embodiments, an automated detection assay is
utilized. Methods for the automation of immunoassays are well known
in the art (e.g., U.S. Pat. Nos. 5,885,530: 4,981,785: 6,159,750:
and 5,358,691, each of which is herein incorporated by reference).
In some embodiments, the analysis and presentation of results are
also automated. For example, in some embodiments, software that
generates a prognosis based on the presence or absence of one or
more antigens can be implemented.
[0242] Competitive binding assays typically rely on the ability of
a labeled standard to compete with a test sample for binding with a
limited amount of antibody. The amount of antigen 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 generally are
insolubilized before or after the competition. As a result, the
standard and test sample that are bound to the antibodies can be
separated from the standard and test sample that remain
unbound.
[0243] Sandwich assays typically involve the use of two antibodies,
each capable of binding to a different immunogenic portion, or
epitope, of the protein to be detected. In typical sandwich assays,
the test sample to be analyzed is bound by a first antibody, which
is immobilized on a solid support, and thereafter a second antibody
binds to the test sample, thus forming an insoluble three-part
complex (e.g., U.S. Pat. No. 4,376,110). The second antibody can
itself be labeled with a detectable moiety (direct sandwich assays)
or can 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.
[0244] Antibodies can also be used for in vivo diagnostic assays.
Generally, an antibody can be labeled with a radionuclide (such as
.sup.111In, .sup.99Tc, .sup.14C, .sup.131I, .sup.3H, .sup.32P or
.sup.35S) so that disease cells or tissues can be localized using
immunoscintiography, for example. In certain embodiment, antibodies
or fragments thereof bind to the extracellular domains of two or
more CAT proteins and the affinity value (Kd) is less than
1.times.10.sup.8 M.
[0245] For immunohistochemistry, a disease tissue sample may be,
for example, fresh or frozen or may be embedded in paraffin and
fixed with a preservative such as formalin. A fixed or embedded
section can be contacted with a labeled primary antibody and
secondary antibody, wherein the antibody is used to detect CAT
protein expression in situ.
[0246] Antibodies can be used to detect a target protein in situ,
in vitro, or in a cell lysate or supernatant in order to evaluate
the abundance and pattern of expression. Also, antibodies can be
used to assess abnormal tissue distribution or abnormal expression
during development or progression of a biological condition.
Antibodies against CAT proteins are useful for detecting the
presence of the proteins in cells or tissues to determine the
pattern of expression of the proteins among various tissues in an
organism and over the course of the organism's development.
[0247] Further, antibodies can be used to assess expression in
disease states such as in active stages of a disease or in an
individual with a predisposition toward disease related to the
protein's function. When a disorder is caused by inappropriate
tissue distribution, developmental expression, or level of
expression of a protein, or expressed/processed form, for example,
an antibody can be prepared against the normal protein. If a
disorder is characterized by a specific mutation in a protein,
antibodies specific for the mutant protein can be used to assay for
the presence of the specific mutant protein and to target the
mutant protein for therapeutic purposes. Antibodies are also useful
as diagnostic tools, as immunological markers for aberrant protein
analyzed by electrophoretic mobility, isoelectric point, tryptic
peptide digest, and other physical assays known in the art.
[0248] Certain exemplary diagnostic methods of the invention can
also include monitoring a treatment modality. Accordingly, where
treatment is ultimately aimed at correcting, for example, the
function, activity, expression level, tissue distribution, or
developmental expression of a protein, antibodies directed against
the protein can be used to monitor therapeutic efficacy and to
modify a treatment regimen as necessary.
[0249] Additionally, antibodies to a target protein are useful in
pharmacogenomic analysis. For example, antibodies prepared against
polymorphic proteins can be used to identify individuals that
require modified treatment modalities. Moreover, the target
proteins and antibodies thereto can be used for clinical trials,
such as to identify individuals that should be included (e.g.,
individuals more likely to respond to a therapy) or excluded (e.g.,
individuals less likely to respond to a therapy, or individuals
more likely to experience harmful side effects from a therapy) from
a clinical trial.
[0250] The invention also encompasses kits for using antibodies to
detect the presence of a target protein in a biological sample. An
exemplary kit can comprise antibodies such as a labeled or
labelable antibody and a compound or agent for detecting protein in
a biological sample; means for determining the amount of protein in
the sample; means for comparing the amount of protein in the sample
with a standard; and instructions for use. Such a kit can be
configured to detect a single target protein or epitope or can be
configured to detect one of a multitude of epitopes, such as in an
antibody detection array.
[0251] LC/MS and ICAT
[0252] In certain exemplary embodiments, the invention provides
detection or diagnostic methods of a CAT by using LC/MS. Proteins
can be prepared from cells by methods known in the art (e.g., Zhang
et al., Nature Biotechnology 21(6):660-666 (2003)). The
differential expression of proteins in disease and healthy (or
drug-resistant and drug-sensitive, for example) samples can be
quantitated using mass spectrometry and ICAT (Isotope Coded
Affinity Tag) labeling, which is known in the art. ICAT is an
isotope label technique that allows for discrimination between two
populations of proteins, such as a healthy and a disease sample.
Over-expression or under-expression of a CAT protein, as measured
by ICAT, can indicate, for example, the likelihood of having or
developing a disease or an associated pathology.
[0253] LC/MS spectra can be collected for labeled samples and
processed as follows. The raw scans from the LC/MS instrument can
be subjected to peak detection and noise reduction software.
Filtered peak lists can then be used to detect `features`
corresponding to specific peptides from the original sample(s).
Features are characterized by their mass/charge ratio, charge,
retention time, isotope pattern, and/or intensity, for example.
[0254] The intensity of a peptide present in both healthy and
disease samples can be used to calculate the differential
expression, or relative abundance, of the peptide. The intensity of
a peptide found exclusively in one sample can be used to calculate
a theoretical expression ratio for that peptide (singleton).
Expression ratios can be calculated for each peptide in an assay or
experiment.
[0255] Statistical tests can be performed to assess the robustness
of the data and select statistically significant differentials. To
ensure the accuracy of data, the following steps can be taken: a)
ensure that similar features are detected in all replicates of an
experiment; b) assess the distribution of the log ratios of all
peptides (a Gaussian is expected); c) calculate the overall pair
wise correlations between ICAT LC/MS maps to ensure that the
expression ratios for peptides are reproducible across multiple
replicates; and d) aggregate multiple experiments in order to
compare the expression ratio of a peptide in multiple diseases or
disease samples.
[0256] 8. Methods of Treatment Based on CAT Proteins
[0257] a. Antibody Therapy
[0258] Antibodies of the invention can be used for therapeutic
purposes. It is contemplated that antibodies of the invention may
be used to treat a mammal, preferably a human, with a disease,
especially cancer, particularly the cancers identified in Table 1
(as indicated for each peptide) and/or Table 3. The antibodies can
be delivered alone, in a pharmaceutical composition (such as with a
carrier), or conjugated to one or more therapeutic agents, for
example.
[0259] Antibodies can be useful for modulating (e.g., agonizing or
antagonizing) protein function, such as for therapeutic purposes.
Antibodies can also be useful for inhibiting protein function by,
for example, blocking the binding of a CAT protein to a binding
partner such as a substrate, which can be useful therapeutically.
Antibodies can be prepared against, for example, specific portions
of a protein that contain domains required for protein function, or
against intact protein that is associated with a cell membrane.
[0260] Antibodies of the invention can also be used for enhancing
the immune response. The antibodies can be administered in amounts
similar to those used for other therapeutic administrations of
antibodies. For example, pooled gamma globulin can be administered
at a range of about 1 mg to about 100 mg per patient.
[0261] Antibodies reactive with CAT proteins can be administered
alone or in conjunction with other therapies, such as anti-cancer
therapies, to a mammal afflicted with cancer or other disease.
Examples of anti-cancer therapies include, but are not limited to,
chemotherapy, radiation therapy, and adoptive immunotherapy therapy
with TIL (tumor infiltrating lymphocytes).
[0262] The selection of an antibody subclass for therapy may depend
upon the nature of the antigen to be acted upon. For example, an
IgM may be preferred in situations where the antigen is highly
specific for the diseased target and rarely occurs on normal cells.
However, where the disease-associated antigen is also expressed in
normal tissues, although at lower levels, the IgG subclass may be
preferred. The IgG subclass may be preferred in these instances
because the binding of at least two IgG molecules in close
proximity is typically required to activate complement, and
therefore less complement-mediated damage may occur in normal
tissues that express smaller amounts of the antigen and thus bind
fewer IgG antibody molecules. Furthermore, IgG molecules, by being
smaller, may be more able than IgM molecules to localize to a
diseased tissue.
[0263] A mechanism for antibody therapy can be that a therapeutic
antibody recognizes a cell surface, secreted, or cytosolic target
protein that is expressed (preferably, over-expressed) in a disease
cell. By NK cell or complement activation, or conjugation of the
antibody with an immunotoxin or radiolabel, the interaction of the
antibody with the target protein can abrogate ligand/receptor
interaction or activation of apoptosis, for example.
[0264] Potential mechanisms of antibody-mediated cytotoxicity of
diseased cells include phagocyte (antibody-dependent cellular
cytotoxicity (ADCC)), complement (complement-dependent cytotoxicity
(CDC)), naked antibody (receptor cross-linking apoptosis and growth
factor inhibition), or targeted payload labeled with a therapeutic
agent, such as a radionuclide, immunotoxin, or
immunochemotherapeutic or other therapeutic agent.
[0265] In certain exemplary embodiments, an antibody is
administered to a nonhuman mammal for the purposes of obtaining
preclinical data, for example. Exemplary nonhuman mammals to be
treated include nonhuman primates, dogs, cats, rodents, and other
mammals in which preclinical studies are performed. Such mammals
may be established animal models for a disease or may be used to
study toxicity of an antibody of interest, for example. Dose
escalation studies may be performed in the mammal, for example.
[0266] An antibody can be administered to an individual by any
suitable means, including parenteral, subcutaneous,
intraperitoneal, intrapulmonary, and intranasal, and, if desired
for local immunomodulatory treatment, intralesional administration.
Parenteral infusions include intramuscular, intravenous,
intraarterial, intraperitoneal, or subcutaneous administration. In
addition, an antibody can be administered by pulse infusion,
particularly with declining doses of the antibody. The dosing can
be given by injections, such as intravenous or subcutaneous
injections, which may depend in part on whether the administration
is brief or chronic.
[0267] For the prevention or treatment of a disease, the
appropriate dosage of an antibody may depend on the type of disease
to be treated, the severity and the course of the disease, whether
the antibody is administered for preventive or therapeutic
purposes, previous therapy, the patient's clinical history and
response to the antibody, and the discretion of the attending
physician.
[0268] Depending on the type and severity of disease, about 1
.mu.g/kg to 150 mg/kg (e.g., 0.1-20 mg/kg) of antibody can be an
initial candidate dosage for administration to a patient, whether,
for example, by one or more separate administrations, or by
continuous infusion. A typical daily dosage may range from about 1
.mu.g/kg to 100 mg/kg or more, depending on such factors as those
mentioned above. An antibody-drug conjugate can be administered
from about 1 .mu.g/kg to 50 mg/kg, typically from about 0.1-20
mg/kg, whether, for example, by one or more separate
administrations, or by continuous infusion. A typical daily dosage
may range from about 0.1 mg/kg to 10 mg/kg, or from about 0.3 mg/kg
to about 7.5 mg/kg, depending on such factors as those mentioned
above. For repeated administrations over several days or longer,
depending on the condition, the treatment can be sustained until a
desired suppression of disease symptoms occurs. However, other
dosage regimens may be useful. Therapy progress can be monitored by
conventional techniques and assays.
[0269] Antibody composition can be formulated, dosed, and
administered in a manner consistent with good medical practice.
Factors for consideration in this context include the particular
disorder being treated, the particular mammal being treated, the
clinical condition of the individual patient, the cause of the
disorder, the site of delivery of the agent, the method of
administration, the scheduling of administration, and other factors
known to medical practitioners.
[0270] An antibody may optionally be formulated with, or
administered with, one or more therapeutic agents used to prevent
or treat the disorder in question. For example, an antibody can be
administered as a co-therapy with a standard of care therapeutic
for the specific disease being treated.
[0271] b. Other Immunotherapy
[0272] An "immunogenic peptide" is a peptide that comprises an
allele-specific motif such that the peptide typically will bind an
MHC allele (HLA in human) and be capable of inducing a CTL
(cytotoxic T-lymphocytes) response. Thus, immunogenic peptides
typically are capable of binding to an appropriate class I or II
MHC molecule and inducing a cytotoxic T cell or T helper cell
response against the antigen from which the immunogenic peptide is
derived.
[0273] Peptides derived from a CAT protein can be modified to
increase their immunogenicity, such as by enhancing the binding of
the peptide to the MHC molecules in which the peptide is presented.
The peptide or modified peptide can be conjugated to a carrier
molecule to enhance the antigenicity of the peptide. Examples of
carrier molecules, include, but are not limited to, human albumin,
bovine albumin, lipoprotein and keyhole limpet hemo-cyanin ("Basic
and Clinical Immunology" (1991) Stites and Terr (eds) Appleton and
Lange, Norwalk Conn., San Mateo, Calif.).
[0274] Further, amino acid sequence variants of a peptide can be
prepared, such as by altering the nucleic acid sequence of the DNA
which encodes the peptide, or by peptide synthesis. At the genetic
level, these variants can be prepared by, for example,
site-directed mutagenesis of nucleotides in the DNA encoding the
peptide, thereby producing DNA encoding the variant, and thereafter
expressing the DNA in recombinant cell culture. The variants can
exhibit the same qualitative biological activity as the nonvariant
peptide.
[0275] Exemplary embodiments of the invention provide peptides or
modified peptides derived from a CAT protein that are
differentially expressed in disease. Examples of peptide
modifications include, but are not limited to, substitutions,
deletions, or additions of one or more amino acids in a given
immunogenic peptide sequence, or mutation of existing amino acids
within a given immunogenic peptide sequence, or derivatization of
existing amino acids within a given immunogenic peptide sequence.
Any amino acid in an immunogenic peptide sequence may be modified.
In some embodiments, at least one amino acid can be substituted or
replaced within the given immunogenic peptide sequence. Any amino
acid may be used to substitute or replace a given amino acid within
the immunogenic peptide sequence. Modified peptides can include any
immunogenic peptide obtained from differentially expressed
proteins, which has been modified and exhibits enhanced binding to
the MHC molecule with which it associates when presented to a
T-cell. These modified peptides can be synthetically or
recombinantly produced by conventional methods, for example.
[0276] In certain exemplary embodiments of the invention, the
peptides comprise, or consist of, sequences of about 5-30 amino
acids in length which are immunogenic (i.e., capable of inducing an
immune response when injected into a subject).
[0277] In certain exemplary embodiments, the peptides may be used,
for example, to treat T cell-mediated pathologies. The term "T
cell-mediated pathologies" refers to any condition in which an
inappropriate T cell response is a component of the pathology. The
term is intended to encompass both T cell mediated diseases and
diseases resulting from unregulated clonal T cell replication.
[0278] Modified (e.g., recombinant) or natural CAT proteins, or
fragments thereof, can be used as a vaccine either prophylactically
or therapeutically. When provided prophylactically, a vaccine can
be provided in advance of any evidence of disease. The prophylactic
administration of a disease vaccine may serve to prevent or
attenuate a disease in a mammal such as a human.
[0279] An exemplary vaccine formulation can comprise an immunogen
that induces an immune response directed against a
disease-associated antigen such as a CAT protein. For example, a
substantially or partially purified CAT protein or fragments
thereof can be administered as a vaccine in a pharmaceutically
acceptable carrier. An immunogen can be administered in a pure or
substantially pure form, or can be administered as a pharmaceutical
composition, formulation, or preparation. Exemplary doses of
protein that can be administered are about 0.001 to about 100 mg
per patient, or about 0.01 to about 100 mg per patient.
Immunization can be repeated as necessary until a sufficient titer
of anti-immunogen antibody or immune cells has been obtained.
[0280] Vaccine can be prepared using, for example, recombinant
protein or expression vectors comprising a nucleic acid sequence
encoding all or part of a CAT protein. Examples of vectors that can
be used in vaccines include, but are not limited to, defective
retroviral vectors, adenoviral vectors vaccinia viral vectors, fowl
pox viral vectors, or other viral vectors (Mulligan, R. C., (1993)
Science 260:926-932). The vectors can be introduced into a mammal
(e.g., a human) either prior to any evidence of a disease or to
mediate regression of a disease in a mammal afflicted with the
disease. Examples of methods for administering a viral vector into
mammals include, but are not limited to, exposure of cells to the
virus ex vivo, or injection of the retrovirus or a producer cell
line of the virus into the affected tissue, or intravenous
administration of the virus. Alternatively, the vector can be
administered locally by direct injection into a disease lesion or
topical application in a pharmaceutically acceptable carrier. The
quantity of viral vector to be administered can be based on the
titer of virus particles. An exemplary range can be about 10.sup.6
to about 10.sup.11 virus particles per mammal.
[0281] After immunization, the efficacy of the vaccine can be
assessed by, for example, the production of antibodies or immune
cells that recognize the antigen, as assessed by specific lytic
activity, specific cytokine production, or disease regression,
which can be measured using conventional methods. If the mammal to
be immunized is already afflicted with a disease, the vaccine can
be administered in conjunction with other therapeutic treatments.
Examples of other therapeutic treatments include, but are not
limited to, adoptive T cell immunotherapy and coadministration of
cytokines or other therapeutic drugs.
[0282] In certain embodiments, mammals, preferably humans, at high
risk for disease, especially cancer, are prophylactically treated
with vaccines of the invention. Examples include, but are not
limited to, individuals with a family history of a disease,
individuals who themselves have a history of disease (e.g., cancer
that has been previously resected and at risk for reoccurrence), or
individuals already afflicted with a disease. When provided
therapeutically, a vaccine can be provided to enhance the patient's
own immune response to a disease antigen. An exemplary vaccine,
which acts as an immunogen, can be a cell, cell lysate from cells
transfected with a recombinant expression vector, or a culture
supernatant containing the expressed protein, for example.
Alternatively, an immunogen can be, for example, a partially or
substantially purified recombinant protein, peptide, or analog
thereof, or a modified protein, peptide, or analog thereof. The
proteins or peptides can be, for example, conjugated with
lipoprotein or administered in liposomal form or with adjuvant.
[0283] Vaccination can be carried out using conventional methods.
For example, an immunogen can be used in a suitable diluent such as
saline or water, or complete or incomplete adjuvants. Further, an
immunogen may or may not be bound to a carrier, including carriers
to increase the immunogenicity of the immunogen. Examples of
carrier molecules include, but are not limited to, bovine serum
albumin (BSA), keyhole limpet hemocyanin (KLH), tetanus toxoid, and
the like. An immunogen also may be coupled with lipoproteins or
administered in liposomal form or with adjuvants. An immunogen can
be administered by any route appropriate for antibody production
such as intravenous, intraperitoneal, intramuscular, subcutaneous,
and the like. An immunogen can be administered once or at periodic
intervals until a significant titer of anti-CAT immune cells or
anti-CAT antibody is produced. The presence of anti-CAT immune
cells can be assessed by measuring the frequency of precursor CTL
(cytotoxic T-lymphocytes) against CAT antigen prior to and after
immunization by a CTL precursor analysis assay (Coulie et al.,
1992, International Journal Of Cancer 50:289-297). An immunoassay
can be used to detect antibody in serum.
[0284] The safety of a vaccine can be determined by examining the
effect of immunization on the general health of an immunized animal
(e.g., weight change, fever, change in appetite or behavior, etc.)
and looking for pathological changes during autopsies. After
initial testing in animals, a vaccine can be tested in patients
having a disease of interest. Conventional methods can be used to
evaluate the immune response of a patient to determine the
efficiency of the vaccine.
[0285] In certain exemplary embodiments of the invention, a CAT
protein or fragments thereof, or a modified CAT protein, can be
exposed to dendritic cells cultured in vitro. The cultured
dendritic cells provide a means of producing T-cell dependent
antigens comprised of dendritic cell-modified antigen or dendritic
cells pulsed with antigen, in which the antigen is processed and
expressed on the antigen-activated dendritic cell. The
antigen-activated dendritic cells or processed dendritic cell
antigens can be used as immunogens for vaccines or for the
treatment of diseases. The dendritic cells can be exposed to the
antigen for sufficient time to allow the antigens to be
internalized and presented on the surface of dendritic cells. The
resulting dendritic cells or the dendritic cell-processed antigens
can then be administered to an individual in need of therapy. Such
methods are described in Steinman et al. (WO93/208185) and in
Banchereau et al. (EPO Application 0563485A1).
[0286] In certain exemplary embodiments of the invention, T-cells
isolated from individuals can be exposed to a CAT protein or
fragment thereof, or a modified CAT protein, in vitro and then
administered in a therapeutically effective amount to a patient in
need of such treatment. Examples of where T-lymphocytes can be
isolated include, but are not limited to, peripheral blood cells
lymphocytes (PBL), lymph nodes, or tumor infiltrating lymphocytes
(TIL). Such lymphocytes can be isolated from the individual to be
treated or from a donor by methods known in the art and cultured in
vitro (Kawakami et al., 1989, J. Immunol. 142: 2453-3461).
Lymphocytes can be cultured in media such as RPMI or RPMI 1640 or
AIM V for 1-10 weeks. Viability can be assessed by trypan blue dye
exclusion assay. Examples of how these sensitized T-cells can be
administered to a mammal include, but are not limited to,
intravenously, intraperitoneally, or intralesionally. Parameters
that can be assessed to determine the efficacy of these sensitized
T-lymphocytes include, but are not limited to, production of immune
cells in the mammal being treated or tumor regression. Conventional
methods can be used to assess these parameters. Such treatment can
be given in conjunction with cytokines or gene-modified cells, for
example (Rosenberg et al., 1992, Human Gene Therapy, 3: 75-90;
Rosenberg et al., 1992, Human Gene Therapy, 3: 57-73).
[0287] 9. Screening Methods Using CAT Proteins
[0288] Exemplary embodiments of the invention provide methods of
screening for agents (interchangeably referred to by such terms as
candidate agents, compounds, or candidate compounds) that modulate
CAT protein activity (interchangeably referred to as protein
function). Examples of candidate agents include, but are not
limited to, proteins, peptides, antibodies, nucleic acids (such as
antisense and RNAi nucleic acid molecules), and small molecules.
Exemplary embodiments of the invention further provide agents
identified by these screening methods, and methods of using these
agents, such as for treating diseases, especially cancer,
particularly the cancers identified in Table 1 (as indicated for
each peptide) and/or Table 3.
[0289] Exemplary screening methods can typically comprise the steps
of (i) contacting a CAT protein with a candidate agent, and (ii)
assaying for CAT protein activity, wherein a change in protein
activity in the presence of the agent relative to protein activity
in the absence of the agent indicates that the agent modulates CAT
protein activity.
[0290] Other exemplary screening methods can determine a candidate
agent's ability to modulate CAT expression. Exemplary methods can
typically comprise the steps of (i) contacting a candidate agent
with a system that is capable of expressing CAT protein or CAT
mRNA, and (ii) assaying for the level of CAT protein or CAT mRNA,
wherein a change in the level in the presence of the agent relative
to the level in the absence of the agent indicates that the agent
modulates CAT expression levels.
[0291] Exemplary embodiments of the invention further provide
methods to screen for agents that bind to CAT proteins. Exemplary
methods can typically comprise the steps of contacting a CAT
protein with a test agent and measuring the extent of binding of
the agent to the CAT protein.
[0292] CAT proteins can be used to identify agents that modulate
activity of a protein in its natural state or an altered form that
causes a specific disease or pathology. CAT proteins and
appropriate variants and fragments can be used in high-throughput
screens to assay candidate compounds for their ability to bind to
CAT. These compounds can be further screened against functional CAT
proteins to determine the effect of the compound on the protein's
activity. Further, these compounds can be tested in animal or
invertebrate systems to determine activity/effectiveness. Compounds
can be identified that activate (agonist) or inactivate
(antagonist) CAT proteins to a desired degree.
[0293] CAT proteins can be used to screen agents for their ability
to stimulate or inhibit interaction between a CAT protein and a
target molecule that normally interacts with the CAT protein (e.g.,
a substrate, an extracellular binding ligand, or a component of a
signal pathway that a CAT protein normally interacts with such as a
cytosolic signal protein). Exemplary assays can include the steps
of combining a CAT protein or fragment thereof with a candidate
compound under conditions that allow the CAT protein (or fragment
thereof) to interact with a target molecule, and detecting the
formation of a complex between the CAT protein and the target
molecule or detecting the biochemical consequence of the
interaction between the CAT protein and the target molecule, such
as any of the associated effects of signal transduction (e.g.,
protein phosphorylation, cAMP turnover, adenylate cyclase
activation, etc.). Any of the biological or biochemical functions
mediated by a CAT protein can be used as an endpoint assay to
identify an agent that modulates CAT activity.
[0294] Candidate compounds or agents include, but are not limited
to, 1) peptides such as soluble peptides, including Ig-tailed
fusion peptides and members of random peptide libraries (see, e.g.,
Lam et al., Nature 354:82-84 (1991); Houghten et al., Nature
354:84-86 (1991)) and combinatorial chemistry-derived molecular
libraries made of D- and/or L-configuration amino acids; 2)
phosphopeptides (e.g., members of random and partially degenerate,
directed phosphopeptide libraries, see, e.g., Songyang et al., Cell
72:767-778 (1993)); 3) antibodies (e.g., polyclonal, monoclonal,
humanized, anti-idiotypic, chimeric, and single chain antibodies as
well as Fab, F(ab').sub.2, Fab expression library fragments, and
epitope-binding fragments of antibodies); and 4) small organic and
inorganic molecules (e.g., molecules obtained from combinatorial
and natural product libraries).
[0295] An exemplary candidate compound or agent is a soluble
fragment of a CAT that competes for substrate binding. Other
exemplary candidate compounds include mutant CAT proteins or
appropriate fragments containing mutations that affect CAT function
and thus compete for substrate. Accordingly, a fragment that
competes for substrate, for example with a higher affinity, or a
fragment that binds substrate but does not allow release, is
encompassed by the invention.
[0296] Compounds can also be screened by using chimeric proteins in
which any portion of a protein such as an amino terminal
extracellular domain, a transmembrane domain (e.g., transmembrane
segments or intracellular or extracellular loops), or a carboxy
terminal intracellular domain can be replaced in whole or part by
heterologous domains or subregions. For example, a
substrate-binding region can be used that interacts with a
different substrate than the substrate that is recognized by a
native target protein. Accordingly, a different set of signal
transduction components can be available as an end-point assay for
activation, thereby allowing assays to be performed in other than
the specific host cell from which a target is derived.
[0297] Competition binding assays can also be used to screen for
compounds that interact with a target protein (e.g., binding
partners and/or ligands). For example, a test compound can be
exposed to a target protein under conditions that allow the test
compound to bind or otherwise interact with the target protein.
Soluble target protein can also be added to the mixture. If the
test compound interacts with the soluble target protein, it can
decrease the amount of complex formed or activity of the target
protein. This type of assay is particularly useful in instances in
which compounds are sought that interact with specific regions of a
target protein. Thus, the soluble target protein that competes with
the target protein can contain peptide sequences corresponding to
the target region of interest.
[0298] To perform cell-free drug screening assays, it may be
desirable to immobilize either a CAT protein (or fragment thereof)
or a molecule that binds the CAT protein (referred to herein as a
"binding partner") to facilitate separation of complexes from
uncomplexed forms, as well as to facilitate automation of the
assays.
[0299] Techniques for immobilizing proteins on matrices can be
utilized in exemplary drug screening assays. In exemplary
embodiments, a fusion protein can be provided which adds a domain
that allows a protein to be bound to a matrix. For example,
glutathione-S-transferase fusion proteins can be adsorbed onto
glutathione SEPHAROSE beads (Sigma Chemical, St. Louis, Mo.) or
glutathione derivatized microtitre plates, which are then combined
with cell lysates (e.g., .sup.35S-labeled) and a candidate
compound, and the mixture incubated under conditions conducive to
complex formation (e.g., at physiological conditions for salt and
pH). Following incubation, the beads can be washed to remove any
unbound label, and the matrix immobilized and radiolabel determined
directly, or in the supernatant after the complexes are
dissociated. Alternatively, the complexes can be dissociated from
the matrix, separated by SDS-PAGE, and the level of a binding
partner found in the bead fraction quantitated from the gel using
standard electrophoretic techniques. For example, either a target
protein or a binding partner can be immobilized by conjugation of
biotin and streptavidin using techniques well known in the art.
Alternatively, antibodies that are reactive with a target protein
but do not interfere with binding of the target protein to its
binding partner can be derivatized to the wells of a plate, and the
target protein trapped in the wells by antibody conjugation.
Preparations of a binding partner and a candidate compound can be
incubated in target protein-presenting wells and the amount of
complex trapped in the well can be quantitated. Methods for
detecting such complexes, in addition to those described for
GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with a binding partner, or which are
reactive with a target protein and compete with the binding
partner, as well as target protein-linked assays which rely on
detecting an enzymatic activity associated with a binding
partner.
[0300] In exemplary embodiments of the invention, a CAT protein can
be used as a "bait protein" in a two-hybrid assay or three-hybrid
assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993)
Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.
268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924;
Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300),
to identify other proteins, which bind to or interact with a CAT
protein and are involved in the protein's activity. The two-hybrid
system is based on the modular nature of most transcription
factors, which typically consist of separable DNA-binding and
activation domains. In exemplary embodiments, the two-hybrid assay
can utilize two different DNA constructs. In one construct, a gene
that encodes a CAT protein can be fused to a gene encoding the DNA
binding domain of a known transcription factor (e.g., GAL-4). In
the other construct, a DNA sequence from a library of DNA sequences
that encode an unidentified protein ("prey" or "sample") can be
fused to a gene that encodes the activation domain of the known
transcription factor. If the "bait" and the "prey" proteins are
able to interact in vivo, forming a CAT-dependent complex, the
DNA-binding and activation domains of the transcription factor are
brought into close proximity. This proximity allows transcription
of a reporter gene (e.g., LacZ), which can be operably linked to a
transcriptional regulatory site responsive to the transcription
factor. Expression of the reporter gene can be detected and cell
colonies containing the functional transcription factor can be
isolated and used to obtain the cloned gene that encodes the
protein that interacts with the CAT protein.
[0301] Agents that modulate a CAT protein can be identified using
one or more of the above assays, alone or in combination. For
example, a cell-based or cell free system can be used for initial
identification of agents, and then activity of the agents can be
confirmed in an animal or other model system. Such model systems
are well known in the art and can readily be employed in this
context.
[0302] 10. Diagnosis, Treatment, and Screening Methods Using CAT
Nucleic Acid Molecules
[0303] The nucleic acid molecules of the invention are useful, for
example, as probes, primers, chemical intermediates, and in
biological assays. The nucleic acid molecules are useful as
hybridization probes for messenger RNA, transcript/cDNA, and
genomic DNA to detect or isolate full-length cDNA and genomic
clones encoding a CAT protein, or variants thereof. The nucleic
acid molecules are also useful as primers for PCR to amplify any
given region of a nucleic acid molecule and are useful to
synthesize antisense molecules of desired length and sequence. The
nucleic acid molecules are also useful for producing ribozymes
corresponding to all, or a part, of the mRNA produced from the
nucleic acid molecules described herein.
[0304] The nucleic acid molecules are also useful for constructing
recombinant vectors. Exemplary vectors include expression vectors
that express a portion of, or all of, a CAT protein. The nucleic
acid molecules are also useful for expressing antigenic portions of
the proteins. The nucleic acid molecules are also useful for
constructing host cells expressing a part, or all, of the proteins.
The nucleic acid molecules are also useful for constructing
transgenic animals expressing all, or a part, of the proteins.
[0305] A primer or probe can correspond to any sequence along the
entire length of a CAT-encoding nucleic acid molecule such as the
nucleic acid molecules of SEQ ID NOS:723-1281, 1976, and 1978.
Accordingly, a primer or probe can be derived from 5' noncoding
regions, coding regions, or 3' noncoding regions, for example.
[0306] Exemplary in vitro techniques for detection of mRNA include
Northern hybridizations and in situ hybridizations. Exemplary in
vitro techniques for detecting DNA include Southern hybridizations
and in situ hybridization. Reverse transcriptase PCR amplification
(RT-PCR) and the like can also be used for detecting RNA
expression. A specific exemplary method of detection comprises
using TaqMan technology (Applied Biosystems, Foster City,
Calif.).
[0307] a. Methods of Diagnosis Using Nucleic Acids
[0308] Nucleic acid molecules of the invention are useful, for
example, as hybridization probes for determining the presence,
level, form, and/or distribution of nucleic acid expression.
Exemplary probes can be used to detect the presence of, or to
determine levels of, a specific nucleic acid molecule in cells,
tissues, and in organisms. Accordingly, probes corresponding to a
CAT described herein can be used to assess expression and/or gene
copy number in a given cell, tissue, or organism, which can be
applied to, for example, diagnosis of disorders involving an
increase or decrease in CAT protein expression relative to normal
CAT protein expression levels.
[0309] Probes can be used as part of a diagnostic test kit for
identifying cells or tissues that express CAT protein
differentially, such as by measuring a level of a CAT-encoding
nucleic acid (e.g., mRNA or genomic DNA) in a sample of cells from
a subject, or determining if a CAT-encoding nucleic acid is
mutated.
[0310] Exemplary embodiments of the invention encompass kits for
detecting the presence of CAT-encoding nucleic acid (e.g., mRNA or
genomic DNA) in a biological sample. For example, an exemplary kit
can comprise reagents such as a labeled or labelable nucleic acid
or agent capable of detecting CAT nucleic acid in a biological
sample; means for determining the amount of CAT nucleic acid in the
sample; and means for comparing the amount of CAT nucleic acid in
the sample with a standard. The compound or agent can be packaged
in a suitable container. The kit can further comprise instructions
for using the kit to detect CAT nucleic acid.
[0311] The nucleic acid molecules are useful in diagnostic assays
for qualitative changes in CAT nucleic acid expression, and
particularly in qualitative changes that lead to pathology. The
nucleic acid molecules can be used to detect mutations in CAT genes
and gene expression products such as mRNA. The nucleic acid
molecules can be used as hybridization probes to detect naturally
occurring genetic mutations in a CAT gene and to determine whether
a subject with the mutation is at risk for a disorder caused by the
mutation. Examples of mutations include deletions, additions, or
substitutions of one or more nucleotides in a gene, chromosomal
rearrangements (such as inversions or transpositions), and
modification of genomic DNA such as aberrant methylation patterns
or changes in gene copy number (such as amplification). Detection
of a mutated form of a CAT gene associated with a dysfunction can
provide a diagnostic tool for an active disease or susceptibility
to disease in instances in which the disease results from
overexpression, underexpression, or altered expression of a CAT
protein, for example.
[0312] Mutations in a CAT gene can be detected at the nucleic acid
level by a variety of techniques. For example, genomic DNA, RNA, or
cDNA can be analyzed directly or can be amplified (e.g., using PCR)
prior to analysis. In certain exemplary embodiments, detection of a
mutation involves the use of a probe/primer in a PCR reaction (see,
e.g. U.S. Pat. No. 4,683,195 and 4,683,202), such as anchor PCR or
RACE PCR, or, alternatively, in a ligation chain reaction (LCR)
(see, e.g., Landegran et al., Science 241:1077-1080 (1988) and
Nakazawa et al., PNAS 91:360-364 (1994)), the latter of which can
be particularly useful for detecting point mutations in a gene (see
Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). Exemplary
methods such as these can include the steps of collecting a sample
of cells from a patient, isolating nucleic acid (e.g., genomic,
mRNA, or both) from the cells of the sample, contacting the nucleic
acid with one or more primers which specifically hybridize to a
target nucleic acid under conditions such that hybridization and
amplification of the target nucleic acid (if present) occurs, and
detecting the presence or absence of an amplification product, or
detecting the size of the amplification product and comparing the
length to a control sample. Deletions and insertions can be
detected by a change in size of the amplified product compared to a
normal genotype. Point mutations can be identified by hybridizing
amplified DNA to normal RNA or antisense DNA sequences, for
example.
[0313] Alternatively, mutations in a CAT gene can be identified,
for example, by alterations in restriction enzyme digestion
patterns as determined by gel electrophoresis. Further,
sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can be used
to identify the presence of specific mutations by development or
loss of a ribozyme cleavage site. Perfectly matched sequences can
be distinguished from mismatched sequences by nuclease cleavage
digestion assays or by differences in melting temperature.
[0314] Sequence changes at specific locations can be assessed by
nuclease protection assays such as RNase and S1 protection, or
chemical cleavage methods. Furthermore, sequence differences
between a mutant CAT gene and a corresponding wild-type gene can be
determined by direct DNA sequencing. A variety of automated
sequencing procedures can be utilized when performing diagnostic
assays (Naeve, C. W., (1995) Biotechniques 19:448), including
sequencing by mass spectrometry (e.g., PCT International
Publication No. WO 94/16101; Cohen et al., Adv. Chromatogr.
36:127-162 (1996); and Griffin et al., Appl. Biochem. Biotechnol.
38:147-159 (1993)).
[0315] Other methods for detecting mutations in a nucleic acid
include methods in which protection from cleavage agents is used to
detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et
al., Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988);
Saleeba et al., Meth. Enzymol. 217:286-295 (1992)), electrophoretic
mobility of mutant and wild type nucleic acid is compared (Orita et
al., PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144
(1993); and Hayashi et al., Genet. Anal. Tech. Appl. 9:73-79
(1992)), and movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed
using denaturing gradient gel electrophoresis (DGGE) (Myers et al.,
Nature 313:495 (1985)). Examples of other techniques for detecting
point mutations include selective oligonucleotide hybridization,
selective amplification, and selective primer extension.
[0316] b. Methods of Monitoring Treatment and Pharmacogenomic
Methods Using Nucleic Acids
[0317] Nucleic acid molecules of the invention are also useful for
monitoring the effectiveness of modulating agents on the expression
or activity of a CAT gene, such as in clinical trials or in a
treatment regimen. For example, the gene expression pattern of a
CAT gene can serve as a barometer for the continuing effectiveness
of treatment with a compound, particularly with compounds to which
a patient can develop resistance. The gene expression pattern can
also serve as a marker indicative of a physiological response of
the affected cells to the compound. For example, based on
monitoring nucleic acid expression, the administration of a
compound can be increased or alternative compounds to which the
patient has not become resistant can be administered instead.
Similarly, if the level of nucleic acid expression falls below a
desirable level, administration of the compound can be
commensurately decreased.
[0318] The nucleic acid molecules are also useful for testing an
individual for a genotype that, while not necessarily causing a
disease, nevertheless affects the treatment modality. Thus, the
nucleic acid molecules can be used to study the relationship
between an individual's genotype and the individual's response to a
compound used for treatment (pharmacogenomic relationship).
Accordingly, the nucleic acid molecules provided herein can be used
to assess the mutation content of a target gene in an individual in
order to select an appropriate compound or dosage regimen for
treatment. For example, target nucleic acid molecules having
genetic variations that affect treatment can provide diagnostic
targets that can be used to tailor treatment to an individual.
Accordingly, the production of recombinant cells and animals having
these genetic variations allows effective clinical design of
treatment compounds and dosage regimens, for example.
[0319] c. Methods of Treatment Using Nucleic Acids
[0320] Nucleic acid molecules of the invention are useful to design
antisense constructs to control CAT gene expression in cells,
tissues, and organisms. An antisense nucleic acid molecule
typically blocks translation of mRNA into CAT protein by
hybridizing to target mRNA in a sequence-specific manner. Nucleic
acid molecules of the invention can also be used to specifically
suppress gene expression by methods such as RNA interference
(RNAi). RNAi and antisense-based gene suppression are well known in
the art (e.g., Science 288:1370-1372, 2000). RNAi typically
operates on a post-transcriptional level and is sequence specific.
RNAi and antisense nucleic acid molecules are useful for treating
diseases, especially cancer. RNAi fragments, particularly
double-stranded (ds) RNAi, as well as antisense nucleic acid
molecules can also be used to generate loss-of-function phenotypes
by suppressing gene expression. Accordingly, exemplary embodiments
of the invention provide RNAi and antisense nucleic acid molecules,
and methods of using these RNAi and antisense nucleic acid
molecules, such as for therapy or for modulating cell function.
Nucleic acid molecules may also be produced that are complementary
to a region of a gene involved in transcription, such as to
hybridize to the gene to prevent transcription.
[0321] Exemplary embodiments of the invention relate to isolated
RNA molecules (double-stranded; single-stranded) that are about 17
to about 29 nucleotides (nt) in length, and more particularly about
21 to about 25 nt in length, which mediate RNAi (e.g., degradation
of mRNA, and such mRNA may be referred to herein as mRNA to be
degraded). With respect to RNAi, the terms RNA, RNA molecule(s),
RNA segment(s), and RNA fragment(s) are used interchangeably to
refer to RNA that mediates RNAi. These terms include
double-stranded RNA, single-stranded RNA, isolated RNA (e.g.,
partially purified RNA, essentially pure RNA, synthetic RNA,
recombinantly produced RNA), as well as altered RNA that differs
from naturally occurring RNA by the addition, deletion,
substitution, and/or alteration of one or more nucleotides. Such
alterations can include, for example, addition of non-nucleotide
material, such as to the end(s) of a 21-25 nt RNA or internally (at
one or more nucleotides of the RNA). Nucleotides in exemplary RNA
molecules of the invention can also comprise non-standard
nucleotides, including non-naturally occurring nucleotides or
deoxyribonucleotides. Collectively, all such altered RNAs are
referred to as analogs or analogs of naturally-occurring RNA. RNA
of 21-25 nt typically need only be sufficiently similar to natural
RNA that it has the ability to mediate RNAi. As used herein, the
phrase "mediates RNAi" refers to the ability to distinguish which
RNAs are to be degraded by RNAi processes. RNA that mediates RNAi
directs degradation of particular mRNAs by RNAi processes. Such RNA
may include RNAs of various structures, including short hairpin
RNA.
[0322] In certain exemplary embodiments, the invention relates to
RNA molecules of about 21 to about 25 nt that direct cleavage of
specific mRNA to which their sequence corresponds. It is not
necessary that there be a perfect correspondence (i.e., match) of
the sequences, but the correspondence must be sufficient to enable
the RNA to direct RNAi cleavage of the target mRNA (Holen et al.,
Nucleic Acids Res. 33:4704-4710 (2005)). In an exemplary
embodiment, the 21-25 nt RNA molecules of the invention comprise a
3' hydroxyl group.
[0323] Certain exemplary embodiments of the invention relate to
21-25 nt RNAs of specific genes, produced by chemical synthesis or
recombinant DNA techniques, that mediate RNAi. As used herein, the
term "isolated RNA" includes RNA obtained by any means, including
processing or cleavage of dsRNA, production by chemical synthetic
methods, and production by recombinant DNA techniques, for example.
Exemplary embodiments of the invention further relate to uses of
the 21-25 nt RNAs, such as for therapeutic or prophylactic
treatment and compositions comprising 21-25 nt RNAs that mediate
RNAi, such as pharmaceutical compositions comprising 21-25 nt RNAs
and an appropriate carrier.
[0324] Further exemplary embodiments of the invention relate to
methods of mediating RNAi of genes of a patient. For example, RNA
of about 21 to about 25 nt which targets a specific mRNA to be
degraded can be introduced into a patient's cells. The cells can be
maintained under conditions allowing degradation of the mRNA,
resulting in RNA-mediated interference of the mRNA of the gene in
the cells of the patient. Treatment of cancer patients, for
example, with RNAi may inhibit the growth and spread of the cancer
and reduce tumor size. Treatment of patients using RNAi can also be
in combination with other therapies. For example, RNAi can be used
in combination with other treatment modalities, such as
chemotherapy, radiation therapy, and other treatments. In an
exemplary embodiment, a chemotherapy agent is used in combination
with RNAi. In a further exemplary embodiment, GEMZAR (gemcitabine
HC1) chemotherapy is used with RNAi.
[0325] Treatment of certain diseases by RNAi may require
introduction of the RNA into the disease cells. RNA can be directly
introduced into a cell, or introduced extracellularly into a
cavity, interstitial space, into the circulation of a patient, or
introduced orally, for example. Physical methods of introducing
nucleic acids, such as injection directly into a cell or
extracellular injection into a patient, may also be used. RNA may
be introduced into vascular or extravascular circulation, the blood
or lymph system, or the cerebrospinal fluid, for example. RNA may
be introduced into an embryonic stem cell or another multipotent
cell, which may be derived from a patient. Physical methods of
introducing nucleic acids include injection of a solution
containing the RNA, bombardment by particles covered by the RNA,
soaking cells or tissue in a solution of the RNA, or
electroporation of cell membranes in the presence of the RNA. A
viral construct packaged into a viral particle may be used to
introduce an expression construct into a cell, with the construct
expressing the RNA. Other methods known in the art for introducing
nucleic acids to cells may be used, such as lipid-mediated carrier
transport, chemical-mediated transport, and the like. The RNA may
be introduced along with components that perform one or more of the
following activities: enhance RNA uptake by the cell, promote
annealing of the duplex strands, stabilize the annealed strands, or
otherwise increase inhibition of the target gene.
[0326] Exemplary RNA of the invention can be used alone or as a
component of a kit having at least one reagent for carrying out in
vitro or in vivo introduction of the RNA to a cell, tissue/fluid,
or patient. Exemplary components of a kit include dsRNA and a
vehicle that promotes introduction of the dsRNA. A kit may also
include instructions for using the kit.
[0327] Certain exemplary embodiments of the invention provide
compositions and methods for cleavage of mRNA by ribozymes having
nucleotide sequences complementary to one or more regions in the
mRNA, thereby attenuating the translation of the mRNA. Examples of
regions in mRNA that can be targeted by ribozymes include coding
regions, particularly coding regions corresponding to catalytic or
other functional activities of a target protein, such as substrate
binding. These compositions and methods may be used to treat a
disorder characterized by abnormal or undesired target nucleic acid
expression.
[0328] In certain exemplary embodiments, nucleic acid molecules of
the invention may be used for gene therapy in individuals having
cells that are aberrant in gene expression of a target. For
example, recombinant cells that have been engineered ex vivo (which
can include an individual's own cells) can be introduced into an
individual where the cells produce the desired target protein to
thereby treat the individual.
[0329] d. Methods of Screening Using Nucleic Acids
[0330] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate CAT nucleic acid
expression.
[0331] Exemplary embodiments of the invention thus provide methods
for identifying a compound that can be used to treat a disease
associated with differential expression of a CAT gene, especially
cancer. Exemplary methods can typically include assaying the
ability of a compound to modulate the expression of a target
nucleic acid to thereby identify a compound that can be used to
treat a disorder characterized by undesired target nucleic acid
expression. The assays can be performed in cell-based or cell-free
systems. Examples of cell-based assays include cells naturally
expressing target nucleic acid or recombinant cells genetically
engineered to express specific target nucleic acid sequences.
[0332] Assays for target nucleic acid expression can involve direct
assay of target nucleic acid levels, such as mRNA levels, or on
collateral compounds involved in a signal pathway. Further, the
expression of genes that are up- or down-regulated in response to a
signal pathway can also be assayed. In these embodiments, the
regulatory regions of these genes can be operably linked to a
reporter gene such as luciferase.
[0333] Thus, in exemplary embodiments, modulators of gene
expression of a target can be identified in methods wherein a cell
is contacted with a candidate agent and the expression of target
mRNA determined. The level of expression of target mRNA in the
presence of the candidate agent is compared to the level of
expression of target mRNA in the absence of the candidate agent.
The candidate agent can then be identified as a modulator of target
nucleic acid expression based on this comparison and may be used,
for example, to treat a disorder characterized by aberrant target
nucleic acid expression. When expression of target mRNA is
statistically significantly greater in the presence of the
candidate agent than in its absence, the candidate agent is
identified as a stimulator (agonist) of nucleic acid expression.
When nucleic acid expression is statistically significantly less in
the presence of the candidate agent than in its absence, the
candidate compound is identified as an inhibitor (antagonist) of
nucleic acid expression.
[0334] 11. Arrays and Expression Analysis
[0335] "Array" (interchangeably referred to as "microarray")
typically refers to an arrangement of at least one, but more
typically at least two, nucleic acid molecules, proteins, or
antibodies on a substrate. In certain exemplary arrangements, at
least one of the nucleic acid molecules, proteins, or antibodies
typically represents a control or standard, and other nucleic acid
molecules, proteins, or antibodies are of diagnostic or therapeutic
interest. In exemplary embodiments, the arrangement of nucleic acid
molecules, proteins, or antibodies on the substrate is such that
the size and signal intensity of each labeled complex (e.g., formed
between each nucleic acid molecule and a complementary nucleic
acid, or between each protein and a ligand or antibody, or between
each antibody and a protein to which the antibody specifically
binds) is individually distinguishable.
[0336] An "expression profile" is a representation of target
expression in a sample. A nucleic acid expression profile can be
produced using, for example, arrays, sequencing, hybridization, or
amplification technologies for nucleic acids from a sample. A
protein expression profile can be produced using, for example,
arrays, gel electrophoresis, mass spectrometry, or antibodies (and,
optionally, labeling moieties) which specifically bind proteins.
Nucleic acids, proteins, or antibodies can be attached to a
substrate or provided in solution, and their detection can be based
on methods well known in the art.
[0337] A substrate includes, but is not limited to, glass, paper,
nylon or other type of membrane, filter, chip, metal, or any other
suitable solid or semi-solid (e.g., gel) support.
[0338] Exemplary arrays can be prepared and used according to the
methods described in U.S. Pat. No. 5,837,832; PCT application
WO95/11995; Lockhart et al., 1996, Nat. Biotech. 14: 1675-1680;
Schena et al., 1996; Proc. Natl. Acad. Sci. 93: 10614-10619; and
U.S. Pat. No. 5,807,522. Exemplary embodiments of the invention
also provide antibody arrays (see, e.g., de Wildt et al. (2000)
Nat. Biotechnol. 18:989-94).
[0339] Certain exemplary embodiments of the invention provide a
nucleic acid array for assaying target expression, which can be
composed of single-stranded nucleic acid molecules, usually either
synthetic antisense oligonucleotides or fragments of cDNAs, fixed
to a solid support. The oligonucleotides can be, for example, about
6-60 nucleotides in length, about 15-30 nucleotides in length, or
about 20-25 nucleotides in length.
[0340] To produce oligonucleotides to a target nucleic acid
molecule for an array, the target nucleic acid molecule of interest
is typically examined using a computer algorithm to identify
oligonucleotides of defined length that are unique to the nucleic
acid molecule, have a GC content within a range suitable for
hybridization, and lack predicted secondary structure that may
interfere with hybridization. In certain instances, it may be
desirable to use pairs of oligonucleotides on an array. In
exemplary embodiments, the "pairs" can be identical, except for one
nucleotide (which can be located in the center of the sequence, for
example). The second oligonucleotide in the pair (mismatched by
one) serves as a control. Any number of oligonucleotide pairs may
be utilized.
[0341] Oligonucleotides can be synthesized on the surface of a
substrate, such as by using a light-directed chemical process or by
using a chemical coupling procedure and an ink jet application
apparatus (e.g., PCT application W095/251116).
[0342] In some exemplary embodiments, an array can be used to
diagnose or monitor the progression of disease, for example, by
assaying target expression.
[0343] For example, an oligonucleotide probe specific for a target
can be labeled by standard methods and added to a biological sample
from a patient under conditions that allow for the formation of
hybridization complexes. After an incubation period, the sample can
be washed and the amount of label (or signal) associated with
hybridization complexes can be quantified and compared with a
standard value. If complex formation in the patient sample is
significantly altered (higher or lower) in comparison to a normal
(e.g., healthy) standard, or is similar to a disease standard, this
differential expression can be diagnostic of a disorder.
[0344] By analyzing changes in patterns of target expression,
disease may be diagnosed at earlier stages before a patient is
symptomatic. In exemplary embodiments of the invention, arrays or
target expression analysis methods can be used to formulate a
diagnosis or prognosis, to design a treatment regimen, and/or to
monitor the efficacy of treatment. For example, a treatment dosage
can be established that causes a change in target expression
patterns indicative of successful treatment, and target expression
patterns associated with the onset of undesirable side effects can
be avoided. In further exemplary embodiments, assays of target
expression can be repeated on a regular basis to determine if the
level of target expression in a patient begins to approximate that
which is observed in a normal subject. The results obtained from
successive assays may be used to show the efficacy of treatment
over a period ranging from several days to years, for example.
[0345] Exemplary arrays of the invention can also be used to screen
candidate agents, such as to identify agents that produce a target
expression profile similar to that caused by known therapeutic
agents, with the expectation that agents that cause a similar
expression profile of a target may have similar therapeutic effects
and/or modes of action on the target.
EXAMPLES
[0346] Exemplary embodiments of the invention are further described
in the following examples, which do not limit the scope of the
invention.
[0347] 1. Tissue Samples and Cell Lines
[0348] Tissue Processing and Preparation of Single Cell Suspensions
from Tissue
[0349] Tissue samples (e.g., normal tissues or disease tissues such
as surgically resected neoplastic or metastatic lesions) can be
procured from clinical sites and transported in transport buffer.
Tissues can be collected as remnant tissues following surgical
resection of cancer (or other disease) tissues. Remnant tissues are
supplied following processing for pathological diagnosis according
to proper standards of patient care. Normal tissue specimens can be
normal tissue adjacent to tumors (or other disease tissue) that is
collected during tumor resection. Normal tissue from healthy
patients not having cancer (or other disease of interest) can also
be included, such as to reduce the contribution from pre-neoplastic
changes that may exist in normal adjacent tissue. Procurement of
tissue samples is carried out in an anonymous manner in compliance
with federally mandated ethical and legal guidelines (HIPAA) and in
accordance with clinical institution ethical review board and
internal institutional review board guidelines.
[0350] Tissue can be crudely minced and incubated for 20-30 minutes
with periodic agitation at 37.degree. C. in Enzyme Combination #1
(200 units collagenase, cat #C5894 Sigma; 126 .mu.g DNAse I, cat
#D4513 Sigma (in 10 mM Tris/HCl pH7.5); 50 mM NaCl; 10 mM MgCl2;
0.05% elastase, cat #E7885 Sigma) (additionally, hyaluronidase
enzyme may also be utilized). D-PBS is added at 3.times. the volume
of the enzyme combination, the tissue finely minced, and
disassociated cells passed through a 200 .mu.m filter. The cells
are washed twice with D-PBS. Red blood cells are lysed with
PharMLyse (BD Biosciences) when necessary. Cell number and
viability are determined by PI exclusion (GUAVA). Cells at a total
cell number greater than 20.times.10.sup.6 are sorted using a
high-speed sorter (MoFlo Cytomation) for epithelial cells (EpCAM
positive).
[0351] The remaining undigested tissue is incubated for 20-30
minutes with periodic agitation at 37.degree. C. in Enzyme
Combination #2 (1.times. Liberase Blendzyme 1, cat #988-417 Roche;
1.times. Liberase Blendzyme 3, cat #814-184 Roche; 0.05% elastase,
cat #E7885 Sigma). D-PBS is added at 3.times. the volume of the
enzyme combination, and the tissue finely minced until tissue is
completely disassociated. The cells are passed through a 200 .mu.m
filter, washed twice with D-PBS, and pooled with cells from the
Enzyme Combination #1 digestion.
[0352] Cells are passed through a 70 .mu.m filter for single cell
suspension, and cell number and viability are determined by PI
exclusion (GUAVA). When needed, red blood cells are lysed with
PharMLyse (BD Biosciences). Cells are incubated in 20 ml of
1.times. PharMLyse in D-PBS for 30 seconds with gentle agitation
and cells pelleted at 300.times.g for 5 minutes at 4.degree. C.
Cells are washed once in D-PBS and cell number and viability are
recalculated by PI exclusion using the GUAVA. Cells at a total cell
number greater than 20.times.10.sup.6 are sorted using a high-speed
sorter (MoFlo Cytomation) for epithelial cells (EpCAM
positive).
[0353] Single cell suspensions can also be prepared from tissue
samples as follows: specimens are washed in DTT for 15 min,
digested with Dispase (30-60 min), then filtered twice (380
.mu.m/74 .mu.m) before red blood cells are removed through addition
of ACK lysis buffer. Epithelial (EpCAM) and leukocyte (CD45)
content and cellular viability (PI exclusion) can be determined
through flow cytometry analysis (LSR I, BD Biosciences, San Jose,
Calif.).
[0354] The epithelial content of both disease and normal specimens
can be enriched through depletion of immune CD45-positive cells by
flow cytometry or purification of Epithelial Cell Surface Antigen
(ECSA/EpCam)-positive cells by bead capture.
[0355] Bead capture of epithelial cells can be performed using a
Dynal CELLection Epithelial Enrich kit (Invitrogen, Carlsbad, CA)
as follows. Dynal CELLection beads at a concentration of
2.times.10.sup.8 beads are incubated with 1.times.10.sup.8 cells in
HBSS with 10% fetal calf serum for 30 minutes at 4.degree. C. Cells
and beads are placed in a magnet system Dynal MPC for 2 minutes.
Bead/cell complexes are washed in RPMI 1640 media with 1% fetal
calf serum. Cells are released from the bead complex with 15 minute
incubation with DNase with agitation in RPMI with 1% fetal calf
serum.
[0356] DynalBead cell depletion of CD45 cells can be carried out as
follows. DynalBead M-450 CD45 beads and cells are incubated at a
concentration of 250 .mu.l beads per 2.times.10.sup.7 cells for 30
minutes at 4.degree. C. Bead/cell complexes are washed in DPBS
buffer with 2% fetal bovine serum. Cells and beads are placed in a
magnet system Dynal MPC for 2 minutes. The supernatant contains
EpCAM enriched cells.
[0357] Cell Line Culture
[0358] Cell lines can be obtained from the American Type Culture
Collection (ATCC, Manassas, Va.). Cell lines can be grown in a
culturing medium that is supplemented as necessary with growth
factors and serum, in accordance with the ATCC guidelines for each
particular cell line. Cultures are established from frozen stocks
in which the cells are suspended in a freezing medium (cell culture
medium with 10% DMSO [v/v]) and flash frozen in liquid nitrogen.
Frozen stocks prepared in this way are stored in liquid nitrogen
vapor. Cell cultures are established by rapidly thawing frozen
stocks at 37.degree. C. Thawed stock cultures are slowly
transferred to a culture vessel containing a large volume of
supplemented culture medium. For maintenance of culture, cells are
seeded at 1.times.10.sup.5 cells/per ml in medium and incubated at
37.degree. C. until confluence of cells in the culture vessel
exceeds 50% by area. At this time, cells are harvested from the
culture vessel using enzymes or EDTA where necessary. The density
of harvested, viable cells is estimated by hemocytometry and the
culture reseeded as above. A passage of this nature is repeated no
more than 25 times, at which point the culture is destroyed and
reestablished from frozen stocks as described above.
[0359] Alternatively, cells (e.g., adipocytes such as
differentiated subcutaneous or visceral adipocytes) can be obtained
from commercial sources, which may provide the cells seeded into
T-75 tissue culture flasks. Upon arrival in the laboratory, the
media is removed and replaced with DMEM/Ham's F-12 medium (1:1 v/v)
supplemented with HEPES pH 7.4, FBS, biotin, pantothenate, human
insulin, dexamethasone, penicillin-streptomycin, and Amphotercin B.
The cells are cultured for two days and then harvested with versene
before enrichment of proteins.
[0360] Alternatively, for secreted protein analysis, cells can be
grown under routine tissue culture conditions in 490 cm.sup.2
roller bottles at an initial seeding density of approximately 15
million cells per roller bottle. When the cells reach .about.70-80%
confluence, the culturing media is removed, the cells are washed 3
times with D-PBS and once with CD293 protein-free media (Invitrogen
cat #11913-019), and the culturing media is replaced with CD293 for
generating conditioned media. Cells are incubated for 72 hours in
CD293 and the media is collected for analysis, such as mass
spectrometry analysis of secreted proteins (30-300 ml). Cell debris
is removed from the conditioned media by centrifugation at 300 g
for 5 minutes and filtering through a 0.2 micron filter prior to
analysis.
[0361] Alternatively, for secreted protein analysis, conditioned
media collected from differentiated cells (e.g., visceral or
subcutaneous adipocytes), can be obtained (e.g., from a commercial
source). Conditioned medium is shipped on dry ice and maintained at
-80.degree. C. ahead of protein capture. Cells are isolated from
tissue and expanded to passage 2 (P2) to passage 4 (P4) prior to
differentiation. Media is changed and cells are grown in
conditioned medium for three days prior to harvesting. Enriching
for proteins such as secreted proteins can then be carried out.
[0362] 2. Cloning and Expression of Target Proteins
[0363] cDNA Retrieval
[0364] Peptide sequences can be searched using the BLAST algorithm
against relevant protein sequence databases to identify the
corresponding full-length protein (reference sequence). Each
full-length protein sequence can then be searched using the BLAST
algorithm against a human cDNA clone collection. For each sequence
of interest, clones can be pulled and streaked onto LB/Ampicillin
(100 .mu.g/ml) plates. Plasmid DNA is isolated using Qiagen spin
mini-prep kit and verified by restriction digest. Subsequently, the
isolated plasmid DNA is sequence verified against the reference
full-length protein sequence. Sequencing reactions are carried out
using Applied Biosystems BigDye Terminator kit followed by ethanol
precipitation. Sequence data is collected using the Applied
Biosystems 3700 Genetic Analyzer and analyzed by alignment to the
reference full-length protein sequence using the Clone Manager
alignment tool.
[0365] PCR
[0366] PCR primers are designed to amplify the region encoding the
full-length protein and/or any regions of the protein that are of
interest for expression (e.g., antigenic or hydrophilic regions as
determined by the Clone Manager sequence analysis tool). Primers
also contain 5' and 3' overhangs to facilitate cloning (see below).
PCR reactions contain 2.5 units Platinum Taq DNA Polymerase High
Fidelity (Invitrogen), 50 ng cDNA plasmid template, 1 .mu.M forward
and reverse primers, 800 .mu.M dNTP cocktail (Applied Biosystems),
and 2 mM MgSO.sub.4. After 20-30 cycles (94.degree. C. for 30
seconds, 55.degree. C. for 1 minute, and 73.degree. C. for 2
minutes), the resulting product is verified by sequence analysis
and quantitated by agarose gel electrophoresis.
[0367] Construction of Entry Clones
[0368] PCR products are cloned into an entry vector for use with
the Gateway recombination based cloning system (Invitrogen). These
vectors include pDonr221, pDonr201, pEntr/D-TOPO, or
pEntr/SD/D-TOPO and are used as described in the cloning methods
below.
[0369] TOPO Cloning into pEntr/D-TOPO or pEntr/SD/D-TOPO
[0370] For cloning using this method, the forward PCR primer
contains a 5' overhang containing the sequence "CACC". PCR products
are generated as described above and cloned into the entry vector
using the Invitrogen TOPO.RTM. cloning kit. Reactions are typically
carried out at room temperature for 10 minutes and subsequently
transformed into TOP10 chemically competent cells (Invitrogen, CA).
Candidate clones are picked, and plasmid DNA is prepared using a
Qiagen spin mini-prep kit and screened by restriction enzyme
digestion. Inserts are subsequently sequence-verified as described
above.
[0371] Gateway Cloning into pDonr201 or pDonr221
[0372] For cloning using this method, PCR primers contain forward
and reverse 5' overhangs. PCR products are generated as described
above. Protein-encoding nucleic acid molecules are recombined into
the entry vector using the Invitrogen Gateway BP Clonase enzyme
mix. Reactions are typically carried out at 25.degree. C. for 1
hour, treated with Proteinase K at 37.degree. C. for 10 minutes,
and transformed into Library Efficiency DH5.alpha. chemically
competent cells (Invitrogen, CA). Candidate clones are picked,
plasmid DNA is prepared using a Qiagen spin mini-prep kit, and
screened by restriction enzyme digestion. Inserts are subsequently
sequence-verified as described above.
[0373] Construction of Expression Clones
[0374] Protein-encoding nucleic acid molecules are transferred from
the entry construct into a series of expression vectors using the
Gateway LR Clonase enzyme mix. Reactions are typically carried out
for 1 hour at 25.degree. C., treated with Proteinase K at
37.degree. C. for 10 minutes, and subsequently transformed into
Library Efficiency DH5a chemically competent cells (Invitrogen).
Candidate clones are picked, plasmid DNA is prepared using a Qiagen
spin mini-prep kit, and screened by restriction enzyme digestion.
Expression vectors include, but are not limited to, pDest14,
pDest15, pDest17, pDest8, pDest10 and pDest20. These vectors allow
expression in systems such as E. coli and recombinant baculovirus.
Other vectors not listed here allow expression in yeast, mammalian
cells, or in vitro.
[0375] Expression of Recombinant Proteins in E. coli
[0376] Constructs are transformed into one or more of the following
host strains: BL21 SI, BL21 AI, (Invitrogen), Origami B (DE3),
Origami B (DE3) pLysS, Rosetta (DE3), Rosetta (DE3) pLysS,
Rosetta-Gami (DE3), Rosetta-Gami (DE3) pLysS, or Rosetta-Gami B
(DE3) pLysS (Novagen). The transformants are grown in LB with or
without NaCl and with appropriate antibiotics, at temperatures in
the range of 20-37.degree. C., with aeration. Expression is induced
with the addition of IPTG (0.03-0.30 mM) or NaCl (75-300 mM) when
the cells are in mid-log growth. Growth is continued for one to 24
hours post-induction. Cells are harvested by centrifugation in a
Sorvall RC-3C centrifuge in a H6000A rotor for 10 minutes at 3000
rpm at 4.degree. C. Cell pellets are stored at -80.degree. C.
[0377] Expression of Recombinant Proteins using Baculovirus
[0378] Recombinant proteins are expressed using baculovirus in Sf21
fall army worm ovarian cells. Recombinant baculoviruses are
prepared using the Bac-to-Bac system (Invitrogen) per the
manufacturer's instructions. Proteins are expressed on the large
scale in Sf900II serum-free medium (Invitrogen) in a 10 L
bioreactor tank (27.degree. C., 130 rpm, 50% dissolved oxygen for
48 hours).
[0379] 3. Recombinant Protein Purification
[0380] Recombinant proteins can be purified from E. coli and/or
insect cells using a variety of standard chromatography methods.
Briefly, cells are lysed using sonication or detergents. The
insoluble material is pelleted by centrifugation at 10,000.times.g
for 15 minutes. The supernatant is applied to an appropriate
affinity column. For example, His-tagged proteins are separated
using a pre-packed chelating sepharose column (Pharmacia) or
GST-tagged proteins are separated using a glutathione sepharose
column (Pharmacia). After using the affinity column, proteins are
further separated using various techniques, such as ion exchange
chromatography (columns from Pharmacia) to separate on the basis of
electrical charge or size exclusion chromatography (columns from
Tosohaas) to separate on the basis of molecular weight, size, and
shape.
[0381] Expression and purification of the protein can also be
achieved using either a mammalian cell expression system or an
insect cell expression system. The pUB6/V5-His vector system
(Invitrogen, CA) can be used to express cDNA in CHO cells. The
vector contains the selectable bsd gene, multiple cloning sites,
the promoter/enhancer sequence from the human ubiquitin C gene, a
C-terminal V5 epitope for antibody detection with anti-V5
antibodies, and a C-terminal polyhistidine (6.times. His) sequence
for rapid purification on PROBOND resin (Invitrogen, CA).
Transformed cells are selected on media containing blasticidin.
[0382] Spodoptera frugiperda (Sf9) insect cells are infected with
recombinant Autographica californica nuclear polyhedrosis virus
(baculovirus). The polyhedrin gene is replaced with the cDNA by
homologous recombination and the polyhedrin promoter drives cDNA
transcription. The protein is synthesized as a fusion protein with
6.times. His which enables purification as described above.
Purified proteins can be used to produce antibodies.
[0383] 4. Chemical Synthesis of Proteins
[0384] Proteins or portions thereof can be produced not only by
recombinant methods (such as described above), but also by using
chemical methods well known in the art. Solid phase peptide
synthesis can be carried out in a batchwise or continuous flow
process which sequentially adds .alpha.-amino- and side
chain-protected amino acid residues to an insoluble polymeric
support via a linker group. A linker group such as
methylamine-derivatized polyethylene glycol is attached to
poly(styrene-co-divinylbenzene) to form the support resin. The
amino acid residues are N-a-protected by acid labile Boc
(t-butyloxycarbonyl) or base-labile Fmoc
(9-fluorenylmethoxycarbonyl) groups. The carboxyl group of the
protected amino acid is coupled to the amine of the linker group to
anchor the residue to the solid phase support resin.
Trifluoroacetic acid or piperidine are used to remove the
protecting group in the case of Boc or Fmoc, respectively. Each
additional amino acid is added to the anchored residue using a
coupling agent or pre-activated amino acid derivative, and the
resin is washed. The full-length peptide is synthesized by
sequential deprotection, coupling of derivitized amino acids, and
washing with dichloromethane and/or N,N-dimethylformamide. The
peptide is cleaved between the peptide carboxy terminus and the
linker group to yield a peptide acid or amide. (Novabiochem 1997/98
Catalog and Peptide Synthesis Handbook, San Diego Calif. pp.
S1-S20).
[0385] Automated synthesis can also be carried out on machines such
as the 431A peptide synthesizer (Applied Bio systems, Foster City,
Calif.). A protein or portion thereof can be purified by
preparative high performance liquid chromatography and its
composition confirmed by amino acid analysis or by sequencing
(Creighton, 1984, Proteins, Structures and Molecular Properties, W
H Freeman, New York N.Y.).
[0386] 5. Antibody Production
[0387] Polyclonal Antibodies
[0388] Polyclonal antibodies against recombinant proteins can be
raised in rabbits (Green Mountain Antibodies, Burlington, Vt.).
Briefly, two New Zealand rabbits are immunized with 0.1 mg of
antigen in complete Freund's adjuvant. Subsequent immunizations are
carried out using 0.05 mg of antigen in incomplete Freund's
adjuvant at days 14, 21, and 49. Bleeds are collected and screened
for recognition of the antigen by solid phase ELISA and Western
blot analysis. The IgG fraction is separated by centrifugation at
20,000.times.g for 20 minutes followed by a 50% ammonium sulfate
cut. The pelleted protein is resuspended in 5 mM Tris and separated
by ion exchange chromatography. Fractions are pooled based on IgG
content. Antigen-specific antibody is affinity purified using
Pierce AminoLink resin coupled to the appropriate antigen.
[0389] Isolation of Antibody Fragments Directed Against a Protein
Target from a Library of scFvs
[0390] Naturally occurring V-genes isolated from human PBLs can be
constructed into a library of antibody fragments which contain
reactivities against a target protein to which the donor may or may
not have been exposed (see, for example, U.S. Pat. No. 5,885,793,
incorporated herein by reference in its entirety).
[0391] Rescue of the library: A library of scFvs is constructed
from the RNA of human PBLs, as described in PCT publication WO
92/01047. To rescue phage displaying antibody fragments,
approximately 10.sup.9 E. coli harboring the phagemid are used to
inoculate 50 ml of 2.times. TY containing 1% glucose and 100
.mu.g/ml of ampicillin (2.times. TY-AMP-GLU) and grown to an O.D.
of 0.8 with shaking. Five ml of this culture is used to innoculate
50 ml of 2.times. TY-AMP-GLU, 2.times.10.sup.8 TU of delta gene 3
helper (M13 delta gene III, see PCT publication WO 92/01047) are
added and the culture incubated at 37.degree. C. for 45 minutes
without shaking and then at 37.degree. C. for 45 minutes with
shaking. The culture is centrifuged at 4000 rpm. for 10 min. and
the pellet resuspended in 2 liters of 2.times. TY containing 100
.mu.g/ml ampicillin and 50 .mu.g/mlkanamycin and grown overnight.
Phage are prepared as described in PCT publication WO 92/01047.
[0392] Preparation of M13 delta gene III: M13 delta gene III helper
phage does not encode gene III protein, hence the phage(mid)
displaying antibody fragments have a greater avidity of binding to
antigen. Infectious M13 delta gene III particles are made by
growing the helper phage in cells harboring a pUC19 derivative
supplying the wild type gene III protein during phage
morphogenesis. The culture is incubated for 1 hour at 37.degree. C.
without shaking and then for a further hour at 37.degree. C. with
shaking. Cells are spun down (IEC-Centra 8,400 rpm for 10 min),
resuspended in 300 ml 2.times. TY broth containing 100 .infin.g
ampicillin/ml and 25 .mu.g kanamycin/ml (2.times. TY-AMP-KAN) and
grown overnight, shaking at 37.degree. C. Phage particles are
purified and concentrated from the culture medium by two
PEG-precipitations (Sambrook et al., 2001, Molecular Cloning: A
Laboratory Manual. 3rd. ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY), resuspended in 2 ml PBS and passed through
a 0.45 .mu.m filter (Minisart NML; Sartorius) to give a final
concentration of approximately 10.sup.13 transducing units/ml
(ampicillin-resistant clones).
[0393] Panning of the library: Immunotubes (Nunc) are coated
overnight in PBS with 4 ml of either 100 .mu.g/ml or 10 .mu.g/ml of
a protein target of interest. Tubes are blocked with 2% Marvel-PBS
for 2 hours at 37.degree. C. and then washed 3 times in PBS.
Approximately 10.sup.13 TU of phage is applied to the tube and
incubated for 30 minutes at room temperature tumbling on an
over-and-under turntable and then left to stand for another 1.5
hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and 10
times with PBS. Phage are eluted by adding 1 ml of 100 mM
triethylamine and rotating 15 minutes on an under-and-over
turntable after which the solution is immediately neutralized with
0.5 ml of 1.0 M Tris-HCl, pH 7.4. Phages are then used to infect 10
ml of mid-log E. coli TG1 by incubating eluted phage with bacteria
for 30 minutes at 37.degree. C. The E. coli are then plated on TYE
plates containing 1% glucose and 100 .mu.g/ml ampicillin. The
resulting bacterial library is then rescued with delta gene 3
helper phage as described above to prepare phage for a subsequent
round of selection. This process is then repeated for a total of 4
rounds of affinity purification with tube-washing increased to 20
times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3
and 4.
[0394] Characterization of binders: Eluted phage from the 3rd and
4th rounds of selection are used to infect E. coli HB 2151 and
soluble scFv is produced (Marks et al., 1991, J. Mol. Biol. 222:
581-597) from single colonies for assay. ELISAs are performed with
microtitre plates coated with either 10 .mu.g/ml of the protein
target of interest in 50 mM bicarbonate pH 9.6. Clones positive in
ELISA are further characterized by PCR fingerprinting (see, e.g.,
PCT publication WO 92/01047) and then by sequence analysis.
[0395] Monoclonal Antibodies
[0396] a) Materials:
[0397] 1. Complete Media No Sera (CMNS) for washing of the myeloma
and spleen cells; Hybridoma medium CM-HAT (Cell Mab (BD), 10% FBS
(or HS); 5% Origen HCF (hybridoma cloning factor) containing 4 mM
L-glutamine and antibiotics) to be used for plating hybridomas
after the fusion.
[0398] 2. Hybridoma medium CM-HT (no aminopterin) (Cell Mab (BD),
10% FBS 5% Origen HCF containing 4 mM L-glutamine and antibiotics)
to be used for fusion maintenance is stored in the refrigerator at
4-6.degree. C. The fusions are fed on days 4, 8, and 12, and
subsequent passages. Inactivated and pre-filtered commercial fetal
bovine serum (FBS) or horse serum (HS) are thawed and stored in the
refrigerator at 4.degree. C. and is pretested for myeloma growth
from single cells prior to use.
[0399] 3. The L-glutamine (200 mM, 100.times. solution), which is
stored at -20.degree. C., is thawed and warmed until completely in
solution. The L-glutamine is dispensed into media to supplement
growth. L-glutamine is added to 2 mM for myelomas and 4 mM for
hybridoma media. Further, the penicillin, streptomycin,
amphotericin (antibacterial-antifungal stored at -20.degree. C.) is
thawed and added to Cell Mab Media to 1%.
[0400] 4. Myeloma growth media is Cell Mab Media (Cell Mab Media,
Quantum Yield, from BD, which is stored in the refrigerator at
4.degree. C. in the dark), to which is added L-glutamine to 2 mM
and antibiotic/antimycotic solution to 1% and is called CMNS.
[0401] 5. One bottle of PEG 1500 in Hepes (Roche, N.J.) is
prepared.
[0402] 6. 8-Azaguanine is stored as the dried powder supplied by
SIGMA at -700.degree. C. until needed. One vial/500 ml of media is
reconstituted and the entire contents are added to 500 ml media
(e.g., 2 vials/liter).
[0403] 7. Myeloma Media is CM which has 10% FBS (or HS) and 8-Aza
(1.times.) stored in the refrigerator at 4.degree. C.
[0404] 8. Clonal cell medium D (Stemcell, Vancouver) contains HAT
and methyl cellulose for semi-solid direct cloning from the fusion.
This comes in 90 ml bottles with a CoA and is melted at 37.degree.
C. in a waterbath in the morning of the day of the fusion. The cap
is loosened and the bottle is left in a CO.sub.2 incubator to
sufficiently gas the medium D and bring the pH down.
[0405] 9. Hybridoma supplements HT [hypoxanthine, thymidine] to be
used in medium for the section of hybridomas and maintenace of
hybridomas through the cloning stages, respectively.
[0406] 10. Origen HCF can be obtained directly from Igen and is a
cell supernatant produced from a macrophage-like cell-line. It can
be thawed and aliqouted to 15 ml tubes at 5 ml per tube and stored
frozen at -20.degree. C. Positive hybridomas are fed HCF through
the first subcloning and are gradually weaned (individual
hybridomas can continue to be supplemented, as needed). This and
other additives are typically more effective in promoting new
hybridoma growth than conventional feeder layers.
[0407] b) Procedure:
[0408] To generate monoclonal antibodies, mice are immunized with
5-50 .mu.g of antigen, either intra-peritoneally (i.p.) or by
intravenous injection in the tail vein (i.v.). The antigen used can
be a recombinant target protein of interest, for example. The
primary immunization takes place two months prior to the harvesting
of splenocytes from the mouse, and the immunization is typically
boosted by i.v. injection of 5-50 .mu.g of antigen every two weeks.
At least one week prior to the expected fusion date, a fresh vial
of myeloma cells is thawed and cultured. Several flasks of
different densities can be maintained so that a culture at the
optimum density is ensured at the time of fusion. An optimum
density can be 3-6.times.10.sup.5 cells/ml, for example. 2-5 days
before the scheduled fusion, a final immunization of approximately
5 .mu.g of antigen in PBS is administered (either i.p. or i.v).
[0409] Myeloma cells are washed with 30 ml serum free media by
centrifugation at 500 g at 4.degree. C. for 5 minutes. Viable cell
density is determined in resuspended cells using hemocytometry and
vital stains. Cells resuspended in complete growth medium are
stored at 37.degree. C. during the preparation of splenocytes.
Meanwhile, to test aminopterin sensitivity, 1.times.10.sup.6
myeloma cells are transferred to a 15 ml conical tube and
centrifuged at 500 g at 4.degree. C. for 5 minutes. The resulting
pellet is resuspended in 15 ml of HAT media and cells plated at 2
drops/well on a 96-well plate.
[0410] To prepare splenocytes from immunized mice, the animals are
euthanised and submerged in 70% ethanol. Under sterile conditions,
the spleen is surgically removed and placed in 10 ml of RPMI medium
supplemented with 20% fetal calf serum in a petri dish. Cells are
extricated from the spleen by infusing the organ with medium >50
times using a 21 g syringe.
[0411] Cells are harvested and washed by centrifugation (at 500 g
at 4.degree. C. for 5 minutes) with 30 ml of medium. Cells are
resuspended in 10 ml of medium and the density of viable cells
determined by hemocytometry using vital stains. The splenocytes are
mixed with myeloma cells at a ratio of 5:1 (spleen cells: myeloma
cells). Both the myeloma and spleen cells are washed twice more
with 30 ml of RPMI-CMNS, and the cells are spun at 800 rpm for 12
minutes.
[0412] Supernatant is removed and cells are resuspended in 5 ml of
RPMI-CMNS and are pooled to fill volume to 30 ml and spun down as
before. Then, the pellet is broken up by gently tapping on the flow
hood surface and resuspending in 1 ml of BMB REG1500 (prewarmed to
37.degree. C.) dropwise with a lcc needle over 1 minute.
[0413] RPMI-CMNS to the PEG cells and RPMI-CMNS are added to slowly
dilute out the PEG. Cells are centrifuged and diluted in 5 ml of
Complete media and 95 ml of Clonacell Medium D (HAT) media (with 5
ml of HCF). The cells are plated out 10 ml per small petri
plate.
[0414] Myeloma/HAT control is prepared as follows: dilute about
1000 P3X63 Ag8.653 myeloma cells into 1 ml of medium D and transfer
into a single well of a 24-well plate. Plates are placed in an
incubator, with two plates inside of a large petri plate, with an
additional petri plate full of distilled water, for 10-18 days
under 5% CO.sub.2 overlay at 37.degree. C. Clones are picked from
semisolid agarose into 96-well plates containing 150-200 .mu.l of
CM-HT. Supernatants are screened 4 days later in ELISA, and
positive clones are moved up to 24-well plates. Heavy growth
requires changing of the media at day 8 (+/-150 ml). The HCF can be
further decreased to 0.5% (gradually -2%, then 1%, then 0.5%) in
the cloning plates.
[0415] 6. Liquid Chromatography and Mass Spectrometry (LC/MS)
[0416] For LC/MS analysis, proteins are reduced in 2.5 mM DTT for 1
hour at 37.degree. C., and alkylated with ICAT.TM. reagent
according to the procedures recommended by the manufacturer
(Applied Biosystems, Framingham, Mass.). The reaction is quenched
by adding excess DTT. Proteins are digested using sequencing grade
modified trypsin overnight at 37.degree. C. followed by desalting
using 3 cc Oasis HLB solid phase extraction columns (Waters,
Milford, MA) and vacuum drying. Cysteine-containing peptides are
purified by avidin column (Applied Biosystems, Framingham, Mass.).
The peptides are reconstituted in buffer A (0.1% formic acid in
water) and separated over a C18 monomeric column (150 mm, 150 .mu.m
i.d., Grace Vydac 238EV5, 5 .mu.m) at a flow rate of 1.5 .mu.l/min
with a trap column. Peptides are eluted from the column using a
gradient, 3%-30% buffer B (0.1% formic acid in 90% acetonitrile) in
215 min, 30%-90% buffer B in 30 min. Eluted peptides are analyzed
using an online QSTAR XL system (MDS/Sciex, Toronto, ON). Peptide
ion peaks from the map are automatically detected with RESPECT.TM.
(PPL Inc., UK).
[0417] The sequence-composition of peptides detected, for example,
at higher levels in disease samples (or drug-resistant samples)
relative to adjacent normal tissue (or drug-sensitive samples) can
be resolved through tandem mass spectrometry and database analysis.
For data analysis, peptide ion peaks of LC/MS maps from normal and
disease samples can be aligned based on mass to charge ratio (m/z),
retention time (Rt), and charge state (z). The list of aligned
peptide ions is loaded into Spotfire.TM. (Spotfire Inc. Somerville,
Mass.). Intensities can be normalized before further differential
analysis between disease and normal samples. Differentially
expressed ions are manually verified before LC-MS/MS-based peptide
sequencing and database searching for protein/protein
identification.
[0418] For intensity normalization and expression analysis, a heat
map can be constructed by sorting the rows by the ratio of the mean
intensity in the disease samples to the mean intensity of the
normal samples. Rows are included if there is at least one MS/MS
identification of an ion in the row. The display colors are
determined for each row separately by assigning black to the median
intensity in the row, green to the lowest intensity in the row, and
red to the highest intensity.
[0419] Using a mass spectrometry procedure such as this, a
comprehensive analysis of proteins differentially expressed by
disease cells (or drug resistant cells, for example) compared with
normal cells (or cells responsive/sensitive to a drug, for example)
can be carried out.
[0420] 7. mRNA Expression Analysis
[0421] Expression of target mRNA can be quantitated by RT-PCR using
TaqMan.RTM. technology. The Taqman.RTM. system couples a 5'
fluorogenic nuclease assay with PCR for real-time quantitation. A
probe is used to monitor the formation of the amplification
product. Total RNA can be isolated from disease model cell lines
using an RNEasy kit.RTM. (Qiagen, Valencia, Calif.) with DNase
treatment (per the manufacturer's instructions). Normal human
tissue RNAs can be acquired from commercial vendors (e.g., Ambion,
Austin, Tex.; Stratagene, La Jolla, Calif.; BioChain Institute,
Newington, N.H.), as well as RNAs from matched disease/normal
tissues.
[0422] Target transcript sequences can be identified for
differentially expressed peptides by database searching using a
search algorithm such as BLAST. TaqMan.RTM. assays (PCR
primer/probe sets) specific for those transcripts can be obtained
from Applied Biosystems (AB) as part of the Assays on Demand.TM.
product line or by custom design through the AB Assays by Design
service. If desired, the assays can be designed to span exon-exon
borders so as not to amplify genomic DNA.
[0423] RT-PCR can be accomplished using AmpliTaq Gold.RTM. and
MultiScribe.TM. reverse transcriptase in the One Step RT-PCR Master
Mix reagent kit (AB) (according to the manufacturer's
instructions). Probe and primer concentrations are 250 nM and 900
nM, respectively, in a 15 .mu.l reaction. For each experiment, a
master mix of the above components is made and aliquoted into each
optical reaction well. Eight nanograms of total RNA is used as
template. Quantitative RT-PCR can be performed using the ABI
Prism.RTM. 7900HT Sequence Detection System (SDS). The following
cycling parameters are used: 48.degree. C. for 30 min. for one
cycle; 95.degree. C. for 10 min for one cycle; and 95.degree. C.
for 15 sec, 60.degree. C. for 1 min. for 40 cycles.
[0424] SDS software can be utilized to calculate the threshold
cycle (C.sub.T) for each reaction, and C.sub.T values are used to
quantitate the relative amount of starting template in the
reaction. The C.sub.T values for each set of reactions can be
averaged for all subsequent calculations
[0425] Data can be analyzed to determine estimated copy number per
cell. Gene expression can be quantitated relative to 18S rRNA
expression and copy number estimated assuming 5.times.10.sup.6
copies of 18S rRNA per cell. Alternatively, data can be analyzed
for fold difference in expression using an endogenous control for
normalization and expressed relative to a normal tissue or normal
cell line reference. The choice of endogenous control can be
determined empirically by testing various candidates against the
cell line and tissue RNA panels and selecting the one with the
least variation in expression. Relative changes in expression can
be quantitated using the 2.sup.-.DELTA..DELTA.CT method (Livak et
al., 2001, Methods 25: 402-408; User bulletin #2: ABI Prism 7700
Sequence Detection System). Alternatively, total RNA can be
quantitated using a RiboGreen RNA Quantitation Kit according to
manufacturer's instructions and the percentage mRNA expression
calculated using total RNA for normalization. Percentage knockdown
can then be calculated relative to a no addition control.
[0426] 8. Flow Cytometry (FACS) Analysis
[0427] Flow cytometry is interchangeably referred to as
fluorescence-activated cell sorting (FACS). Quantitative flow
cytometry can be used to compare the level of expression of a
protein on disease cells to the level found on normal cells, for
example.
[0428] Expression levels of a target protein on primary tissue
samples can be quantified using the Quantum Simply Cellular System
(Bangs Laboratories, Fishers, IN) and a target-specific antibody.
Normal adjacent and disease tissues can be processed into single
cell suspensions, as described above, which can be stained for
various markers (e.g., the epithelial marker EpCam) and the
target-specific antibody. At least 0.5.times.10.sup.6 cells are
typically used for each analysis. Cells are washed once with Flow
Staining Buffer (0.5% BSA, 0.05% NaN3 in D-PBS). To the cells, 20
.mu.l of each target-specific antibody are added. An additional 5
.mu.l of anti-EpCam antibody conjugated to APC can be added when
unsorted cells are used. Cells are incubated with antibodies for 30
minutes at 4.degree. C. Cells are washed once with Flow Staining
Buffer and either analyzed immediately on an LSR flow cytometry
apparatus or fixed in 1% formaldehyde and stored at 4.degree. C.
until LSR analysis. Antibodies used to detect a target can be
PE-conjugated. PE-conjugated mouse IgG1k can used as an isotype
control antibody. Cells are analyzed by flow cytometry and epitope
copy number and the percentage of viable epithelial cells positive
for target expression can be measured. Cell numbers and viability
can be determined by PI exclusion (GUAVA) for cells isolated from
both normal and disease tissue. Standard curve and samples can be
analyzed on a LSR I (BDBiosciences, San Jose Calif.) flow
cytometer. Antibody binding capacity for each lineage population
can be calculated using geometric means and linear regression.
[0429] Expression levels of a target protein can be quantified in
cell lines with QIFIKIT flow cytometric indirect immunofluorescence
assay (Dako A/S) using a primary antibody to the target. Briefly,
cells are detached with versene or trypsin and washed once with
complete media and then PBS. 5.times.10.sup.5 cells/sample are
incubated with saturating concentration (10 .mu.g/ml) of primary
antibody for 60 minutes at 4.degree. C. After washes, a
FITC-conjugated secondary antibody (1:50 dilution) is added for 45
minutes at 4.degree. C. QIFIKIT standard beads are simultaneously
labeled with the secondary antibody. Binding of antibodies is
analyzed by flow cytometry and specific antigen density is
calculated by subtracting background antibody equivalent from
antibody-binding capacity based on a standard curve of log mean
fluorescence intensity versus log antigen binding capacity.
[0430] Cells can also be prepared for flow cytometry analysis (as
well as other types of analysis) as follows: cells are incubated
with 1:100 dilution of BrdU in culturing media for 2-4 hours (BrdU
Flow Kit, cat #559619 BD Biosciences). Cells are washed 3 times
with D-PBS and disassociated from the flask with versene. Cell
numbers and viability can be determined by PI exclusion (GUAVA).
Cells are washed once with Flow Staining Buffer (0.5% BSA, 0.05%
NaN.sub.3 in D-PBS). Cells are incubated with 400 .mu.l of
Cytofix/Cytoperm Buffer (BrdU Flow Kit, BD Biosciences) for 15-30
minutes at 4.degree. C. Cells are washed once with Flow Staining
Buffer and resuspended in 400 .mu.l Cytoperm Plus Buffer (BrdU Flow
Kit BD Biosciences). Cells are incubated for 10 minutes at
4.degree. C. and washed once with 1.times. Perm/Wash Buffer (BrdU
Flow Kit, BD Biosciences). Cells are incubated for 1 hour at
37.degree. C. protected from light in DNAse solution (BrdU Flow
Kit, BD Biosciences). Cells are washed once with 1.times. Perm/Wash
Buffer and incubated for 20 min at room temperature with anti-BrdU
FITC-conjugated antibody (BrdU Flow Kit, BD Biosciences),
PE-conjugated active caspase 3 (BD Biosciences cat #550821), and PE
mouse IgG2B isotype control. Cells are washed once with 1.times.
Perm/Wash Buffer and resuspended in DAPI for LSR flow cytometry
analysis.
[0431] 9. Immunohistochemistry (IHC)
[0432] IHC of Tissue Sections
[0433] Paraffin embedded, fixed tissue sections (e.g., from disease
tissue samples such as solid tumors or other cancer tissues) can be
obtained from a panel of normal tissues as well as tumor (or other
disease) samples with matched normal adjacent tissues, along with
replicate sections (if desired). For example, for an initial
survery of target expression, a panel of common cancer
formalin-fixed paraffin-embedded (FFPE) tissue microarrays (TMAs)
can be used for analysis, and such TMAs can be obtained from
commercial sources (TriStar, Rockville, Md.; USBiomax, Rockville,
Md.; Imgenex, San Diego, Calif.; Petagen/Abxis, Seoul, Korea).
Sections can be stained with hemotoxylin and eosin and
histologically examined to ensure adequate representation of cell
types in each tissue section.
[0434] An identical set of tissues can be obtained from frozen
sections for use in those instances where it is not possible to
generate antibodies that are suitable for fixed sections. Frozen
tissues do not require an antigen retrieval step.
[0435] Paraffin Fixed Tissue Sections
[0436] An exemplary protocol for hemotoxylin and eosin staining of
paraffin embedded, fixed tissue sections is as follows. Sections
are deparaffinized in three changes of xylene or xylene substitute
for 2-5 minutes each. Sections are rinsed in two changes of
absolute alcohol for 1-2 minutes each, in 95% alcohol for 1 minute,
followed by 80% alcohol for 1 minute. Slides are washed in running
water and stained in Gill solution 3 hemotoxylin for 3-5 minutes.
Following a vigorous wash in running water for 1 minute, sections
are stained in Scott's solution for 2 minutes. Sections are washed
for 1 minute in running water and then counterstained in eosin
solution for 2-3 minutes, depending upon the desired staining
intensity. Following a brief wash in 95% alcohol, sections are
dehydrated in three changes of absolute alcohol for 1 minute each
and three changes of xylene or xylene substitute for 1-2 minutes
each. Slides are coverslipped and stored for analysis.
[0437] Optimization of Antibody Staining
[0438] For each antibody, a positive and negative control sample
can be generated using data from ICAT analysis of disease cell
lines or tissues. Cells can be selected that are known to express
low levels of a particular target as determined from the ICAT data,
and this cell line can be used as a reference normal control.
Similarly, a disease cell line that is determined to over-express
the target can also be selected.
[0439] Antigen Retrieval
[0440] Sections are deparaffinized and rehydrated by washing 3
times for 5 minutes in xylene, two times for 5 minutes in 100%
ethanol, two times for 5 minutes in 95% ethanol, and once for 5
minutes in 80% ethanol. Sections are then placed in endogenous
blocking solution (methanol+2% hydrogen peroxide) and incubated for
20 minutes at room temperature. Sections are rinsed twice for 5
minutes each in deionized water and twice for 5 minutes in
phosphate buffered saline (PBS), pH 7.4.
[0441] Alternatively, where necessary, sections are de-parrafinized
by High Energy Antigen Retrieval as follows: sections are washed
three times for 5 minutes in xylene, two times for 5 minutes in
100% ethanol, two times for 5 minutes in 95% ethanol, and once for
5 minutes in 80% ethanol. Sections are placed in a Coplin jar with
dilute antigen retrieval solution (10 mM citrate acid, pH 6). The
Coplin jar containing slides is placed in a vessel filled with
water and microwaved on high for 2-3 minutes (700 watt oven).
Following cooling for 2-3 minutes, steps 3 and 4 are repeated four
times (depending on the tissue), followed by cooling for 20 minutes
at room temperature. Sections are then rinsed in deionized water
(two times for 5 minutes), placed in modified endogenous oxidation
blocking solution (PBS+2% hydrogen peroxide), and rinsed for 5
minutes in PBS.
[0442] Alternatively, formalin fixed paraffin embedded tissues can
be deparaffinized and processed for antigen retrieval using the
EZ-retriever system (BioGenex, San Ramon, Calif.). EZ-antigen
Retrieval common solution is used for deparaffinization and
EZ-retrieval citrate-based buffer used for antigen retrieval.
Samples are pre-blocked with non-serum protein block (Dako A/S,
Glostrup, Denmark) for 15 min. Primary antibodies (at 2.5-5.0
.mu.g/ml, for example) are incubated overnight at room temperature.
Envision Plus system HRP (Dako A/S) is used for detection with
diaminobenzidine (DAB) as substrate for horseradish peroxidase.
[0443] Blocking and Staining
[0444] Sections are blocked with PBS/1% bovine serum albumin (PBA)
for 1 hour at room temperature followed by incubation in normal
serum diluted in PBA (2%) for 30 minutes at room temperature to
reduce non-specific binding of antibody. Incubations are performed
in a sealed humidity chamber to prevent air-drying of the tissue
sections. The choice of blocking serum is typically the same as the
species of the biotinylated secondary antibody. Excess antibody is
gently removed by shaking and sections covered with primary
antibody diluted in PBA and incubated either at room temperature
for 1 hour or overnight at 4.degree. C. (care is taken that the
sections do not touch during incubation). Sections are rinsed twice
for 5 minutes in PBS, shaking gently. Excess PBS is removed by
gently shaking. The sections are covered with diluted biotinylated
secondary antibody in PBA and incubated for 30 minutes to 1 hour at
room temperature in the humidity chamber. If using a monoclonal
primary antibody, addition of 2% rat serum can be used to decrease
the background on rat tissue sections. Following incubation,
sections are rinsed twice for 5 minutes in PBS, shaking gently.
Excess PBS is removed and sections incubated for 1 hour at room
temperature in Vectastain ABC reagent (as per kit instructions).
The lid of the humidity chamber is secured during all incubations
to ensure a moist environment. Sections are rinsed twice for 5
minutes in PBS, shaking gently.
[0445] Developing and Counterstaining
[0446] Sections are incubated for 2 minutes in peroxidase substrate
solution that is made up immediately prior to use as follows: 10 mg
diaminobenzidine (DAB) dissolved in 10 ml of 50 mM sodium phosphate
buffer, pH 7.4; 12.5 microliters 3% CoCl.sub.2/NiCl.sub.2 in
deionized water; and 1.25 microliters hydrogen peroxide.
[0447] Slides are rinsed well three times for 10 minutes in
deionized water and counterstained with 0.01% Light Green acidified
with 0.01% acetic acid for 1-2 minutes, depending on the desired
intensity of counterstain.
[0448] Slides are rinsed three times for 5 minutes with deionized
water and dehydrated two times for 2 minutes in 95% ethanol; two
times for 2 minutes in 100% ethanol; and two times for 2 minutes in
xylene. Stained slides are mounted for visualization by
microscopy.
[0449] Slides are scored manually using a microscope such as the
Zeiss Axiovert 200M microscope (Carl Zeiss Microimaging, Thornwood,
NY). Representative images are acquired using 40.times. objective
(400.times. magnification).
[0450] IHC Staining of Frozen Tissue Sections
[0451] For IHC staining of frozen tissue sections, fresh tissues
are embedded in OCT in plastic mold, without trapping air bubbles
surrounding the tissue. Tissues are frozen by setting the mold on
top of liquid nitrogen until 70-80% of the block turns white at
which point the mold is placed on dry ice. The frozen blocks are
stored at -80.degree. C. Blocks are sectioned with a cryostat with
care taken to avoid warming to greater than -10.degree. C.
Initially, the block is equilibrated in the cryostat for about 5
minutes and 6-10 mm sections are cut sequentially. Sections are
allowed to dry for at least 30 minutes at room temperature.
Following drying, tissues are stored at 4.degree. C. for short term
and -80.degree. C. for long term storage.
[0452] Sections are fixed by immersing in an acetone jar for 1-2
minutes at room temperature, followed by drying at room
temperature. Primary antibody is added (diluted in 0.05 M
Tris-saline [0.05 M Tris, 0.15 M NaCl, pH 7.4], 2.5% serum)
directly to the sections by covering the section dropwise to cover
the tissue entirely. Binding is carried out by incubation in a
chamber for 1 hour at room temperature. Without letting the
sections dry out, the secondary antibody (diluted in
Tris-saline/2.5% serum) is added in a similar manner to the primary
antibody and incubated as before (at least 45 minutes).
[0453] Following incubation, the sections are washed gently in
Tris-saline for 3-5 minutes and then in Tris-saline/2.5% serum for
another 3-5 minutes. If a biotinylated primary antibody is used, in
place of the secondary antibody incubation, slides are covered with
100 .mu.l of diluted alkaline phosphatase conjugated streptavidin,
incubated for 30 minutes at room temperature and washed as above.
Sections are incubated with alkaline phosphatase substrate (1 mg/ml
Fast Violet; 0.2 mg/ml Napthol AS-MX phosphate in Tris-Saline pH
8.5) for 10-20 minutes until the desired positive staining is
achieved at which point the reaction is stopped by washing twice
with Tris-saline. Slides are counter-stained with Mayer's
hematoxylin for 30 seconds and washed with tap water for 2-5
minutes. Sections are mounted with Mount coverslips and mounting
media.
[0454] 10. RNAi Assays in Cell Lines
[0455] RNAi Transfections
[0456] Expression of a target can be knocked down by transfection
with small interfering RNA (siRNA) to that target. Synthetic siRNA
oligonucleotides can be obtained from Dharmacon (Lafayette, Colo.)
or Qiagen (Valencia, Calif.). For siRNA transfection, cells (e.g.,
disease cells) can be seeded into 96 well tissue culture plates at
a density of 2,500 cells per well 24 hours before transfection.
Culture medium is removed and 50 .mu.l of reaction mix containing
siRNA (final concentration 1 to 100 nM) and 0.4 .mu.l of
DharmaFECT4 (Dharmacon, Lafayette, Colo.) diluted in Opti-MEM is
added to each well. An equal volume of complete medium follows and
the cells are then incubated at 5% CO.sub.2 at 37.degree. C. for 1
to 4 days.
[0457] Alternatively, in the initial screening phase, RNAi can be
performed using 100 nM (final) of Smartpools (Dharmacon, Lafayette,
Colo.), pool of 4--for Silencing siRNA duplexes (Qiagen, Valencia,
Calif.), or non-targeting negative control siRNA (Dharmacon or
Qiagen). In the breakout phase, each individual duplex is used at
100 nM (final). In the titration phase, individual duplex is used
at 0.1-100 nM (final). Transient transfections are carried out
using either Lipofectamine 2000 from Invitrogen (Carlsbad, Calif.)
or GeneSilencer from Gene Therapy Systems (San Diego, Calif.) (see
below). One day after transfections, total RNA is isolated using
the RNeasy 96 Kit (Qiagen) according to manufacturer's instructions
and expression of mRNA is quantitated using TaqMan technology.
Apoptosis and cell proliferation assays can be performed daily
using Apop-one homogeneous caspase-3/7 kit and Alamar Blue or
CellTiter 96 AQueous One Solution Cell Proliferation Assays (see
below).
[0458] RNAi Transfections--Lipofectamine 2000 and GeneSilencer
[0459] Transient RNAi transfections can be carried out using
Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) or GeneSilencer
(Gene Therapy Systems, San Diego, Calif.), such as on sub-confluent
disease cell lines, as described elsewhere (Elbashir et al., 2001,
Nature 411: 494-498; Caplen et al., 2001, Proc Natl Acad Sci USA
98: 9742-9747; Sharp, 2001, Genes and Development 15: 485-490).
Synthetic RNA to a gene of interest or non-targeting negative
control siRNA are transfected using Lipofectamine 2000 or
GeneSilencer according to manufacturer's instructions. Cells are
plated in 96-well plates in antibiotic-free medium. The next day,
the transfection reagent and siRNA are prepared for transfections
as follows.
[0460] 0.1-100 nM siRNA is resuspended in 20-25 .mu.l serum-free
media in each well (with Plus for Lipofectamine 2000) and incubated
at room temperature for 15 minutes. 0.1-1 .mu.l of Lipofectamine
2000 or 1-1.5 .mu.l of GeneSilencer is also resuspended in
serum-free medium to a final volume of 20-25 .mu.l per well. After
incubation, the diluted siRNA and either the Lipofectamine 2000 or
the GeneSilencer are combined and incubated for 15 minutes
(Lipofectamine 2000) or 5-20 minutes (GeneSilencer) at room
temperature. Media is then removed from the cells and the combined
siRNA-Lipofectamine 2000 reagent or siRNA-GeneSilencer reagent is
added to a final volume of 50 .mu.l per well. After further
incubation at 37.degree. C. for 4 hours, 50 .mu.l serum-containing
medium is added back to the cells. 1-4 days after transfection,
expression of mRNA can be quantitated by RT-PCR using TaqMan
technology, and protein expression levels can be measured by flow
cytometry. Apoptosis and proliferation assays can be performed
daily using Apop-one homogeneous caspase-3/7 kit and Alamar Blue or
CellTiter 96 AQueous One Solution Cell Proliferation Assays (see
below).
[0461] mRNA and Protein Knockdowns
[0462] Knockdown of target mRNA levels can be monitored by Q-PCR
one day after siRNA transfection by using a TaqMan.RTM. assay
(Applied Biosystems, Foster City, Calif.). RT-PCR is accomplished
in a one-step reaction by using M-MLV reverse transcriptase
(Promega, Madison, Wis.) and AmpliTaq Gold.RTM. (ABI) and analyzed
on the ABI Prism.RTM. 7900HT Sequence Detection System (ABI).
Relative gene expression can be quantitated by the .DELTA..DELTA.Ct
method (User Bulletin #2, ABI) with 18S rRNA serving as the
endogenous control.
[0463] Protein knockdown can be monitored by FACS four days after
transfection by using an antibody to the target. The samples can be
run on a LSR flow cytometer (BD Biosciences, San Jose, Calif.) and
live cells monitored by using PI exclusion (50 .mu.g/ml PI, 2.5
units/ml RNase A, 0.1% Triton X-100 in D-PBS). The data can be
analyzed using CellQuest software.
[0464] Cell Proliferation--Alamar Blue
[0465] Cell growth can be assessed four days after transfection by
adding a 1:10 dilution of Alamar blue reagent (Invitrogen,
Carlsbad, CA or Biosource, Camarillo, Calif.) and incubated for 2
hours at 37.degree. C. Analysis can be performed on a Spectrafluor
Plus (Tecan, Durham, N.C.) set at excitation wavelength of 530 nm
and emission wavelength of 595 nm.
[0466] Cell Proliferation--MTS
[0467] Alternatively, cell proliferation assays can be performed
using a CellTiter 96 AQueous One Solution Cell Proliferation Assay
kit (Promega, Madison, WI). 20 .mu.l of CellTiter 96 AQueous One
Solution is added to 100 .mu.l of culture medium. The plates are
then incubated for 1-4 hours at 37.degree. C. in a humidified 5%
CO.sub.2 incubator. After incubation, the change in absorbance is
read at 490 nm.
[0468] Apoptosis
[0469] Apoptosis assays can be performed using the Apop-one
homogeneous caspase-3/7 kit (Promega, Madison, Wis.). Briefly, the
caspase-3/7 substrate is thawed to room temperature and diluted
1:100 with buffer. The diluted substrate is then added 1:1 to
cells, control, or blank. The plates are then placed on a plate
shaker for 30 minutes to 18 hours at 300-500rpm. The fluorescence
of each well is then measured using an excitation wavelength of
485+/-20 nm and an emission wavelength of 530+/-25 nm.
[0470] 11. Antibody Assays in Cell Lines
[0471] Cytotoxicity Assays
[0472] Cytotoxicity can be measured using a Resazurin (Sigma, Mo.)
dye reduction assay (McMillian et al., 2002, Cell Biol. Toxicol.
18:157-173). Briefly, cells are plated at 1,000-5,500 cells/well in
96 well plates, allowed to attach to the plates for 18 hours before
addition of fresh media with or without antibody. After 96-144
hours of exposure to antibody, resazurin is added to cells to a
final concentration of 50 Cells are incubated for 2-6 hours
depending on dye conversion of cell lines, and dye reduction is
measured on a Fusion HT fluorescent plate reader (Packard
Instruments, Meridien, Conn.) with excitation and emission
wavelengths of 530 nm and 590 nm, respectively. The IC.sub.50 value
is defined here as the drug concentration that results in 50%
reduction in growth or viability as compared with untreated control
cultures.
[0473] Assays for Antibody-Dependent Cellular Cytotoxicity
[0474] Antibody-dependent cellular cytotoxicity (ADCC) assays can
be carried out as follows. Cultured disease cells (e.g., tumor
cells) are labeled with 100 .mu.Ci .sup.51Cr for 1 hour (Livingston
et al., 1997, Cancer Immunol. Immunother. 43, 324-330). After being
washed three times with culture medium, cells are resuspended at
10.sup.5/ml, and 100 .mu.l/well are plated onto 96-well
round-bottom plates. A range of antibody concentrations are applied
to the wells, including an isotype control together with donor
peripheral blood mononuclear cells that are plated at a 100:1 and
50:1 ratio. After an 18 hour incubation at 37.degree. C.,
supernatant (30 .mu.l/well) is harvested and transferred onto
Lumaplate 96 (Packard), dried, and read in a Packard Top-Count NXT
.gamma. counter. Spontaneous release is determined by cpm of
disease cells incubated with medium and maximum release by cpm of
disease cells plus 1% Triton X-100 (Sigma). Specific lysis is
defined as: % specific lysis=[(experimental release-spontaneous
release)/(maximum release-spontaneous release)].times.100. The
percent ADCC is expressed as peak specific lysis postimmune
subtracted by preimmune percent specific lysis. A doubling of the
ADCC to >20% can typically be considered significant.
[0475] Assays for Complement Dependent Cytotoxicity
[0476] Chromium release assays to assess complement dependent
cytotoxicity (CDC) can be carried out as follows (Dickler et al.,
1999, Clin. Cancer Res. 5, 2773-2779). Cultured disease cells
(e.g., tumor cells) are washed in FCS-free media two times,
resuspended in 500 .mu.l of media, and incubated with 100 .mu.Ci
.sup.51Cr per 10 million cells for 2 hours at 37.degree. C. The
cells are then shaken every 15 min for 2 hours, washed 3 times in
media to achieve a concentration of approximately 20,000
cells/well, and then plated in round-bottom plates. The plates
contain either 50 .mu.l cells plus 50 .mu.l monoclonal antibody, 50
.mu.l cells plus serum (pre- and post-therapy), or 50 .mu.l cells
plus mouse serum as a control. The plates are incubated in a cold
room on a shaker for 45 min. Human complement of a 1:5 dilution
(resuspended in 1 ml of ice-cold water and diluted with 3% human
serum albumin) is added to each well at a volume of 100 .mu.l.
Control wells include those for maximum release of isotope in 10%
Triton X-100 (Sigma) and for spontaneous release in the absence of
complement with medium alone. The plates are incubated for 2 hours
at 37.degree. C., centrifuged for 3 min, and then 100 .mu.l of
supernatant is removed for radioactivity counting. The percentage
of specific lysis is calculated as follows: %
cytotoxicity=[(experimental release-spontaneous release)/(maximum
release-spontaneous release)].times.100. A doubling of the CDC to
>20% can typically be considered significant.
[0477] Cell Proliferation Assays
[0478] To measure cell proliferation, cells can be plated, grown
and treated as for the cytotoxicity assay (above) in 96 well
plates. After 96-144 hours of treatment, 0.5 .mu.Ci/well
.sup.3H-Thymidine (PerkinElmer, 6.7 Ci/mmol) is added to cells and
incubated for 4-6 hours at 37.degree. C., 5% CO.sub.2 in an
incubator. To lyse cells, plates are frozen overnight at
-20.degree. C. and then cell lysates are harvested using FilterMate
(Packard Instrument, Meridien, Conn.) into 96 well filter plates.
Radioactivity associated with cells is measured on a TopCount
(Packard) scintillation counter.
[0479] Other cell assays (e.g., proliferation assays such as Alamar
blue and MTS, and apoptosis assays) can be carried out using
antibodies, as described above for RNAi.
[0480] Testing of Function-Blocking Antibodies
[0481] For testing of function-blocking antibodies, sub-confluent
disease cell lines are serum-starved overnight. The next day,
serum-containing media is added back to the cells in the presence
of 5-50 ng/ml of function-blocking antibodies. After 2 or 5 days
incubation at 37.degree. C. 5% CO.sub.2, antibody binding is
examined by flow cytometry, and apoptosis and proliferation are
measured.
[0482] Cell Invasion
[0483] Cell invasion assays can be performed using a 96-well cell
invasion assay kit (Chemicon). After the cell invasion chamber
plates are adjusted to room temperature, 100 .mu.l serum-free media
is added to the interior of the inserts. 1-2 hours later, cell
suspensions of 1.times.10.sup.6 cells/ml are prepared. Media is
then carefully removed from the inserts and 100 .mu.l of prepared
cells are added into the insert +/-0 to 50 ng function blocking
antibodies. The cells are pre-incubated for 15 minutes at
37.degree. C. before 150 .mu.l of media containing 10% FBS is added
to the lower chamber. The cells are then incubated for 48 hours at
37.degree. C. After incubation, the cells from the top side of the
insert are discarded and the invasion chamber plates are then
placed on a new 96-well feeder tray containing 150 .mu.l of
pre-warmed cell detachment solution in the wells. The plates are
incubated for 30 minutes at 37.degree. C. and are periodically
shaken. Lysis buffer/dye solution (4 .mu.l CyQuant Dye/300 .mu.l
4.times. lysis buffer) is prepared and added to each well of
dissociation buffer/cells on feeder tray. The plates are incubated
for 15 minutes at room temperature before 150 .mu.l is transferred
to a new 96-well plate. Fluorescence of invading cells is then read
at 480 nm excitation and 520 nm emission.
[0484] Receptor Internalization
[0485] For quantification of receptor internalization, ELISA assays
can be performed essentially as described by Daunt et al. (Daunt et
al., 1997, Mol. Pharmacol. 51, 711-720). Cell lines are plated at
6.times.10.sup.5 cells per in a 24-well tissue culture dishes that
have previously been coated with 0.1 mg/ml poly-L-lysine. The next
day, the cells are washed once with PBS and incubated in DMEM at
37.degree. C. for several minutes. Agonist to the cell surface
target of interest is then added to the wells at a pre-determined
concentration in prewarmed DMEM. The cells are then incubated for
various times at 37.degree. C. and reactions are stopped by
removing the media and fixing the cells in 3.7% formaldehyde/TBS
for 5 min at room temperature. The cells are then washed three
times with TBS and nonspecific binding blocked with TBS containing
1% BSA for 45 min at room temperature. The first antibody is added
at a pre-determined dilution in TBS/BSA for 1 hr at room
temperature. Three washes with TBS follow, and cells are briefly
reblocked for 15 min at room temperature. Incubation with goat
anti-mouse conjugated alkaline phosphatase (Bio-Rad) diluted 1:1000
in TBS/BSA is carried out for 1 hr at room temperature. The cells
are washed three times with TBS and a colorimetric alkaline
phosphatase substrate is added. When the adequate color change is
reached, 100 .mu.l samples are taken for colorimetric readings.
[0486] 12. Treatment with Antibodies
[0487] Treatment of Disease Cells with Monoclonal Antibodies.
[0488] Disease cells (e.g., cancer cells), or cells such as NIH 3T3
cells that express a target of interest, are seeded at a density of
4.times.10.sup.4 cells per well in 96-well microtiter plates and
allowed to adhere for 2 hours. The cells are then treated with
different concentrations of monoclonal antibody (Mab) specific for
the protein target of interest, or irrelevant isotype matched
(e.g., anti-rHuIFN-gamma) Mab, at 0.05, 0.5 or 5.0 .mu.g/ml. After
a 72 hour incubation, the cell monolayers are stained with crystal
violet dye for determination of relative percent viability (RPV)
compared to control (untreated) cells. Each treatment group can
have replicates. Cell growth inhibition is monitored.
[0489] In vivo Treatment with Monoclonal Antibodies.
[0490] NIH 3T3 cells transfected with either an expression plasmid
that expresses the target of interest or a neo-DHFR vector are
injected into nu/nu (athymic) mice subcutaneously at a dose of
10.sup.6 cells in 0.1 ml of phosphate-buffered saline. On days 0,
1, 5, and every 4 days thereafter, 100 .mu.g (0.1 ml in PBS) of a
Mab specific for the protein target of interest, or an irrelevant
Mab, of the IgA2 subclass is injected intraperitoneally. Disease
progression (e.g., tumor occurrence and size) can be monitored for
a one month period of treatment, for example.
[0491] 13. Examples of Results from Experimental Validation
[0492] The Genbank protein accession numbers, and protein SEQ ID
NOS corresponding to Table 1, for the four targets described below
are as follows: Angiopoietin-like 4 (ANGPTL4)=Q9BY76 (protein SEQ
ID NOS:476-478 in Table 1), Tweety Homolog 3 (TTYH3)=NP_079526
(exemplary TTYH3 protein and encoding transcript sequences are
provided in the sequence listing as SEQ ID NOS:1977-1978), CEACAM6
(carcinoembryonic antigen-related cell adhesion molecule 6
precursor)=P40199 or NP_002474 (exemplary CEACAM6 protein and
encoding transcript sequences are provided in the sequence listing
as SEQ ID NOS:1975-1976), and protein F1111273=gi120380426 (protein
SEQ ID NOS:713-714 in Table 1).
[0493] Angiopoietin-Like 4 (ANGPTL4)
[0494] ANGPTL4 peptides were identified by mass spec to be
over-expressed by 36-fold in spheroid cells (i.e., cancer stem
cells) compared with parental adherent cells from lung cancer cell
line H1299.
[0495] See FIG. 1 for further results from experimental validation
of ANGPTL4.
[0496] Tweety Homolog 3 (TTYH3)
[0497] A TTYH3 peptide was identified by mass spec as
over-expressed by the following ratios: 6.8-fold in spheroid cells
(i.e., cancer stem cells) compared with differentiated cells from a
colon cancer cell line (referred to as CBT026T), 9.9-fold in a
colon tumor tissue sample, and 6.5-fold in a liver cancer cell
line.
[0498] See FIG. 2 for further results from experimental validation
of TTYH3.
[0499] CEACAM6 (Carcinoembryonic Antigen-Related Cell Adhesion
Molecule 6 Precursor)
[0500] CEACAM6 peptides were identified by mass spec as
over-expressed by the following ratios: 5.5-fold in spheroid cells
(i.e., cancer stem cells) compared with differentiated cells from a
colon cancer cell line (referred to as CBT026T), 9.4-49.0 fold in a
colon tumor tissue sample, 4.6-5.3 fold in a liver tumor tissue
sample, and 9.1-24.0 fold in a pancreatic cancer cell line.
[0501] See FIG. 3 for further results from experimental validation
of CEACAM6.
[0502] Protein FLJ11273
[0503] An FLJ11273 peptide was identified by mass spec as
over-expressed by 5.5-fold in spheroid cells (i.e., cancer stem
cells) compared with differentiated cells from a colon cancer cell
line (referred to as CBT026T).
[0504] See FIG. 4 for further results from experimental validation
of FLJ11273.
[0505] 14. Examples of Results of RNAi Assays in Cancer Stem Cell
Lines
[0506] RNAi knockdown of the prominin 1 (PROM1) target in colon
cancer stem cell line CBT026T-HES induced apoptosis and inhibited
cell proliferation in this cell line (prominin 1 is represented in
Table 1 by protein SEQ ID NOS:411-413 and 700).
[0507] RNAi knockdown of the integrin beta-6 (ITGB6) target in the
HT29 colon cancer stem cell line inhibited cell proliferation in
this cell line (ITGB6 is represented in Table 1 by protein SEQ ID
NOS:379-380).
[0508] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described methods and
compositions of the invention will be apparent to those skilled in
the art without departing from the scope and spirit of the
invention. Although the invention has been described in connection
with specific exemplary embodiments, it should be understood that
the invention as claimed should not be unduly limited to such
specific embodiments. Indeed, various modifications of the
above-described modes for carrying out the invention, which are
obvious to those skilled in the field of molecular biology or
related fields, are intended to be within the scope of the
following claims.
TABLE-US-00001 TABLE 3 Subcellular Target Symbol IHC Apoptosis
Proliferation Matrix Location ITAV_HUMAN H1299_StemCell_Cys_Only
Cell Surface SLC1A5 30% Ovarian Tumors Colon (++)
CBT026_HES_Primary Cell Surface 40% Prostate Tumors Pancreatic (++)
40% Met Panc Tumors Prostate (++) 20% Panc Tumors ATP1B3 100%
Glioblastoma Kidney (++) Pancreatic (+++) HT29_Stemcell Cell
Surface 80% NSC Lung Tumors Melanoma (++) Gastric (++)
CBT026_HES_Primary 71% Panc Tumors Pancreatic (++) Kidney (++) 70%
Breast Gastric (+) Lung (++) 70% Melanoma Melanoma (++) 70%
Melanoma Node 67% Met Panc Tumors 70% Squ Lung 50% Colon 50% Ovary
38% Liver Tumors 30% Gastric Tumors TSN1_HUMAN 50% Liver Tumors
Colon (++) CBT026_T Cell Surface 40% Melanoma Node Gastric (++)
CBT026_HES_Primary 100% Pancreas Tumors Melanoma (++) 100% Met
Pancreas Tumors Pancreatic (+) Prostate (+) TFR1_HUMAN CBT026_N
Cell Surface CBT026_HES_Primary T4S3_HUMAN Pancreatic (++) CBT026_N
Cell Surface CBT026_T M6PR Lung (++) HT29_Stemcell Cell Surface
Pancreatic (++) CBT026_N Gastric (+) CBT026_HES_Primary BST2 Colon
(++) Colon (++) CBT026_N Cell Surface Liver (++) Liver (++) RKO
Sphere vs Lung (++) Lung (++) Adherent PNGF Melanoma (++) Melanoma
(++) Ovary (+) CEACAM5 CBT026_N Cell Surface CBT026_T
CBT026_HES_Primary ITGA6 100% Kidney Tumors Colon (++) Lung (+++)
CBT026_N Cell Surface 80% NSC Lung Tumors Colon (++) CBT026_T 70%
Squ Lung Tumors Gastric (++) CBT026_HES_Primary 67% Met Panc Tumors
60% Melanoma Node 50% Melanoma 50% Glioblastoma 40% Gastric Tumors
63% Liver Tumors 25% Panc Tumors 20% Ovary Tumors 20% Colon Tumors
ITGB4 40% Breast Tumors Colon (++) CBT026_N Cell Surface CBT026_T
CBT026_HES_Primary C20orf3 80% Melanoma Colon (++) CBT026_N Cell
Surface 70% Melanoma Node Liver (++) CBT026_T 50% NHL, Lymph Node
Lung (++) CBT026_HES_Primary 50% Colon Tumors RKO Sphere vs 50%
Kidney Tumors Adherent PNGF 83% Glioblastoma 33% Met Panc Tumors
29% Panc Tumors 20% Prostate Tumors 25% Liver Tumors TACSTD1
CBT026_N Cell Surface CBT026_T CBT026_HES_Primary TM9SF3 90%
Melanoma Pancreatic (++) HT29_Stemcell Cell Surface 50%
Glioblastoma CBT026_N 40% Colon Tumors CNT026_HES_Primary 60%
Melanoma Node 33% NHL Node 20% Prostate Tumors ECE1
H1299_StemCell_Glyco Cell Surface CALR CBT026_N Cell Surface ICAM1
RKO Sphere vs Cell Surface Adherent PNGF ADAM10 CBT026_N Cell
Surface SYPL CBT026_HES_Primary Cell Surface CBT026_T Melanoma
Stemcell_EGT003D LMAN2 HT29_StemCell Cell Surface CBT026_N
H1299_StemCell_Glyco ITM1 Colon (++) Colon (++) HT29_StemCell Cell
Surface CBT026_N CBT026_T MUC13 100% Glioblastoma Pancreatic (++)
Gastric (++) CBT026_N Cell Surface 100% Colon Tumors Colon (+)
CBT026_HES_Primary 30% Esoph Tumors 100% Liver Tumors 30% Melanoma
Node 70% Pancreas Tumors 70% Melanoma 30% Gastric Tumors LTF
Melanoma Secreted StemCell_EGT003D SLC5A1 33% Glioblastoma Colon
(++) Colon (++) CBT026_N Cell Surface 100% Colon Tumors Gastric
(++) 90% Kidney Tumors Pancreatic (++) 88% Liver Tumors 70% NSC
Lung Tumors 20% Squ Lung Tumors 50% Melanoma Node 50% NHL Node 86%
Panc Tumors 100% Met Panc Tumors 50% Melanoma PROM1 60% Colon Lung
(++) Colon (++) CBT026_N Cell Surface Lung (++) CBT026_T
CBT026_HES_Primary NPC2 70% Breast Tumors Colon (++) Melanoma
Secreted 50% Colon Tumors Stemcell_EGT003D 70% NSC Lung Tumors 43%
Panc Tumors 66% Met Panc Tumors 20% Prostate Tumors CD68 Kidney
(++) Liver (++) CSC Overlap Cell Surface Gastric (+) CBT026_N CTSB
Melanoma Secreted StemCell_EGT003D CTSD Gastric (++) CBT026_N
Secreted Liver (++) Melanoma Stemcell_EGT003D OLFM4 70% Bladder
Tumors CBT026_N Secreted 20% Colon Tumors 100% Kidney Tumors 38%
Liver Tumors 40% Melanoma Node 33% NHL Node 50% Gastric Tumors
CDH17 CBT026_N Cell Surface CBT026_T CBT026_HES_Primary ITGB6 Colon
(++) Colon (++) HT29_StemCell Cell Surface Kidney (++) Gastric (++)
Lung (++) Lung (++) Pancreatic (++) Pancreatic (++) Gastric (+)
Liver (+) Liver (+) ST14 70% Colon Tumors Colon (++) CBT026_T Cell
Surface 20% Squ Lung Tumors Lung (++) CBT026_HES_Primary 80% NHL
Node Pancreatic (++) 60% Ovary Tumors Gastric (+) 38% Panc Tumors
XP_114346 Colon (++) Colon (++) HT29_StemCell Cell Surface Lung
(++) Lung (++) CBT026_N CBT026_T CBT026_HES_Primary FLJ14681
H1299_StemCell_Glyco Cell Surface SERPINF2 Melanoma Secreted
Stemcell_EGT003D DPEP1 38% Liver Colon (++) Colon (+++) CBT026_N
Cell Surface 20% Colon 20% Prostate 10% Gastric GP25L2 CBT026_N
Cell Surface GPC1 Gastric (++) RKO Sphere vs Cell Surface Liver
(++) Adherent PNGF Breast (+) Lung (+) gi|23268459 Melanoma
Secreted StemCell_EGT003D NCSTN CBT026_N Cell Surface CBT026_T
CBT026_HES_Primary CD97 CBT026_N Cell Surface LY75 Colon (++)
Kidney (++) HT29_Stemcell Cell Surface Kidney (++) SLC11A2
HT29_StemCell Cell Surface ANPEP H1299_StemCell_Glyco Secreted GGT1
CBT026_N Cell Surface CEACAM1 HT29_StemCell Cell Surface CBT026_N
CBT026_T CBT026_HES_Primary PGLYRP2 Melanoma Secreted
Stemcell_EGT003D PZP LS123_StemCell Secreted Q9Y3B3 Melanoma Cell
Surface Stemcell_EGT003D BGN CBT026_12Maps Secreted CBT026_N
CBT026_T CBT026_HES_Primary HT29_StemCell RKO Sphere vs Adherent
PNGF H1299_Stemcell_Glyco Melanoma Stemcell_EGT003D PIGR Colon (++)
HT29_Stemcell Cell Surface Lung (++) DSG2 Kidney (++) Kidney (++)
HT29_Stemcell Cell Surface Pancreatic (+) gi|22859168 Colon (++)
HT29_Stemcell Cell Surface Pancreatic (++) Gastric (+) Kidney (+)
Melanoma (+) PTGFRN CBT026_N Cell Surface CBT026_HES_Primary IGSF8
66% NHL Node Colon (++) CBT026_HES_Primary Cell Surface 20% Ovary
Tumors Gastric (++) 30% Melanoma Lung (++) gi|41352831 Melanoma
Cell Surface StemCell_EGT003D CDCP1 Breast (+++) CBT026_N Cell
Surface Colon (+++) Lung (+++) Gastric (++) Liver (++) Melanoma
(++) Pancreatic (++) ATP1B1 70% Melanoma node Lung (++) Breast (++)
CBT026_N Cell Surface 70% Breast Tumors Melanoma (++) Colon (++)
CBT026_T 67% Glioblastoma Melanoma (++) CBT026_HES_Primary 50%
Melanoma HT29_Stemcell 20% Esoph Tumors CLIC1 Melanoma (++) Gastric
(++) H1299_Stemcell_Cys_Only Cell Surface Melanoma (++) CLU 30%
Breast Tumors Colon (++) CBT026_N Secreted 60% Ovary Tumors
Melanoma (+) CBT026_T 30% Prostate Tumors CBT026_HES_Primary
H1299_Stemcell_Glyco TIMP1 RKO Sphere vs Secreted Adherent PNGF
TM9SF1 50% Panc Tumors Kidney (++) HT29_Stemcell Cell Surface 20%
Prostate Tumors Lung (++) LGALS3BP Melanoma Secreted
Stemcell_EGT003D CBT026_T CBT026_N CBT026_HES_Primary H1299_Stem
cell_Glyco FAT Gastric (++) Colon (++) CBT026_N Cell Surface
Gastric (++) Liver (+) HLA-C CBT026_N Cell Surface ENPP1 Colon (+)
H1299_Stemcell_Cys_Only Cell Surface H1299_Stemcell_Glyco HSPG2
HT29_StemCell Secreted H1299_StemCell_Glyco CBT026_N
PLTP Lung (+) Colon (++) CBT026_12Maps Secreted Gastric (+)
Melanoma (+) PON1 RKO Sphere vs Secreted Adherent PNGF HLA-C
CBT026_N Cell Surface MUCDHL Colon (++) CBT026_N Cell Surface
CBT026_T SLC7A1 Colon (++) CSC Overlap Cell Surface Liver (++)
CBT026_HES_Primary Pancreatic (++) Gastric (+) GPR56 CBT026_N Cell
Surface P2RX4 Kidney (++) Colon (++) CBT026_N Cell Surface Kidney
(++) Gastric (+) Lung (+) gi|21361918 Melanoma (++) Melanoma
Secreted Colon (+) StemCell_EGT003D Gastric (+) HLA-A CBT026_12Maps
Cell Surface ATP6V0A1 CBT026_T Cell Surface CBT026_HES_Primary
TM9SF4 40% Colon Tumors Colon (++) Melanoma Cell Surface 100% Liver
Tumors Stemcell_EGT003D 56% NSC Lung Tumors 64% Squ Lung Tumors 80%
Melanoma Node 50% NHL Node 88% Panc Tumors 50% Met Panc Tumors 70%
Melanoma TMED7 Melanoma Cell Surface Stemcell_EGT003D KIAA1815 33%
Glioblastoma Breast (++) CBT026_N Cell Surface 38% Liver Tumors
Lung (++) HT29_Stemcell 73% Squ Lung Tumors 50% Panc Tumors 30%
Prostate Tumors CD82 Lung (+) Colon (++) CBT026_HES_Primary Cell
Surface Ovary (+) Pancreatic (++) LAMA4 CBT026_12Maps Secreted
SERPINE2 Kidney (++) Lung (++) H1299_Stemcell_Glyco Secreted Lung
(++) Ovary (+) GOLPH2 HT29_StemCell Secreted CBT026_N BMP1
H1299_Stemcell_Glyco Secreted EVA1 HT29_Stem cell Cell Surface
SERPINF1 CBT026_12 maps Secreted HT29_Stemcell CBT026_HES_primary
GREM2 HT29_Stemcell Secreted FAM38A HT29_Stemcell Cell Surface LIPG
HT29_stem cell Secreted ST3GAL1 Melanoma (++) CBT026_N Secreted
LNPEP CBT026_N Cell Surface MFAP4 CBT026_HES_primary Secreted
CBT026_N CBT026_T DKFZP564G2022 CBT026_N Cell Surface CEACAM6
Gastric (+) Prostate (+) CBT026_N Cell Surface CBT026_T
CBT026_HES_Primary FLJ11273 Colon (+) Pancreatic (++) CBT026_T Cell
Surface Colon (+) CBT026_N CBT026_HES_Primary CEACAM7 CBT026_N Cell
Surface CANT1 CBT026_N Secreted SSR1 Ovary (+) Ovary (+) CBT026_N
Cell Surface Pancreatic (+) CBT026_T CBT026_HES_Primary TTYH3 Colon
(++) Colon (++) CBT026_N Cell Surface Lung (++) Gastric (++)
CBT026_T Melanoma (++) Liver (++) CBT026_HES_Primary Ovary (+) Lung
(++) Prostate (+) Melanoma (++) Kidney (+) Pancreatic (+) CLPTM1
CBT026_N Cell Surface CBT026_HES_Primary FAM3D Colon (++) CBT026_N
Secreted LOC284361 Colon (++) Colon (++) CBT026_N Cell Surface Lung
(++) Lung (++) Kidney (+) Pancreatic (+) Liver (+) KIAA0090 Colon
(++) Colon (++) CBT026_N Secreted Breast (+) Lung (++) CBT026_T
Pancreatic (+) CBT026_HES_Primary LAMA5 H1299_StemCell_Glyco
Secreted ATP6AP1 CBT026_HES_Primary Cell Surface PTPRK Lung (+)
Colon (++) CBT026_HES_Primary Cell Surface Ovary (+) Pancreatic
(++) Prostate (+) NCLN H1299_Stem Cell_Glyco Cell Surface ANGPTL4
Colon (++) Colon (++) H1299_StemCell_Glyco Secreted Kidney (++)
Kidney (++) Pancreatic (++) MFGE8 H1299_StemCell_Glyco Cell Surface
COMT 100% Kidney Tumors Breast (++) Colon (+++) LS123_Stemcell Cell
Surface 100% Ovary Tumors Colon (++) Lung (+++) 60% Melanoma Node
Gastric (++) Melanoma (+++) 50% Melanoma Breast (++) 43% Panc
Tumors Gastric (++) 50% Colon Tumors Kidney (++) 30% Bladder Tumors
Liver (++) 50% Glioblastoma Prostate (++) 30% Squ Lung Tumors 30%
Prostate Tumors 25% Liver Tumors GLG1 30% Squ Lung Tumors Liver
(++) H1299_Stemcell_Glyco Cell Surface 50% Melanoma Node Breast (+)
Melanoma 43% Panc Tumors Stemcell_EGT003D 80% Melanoma CD44
CBT026_HES_Primary Cell Surface LAMP1 Prostate (++) CBT026_N Cell
Surface CBT026_T CBT026_HES_Primary SLC3A2 100% NSC Lung Tumors
Pancreatic (++) Lung (+++) CBT026_HES_Primary Cell Surface 100% Squ
Lung Tumors Colon (++) 90% Melanoma Gastric (++) 83% Glioblastoma
Pancreatic (++) 80% Colon Tumors Prostate (++) 50% Breast Tumors
SCARB2 HT29_StemCell Cell Surface CBT026_N CBT026_T
CBT026_HES_Primary ITGA3 Pancreatic (++) Pancreatic (++) CBT026_N
Cell Surface CBT026_HES_Primary PLXNB2 CBT026_N Cell Surface
CBT026_HES_Primary LAMP2 Lung (++) Liver (++) CBT026_N Cell Surface
Melanoma (++) Lung (++) CBT026_T Pancreatic (++) Colon (+)
CBT026_HES_Primary Colon (+) AADACL1 60% Colon Tumors CBT026_N Cell
Surface 80% Melanoma Node CBT026_T 33% NHL Node CBT026_HES_Primary
100% Panc Tumors 75% Met Panc Tumors 80% Melanoma ALCAM 40% Breast
Tumors Colon (++) Breast (++) CBT026_ five sets with Cell Surface
30% Bladder Tumors Gastric (++) Colon (++) N primary 20% Colon
Tumors Kidney (++) Gastric (++) Liver (++) Kidney (++) Melanoma (+)
Liver (++) Pancreatic (++) Melanoma (+) SLC2A1 Colon (-) Breast (-)
CBT026_T Cell Surface Pancreatic (-) Colon (-) CBT026_N Lung (-)
Gastric (-) CBT026_HES_Primary Breast (-) Kidney (-) Kidney (-)
Liver (-) Gastric (-) Lung (+) Prostate (-) Melanoma (-) Melanoma
(-) Pancreatic (-) Liver (-) Prostate (-) ITGB1 CBT026_HES_Primary
Cell Surface BSG Lung (-) Lung (-) CBT026_HES_Primary Cell Surface
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20180155421A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20180155421A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References