U.S. patent application number 13/285758 was filed with the patent office on 2012-02-23 for detecting bcl-b expression in cancer and uses thereof.
This patent application is currently assigned to Sanford-Burnham Medical Research Institute. Invention is credited to Maryla Krajewska, John C. Reed.
Application Number | 20120046195 13/285758 |
Document ID | / |
Family ID | 41257349 |
Filed Date | 2012-02-23 |
United States Patent
Application |
20120046195 |
Kind Code |
A1 |
Reed; John C. ; et
al. |
February 23, 2012 |
DETECTING BCL-B EXPRESSION IN CANCER AND USES THEREOF
Abstract
Provided herein are compositions and methods of detecting Bcl-B
expression in cancer cells to prognose, monitor, or select
therapies for cancers such as breast cancer, prostate cancer, lung
cancer, or gastric cancer.
Inventors: |
Reed; John C.; (Rancho Santa
Fe, CA) ; Krajewska; Maryla; (Oceanside, CA) |
Assignee: |
Sanford-Burnham Medical Research
Institute
|
Family ID: |
41257349 |
Appl. No.: |
13/285758 |
Filed: |
October 31, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12433481 |
Apr 30, 2009 |
8071315 |
|
|
13285758 |
|
|
|
|
61048995 |
Apr 30, 2008 |
|
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Current U.S.
Class: |
506/9 ; 435/6.11;
435/6.12; 435/7.23 |
Current CPC
Class: |
C12Q 2600/118 20130101;
C12Q 1/6886 20130101; G01N 33/57484 20130101; C12Q 2600/106
20130101 |
Class at
Publication: |
506/9 ; 435/6.11;
435/6.12; 435/7.23 |
International
Class: |
C40B 30/04 20060101
C40B030/04; G01N 33/566 20060101 G01N033/566; C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under Grant
CA-113318, GM-60554, CA-81534, P30CA06055, and CA114810 awarded by
the National Institutes of Health. The government has certain
rights in the invention.
Claims
1. A method of determining the severity of a breast cancer,
prostate cancer, or lung cancer, comprising examining the
expression of Bcl-B in one or more cells of said cancer, wherein an
increase in the amount of Bcl-B expression in the cancer cells or
an increase in the number or percentage of cells with detectable
Bcl-B expression as compared to that of normal reference cells
indicates an increase in cancer severity.
2. The method of claim 1, wherein an increase in the amount of
Bcl-B expression in the cancer cells as compared to that of normal
reference cells indicates a higher tumor grade.
3. The method of claim 1, wherein an increase in the amount of
Bcl-B expression in the cancer cells as compared to that of normal
reference cells indicates a greater likelihood of death.
4. The method of claim 1, wherein an increase in the amount of
Bcl-B expression in the cancer cells as compared to that of normal
reference cells indicates metastasis.
5. The method of claim 1, wherein an increase in the number or
percentage of cells with detectable Bcl-B expression as compared to
that of normal reference cells indicates a higher tumor grade.
6. The method of claim 1, wherein an increase in the number or
percentage of cells with detectable Bcl-B expression as compared to
that of normal reference cells indicates a greater likelihood of
death.
7. The method of claim 1, wherein an increase in the number or
percentage of cells with detectable Bcl-B expression as compared to
that of normal reference cells indicates metastasis.
8. The method of claim 1, wherein the cancer is breast cancer.
9. The method of claim 1, wherein the cancer is prostate
cancer.
10. The method of claim 1, wherein the cancer is lung cancer.
11. The method of claim 1, wherein the normal reference cells
comprise healthy cells from the same tissue of the subject as the
cancer cells.
12. The method of claim 1, wherein the normal reference cells
comprise non-cancerous cells of similar tissue from the
subject.
13. The method of claim 1, wherein the normal reference cells
comprise non-cancerous cells of similar tissue from one or more
unrelated individuals.
14. A method of determining the severity of a gastric cancer,
comprising examining the expression of Bcl-B in one or more cells
of said gastric cancer, wherein a decrease in the amount of Bcl-B
expression in the cancer cells or a decline in the number or
percentage of cells with detectable Bcl-B expression as compared to
that of normal reference cells indicates an increase in cancer
severity.
15. A method of monitoring the progression of a breast cancer,
prostate cancer, or lung cancer, comprising examining the
expression of Bcl-B in one or more cells of said cancer, wherein an
increase in the amount of Bcl-B expression in the cancer cells or
an increase in the number or percentage of cells with detectable
Bcl-B expression as compared to that of normal reference cells
indicates an increased risk of cancer progression.
16. A method of monitoring the progression of a gastric cancer,
comprising examining the expression of Bcl-B in one or more cells
of said cancer, wherein a decrease in the amount of Bcl-B
expression in the cancer cells or a decrease in the number or
percentage of cells with detectable Bcl-B expression as compared to
that of normal reference cells indicates an increased risk of
cancer progression.
17-18. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of U.S. application Ser. No.
12/433,481, filed Apr. 30, 2009, which claims benefit of U.S.
Provisional Application No. 61/048,995, filed Apr. 30, 2008.
application Ser. No. 12/433,481, filed Apr. 30, 2009, and
Application No. 61/048,995, filed Apr. 30, 2008, are hereby
incorporated herein by reference in their entirety.
REFERENCE TO SEQUENCE LISTING
[0003] The Sequence Listing submitted Oct. 31, 2011 as a text file
named "SBMRI.sub.--38.sub.--8403_AMD_AFD_Sequence_Listing.txt,"
created on Oct. 31, 2011, and having a size of 3,417 bytes is
hereby incorporated by reference pursuant to 37 C.F.R.
.sctn.1.52(e)(5).
BACKGROUND
[0004] Defective apoptosis represents one of the six recognized
cardinal features of cancer (Hanahan D, et al. 2000). Bcl-2-family
proteins are evolutionarily conserved regulators of cell life and
death. In humans, six anti-apoptotic members of the Bcl-2 family
have been identified, including Bcl-2, Bcl-X.sub.L, Mcl-1, Bcl-W,
Bfl-1, and Bcl-B (Reed J C. 2000). Over-expression of Bcl-2 and
some other anti-apoptotic members of the Bcl-2 family has been
documented in human cancers (Reed J C. 1996). Given that
investigational therapies targeting specific Bcl-2-family proteins
or their encoding mRNAs are now in clinical trials, it is important
to define which Bcl-2 family proteins are over-expressed in various
types of cancer, so that appropriate targeted therapies can be
matched to specific malignancies.
BRIEF SUMMARY
[0005] In accordance with the purpose of this invention, as
embodied and broadly described herein, this invention relates to
compositions and methods of detecting Bcl-B expression to prognose,
monitor, or select therapies for cancers.
[0006] Additional advantages of the disclosed method and
compositions will be set forth in part in the description which
follows, and in part will be understood from the description, or
may be learned by practice of the disclosed method and
compositions. The advantages of the disclosed method and
compositions will be realized and attained by means of the elements
and combinations particularly pointed out in the appended claims.
It is to be understood that both the foregoing general description
and the following detailed description are exemplary and
explanatory only and are not restrictive of the invention as
claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate several
embodiments of the disclosed method and compositions and together
with the description, serve to explain the principles of the
disclosed method and compositions.
[0008] FIGS. 1A, 1B, and 1C show characterization of Bcl-B
antibodies and immunodetection of Bcl-B protein in human tissue
lysates. FIG. 1A shows GST fusion proteins containing Bcl-XL,
Bfl-1, Bcl-2, Mcl-1, Bcl-W, and Bcl-B (0.1 .mu.g/lane) analyzed by
immunoblotting using AR-77 antiserum (top). The blot was reprobed
with anti-GST (bottom). Cell lysates from HeLa cells with
tetracycline-inducible Bcl-B are also included (tet on/off). FIGS.
1B and 1C show selected GST-fusion proteins (0.05 .mu.g/lane) and
human tissue lysates normalized for total protein content (50
.mu.g/lane) subjected to SDS-PAGE/immunoblot analysis, using AR-77
[1:2,000 (v/v) (B) or BR-49 [1:3,000 (v/v)] (C) antibodies to Bcl-B
(top). Blots were reprobed with anti-HSP60 and anti-.beta.-actin
antibodies (bottom). Antibody detection was accomplished using an
enhanced chemiluminescence method. Black arrows indicated Bcl-B
momers or SDS-resistant Bcl-B dimers. White arrows indicate
GST-Bcl-B fusion protein.
[0009] FIGS. 2A-2L show immunohistochemical detection of Bcl-B
expression in B cells and plasma cells. FIGS. 2A-2D show serial
sections of normal human lymph node specimen stained with (A)
anti-Bcl-B antiserum (raised against recombinant Bcl-B protein)
(400.times.), (B) preimmune serum (100.times.), (C) anti-Bcl-B
antiserum preabsorbed with GST-Bcl-B (200.times.), and (D)
anti-Bcl-B antiserum preabsorbed with GST-Bcl-xL (200.times.).
Specimens were counterstained with hematoxylin. FIGS. 2E-2H show
human lymph node (E-F) and spleen (G-H) sections containing
secondary follicles (E, H) stained with the Bcl-B antibody to
visualize immunopositive B cells in germinal centers (E, H) and
plasma cells in medullary cords (F) and red pulp (G).
Photomicrographs were taken at original magnifications ranging from
100.times. to 400.times.. FIGS. 2I-2L show TMA containing gut
specimens from patients with Crohn's disease were double stained
with the Bcl-B (DAB, brown) and CD138 (SG, black) antibodies and
counterstained with Nuclear Red. The brown (I) and black (J) colors
were separated in the annotated regions using a color deconvolution
algorithm (Aperio). Quantification of immunohistochemical staining
for Bcl-B (K) and CD138 (L) was performed using color translation
and an automated thresholding algorithm (Aperio). Shown is
colocalization of Bcl-B and CD138 cells. Original magnifications
are 100.times. (I, J) and 400.times. (K, L).
[0010] FIGS. 3A-3H show immunohistochemical detection of Bcl-B
expression in human hematolymphoid malignancies. Bone marrow
biopsies from multiple myeloma patients (A-D), and lymph node
specimens from DLBCL (E, F) and FL (G, H) cases were immunostained
using Bcl-B antiserum. Original magnifications are 150.times. (G),
200.times. (A-C, E), 1000.times. (D, F, H) and 800-1200.times.
(insets).
[0011] FIGS. 4A-4H show distribution of Bcl-B immunostainings in
human nonlymphoid malignancies. Representative Bcl-B immunostaining
results are presented for microarrays of (A-C) breast specimens in
normal mammary epithelium (A, 60.times.), DCIS (B, 200.times.), and
ductal adenocarcinoma (C, 60.times.), (D-E) uterine cervix
specimens in normal cervix (D, 60.times.) and squamous cervical
carcinoma (E, 200.times.), (F-H) gastric specimens in normal
gastric epithelium (F, .times.100; inset .times.300) and gastric
cancers (G, H, 60.times.), (I-J) colon specimens in normal colonic
epithelium (I, 200.times.) and colon cancer (J, 60.times.), and
(K-L) SCLC in primary tumor (K, 200.times.) and LN metastasis (L,
200.times.). Note Bcl-B immunopositive plasma cells in normal (F,
I) and malignant (E, J-L) tissues.
[0012] FIGS. 5A-5F show graphic presentation of Bcl-B IHC results
in human nonlymphoid malignancies. Box and whisker plots display
the distribution of immunopercentage data for Bcl-B in normal
mammary epithelium (NE) vs in situ (IS) and invasive (INV) breast
carcinoma (panel A), and in normal prostatic epithelium (NE) vs
benign prostatic hyperplasia (BPH), prostatic intraepithelial
neoplasia (PIN) and prostate cancer (CA) (panel C). Box and whisker
plots depict Bcl-B immunoscore data for low vs high grade breast
cancers (panel B), for prostate tumors from patients who survived
(S) or died from cancer (D) (panel D), and for well (W), moderately
(M) and poorly (P) differentiated gastric cancers (panel E). The
mean immunopercentage/immunoscore is plotted as a marker; whiskers
reflect .+-.1.96 SE from the mean. FIG. 5F shows Bcl-B
immunopercentage data for SCLC dichotomized into high versus low
expression groups based on the median values. The percentage of
patients remaining alive (ordinate) was compounded over time
(abscissa) (in years), by the Kaplan-Meier method. The log-rank
test was used for correlating the immunostaining data with the
patient survival.
[0013] FIGS. 6A, 6B, and 6C show immunoblot analysis of plasma
Cells confirms Bcl-B expression. Plasma cells (PC) were isolated
from a human normal bone marrow suspension using CD138.sup.+ human
plasma cell isolation kit from Miltenyi Biotec according to the
manufacturer's instruction. Briefly, CD138-expressing cells were
separated by magnetic labeling with CD138 microbeads and enrichment
of labeled cells using magnetic cell sorting (MACS). Giemsa
staining showed enrichment at 86.1.+-.7.3% from 3 experiments. In
addition, plasma cell fractions were isolated from bone marrow
aspirates derived from 4 patients with multiple myeloma. Cell
lysates were analyzed by immunoblotting as described in the
manuscript, probing blots with rabbit anti-Bcl-B antibodies (whole
serum or affinity purified) [top] or with mouse monoclonal
antibodies directed against Hsp60 or .beta.-actin [bottom].
Molecular weight (MW) markers are shown in kilo-Daltons (kDa). FIG.
6A shows immunoblot data for (1) recombinant His6-Bcl-GL; (2)
recombinant GST-Bcl-B, showing bands corresponding to Bcl-B,
GST-Bcl-B, dimers and oligomers of these proteins; and (C) plasma
cell specimen. FIG. 6B shows immunoblot data for (1) GST-Bcl-B
protein, (2-4) plasma cell preparations from normal bone marrow;
and (5) plasma cells isolated from multiple myeloma bone marrow
specimen. FIG. 6C shows immunoblot data for (1) plasma cell
preparation from normal bone marrow and (2) plasma cells isolated
from multiple myeloma bone marrow specimen. Unmodified Bcl-B (arrow
head) and modified (possibly ubiquitinylated) or SDS-resistant
oligomerized forms of Bcl-B protein are seen.
[0014] FIGS. 7A-7D show immunohistochemical analysis of Bcl-B
immunoreactivity in plasma cells. Serial sections of a specimen of
colon from a patient with Crohn's disease were stained with (A)
anti-Bcl-B antiserum (AR-77), (B) preimmune serum, (C) anti-Bcl-B
antiserum preadsorbed with GST-Bcl-B, and (D) anti-Bcl-B antiserum
preadsorbed with GST-Bcl-XL proteins. Antibody detection was
accomplished with diaminobenzidine, followed by hematoxylin
counterstaining of nuclei (400.times. original magnification). Note
that the AR-77 anti-Bcl-B antiserum stains plasma cells (arrows
highlight some examples) and that Bcl-B protein preadsorption
neutralizes staining, unlike Bcl-XL preadsorption.
[0015] FIGS. 8A-8D show Bcl-B expression in secretory cells of
prostate gland. Prostate tissue speciments (A--Normal gland;
B--Adenocarcinoma; C-D--BPH) were stained with anti-Bcl-B
antiserum. Antibody detection was with diaminobenzidine, followed
by hematoxylin counterstaining of nuclei. Shown are various
magnifications (length bars=micrometers), including an inset with
higher magnification in (C). Note that luminal secretory cells are
weakly (normal gland) or strongly (BPH) stained, while basal cells
show no staining (arrows).
DETAILED DESCRIPTION
[0016] The disclosed method and compositions may be understood more
readily by reference to the following detailed description of
particular embodiments and the Example included therein and to the
Figures and their previous and following description.
[0017] Disclosed are materials, compositions, and components that
can be used for, can be used in conjunction with, can be used in
preparation for, or are products of the disclosed method and
compositions. These and other materials are disclosed herein, and
it is understood that when combinations, subsets, interactions,
groups, etc. of these materials are disclosed that while specific
reference of each various individual and collective combinations
and permutation of these compounds may not be explicitly disclosed,
each is specifically contemplated and described herein. For
example, if an antibody is disclosed and discussed and a number of
modifications that can be made to a number of molecules including
the antibody are discussed, each and every combination and
permutation of antibody and the modifications that are possible are
specifically contemplated unless specifically indicated to the
contrary. Thus, if a class of molecules A, B, and C are disclosed
as well as a class of molecules D, E, and F and an example of a
combination molecule, A-D is disclosed, then even if each is not
individually recited, each is individually and collectively
contemplated. Thus, in this example, each of the combinations A-E,
A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated
and should be considered disclosed from disclosure of A, B, and C;
D, E, and F; and the example combination A-D. Likewise, any subset
or combination of these is also specifically contemplated and
disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E
are specifically contemplated and should be considered disclosed
from disclosure of A, B, and C; D, E, and F; and the example
combination A-D. This concept applies to all aspects of this
application including, but not limited to, steps in methods of
making and using the disclosed compositions. Thus, if there are a
variety of additional steps that can be performed it is understood
that each of these additional steps can be performed with any
specific embodiment or combination of embodiments of the disclosed
methods, and that each such combination is specifically
contemplated and should be considered disclosed.
[0018] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the method and
compositions described herein. Such equivalents are intended to be
encompassed by the following claims.
[0019] It is understood that the disclosed method and compositions
are not limited to the particular methodology, protocols, and
reagents described as these may vary. It is also to be understood
that the terminology used herein is for the purpose of describing
particular embodiments only, and is not intended to limit the scope
of the present invention which will be limited only by the appended
claims.
A. GENERAL
[0020] Provided herein are methods relating to examining the
expression of Bcl-B in one or more cells of a cancer, such as
breast cancer, prostate cancer, lung cancer, or gastric cancer.
[0021] Bcl-B (a.k.a. Bcl2-L-10) was the last anti-apoptotic member
of the human Bcl-2 family to be identified (Ke N, et al. 2001;
Zhang H, et al. 2001). Bcl-B contains conserved BH1, BH2, BH3-like,
and BH4 domains, as well as a C-terminal transmembrane domain,
typical of anti-apoptotic Bcl-2-family proteins that target
intracellular membranes of mitochondria (Ke N, et al. 2001; Zhang
H, et al. 2001). Dimerization of pro- and anti-apoptotic
Bcl-2-family proteins plays an important role in controlling their
activity (Chao D T, et al. 1998; Wang H-G, et al. 1998). The Bcl-B
protein was shown to differentially bind pro-apoptotic Bcl-2-family
members (Ke N, et al. 2001). Thus, Bcl-B can have a unique pattern
of selectivity for binding to various pro-apoptotic members of the
Bcl-2 family, indicating a specific role for this protein in
controlling cell life and death.
[0022] Though initially recognized for its anti-apoptotic activity,
the mouse ortholog of Bcl-B reportedly displays either
anti-apoptotic or pro-apoptotic activity, depending on cellular
context (Inohara N, et al. 1998; Song Q, et al. 1999). Bcl-B binds
orphan nuclear receptor Nur77/TR3, which converts the phenotype of
Bcl-B from anti-apoptotic to pro-apoptotic (Luciano F, et al.
2007). Thus, Bcl-B is similar to Bcl-2 in its ability to display
opposing effects on apoptosis, based on protein interactions and
other factors (Lin B, et al. 2004).
[0023] Bcl-B protein expression was investigated in normal human
tissues and in several types of human malignancy by
immunohistochemistry using monospecific antibodies that recognize
Bcl-B and the expression results correlated with clinically
relevant variables.
[0024] Provided herein is a method of determining the severity of a
breast cancer, prostate cancer, or lung cancer comprising examining
the expression of Bcl-B in one or more cells of said cancer. In
some aspects, an increase in the relative amount of Bcl-B
expression in the cancer cells or as compared to that of normal
reference cells indicates an increase in cancer severity. In some
aspects of the method an increase in the amount of Bcl-B expression
in the cancer cells or an increase in the number or percentage of
cells with detectable Bcl-B expression as compared to that of
normal reference cells indicates a higher tumor grade. In some
aspects of the method an increase in the amount of Bcl-B expression
in the cancer cells or an increase in the number or percentage of
cells with detectable Bcl-B expression as compared to that of
normal reference cells indicates a greater likelihood of death. In
some aspects of the method an increase in the amount of Bcl-B
expression in the cancer cells or an increase in the number or
percentage of cells with detectable Bcl-B expression as compared to
that of normal reference cells indicates metastasis. In some
aspects, an increase in the amount of Bcl-B expression in the
breast, prostate or lung cancer cells or an increase in the number
or percentage of breast, prostate or lung cells with detectable
Bcl-B expression as compared to that of normal reference cells
indicates an increase in cancer severity. In some aspects of the
method an increase in the amount of Bcl-B expression in the breast,
prostate of lung cancer cells or an increase in the number or
percentage of breast, prostate or lung cells with detectable Bcl-B
expression as compared to that of normal reference cells indicates
a higher tumor grade. In some aspects of the method an increase in
the amount of Bcl-B expression in the breast, prostate or lung
cancer cells or an increase in the number or percentage of breast,
prostate or lung cells with detectable Bcl-B expression as compared
to that of normal reference cells indicates a greater likelihood of
death. In some aspects of the method an increase in the amount of
Bcl-B expression in the breast, prostate or lung cancer cells or an
increase in the number or percentage of breast, prostate or lung
cells with detectable Bcl-B expression as compared to that of
normal reference cells indicates metastasis.
[0025] Also provided herein is a method of determining the severity
of a gastric cancer comprising examining the expression of Bcl-B in
one or more cells of said cancer. In some aspects of the method a
decrease in the amount of Bcl-B expression in the cancer cells or a
decline in the number or percentage of cells with detectable Bcl-B
expression as compared to that of normal reference cells indicates
an increase in cancer severity. In some aspects of the method a
decrease in the amount of Bcl-B expression in the cancer cells or a
decline in the number or percentage of cells with detectable Bcl-B
expression as compared to that of normal reference cells indicates
a higher tumor grade. In some aspects of the method a decrease in
the amount of Bcl-B expression in the cancer cells or a decline in
the number or percentage of cells with detectable Bcl-B expression
as compared to that of normal reference cells indicates a greater
likelihood of death. In some aspects of the method a decrease in
the amount of Bcl-B expression in the cancer cells or a decline in
the number or percentage of cells with detectable Bcl-B expression
as compared to that of normal reference cells indicates metastasis.
In some aspects of the method a decrease in the amount of Bcl-B
expression in the gastric cancer cells or a decline in the number
or percentage of gastric cells with detectable Bcl-B expression as
compared to that of normal reference cells indicates an increase in
cancer severity. In some aspects of the method a decrease in the
amount of Bcl-B expression in the gastric cancer cells or a decline
in the number or percentage of gastric cells with detectable Bcl-B
expression as compared to that of normal reference cells indicates
a higher tumor grade. In some aspects of the method a decrease in
the amount of Bcl-B expression in the gastric cancer cells or a
decline in the number or percentage of gastric cells with
detectable Bcl-B expression as compared to that of normal reference
cells indicates a greater likelihood of death. In some aspects of
the method a decrease in the amount of Bcl-B expression in the
gastric cancer cells or a decline in the number or percentage of
gastric cells with detectable Bcl-B expression as compared to that
of normal reference cells indicates metastasis.
[0026] Disclosed herein is a method of monitoring the progression
of a breast cancer, prostate cancer, or lung cancer comprising
examining the expression of Bcl-B in one or more cells of said
cancer. In some aspects, an an increase in the amount of Bcl-B
expression in the cancer cells or an increase in the number or
percentage of cells with detectable Bcl-B expression as compared to
that of normal reference cells indicates an increased risk of
cancer progression. In some aspects, an an increase in the amount
of Bcl-B expression in the breast, prostate or lung cancer cells or
an increase in the number or percentage of breast, prostate or lung
cells with detectable Bcl-B expression as compared to that of
normal reference cells indicates an increased risk of cancer
progression.
[0027] Also disclosed herein is a method of monitoring the
progression of a gastric cancer, comprising examining the
expression of Bcl-B in one or more cells of said cancer. In some
aspects, a decrease in the amount of Bcl-B expression in the cancer
cells or a decrease in the number or percentage of cells with
detectable Bcl-B expression as compared to that of normal reference
cells indicates an increased risk of cancer progression. In some
aspects, a decrease in the amount of Bcl-B expression in the
gastric cancer cells or a decrease in the number or percentage of
gastric cells with detectable Bcl-B expression as compared to that
of normal reference cells indicates an increased risk of cancer
progression.
[0028] Reference herein to an increase in the amount of Bcl-B
expression or to an increase in the number or percentage of cells
with detectable Bcl-B includes an at least 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,
110, 120, 130, 140, 150, 160, 170, 180, 190, or 200% increase as
compared to that of normal reference cells.
[0029] Reference herein to an decrease in the amount of Bcl-B
expression or to an decrease in the number or percentage of cells
with detectable Bcl-B includes an at least 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,
110, 120, 130, 140, 150, 160, 170, 180, 190, or 200% decrease as
compared to that of normal reference cells.
[0030] Also disclosed herein is a method of selecting a therapy for
treating a subject with breast cancer or prostate cancer,
comprising examining the expression of Bcl-B in one or more cells
of said cancer. In this regard, it has been controversial how to
optimally treat patients with early stage (stage I/II) prostate and
breast cancer, because staging methods do not detect
micrometastatic disease. If tumor cells have not yet acquired
metastatic competency, then local/regional therapy can be effective
(e.g., surgery, radiotherapy). However, if tumor cells have already
metastasized, then the patient should receive systemic therapy,
such as chemotherapy or anti-hormonal therapy. Testing for Bcl-B
can discriminate those patients at risk of having tumor
micrometastaces and those who need systemic therapy.
[0031] Accordingly, in some aspects, a higher amount of Bcl-B
expression in the cancer cells or a higher number or percentage of
cancer cells with detectable Bcl-B expression as compared to that
of non-metastatic and/or non-neoplastic reference cells indicates
that the selected treatment is a systemic therapy. For example, a
higher amount of Bcl-B expression in the breast or prostate cancer
cells or a higher number or percentage of breast or prostate cancer
cells with detectable Bcl-B expression as compared to that of
non-metastatic and/or non-neoplastic reference cells can indicate
that the selected treatment is adjuvant chemotherapy, hormonal
therapy, or a combination thereof. Other systemic therapies are
known in the art and can be selected by the physician for use in
the disclosed method.
[0032] In some aspects, a lower or equivalent amount of Bcl-B
expression in the cancer cells or a lower or equivalent number or
percentage of cancer cells with detectable Bcl-B expression as
compared to that of non-metastatic and/or non-neoplastic reference
cells indicates that the selected treatment is a local, regional,
or targeted therapy. For example, a lower or equivalent amount of
Bcl-B expression in the breast or prostate cancer cells or a lower
or equivalent number or percentage of breast or prostate cancer
cells with detectable Bcl-B expression as compared to that of
non-metastatic and/or non-neoplastic reference cells can indicate
that the selected treatment is surgery, local therapy, or a
combination thereof. Other targeted therapies, such as
anti-angiogenesis therapy, are known in the art and can be selected
by the physician for use in the disclosed method.
[0033] Also disclosed herein is a method of selecting a therapy for
treating a subject with lung cancer, comprising examining the
expression of Bcl-B in one or more cells of said cancer, wherein a
higher amount of Bcl-B expression in the cancer cells or a higher
number or percentage of cancer cells with detectable Bcl-B
expression as compared to that of non-metastatic and/or
non-neoplastic reference cells indicates that the selected
treatment is aggressive chemotherapy, investigational drugs, or a
combination thereof.
[0034] Reference herein a higher amount of Bcl-B expression or to a
higher number or percentage of cells with detectable Bcl-B includes
at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170,
180, 190, or 200% higher as compared to that of normal reference
cells.
[0035] Reference herein a lower amount of Bcl-B expression or to a
lower number or percentage of cells with detectable Bcl-B includes
at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170,
180, 190, or 200% lower as compared to that of normal reference
cells.
[0036] As used herein, "normal reference cells" can refer to
healthy cells from the same tissue of the subject as the cancer
cells. Alternatively, normal reference cells can refer to
non-cancerous cells of similar tissue from one or more unrelated
individuals.
[0037] As used herein, "non-metastatic reference cells" can refer
to primary cancer cells from the subject. Alternatively,
non-metastatic reference cells can refer to primary cancer cells of
a histologically similar cancer from one or more unrelated
individuals.
[0038] In some aspects, it is not necessary to measure Bcl-B
expression in normal (non-neoplastic) reference cells or
non-metastatic reference cells to practice the methods. In these
aspects, it is sufficient to compare the amount, number, or
percentage identified for the cancer cells of the subject to an
amount, number, or percentage documented for the normal
(non-neoplastic) reference cells or non-metastatic reference
cells.
[0039] The amount of Bcl-B expression in the cancer cells or the
number, or percentage of cells with detectable Bcl-B expression can
be evaluated or measured using standard methods known in the art,
including those disclosed herein. For example, Bcl-B expression can
be detected using immunoassays or nucleic acid detection methods.
The number or percentage of cells with detectable Bcl-B expression
can be evaluated, for example, using cell separation methods.
[0040] 1. Breast Cancer
[0041] The tumor of the disclosed method can be a breast cancer.
Thus, the breast tumor can be ductal carcinoma in situ (DCIS),
lobular carcinoma in situ (LCIS), infiltrating ductal carcinoma
(IDC), infiltrating lobular carcinoma (ILC), medullary carcinoma,
inflammatory breast cancer (IBC), tubular carcinoma (TC), colloid
carcinoma, metaplastic carcinoma, papillary carcinoma, adenoid
cystic carcinoma (ACC), secretory carcinoma, or Paget's disease of
the breast. The breast tumor can be estrogen receptor-negative,
progesterone receptor-negative, and HER2-negative (triple-negative
breast cancer).
[0042] Breast cancers are described along four different
classification schemes, or groups, each based on different criteria
and serving a different purpose: pathology, grade of tumor, protein
& gene expression status, and stage of a tumor.
[0043] A pathologist can categorize each tumor based on its
histological (microscopic anatomy) appearance and other criteria.
The most common pathologic types of breast cancer are invasive
ductal carcinoma, malignant cancer in the breast's ducts, and
invasive lobular carcinoma, malignant cancer in the breast's
lobules. The histological grade of a tumor is determined by a
pathologist under a microscope. A well-differentiated (low grade)
tumor resembles normal tissue. A poorly differentiated (high grade)
tumor is composed of disorganized cells and, therefore, does not
look like normal tissue. Moderately differentiated (intermediate
grade) tumors are somewhere in between.
[0044] Breast cancers can be tested for expression, or detectable
effect, of the estrogen receptor (ER), progesterone receptor (PR)
and HER2/neu proteins. These tests can be done by
immunohistochemistry. The profile of expression of a given tumor
helps predict its prognosis, or outlook, and helps an oncologist
choose the most appropriate treatment. As disclosed herein, the
amount of Bcl-B expression in the cancer cells or the number, or
percentage of cells with detectable Bcl-B expression can further be
used to predict the prognosis and guide the oncologist to choose
the most appropriate treatment.
[0045] The currently accepted staging scheme for breast cancer is
the TNM classification: Tumor, lymph Node, and Metastases. There
are five tumor classification values (Tis, T1, T2, T3 or T4) which
depend on the presence or absence of invasive cancer, the
dimensions of the invasive cancer, and the presence or absence of
invasion outside of the breast (e.g. to the skin of the breast, to
the muscle or to the rib cage underneath). There are four lymph
node classification values (N0, N1, N2 or N3) which depend on the
number, size and location of breast cancer cell deposits in lymph
nodes. There are two metastatic classification values (M0 or M1)
which depend on the presence or absence of breast cancer cells in
locations other than the breast and lymph nodes (so-called distant
metastases, e.g. to bone, brain, lung).
[0046] Breast cancer is diagnosed by the examination of surgically
removed breast tissue. A number of procedures can obtain tissue or
cells prior to definitive treatment for histological or cytological
examination. Such procedures include fine-needle aspiration, nipple
aspirates, ductal lavage, core needle biopsy, and local surgical
excision. These diagnostic steps, when coupled with radiographic
imaging, are usually accurate in diagnosing a breast lesion as
cancer. Occasionally, pre-surgical procedures such as fine needle
aspirate may not yield enough tissue to make a diagnosis, or may
miss the cancer entirely. Imaging tests are sometimes used to
detect metastasis and include chest X-ray, bone scan, Cat scan,
MRI, and PET scanning While imaging studies are useful in
determining the presence of metastatic disease, they are not in and
of themselves diagnostic of cancer. Only microscopic evaluation of
a biopsy specimen can yield a cancer diagnosis. Ca 15.3
(carbohydrate antigen 15.3, epithelial mucin) is a tumor marker
determined in blood which can be used to follow disease activity
over time after definitive treatment. Blood tumor marker testing is
not routinely performed for the screening of breast cancer, and has
poor performance characteristics for this purpose.
[0047] The mainstay of breast cancer treatment is surgery when the
tumor is localized, with possible adjuvant hormonal therapy (with
tamoxifen or an aromatase inhibitor), chemotherapy, and/or
radiotherapy. At present, the treatment recommendations after
surgery (adjuvant therapy) follow a pattern. Depending on clinical
criteria (age, type of cancer, size, metastasis) patients are
roughly divided to high risk and low risk cases, with each risk
category following different rules for therapy. Treatment
possibilities include radiation therapy, chemotherapy, hormone
therapy, and immune therapy.
[0048] Radiation has dramatically altered the management of primary
breast cancer. Breast conservation, using lumpectomy and radiation
therapy, is the treatment of choice in early-stage breast cancer.
Cosmetic results are good in most patients, and survival is not
compromised. Many patients with locally advanced breast cancer show
improvement in local control with radiotherapy, and there is
increased survival following radiation. The disclosed methods can
be used to guide the selection of the appropriate therapy.
[0049] 2. Prostate Cancer
[0050] Prostate cancer is most often discovered by physical
examination or by screening blood tests, such as the PSA (prostate
specific antigen) test. There is some current concern about the
accuracy of the PSA test and its usefulness. Suspected prostate
cancer is typically confirmed by removing a piece of the prostate
(biopsy) and examining it under a microscope. Further tests, such
as X-rays and bone scans, can be performed to determine whether
prostate cancer has spread.
[0051] Prostate cancer can be treated with surgery, radiation
therapy, hormonal therapy, occasionally chemotherapy, proton
therapy, or some combination of these. The age and underlying
health of the man as well as the extent of spread, appearance under
the microscope, and response of the cancer to initial treatment are
important in determining the outcome of the disease. Since prostate
cancer is a disease of older men, many will die of other causes
before a slowly advancing prostate cancer can spread or cause
symptoms. This makes treatment selection difficult. The decision
whether or not to treat localized prostate cancer (a tumor that is
contained within the prostate) with curative intent is a patient
trade-off between the expected beneficial and harmful effects in
terms of patient survival and quality of life. The disclosed
methods can be used to guide the selection of the appropriate
therapy.
[0052] The only test which can fully confirm the diagnosis of
prostate cancer is a biopsy, the removal of small pieces of the
prostate for microscopic examination. However, prior to a biopsy,
several other tools may be used to gather more information about
the prostate and the urinary tract. Cystoscopy shows the urinary
tract from inside the bladder, using a thin, flexible camera tube
inserted down the urethra. Transrectal ultrasonography creates a
picture of the prostate using sound waves from a probe in the
rectum.
[0053] If cancer is suspected, a biopsy is offered. During a
biopsy, a urologist obtains tissue samples from the prostate via
the rectum. The tissue samples are then examined under a microscope
to determine whether cancer cells are present, and to evaluate the
microscopic features (or Gleason score) of any cancer found. Tissue
samples can be stained for the presence of PSA and other tumor
markers in order to determine the origin of maligant cells that
have metastasized.
[0054] An important part of evaluating prostate cancer is
determining the stage, or how far the cancer has spread. Knowing
the stage helps define prognosis and is useful when selecting
therapies. The most common system is the four-stage TNM system
(abbreviated from Tumor/Nodes/Metastases). Its components include
the size of the tumor, the number of involved lymph nodes, and the
presence of any other metastases.
[0055] The most important distinction made by any staging system is
whether or not the cancer is still confined to the prostate. In the
TNM system, clinical T1 and T2 cancers are found only in the
prostate, while T3 and T4 cancers have spread elsewhere. Several
tests can be used to look for evidence of spread. These include
computed tomography to evaluate spread within the pelvis, bone
scans to look for spread to the bones, and endorectal coil magnetic
resonance imaging to closely evaluate the prostatic capsule and the
seminal vesicles. Bone scans should reveal osteoblastic appearance
due to increased bone density in the areas of bone
metastisis--opposite to what is found in many other cancers that
metastasize.
[0056] After a prostate biopsy, a pathologist identifies the grade
of the tumor. The Gleason system can be used to grade prostate
tumors from 2 to 10, where a Gleason score of 10 indicates the most
abnormalities. The pathologist assigns a number from 1 to 5 for the
most common pattern observed under the microscope, then does the
same for the second most common pattern. The sum of these two
numbers is the Gleason score. The Whitmore-Jewett stage is another
method sometimes used. Proper grading of the tumor is critical,
since the grade of the tumor is one of the major factors used to
determine the treatment recommendation.
[0057] Treatment for prostate cancer can involve watchful waiting,
surgery, radiation therapy, High Intensity Focused Ultrasound
(HIFU), chemotherapy, cryosurgery, hormonal therapy, or some
combination. Which option is generally decided based on the stage
of the disease, the Gleason score, and the PSA level. The disclosed
methods can be used to further discriminate between therapeutic
options based on Bcl-B expression.
[0058] If the cancer has spread beyond the prostate, treatment
options significantly change, so most doctors who treat prostate
cancer use a variety of nomograms to predict the probability of
spread. Treatment by watchful waiting, HIFU, radiation therapy,
cryosurgery, and surgery are generally offered to men whose cancer
remains within the prostate. Hormonal therapy and chemotherapy are
often reserved for disease which has spread beyond the prostate.
However, there are exceptions: radiation therapy may be used for
some advanced tumors, and hormonal therapy is used for some early
stage tumors. Cryotherapy, hormonal therapy, and chemotherapy can
also be offered if initial treatment fails and the cancer
progresses.
[0059] Watchful waiting, also called "active surveillance," refers
to observation and regular monitoring without invasive treatment.
Watchful waiting is often used when an early stage, slow-growing
prostate cancer is found in an older man. Watchful waiting may also
be suggested when the risks of surgery, radiation therapy, or
hormonal therapy outweigh the possible benefits. Other treatments
can be started if symptoms develop, or if there are signs that the
cancer growth is accelerating (e.g., rapidly rising PSA, increase
in Gleason score on repeat biopsy, or Bcl-B expression as disclosed
herein).
[0060] Surgical removal of the prostate, or prostatectomy, is a
common treatment either for early stage prostate cancer, or for
cancer which has failed to respond to radiation therapy. The most
common type is radical retropubic prostatectomy, when the surgeon
removes the prostate through an abdominal incision. Another type is
radical perineal prostatectomy, when the surgeon removes the
prostate through an incision in the perineum, the skin between the
scrotum and anus. Radical prostatectomy can also be performed
laparoscopically, through a series of small (1 cm) incisions in the
abdomen, with or without the assistance of a surgical robot.
[0061] Radiation therapy (radiotherapy), can be used to treat all
stages of prostate cancer, or when surgery fails. Radiotherapy uses
ionizing radiation to kill prostate cancer cells. When absorbed in
tissue, Ionizing radiation such as Gamma and x-rays damage the DNA
in cells, which increases the probability of apoptosis (cell
death). Two different kinds of radiation therapy are used in
prostate cancer treatment: external beam radiation therapy and
brachytherapy.
[0062] External beam radiation therapy uses a linear accelerator to
produce high-energy x-rays which are directed in a beam towards the
prostate. A technique called Intensity Modulated Radiation Therapy
(IMRT) can be used to adjust the radiation beam to conform with the
shape of the tumor, allowing higher doses to be given to the
prostate and seminal vesicles with less damage to the bladder and
rectum. External beam radiation therapy is generally given over
several weeks, with daily visits to a radiation therapy center. New
types of radiation therapy can have fewer side effects then
traditional treatment, one of these is Tomotherapy.
[0063] Permanent implant brachytherapy is a popular treatment
choice for patients with low to intermediate risk features, can be
performed on an outpatient basis, and is associated with good
10-year outcomes with relatively low morbidity. It involves the
placement of about 100 small "seeds" containing radioactive
material with a needle through the skin of the perineum directly
into the tumor while under spinal or general anesthetic. These
seeds emit lower-energy X-rays which are only able to travel a
short distance. Although the seeds eventually become inert, they
remain in the prostate permanently. The risk of exposure to others
from men with implanted seeds is generally accepted to be
insignificant.
[0064] Cryosurgery is another method of treating prostate cancer.
It is less invasive than radical prostatectomy, and general
anesthesia is less commonly used. Under ultrasound guidance, metal
rods are inserted through the skin of the perineum into the
prostate. Highly purified Argon gas is used to cool the rods,
freezing the surrounding tissue at -196.degree. C. (-320.degree.
F.). As the water within the prostate cells freeze, the cells die.
The urethra is protected from freezing by a catheter filled with
warm liquid.
[0065] Hormonal therapy uses medications or surgery to block
prostate cancer cells from getting dihydrotestosterone (DHT), a
hormone produced in the prostate and required for the growth and
spread of most prostate cancer cells. Blocking DHT often causes
prostate cancer to stop growing and even shrink. However, hormonal
therapy rarely cures prostate cancer because cancers which
initially respond to hormonal therapy typically become resistant
after one to two years. Hormonal therapy is therefore usually used
when cancer has spread from the prostate. It can also be given to
certain men undergoing radiation therapy or surgery to help prevent
return of their cancer.
[0066] Hormonal therapy for prostate cancer targets the pathways
the body uses to produce DHT. A feedback loop involving the
testicles, the hypothalamus, and the pituitary, adrenal, and
prostate glands controls the blood levels of DHT. First, low blood
levels of DHT stimulate the hypothalamus to produce gonadotropin
releasing hormone (GnRH). GnRH then stimulates the pituitary gland
to produce luteinizing hormone (LH), and LH stimulates the
testicles to produce testosterone. Finally, testosterone from the
testicles and dehydroepiandrosterone from the adrenal glands
stimulate the prostate to produce more DHT. Hormonal therapy can
decrease levels of DHT by interrupting this pathway at any
point.
[0067] There are several forms of hormonal therapy. Orchiectomy is
surgery to remove the testicles. Because the testicles make most of
the body's testosterone, after orchiectomy testosterone levels
drop. Now the prostate not only lacks the testosterone stimulus to
produce DHT, but also it does not have enough testosterone to
transform into DHT.
[0068] Antiandrogens are medications such as flutamide,
bicalutamide, nilutamide, and cyproterone acetate which directly
block the actions of testosterone and DHT within prostate cancer
cells. Medications which block the production of adrenal androgens
such as DHEA include ketoconazole and aminoglutethimide. Because
the adrenal glands only make about 5% of the body's androgens,
these medications are generally used only in combination with other
methods that can block the 95% of androgens made by the testicles.
These combined methods are called total androgen blockade (TAB).
TAB can also be achieved using antiandrogens.
[0069] GnRH action can be interrupted in one of two ways. GnRH
antagonists suppress the production of LH directly, while GnRH
agonists suppress LH through the process of downregulation after an
initial stimulation effect. Abarelix is an example of a GnRH
antagonist, while the GnRH agonists include leuprolide, goserelin,
triptorelin, and buserelin. Initially, GnRH agonists increase the
production of LH. However, because the constant supply of the
medication does not match the body's natural production rhythm,
production of both LH and GnRH decreases after a few weeks.
[0070] HIFU for prostate cancer utilizes high intensity focused
ultrasound (HIFU) to ablate/destroy the tissue of the prostate.
During the HIFU procedure, sound waves are used to heat the
prostate tissue thus destroying the cancerous cells. Essentially,
ultrasonic waves are precisely focused on specific areas of the
prostate to eliminate the prostate cancer with minimal risks of
affecting other tissue or organs. Temperatures at the focal point
of the sound waves can exceed 100.degree. C. HIFU procedure for
prostate cancer is performed using a transrectal probe.
[0071] 3. Gastric Cancer
[0072] Stomach cancer can develop in any part of the stomach and
may spread throughout the stomach and to other organs; particularly
the esophagus and the small intestine. Stomach cancer causes nearly
one million deaths worldwide per year. To find the cause of
symptoms, the doctor asks about the patient's medical history, does
a physical exam, and may order laboratory studies. The patient may
also have one or all of the following exams: gastroscopic exam,
upper GI series (may be called barium roentgenogram), and fecal
occult blood test. Abnormal tissue seen in a gastroscope
examination can be biopsied by the surgeon or gastroenterologist.
This tissue can then be sent to a pathologist for histological
examination under a microscope to check for the presence of
cancerous cells. A biopsy, with subsequent histological analysis
can confirm the presence of cancer cells.
[0073] A condition of darkened hyperplasia of the skin, frequently
of the axilla and groin, known as acanthosis nigricans, commonly
prompts a study into gastric carcinoma. It should be noted that
this hyperplasia can be found in obese individuals with no
underlying cancer.
[0074] If cancer cells are found in the tissue sample, the next
step is to stage, or find out the extent of the disease. Various
tests determine whether the cancer has spread and, if so, what
parts of the body are affected. Because stomach cancer can spread
to the liver, the pancreas, and other organs near the stomach as
well as to the lungs, the doctor may order a CT scan, a PET scan,
an endoscopic ultrasound exam, or other tests to check these areas.
Blood tests for tumor markers, such as carcinoembryonic antigen
(CEA) and carbohydrate antigen (CA) can be ordered, as their levels
correlate to extent of metastasis, especially to the liver, and the
cure rate.
[0075] Like any cancer, treatment is adapted to fit each person's
individual needs and depends on the size, location, and extent of
the tumor, the stage of the disease, and general health. Cancer of
the stomach is difficult to cure unless it is found in an early
stage (before it has begun to spread). Unfortunately, because early
stomach cancer causes few symptoms, the disease is usually advanced
when the diagnosis is made. Treatment for stomach cancer may
include surgery, chemotherapy, and/or radiation therapy. New
treatment approaches such as biological therapy and improved ways
of using current methods are being studied in clinical trials.
[0076] Surgery is the most common treatment for stomach cancer. The
surgeon removes part or all of the stomach, as well as some of the
tissue around the stomach, with the basic goal of removing all
cancer and a margin of normal tissue. Depending on the extent of
invasion and the location of the tumor, surgery may also include
removal of part of the intestine or pancreas. Tumors in the lower
parts of the stomach can call for a Billroth I or Billroth II
procedure. Endoscopic mucosal resection is a treatment for early
gastric cancer that has been pioneered in Japan, but is available
in the United States at some centers. In this procedure, the tumor
is removed from the wall of the stomach using an endoscope, with
the advantage in that it is a smaller operation than removing the
stomach. Surgical interventions are currently curative in less than
40% of cases, and, in cases of metastasis, may only be
palliative.
[0077] Some chemotherapy drugs used in stomach cancer treatment
include: 5-FU (fluorouracil), BCNU (carmustine), methyl-CCNU
(Semustine), and doxorubicin (Adriamycin), as well as Mitomycin C,
and more recently cisplatin and taxotere in various combinations.
Chemotherapy can be given before surgery to shrink the tumor, or as
adjuvant therapy after surgery to destroy remaining cancer cells.
Also available are combination treatments with chemotherapy and
radiation therapy. The anticancer drugs can be put directly into
the abdomen (intraperitoneal hyperthermic chemoperfusion).
[0078] Radiation therapy can be used in combination with surgery
and chemotherapy, or used only with chemotherapy in cases where the
individual is unable to undergo surgery. Radiation therapy can also
be used to relieve pain or blockage by shrinking the tumor for
palliation of incurable disease.
[0079] 4. Lung Cancer
[0080] The vast majority of primary lung cancers are carcinomas of
the lung, derived from epithelial cells. Lung cancer, the most
common cause of cancer-related death in men and the second most
common in women, is responsible for 1.3 million deaths worldwide
annually. The most common symptoms are shortness of breath,
coughing (including coughing up blood), and weight loss.
[0081] The main types of lung cancer are small cell lung carcinoma
and non-small cell lung carcinoma. This distinction is important
because the treatment varies; non-small cell lung carcinoma (NSCLC)
is sometimes treated with surgery, while small cell lung carcinoma
(SCLC) usually responds better to chemotherapy and radiation. The
most common cause of lung cancer is long term exposure to tobacco
smoke. The occurrence of lung cancer in non-smokers, who account
for fewer than 10% of cases, appears to be due to a combination of
genetic factors, radon gas, asbestos, and air pollution, including
second-hand smoke.
[0082] Lung cancer can be seen on chest x-ray and computed
tomography (CT scan). The diagnosis is confirmed with a biopsy.
This is usually performed via bronchoscopy or CT-guided biopsy.
Treatment and prognosis depend upon the histological type of
cancer, the stage (degree of spread), and the patient's performance
status. Possible treatments include surgery, chemotherapy, and
radiotherapy. With treatment, the five-year survival rate is
14%.
[0083] The vast majority of lung cancers are
carcinomas--malignancies that arise from epithelial cells. There
are two main types of lung carcinoma, categorized by the size and
appearance of the malignant cells seen by a histopathologist under
a microscope: non-small cell (80.4%) and small-cell (16.8%) lung
carcinoma. This classification, based on histological criteria, has
important implications for clinical management and prognosis of the
disease.
[0084] The non-small cell lung carcinomas are grouped together
because their prognosis and management are similar. There are three
main sub-types: squamous cell lung carcinoma, adenocarcinoma and
large cell lung carcinoma. Accounting for 31.1% of lung cancers,
squamous cell lung carcinoma usually starts near a central
bronchus. Cavitation and necrosis within the center of the cancer
is a common finding. Well-differentiated squamous cell lung cancers
often grow more slowly than other cancer types. Adenocarcinoma
accounts for 29.4% of lung cancers. It usually originates in
peripheral lung tissue. Most cases of adenocarcinoma are associated
with smoking. However, among people who have never smoked
("never-smokers"), adenocarcinoma is the most common form of lung
cancer. A subtype of adenocarcinoma, the bronchioloalveolar
carcinoma, is more common in female never-smokers, and may have
different responses to treatment. Accounting for 10.7% of lung
cancers, large cell lung carcinoma is a fast-growing form that
develops near the surface of the lung. It is often poorly
differentiated and tends to metastasize early.
[0085] Small cell lung carcinoma (SCLC, also called "oat cell
carcinoma") is less common. It tends to arise in the larger airways
(primary and secondary bronchi) and grows rapidly, becoming quite
large. The "oat" cell contains dense neurosecretory granules
(vesicles containing neuroendocrine hormones) which give this an
endocrine/paraneoplastic syndrome association. While initially more
sensitive to chemotherapy, it ultimately carries a worse prognosis
and is often metastatic at presentation. Small cell lung cancers
are divided into Limited stage and Extensive stage disease. This
type of lung cancer is strongly associated with smoking
[0086] The lung is a common place for metastasis from tumors in
other parts of the body. These cancers are identified by the site
of origin, thus a breast cancer metastasis to the lung is still
known as breast cancer. They often have a characteristic round
appearance on chest x-ray. Primary lung cancers themselves most
commonly metastasize to the adrenal glands, liver, brain, and
bone.
[0087] Lung cancer staging is an assessment of the degree of spread
of the cancer from its original source. It is an important factor
affecting the prognosis and potential treatment of lung cancer.
Non-small cell lung carcinoma is staged from IA ("one A", best
prognosis) to IV ("four", worst prognosis). Small cell lung
carcinoma is classified as limited stage if it is confined to one
half of the chest and within the scope of a single radiotherapy
field. Otherwise it is extensive stage.
[0088] Performing a chest x-ray is the first step if a patient
reports symptoms that may be suggestive of lung cancer. This can
reveal an obvious mass, widening of the mediastinum (suggestive of
spread to lymph nodes there), atelectasis (collapse), consolidation
(pneumonia), or pleural effusion. If there are no x-ray findings
but the suspicion is high (such as a heavy smoker with
blood-stained sputum), bronchoscopy and/or a CT scan can provide
the necessary information. Bronchoscopy or CT-guided biopsy is
often used to identify the tumor type. Treatment for lung cancer
depends on the cancer's specific cell type, how far it has spread,
and the patient's performance status. Common treatments include
surgery, chemotherapy, and radiation therapy. If investigations
confirm lung cancer, CT scan and often positron emission tomography
(PET) can be used to determine whether the disease is localised and
amenable to surgery or whether it has spread to the point where it
cannot be cured surgically. Blood tests and spirometry (lung
function testing) can be used to assess whether the patient is well
enough to be operated on. If spirometry reveals poor respiratory
reserve (often due to chronic obstructive pulmonary disease),
surgery may be contraindicated.
[0089] Surgery itself has an operative death rate of about 4.4%,
depending on the patient's lung function and other risk factors.
Surgery is usually only an option in non-small cell lung carcinoma
limited to one lung, up to stage IIIA. This can be assessed with
medical imaging (computed tomography, positron emission
tomography). A sufficient pre-operative respiratory reserve must be
present to allow adequate lung function after the tissue is
removed.
[0090] Procedures include wedge resection (removal of part of a
lobe), segmentectomy (removal of an anatomic division of a
particular lobe of the lung), lobectomy (one lobe), bilobectomy
(two lobes) or pneumonectomy (whole lung). In patients with
adequate respiratory reserve, lobectomy is generally the preferred
option, as this minimizes the chance of local recurrence. If the
patient does not have enough functional lung for this, wedge
resection can be performed. Radioactive iodine brachytherapy at the
margins of wedge excision can reduce recurrence to that of
lobectomy.
[0091] Small cell lung carcinoma can be treated primarily with
chemotherapy and radiation. Primary chemotherapy can also be given
in metastatic non-small cell lung carcinoma.
[0092] The combination regimen depends on the tumor type. Non-small
cell lung carcinoma can be treated with cisplatin or carboplatin,
in combination with gemcitabine, paclitaxel, docetaxel, etoposide
or vinorelbine. In small cell lung carcinoma, cisplatin and
etoposide can be used. Combinations with carboplatin, gemcitabine,
paclitaxel, vinorelbine, topotecan and irinotecan can also be
used.
[0093] Adjuvant chemotherapy refers to the use of chemotherapy
after surgery to improve the outcome. During surgery, samples are
taken from the lymph nodes. If these samples contain cancer, then
the patient has stage II or III disease. In this situation,
adjuvant chemotherapy can improve survival by up to 15%. For
example, the patient can be treated with platinum-based
chemotherapy (including either cisplatin or carboplatin).
[0094] Radiotherapy is often given together with chemotherapy, and
can be used with curative intent in patients with non-small cell
lung carcinoma who are not eligible for surgery. This form of high
intensity radiotherapy is called radical radiotherapy. A refinement
of this technique is continuous hyperfractionated accelerated
radiotherapy (CHART), where a high dose of radiotherapy is given in
a short time period. For small cell lung carcinoma cases that are
potentially curable, in addition to chemotherapy, chest radiation
is often recommended.
[0095] For both non-small cell lung carcinoma and small cell lung
carcinoma patients, smaller doses of radiation to the chest may be
used for symptom control (palliative radiotherapy). Unlike other
treatments, it is possible to deliver palliative radiotherapy
without confirming the histological diagnosis of lung cancer.
[0096] Patients with limited stage small cell lung carcinoma are
usually given prophylactic cranial irradiation (PCI). This is a
type of radiotherapy to the brain, used to reduce the risk of
metastasis. More recently, PCI has also been shown to be beneficial
in those with extensive small cell lung cancer. In patients whose
cancer has improved following a course of chemotherapy, PCI has
been shown to reduce the cumulative risk of brain metastases within
one year from 40.4% to 14.6%.
[0097] Extracranial stereotactic radiation can be used in the
treatment of early-stage lung cancer. In this form of radiation
therapy, very high doses are delivered in a small number of
sessions using stereotactic targeting techniques. Its use is
primarily in patients who are not surgical candidates due to
medical comorbidities.
[0098] Radiofrequency ablation can be used in the treatment of
bronchogenic carcinoma. It is done by inserting a small heat probe
into the tumor to kill the tumor cells.
[0099] Various molecular targeted therapies have been developed for
the treatment of advanced lung cancer. Gefitinib (Iressa) is one
such drug, which targets the tyrosine kinase domain of the
epidermal growth factor receptor (EGF-R) which is expressed in many
cases of non-small cell lung carcinoma. Erlotinib (Tarceva),
another tyrosine kinase inhibitor, has been shown to increase
survival in lung cancer patients and has recently been approved by
the FDA for second-line treatment of advanced non-small cell lung
carcinoma. The angiogenesis inhibitor bevacizumab (in combination
with paclitaxel and carboplatin) can be used to improve the
survival of patients with advanced non-small cell lung
carcinoma.
[0100] Other treatments include cyclo-oxygenase-2 inhibitors, the
apoptosis promoter exisulind, proteasome inhibitors, bexarotene,
vaccines, ras proto-oncogene inhibition, phosphoinositide 3-kinase
inhibition, histone deacetylase inhibition, and tumor suppressor
gene replacement.
B. METHODS AND COMPOSITIONS
[0101] Bcl-B can be detected using any suitable method, technique
and compositions. Many general assays and assay compositions are
known and can be used or adapted to detect Bcl-B. Certain methods
and compositions may be better suited to detecting Bcl-B
expression, Bcl-B expression levels, or cells expressing Bcl-B.
Those of skill in the art are aware of how to apply known
techniques for these purposes. Some useful methods, techniques and
compositions are discussed below.
[0102] 1. Immunohistochemistry
[0103] Disclosed herein are immunohistochemistry methods that can
be used to detect Bcl-B in cancer cells for use in the methods
disclosed herein. In some aspects, the method comprises
immunolabeling Bcl-B in cancer tissue and creating a digital image
of the immunolabeling. In some aspects, the digital image is
analyzed to count the percentage of immunolabled cells. Other such
adaptations of standard immunohistochemistry methods are known and
can be used in the disclosed methods.
[0104] Immunohistochemistry or IHC refers to the process of
localizing proteins in cells of a tissue section exploiting the
principle of antibodies binding specifically to antigens in
biological tissues. It takes its name from the roots "immuno," in
reference to antibodies used in the procedure, and "histo," meaning
tissue (compare to immunocytochemistry). Immunohistochemical
staining is widely used in the diagnosis of abnormal cells such as
those found in cancerous tumors. Specific molecular markers are
characteristic of particular cellular events such as proliferation
or cell death (apoptosis). IHC is also widely used in basic
research to understand the distribution and localization of
biomarkers and differentially expressed proteins in different parts
of a biological tissue.
[0105] Visualising an antibody-antigen interaction can be
accomplished in a number of ways. In the most common instance, an
antibody is conjugated to an enzyme, such as peroxidase, that can
catalyse a colour-producing reaction (see immunoperoxidase
staining). Alternatively, the antibody can also be tagged to a
fluorophore, such as FITC, rhodamine, Texas Red, Alexa Fluor, or
DyLight Fluor(see immunofluorescence). The latter method is of
great use in confocal laser scanning microscopy, which is highly
sensitive and can also be used to visualise interactions between
multiple proteins.
[0106] There are two strategies used for the immmunohistochemical
detection of antigens in tissue, the direct method and the indirect
method. In both cases, the tissue is treated to rupture the
membranes, usually by using a kind of detergent such as Triton
X-100. Some antigen also need additional step for unmasking,
resulting in better detection results.
[0107] The direct method is a one-step staining method, and
involves a labeled antibody (e.g. FITC conjugated antiserum)
reacting directly with the antigen in tissue sections. This
technique utilizes only one antibody and the procedure is therefore
simple and rapid. However, it can suffer problems with sensitivity
due to little signal amplification and is in less common use than
indirect methods.
[0108] The indirect method involves an unlabeled primary antibody
(first layer) which reacts with tissue antigen, and a labeled
secondary antibody (second layer) which reacts with the primary
antibody. (The secondary antibody must be against the IgG of the
animal species in which the primary antibody has been raised.) This
method is more sensitive due to signal amplification through
several secondary antibody reactions with different antigenic sites
on the primary antibody. The second layer antibody can be labeled
with a fluorescent dye or an enzyme.
[0109] 2. Immunoassay
[0110] Disclosed herein are immunoassays that can be used to detect
Bcl-B in cancer cells for use in the methods disclosed herein. The
steps of various useful immunodetection methods have been described
in the scientific literature, such as, e.g., Maggio et al.,
Enzyme-Immunoassay, (1987) and Nakamura, et al., Enzyme
Immunoassays: Heterogeneous and Homogeneous Systems, Handbook of
Experimental Immunology, Vol. 1: Immunochemistry, 27.1-27.20
(1986), each of which is incorporated herein by reference in its
entirety and specifically for its teaching regarding
immunodetection methods. Immunoassays, in their most simple and
direct sense, are binding assays involving binding between
antibodies and antigen. Many types and formats of immunoassays are
known and all are suitable for detecting the disclosed biomarkers.
Examples of immunoassays are enzyme linked immunosorbent assays
(ELISAs), radioimmunoassays (RIA), radioimmune precipitation assays
(RIPA), immunobead capture assays, Western blotting, dot blotting,
gel-shift assays, Flow cytometry, protein arrays, multiplexed bead
arrays, magnetic capture, in vivo imaging, fluorescence resonance
energy transfer (FRET), and fluorescence recovery/localization
after photobleaching (FRAP/FLAP).
[0111] In general, immunoassays involve contacting a sample
suspected of containing a molecule of interest (such as the
disclosed biomarkers) with an antibody to the molecule of interest
or contacting an antibody to a molecule of interest (such as
antibodies to the disclosed biomarkers) with a molecule that can be
bound by the antibody, as the case may be, under conditions
effective to allow the formation of immunocomplexes. Contacting a
sample with the antibody to the molecule of interest or with the
molecule that can be bound by an antibody to the molecule of
interest under conditions effective and for a period of time
sufficient to allow the formation of immune complexes (primary
immune complexes) is generally a matter of simply bringing into
contact the molecule or antibody and the sample and incubating the
mixture for a period of time long enough for the antibodies to form
immune complexes with, i.e., to bind to, any molecules (e.g.,
antigens) present to which the antibodies can bind. In many forms
of immunoassay, the sample-antibody composition, such as a tissue
section, ELISA plate, dot blot or Western blot, can then be washed
to remove any non-specifically bound antibody species, allowing
only those antibodies specifically bound within the primary immune
complexes to be detected.
[0112] Immunoassays can include methods for detecting or
quantifying the amount of a molecule of interest (such as the
disclosed biomarkers or their antibodies) in a sample, which
methods generally involve the detection or quantitation of any
immune complexes formed during the binding process. In general, the
detection of immunocomplex formation is well known in the art and
can be achieved through the application of numerous approaches.
These methods are generally based upon the detection of a label or
marker, such as any radioactive, fluorescent, biological or
enzymatic tags or any other known label. See, for example, U.S.
Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;
4,275,149 and 4,366,241, each of which is incorporated herein by
reference in its entirety and specifically for teachings regarding
immunodetection methods and labels.
[0113] As used herein, a label can include a fluorescent dye, a
member of a binding pair, such as biotinistreptavidin, a metal
(e.g., gold), or an epitope tag that can specifically interact with
a molecule that can be detected, such as by producing a colored
substrate or fluorescence. Substances suitable for detectably
labeling proteins include fluorescent dyes (also known herein as
fluorochromes and fluorophores) and enzymes that react with
colorometric substrates (e.g., horseradish peroxidase). The use of
fluorescent dyes is generally preferred in the practice of the
invention as they can be detected at very low amounts. Furthermore,
in the case where multiple antigens are reacted with a single
array, each antigen can be labeled with a distinct fluorescent
compound for simultaneous detection. Labeled spots on the array are
detected using a fluorimeter, the presence of a signal indicating
an antigen bound to a specific antibody.
[0114] Fluorophores are compounds or molecules that luminesce.
Typically fluorophores absorb electromagnetic energy at one
wavelength and emit electromagnetic energy at a second wavelength.
Representative fluorophores include, but are not limited to, 1,5
IAEDANS; 1,8-ANS; 4-Methylumbelliferone;
5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM);
5-Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine
(5-TAMRA); 5-Hydroxy Tryptamine (5-HAT); 5-ROX
(carboxy-X-rhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE;
7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD);
7-Hydroxy-4-1 methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine
(ACMA); ABQ; Acid Fuchsin; Acridine Orange; Acridine Red; Acridine
Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin
(Photoprotein); AFPs--AutoFluorescent Protein--(Quantum
Biotechnologies) see sgGFP, sgBFP; Alexa Fluor 350.TM.; Alexa Fluor
430.TM.; Alexa Fluor 488.TM.; Alexa Fluor 532.TM.; Alexa Fluor
546.TM.; Alexa Fluor 568.TM.; Alexa Fluor 594.TM.; Alexa Fluor
633.TM.; Alexa Fluor 647.TM.; Alexa Fluor 660.TM.; Alexa Fluor
680.TM.; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC);
AMC, AMCA-S; Aminomethylcoumarin (AMCA); AMCA-X; Aminoactinomycin
D; Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7;
APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R;
Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG.TM.
CBQCA; ATTO-TAG.TM. FQ; Auramine; Aurophosphine G; Aurophosphine;
BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH);
Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP (Y66H);
Blue Fluorescent Protein; BFP/GFP FRET; Bimane; Bisbenzemide;
Bisbenzimide (Hoechst); bis-BTC; Blancophor FFG; Blancophor SV;
BOBO.TM.-1; BOBO.TM.-3; Bodipy492/515; Bodipy493/503;
Bodipy500/510; Bodipy; 505/515; Bodipy 530/550; Bodipy 542/563;
Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591;
Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy Fl;
Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR;
Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP;
Bodipy TR-X SE; BO-PRO.TM.-1; BO-PRO.TM.-3; Brilliant Sulphoflavin
FF; BTC; BTC-5N; Calcein; Calcein Blue; Calcium Crimson- ; Calcium
Green; Calcium Green-1 Ca.sup.2+ Dye; Calcium Green-2 Ca.sup.2+;
Calcium Green-5N Ca.sup.2+; Calcium Green-C18 Ca.sup.2+; Calcium
Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade
Blue.TM.; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA;
CFP (Cyan Fluorescent Protein); CFP/YFP FRET; Chlorophyll;
Chromomycin A; Chromomycin A; CL-NERF; CMFDA; Coelenterazine;
Coelenterazine cp; Coelenterazine f; Coelenterazine fcp;
Coelenterazine h; Coelenterazine hcp; Coelenterazine ip;
Coelenterazine n; Coelenterazine O; Coumarin Phalloidin;
C-phycocyanine; CPM I Methylcoumarin; CTC; CTC Formazan; Cy2.TM.;
Cy3.1 8; Cy3.5.TM.; Cy3.TM.; Cy5.1 8; Cy5.5.TM.; Cy5.TM.; Cy7.TM.;
Cyan GFP; cyclic AMP Fluorosensor (FiCRhR) Dabcyl; Dansyl; Dansyl
Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl
fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3'DCFDA; DCFH
(Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine
123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di 16-ASP);
Dichlorodihydrofluorescein Diacetate (DCFH); DiD-Lipophilic Tracer;
DiD (DilC18(5)); DIDS; Dihydorhodamine 123 (DHR); Dil (DilC18(3));
I Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DilC18(7)); DM-NERF
(high pH); DNP; Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS;
EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC;
Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin;
EukoLight; Europium (111) chloride; EYFP; Fast Blue; FDA; Feulgen
(Pararosaniline); FIF (Formaldehyd Induced Fluorescence); FITC;
Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein
Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine);
Fluor-Ruby; FluorX; FM 1-43.TM.; FM 4-46; Fura Red.TM. (high pH);
Fura Red.TM./Fluo-3; Fura-2; Fura-2/BCECF; Genacryl Brilliant Red
B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl
Yellow 5GF; GeneBlazer; (CCF2); GFP (S65T); GFP red shifted
(rsGFP); GFP wild type' non-UV excitation (wtGFP); GFP wild type,
UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular blue;
Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580;
HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold);
Hydroxytryptamine; Indo-1, high calcium; Indo-1 low calcium;
Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf;
JC-1; JO JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751
(RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine
Rhodamine; Lissamine Rhodamine B; Calcein/Ethidium homodimer;
LOLO-1; LO-PRO-1; ; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker
Blue-White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker
Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow/Blue;
Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2;
Mag-Fura-5; Mag-Indo-1; Magnesium Green; Magnesium Orange;
Malachite Green; Marina Blue; I Maxilon Brilliant Flavin 10 GFF;
Maxilon Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin;
Mitotracker Green FM; Mitotracker Orange; Mitotracker Red;
Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH);
Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD
Amine; Nile Red; Nitrobenzoxedidole; Noradrenaline; Nuclear Fast
Red; i Nuclear Yellow; Nylosan Brilliant lavin EBG; Oregon
Green.TM.; Oregon Green.TM. 488; Oregon Green.TM. 500; Oregon
Green.TM. 514; Pacific Blue; Pararosaniline (Feulgen); PBFI;
PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed (Red 613); Phloxin
B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite
RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin
R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1;
POPO-3; PO-PRO-1; PO-I PRO-3; Primuline; Procion Yellow; Propidium
Iodid (P1); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant
Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin; RH 414; Rhod-2;
Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine
6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB;
Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine:
Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal;
R-phycocyanine; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L;
S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B;
Sevron Brilliant Red 4G; Sevron I Brilliant Red B; Sevron Orange;
Sevron Yellow L; sgBFP.TM. (super glow BFP); sgGFP.TM. (super glow
GFP); SITS (Primuline; Stilbene Isothiosulphonic Acid); SNAFL
calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; Sodium Green;
SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ
(6-methoxy-N-(3 sulfopropyl) quinolinium); Stilbene;
Sulphorhodamine B and C; Sulphorhodamine Extra; SYTO 11; SYTO 12;
SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO
21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42;
SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO
63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85;
SYTOX Blue; SYTOX Green; SYTOX Orange; Tetracycline;
Tetramethylrhodamine (TRITC); Texas Red.TM.; Texas Red-X.TM.
conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole
Orange; Thioflavin 5; Thioflavin S; Thioflavin TON; Thiolyte;
Thiozole Orange; Tinopol CBS (Calcofluor White); TIER; TO-PRO-1;
TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC
TetramethylRodaminelsoThioCyanate; True Blue; Tru Red; Ultralite;
Uranine B; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; Xylene
Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO 3;
YOYO-1;YOYO-3; Sybr Green; Thiazole orange (interchelating dyes);
semiconductor nanoparticles such as quantum dots; or caged
fluorophore (which can be activated with light or other
electromagnetic energy source), or a combination thereof.
[0115] A modifier unit such as a radionuclide can be incorporated
into or attached directly to any of the compounds described herein
by halogenation. Examples of radionuclides useful in this
embodiment include, but are not limited to, tritium, iodine-125,
iodine-131, iodine-123, iodine-124, astatine-210, carbon-11,
carbon-14, nitrogen-13, fluorine-18. In another aspect, the
radionuclide can be attached to a linking group or bound by a
chelating group, which is then attached to the compound directly or
by means of a linker. Examples of radionuclides useful in the apset
include, but are not limited to, Tc-99m, Re-186, Ga-68, Re-188,
Y-90, Sm-153, Bi-212, Cu-67, Cu-64, and Cu-62. Radiolabeling
techniques such as these are routinely used in the
radiopharmaceutical industry.
[0116] The radiolabeled compounds are useful as imaging agents to
diagnose neurological disease (e.g., a neurodegenerative disease)
or a mental condition or to follow the progression or treatment of
such a disease or condition in a mammal (e.g., a human). The
radiolabeled compounds described herein can be conveniently used in
conjunction with imaging techniques such as positron emission
tomography (PET) or single photon emission computerized tomography
(SPECT).
[0117] Labeling can be either direct or indirect. In direct
labeling, the detecting antibody (the antibody for the molecule of
interest) or detecting molecule (the molecule that can be bound by
an antibody to the molecule of interest) include a label. Detection
of the label indicates the presence of the detecting antibody or
detecting molecule, which in turn indicates the presence of the
molecule of interest or of an antibody to the molecule of interest,
respectively. In indirect labeling, an additional molecule or
moiety is brought into contact with, or generated at the site of,
the immunocomplex. For example, a signal-generating molecule or
moiety such as an enzyme can be attached to or associated with the
detecting antibody or detecting molecule. The signal-generating
molecule can then generate a detectable signal at the site of the
immunocomplex. For example, an enzyme, when supplied with suitable
substrate, can produce a visible or detectable product at the site
of the immunocomplex. ELISAs use this type of indirect
labeling.
[0118] As another example of indirect labeling, an additional
molecule (which can be referred to as a binding agent) that can
bind to either the molecule of interest or to the antibody (primary
antibody) to the molecule of interest, such as a second antibody to
the primary antibody, can be contacted with the immunocomplex. The
additional molecule can have a label or signal-generating molecule
or moiety. The additional molecule can be an antibody, which can
thus be termed a secondary antibody. Binding of a secondary
antibody to the primary antibody can form a so-called sandwich with
the first (or primary) antibody and the molecule of interest. The
immune complexes can be contacted with the labeled, secondary
antibody under conditions effective and for a period of time
sufficient to allow the formation of secondary immune complexes.
The secondary immune complexes can then be generally washed to
remove any non-specifically bound labeled secondary antibodies, and
the remaining label in the secondary immune complexes can then be
detected. The additional molecule can also be or include one of a
pair of molecules or moieties that can bind to each other, such as
the biotin/avadin pair. In this mode, the detecting antibody or
detecting molecule should include the other member of the pair.
[0119] Other modes of indirect labeling include the detection of
primary immune complexes by a two step approach. For example, a
molecule (which can be referred to as a first binding agent), such
as an antibody, that has binding affinity for the molecule of
interest or corresponding antibody can be used to form secondary
immune complexes, as described above. After washing, the secondary
immune complexes can be contacted with another molecule (which can
be referred to as a second binding agent) that has binding affinity
for the first binding agent, again under conditions effective and
for a period of time sufficient to allow the formation of immune
complexes (thus forming tertiary immune complexes). The second
binding agent can be linked to a detectable label or
signal-generating molecule or moiety, allowing detection of the
tertiary immune complexes thus formed. This system can provide for
signal amplification.
[0120] Immunoassays that involve the detection of as substance,
such as a protein or an antibody to a specific protein, include
label-free assays, protein separation methods (i.e.,
electrophoresis), solid support capture assays, or in vivo
detection. Label-free assays are generally diagnostic means of
determining the presence or absence of a specific protein, or an
antibody to a specific protein, in a sample. Protein separation
methods are additionally useful for evaluating physical properties
of the protein, such as size or net charge. Capture assays are
generally more useful for quantitatively evaluating the
concentration of a specific protein, or antibody to a specific
protein, in a sample. Finally, in vivo detection is useful for
evaluating the spatial expression patterns of the substance, i.e.,
where the substance can be found in a subject, tissue or cell.
[0121] Provided that the concentrations are sufficient, the
molecular complexes ([Ab-Ag]n) generated by antibody-antigen
interaction are visible to the naked eye, but smaller amounts may
also be detected and measured due to their ability to scatter a
beam of light. The formation of complexes indicates that both
reactants are present, and in immunoprecipitation assays a constant
concentration of a reagent antibody is used to measure specific
antigen ([Ab-Ag]n), and reagent antigens are used to detect
specific antibody ([Ab-Ag]n). If the reagent species is previously
coated onto cells (as in hemagglutination assay) or very small
particles (as in latex agglutination assay), "clumping" of the
coated particles is visible at much lower concentrations. A variety
of assays based on these elementary principles are in common use,
including Ouchterlony immunodiffusion assay, rocket
immunoelectrophoresis, and immunoturbidometric and nephelometric
assays. The main limitations of such assays are restricted
sensitivity (lower detection limits) in comparison to assays
employing labels and, in some cases, the fact that very high
concentrations of analyte can actually inhibit complex formation,
necessitating safeguards that make the procedures more complex.
Some of these Group 1 assays date right back to the discovery of
antibodies and none of them have an actual "label" (e.g. Ag-enz).
Other kinds of immunoassays that are label free depend on
immunosensors, and a variety of instruments that can directly
detect antibody-antigen interactions are now commercially
available. Most depend on generating an evanescent wave on a sensor
surface with immobilized ligand, which allows continuous monitoring
of binding to the ligand. Immunosensors allow the easy
investigation of kinetic interactions and, with the advent of
lower-cost specialized instruments, may in the future find wide
application in immunoanalysis.
[0122] The use of immunoassays to detect a specific protein can
involve the separation of the proteins by electophoresis.
Electrophoresis is the migration of charged molecules in solution
in response to an electric field. Their rate of migration depends
on the strength of the field; on the net charge, size and shape of
the molecules and also on the ionic strength, viscosity and
temperature of the medium in which the molecules are moving. As an
analytical tool, electrophoresis is simple, rapid and highly
sensitive. It is used analytically to study the properties of a
single charged species, and as a separation technique.
[0123] An immunoassay that uses electrophoresis that is
contemplated in the current methods is Western blot analysis.
Western blotting or immunoblotting allows the determination of the
molecular mass of a protein and the measurement of relative amounts
of the protein present in different samples. Detection methods
include chemiluminescence and chromagenic detection. Standard
methods for Western blot analysis can be found in, for example, D.
M. Bollag et al., Protein Methods (2d edition 1996) and E. Harlow
& D. Lane, Antibodies, a Laboratory Manual (1988), U.S. Pat.
No. 4,452,901, each of which is herein incorporated by reference in
their entirety for teachings regarding Western blot methods.
Generally, proteins are separated by gel electrophoresis, usually
SDS-PAGE. The proteins are transferred to a sheet of special
blotting paper, e.g., nitrocellulose, though other types of paper,
or membranes, can be used. The proteins retain the same pattern of
separation they had on the gel. The blot is incubated with a
generic protein (such as milk proteins) to bind to any remaining
sticky places on the nitrocellulose. An antibody is then added to
the solution which is able to bind to its specific protein.
[0124] The attachment of specific antibodies to specific
immobilized antigens can be readily visualized by indirect enzyme
immunoassay techniques, usually using a chromogenic substrate (e.g.
alkaline phosphatase or horseradish peroxidase) or chemiluminescent
substrates. Other possibilities for probing include the use of
fluorescent or radioisotope labels (e.g., fluorescein, .sup.125I).
Probes for the detection of antibody binding can be conjugated
anti-immunoglobulins, conjugated staphylococcal Protein A (binds
IgG), or probes to biotinylated primary antibodies (e.g.,
conjugated avidin/streptavidin).
[0125] The power of the technique lies in the simultaneous
detection of a specific protein by means of its antigenicity, and
its molecular mass. Proteins are first separated by mass in the
SDS-PAGE, then specifically detected in the immunoassay step. Thus,
protein standards (ladders) can be run simultaneously in order to
approximate molecular mass of the protein of interest in a
heterogeneous sample.
[0126] Radioimmune Precipitation Assay (RIPA) is a sensitive assay
using radiolabeled antigens to detect specific antibodies in serum.
The antigens are allowed to react with the serum and then
precipitated using a special reagent such as, for example, protein
A sepharose beads. The bound radiolabeled immunoprecipitate is then
commonly analyzed by gel electrophoresis. Radioimmunoprecipitation
assay (RIPA) is often used as a confirmatory test for diagnosing
the presence of HIV antibodies. RIPA is also referred to in the art
as Farr Assay, Precipitin Assay, Radioimmune Precipitin Assay;
Radioimmunoprecipitation Analysis; Radioimmunoprecipitation
Analysis, and Radioimmunoprecipitation Analysis.
[0127] Also contemplated are immunoassays wherein the protein or
antibody specific for the protein is bound to a solid support
(e.g., tube, well, bead, or cell) to capture the antibody or
protein of interest, respectively, from a sample, combined with a
method of detecting the protein or antibody specific for the
protein on the support. Examples of such immunoassays include
Radioimmunoassay (RIA), Enzyme-Linked Immunosorbent Assay (ELISA),
Flow cytometry, protein array, multiplexed bead assay, and magnetic
capture.
[0128] Radioimmunoassay (RIA) is a classic quantitative assay for
detection of antigen-antibody reactions using a radioactively
labeled substance (radioligand), either directly or indirectly, to
measure the binding of the unlabeled substance to a specific
antibody or other receptor system. Radioimmunoassay is used, for
example, to test hormone levels in the blood without the need to
use a bioassay. Non-immunogenic substances (e.g., haptens) can also
be measured if coupled to larger carrier proteins (e.g., bovine
gamma-globulin or human serum albumin) capable of inducing antibody
formation. RIA involves mixing a radioactive antigen (because of
the ease with which iodine atoms can be introduced into tyrosine
residues in a protein, the radioactive isotopes .sup.125I or
.sup.131I are often used) with antibody to that antigen. The
antibody is generally linked to a solid support, such as a tube or
beads. Unlabeled or "cold" antigen is then adding in known
quantities and measuring the amount of labeled antigen displaced.
Initially, the radioactive antigen is bound to the antibodies. When
cold antigen is added, the two compete for antibody binding
sites--and at higher concentrations of cold antigen, more binds to
the antibody, displacing the radioactive variant. The bound
antigens are separated from the unbound ones in solution and the
radioactivity of each used to plot a binding curve. The technique
is both extremely sensitive, and specific.
[0129] Enzyme-Linked Immunosorbent Assay (ELISA), or more
generically termed EIA (Enzyme ImmunoAssay), is an immunoassay that
can detect an antibody specific for a protein. In such an assay, a
detectable label bound to either an antibody-binding or
antigen-binding reagent is an enzyme. When exposed to its
substrate, this enzyme reacts in such a manner as to produce a
chemical moiety which can be detected, for example, by
spectrophotometric, fluorometric or visual means. Enzymes which can
be used to detectably label reagents useful for detection include,
but are not limited to, horseradish peroxidase, alkaline
phosphatase, glucose oxidase, .beta.-galactosidase, ribonuclease,
urease, catalase, malate dehydrogenase, staphylococcal nuclease,
asparaginase, yeast alcohol dehydrogenase, alpha.-glycerophosphate
dehydrogenase, triose phosphate isomerase, glucose-6-phosphate
dehydrogenase, glucoamylase and acetylcholinesterase. For
descriptions of ELISA procedures, see Voller, A. et al., J. Clin.
Pathol. 31:507-520 (1978); Butler, J. E., Meth. Enzymol. 73:482-523
(1981); Maggio, E. (ed.), Enzyme Immunoassay, CRC Press, Boca
Raton, 1980; Butler, J. E., In: Structure of Antigens, Vol. 1 (Van
Regenmortel, M., CRC Press, Boca Raton, 1992, pp. 209-259; Butler,
J. E., In: van Oss, C. J. et al., (eds), Immunochemistry, Marcel
Dekker, Inc., New York, 1994, pp. 759-803; Butler, J. E. (ed.),
Immunochemistry of Solid-Phase Immunoassay, CRC Press, Boca Raton,
1991); Crowther, "ELISA: Theory and Practice," In: Methods in
Molecule Biology, Vol. 42, Humana Press; New Jersey, 1995;U.S. Pat.
No. 4,376,110, each of which is incorporated herein by reference in
its entirety and specifically for teachings regarding ELISA
methods.
[0130] Variations of ELISA techniques are know to those of skill in
the art. In one variation, antibodies that can bind to proteins can
be immobilized onto a selected surface exhibiting protein affinity,
such as a well in a polystyrene microtiter plate. Then, a test
composition suspected of containing a marker antigen can be added
to the wells. After binding and washing to remove non-specifically
bound immunocomplexes, the bound antigen can be detected. Detection
can be achieved by the addition of a second antibody specific for
the target protein, which is linked to a detectable label. This
type of ELISA is a simple "sandwich ELISA." Detection also can be
achieved by the addition of a second antibody, followed by the
addition of a third antibody that has binding affinity for the
second antibody, with the third antibody being linked to a
detectable label.
[0131] Another variation is a competition ELISA. In competition
ELISA's, test samples compete for binding with known amounts of
labeled antigens or antibodies. The amount of reactive species in
the sample can be determined by mixing the sample with the known
labeled species before or during incubation with coated wells. The
presence of reactive species in the sample acts to reduce the
amount of labeled species available for binding to the well and
thus reduces the ultimate signal.
[0132] Regardless of the format employed, ELISAs have certain
features in common, such as coating, incubating or binding, washing
to remove non-specifically bound species, and detecting the bound
immunecomplexes. Antigen or antibodies can be linked to a solid
support, such as in the form of plate, beads, dipstick, membrane or
column matrix, and the sample to be analyzed applied to the
immobilized antigen or antibody. In coating a plate with either
antigen or antibody, one will generally incubate the wells of the
plate with a solution of the antigen or antibody, either overnight
or for a specified period of hours. The wells of the plate can then
be washed to remove incompletely adsorbed material. Any remaining
available surfaces of the wells can then be "coated" with a
nonspecific protein that is antigenically neutral with regard to
the test antisera. These include bovine serum albumin (BSA), casein
and solutions of milk powder. The coating allows for blocking of
nonspecific adsorption sites on the immobilizing surface and thus
reduces the background caused by nonspecific binding of antisera
onto the surface.
[0133] In ELISAs, a secondary or tertiary detection means rather
than a direct procedure can also be used. Thus, after binding of a
protein or antibody to the well, coating with a non-reactive
material to reduce background, and washing to remove unbound
material, the immobilizing surface is contacted with the control
clinical or biological sample to be tested under conditions
effective to allow immunecomplex (antigen/antibody) formation.
Detection of the immunecomplex then requires a labeled secondary
binding agent or a secondary binding agent in conjunction with a
labeled third binding agent.
[0134] "Under conditions effective to allow immunecomplex
(antigen/antibody) formation" means that the conditions include
diluting the antigens and antibodies with solutions such as BSA,
bovine gamma globulin (BGG) and phosphate buffered saline
(PBS)/Tween so as to reduce non-specific binding and to promote a
reasonable signal to noise ratio.
[0135] The suitable conditions also mean that the incubation is at
a temperature and for a period of time sufficient to allow
effective binding. Incubation steps can typically be from about 1
minute to twelve hours, at temperatures of about 20.degree. to
30.degree. C., or can be incubated overnight at about 0.degree. C.
to about 10.degree. C.
[0136] Following all incubation steps in an ELISA, the contacted
surface can be washed so as to remove non-complexed material. A
washing procedure can include washing with a solution such as
PBS/Tween or borate buffer. Following the formation of specific
immunecomplexes between the test sample and the originally bound
material, and subsequent washing, the occurrence of even minute
amounts of immunecomplexes can be determined.
[0137] To provide a detecting means, the second or third antibody
can have an associated label to allow detection, as described
above. This can be an enzyme that can generate color development
upon incubating with an appropriate chromogenic substrate. Thus,
for example, one can contact and incubate the first or second
immunecomplex with a labeled antibody for a period of time and
under conditions that favor the development of further
immunecomplex formation (e.g., incubation for 2 hours at room
temperature in a PBS-containing solution such as PBS-Tween).
[0138] After incubation with the labeled antibody, and subsequent
to washing to remove unbound material, the amount of label can be
quantified, e.g., by incubation with a chromogenic substrate such
as urea and bromocresol purple or
2,2'-azido-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS] and
H.sub.2O.sub.2, in the case of peroxidase as the enzyme label.
Quantitation can then be achieved by measuring the degree of color
generation, e.g., using a visible spectra spectrophotometer.
[0139] Protein arrays are solid-phase ligand binding assay systems
using immobilized proteins on surfaces which include glass,
membranes, microtiter wells, mass spectrometer plates, and beads or
other particles. The assays are highly parallel (multiplexed) and
often miniaturized (microarrays, protein chips). Their advantages
include being rapid and automatable, capable of high sensitivity,
economical on reagents, and giving an abundance of data for a
single experiment. Bioinformatics support is important; the data
handling demands sophisticated software and data comparison
analysis. However, the software can be adapted from that used for
DNA arrays, as can much of the hardware and detection systems.
[0140] One of the chief formats is the capture array, in which
ligand-binding reagents, which are usually antibodies but can also
be alternative protein scaffolds, peptides or nucleic acid
aptamers, are used to detect target molecules in mixtures such as
plasma or tissue extracts. In diagnostics, capture arrays can be
used to carry out multiple immunoassays in parallel, both testing
for several analytes in individual sera for example and testing
many serum samples simultaneously. In proteomics, capture arrays
are used to quantitate and compare the levels of proteins in
different samples in health and disease, i.e. protein expression
profiling. Proteins other than specific ligand binders are used in
the array format for in vitro functional interaction screens such
as protein-protein, protein-DNA, protein-drug, receptor-ligand,
enzyme-substrate, etc. The capture reagents themselves are selected
and screened against many proteins, which can also be done in a
multiplex array format against multiple protein targets.
[0141] For construction of arrays, sources of proteins include
cell-based expression systems for recombinant proteins,
purification from natural sources, production in vitro by cell-free
translation systems, and synthetic methods for peptides. Many of
these methods can be automated for high throughput production. For
capture arrays and protein function analysis, it is important that
proteins should be correctly folded and functional; this is not
always the case, e.g. where recombinant proteins are extracted from
bacteria under denaturing conditions. Nevertheless, arrays of
denatured proteins are useful in screening antibodies for
cross-reactivity, identifying autoantibodies and selecting ligand
binding proteins.
[0142] Protein arrays have been designed as a miniaturization of
familiar immunoassay methods such as ELISA and dot blotting, often
utilizing fluorescent readout, and facilitated by robotics and high
throughput detection systems to enable multiple assays to be
carried out in parallel. Commonly used physical supports include
glass slides, silicon, microwells, nitrocellulose or PVDF
membranes, and magnetic and other microbeads. While microdrops of
protein delivered onto planar surfaces are the most familiar
format, alternative architectures include CD centrifugation devices
based on developments in microfluidics (Gyros, Monmouth Junction,
N.J.) and specialised chip designs, such as engineered
microchannels in a plate (e.g., The Living Chip.TM., Biotrove,
Woburn, Mass.) and tiny 3D posts on a silicon surface (Zyomyx,
Hayward Calif.). Particles in suspension can also be used as the
basis of arrays, providing they are coded for identification;
systems include colour coding for microbeads (Luminex, Austin,
Tex.; Bio-Rad Laboratories) and semiconductor nanocrystals (e.g.,
QDots.TM., Quantum Dot, Hayward, Calif.), and barcoding for beads
(UltraPlex.TM., SmartBead Technologies Ltd, Babraham, Cambridge,
UK) and multimetal microrods (e.g., Nanobarcodes.TM. particles,
Nanoplex Technologies, Mountain View, Calif.). Beads can also be
assembled into planar arrays on semiconductor chips (LEAPS
technology, BioArray Solutions, Warren, N.J.).
[0143] Immobilization of proteins involves both the coupling
reagent and the nature of the surface being coupled to. A good
protein array support surface is chemically stable before and after
the coupling procedures, allows good spot morphology, displays
minimal nonspecific binding, does not contribute a background in
detection systems, and is compatible with different detection
systems. The immobilization method used are reproducible,
applicable to proteins of different properties (size, hydrophilic,
hydrophobic), amenable to high throughput and automation, and
compatible with retention of fully functional protein activity.
Orientation of the surface-bound protein is recognized as an
important factor in presenting it to ligand or substrate in an
active state; for capture arrays the most efficient binding results
are obtained with orientated capture reagents, which generally
require site-specific labeling of the protein.
[0144] Both covalent and noncovalent methods of protein
immobilization are used and have various pros and cons. Passive
adsorption to surfaces is methodologically simple, but allows
little quantitative or orientational control; it may or may not
alter the functional properties of the protein, and reproducibility
and efficiency are variable. Covalent coupling methods provide a
stable linkage, can be applied to a range of proteins and have good
reproducibility; however, orientation may be variable, chemical
derivatization may alter the function of the protein and requires a
stable interactive surface. Biological capture methods utilizing a
tag on the protein provide a stable linkage and bind the protein
specifically and in reproducible orientation, but the biological
reagent must first be immobilized adequately and the array may
require special handling and have variable stability.
[0145] Several immobilization chemistries and tags have been
described for fabrication of protein arrays. Substrates for
covalent attachment include glass slides coated with amino- or
aldehyde-containing silane reagents. In the Versalinx.TM. system
(Prolinx, Bothell, Wash.) reversible covalent coupling is achieved
by interaction between the protein derivatised with phenyldiboronic
acid, and salicylhydroxamic acid immobilized on the support
surface. This also has low background binding and low intrinsic
fluorescence and allows the immobilized proteins to retain
function. Noncovalent binding of unmodified protein occurs within
porous structures such as HydroGel.TM. (PerkinElmer, Wellesley,
Mass.), based on a 3-dimensional polyacrylamide gel; this substrate
is reported to give a particularly low background on glass
microarrays, with a high capacity and retention of protein
function. Widely used biological coupling methods are through
biotin/streptavidin or hexahistidine/Ni interactions, having
modified the protein appropriately. Biotin may be conjugated to a
poly-lysine backbone immobilised on a surface such as titanium
dioxide (Zyomyx) or tantalum pentoxide (Zeptosens, Witterswil,
Switzerland).
[0146] Array fabrication methods include robotic contact printing,
ink-jetting, piezoelectric spotting and photolithography. A number
of commercial arrayers are available [e.g. Packard Biosciences] as
well as manual equipment [V & P Scientific]. Bacterial colonies
can be robotically gridded onto PVDF membranes for induction of
protein expression in situ.
[0147] At the limit of spot size and density are nanoarrays, with
spots on the nanometer spatial scale, enabling thousands of
reactions to be performed on a single chip less than 1 mm square.
BioForce Laboratories have developed nanoarrays with 1521 protein
spots in 85 sq microns, equivalent to 25 million spots per sq cm,
at the limit for optical detection; their readout methods are
fluorescence and atomic force microscopy (AFM).
[0148] Fluorescence labeling and detection methods are widely used.
The same instrumentation as used for reading DNA microarrays is
applicable to protein arrays. For differential display, capture
(e.g., antibody) arrays can be probed with fluorescently labeled
proteins from two different cell states, in which cell lysates are
directly conjugated with different fluorophores (e.g. Cy-3, Cy-5)
and mixed, such that the color acts as a readout for changes in
target abundance. Fluorescent readout sensitivity can be amplified
10-100 fold by tyramide signal amplification (TSA) (PerkinElmer
Lifesciences). Planar waveguide technology (Zeptosens) enables
ultrasensitive fluorescence detection, with the additional
advantage of no intervening washing procedures. High sensitivity
can also be achieved with suspension beads and particles, using
phycoerythrin as label (Luminex) or the properties of semiconductor
nanocrystals (Quantum Dot). A number of novel alternative readouts
have been developed, especially in the commercial biotech arena.
These include adaptations of surface plasmon resonance (HTS
Biosystems, Intrinsic Bioprobes, Tempe, Ariz.), rolling circle DNA
amplification (Molecular Staging, New Haven Conn.), mass
spectrometry (Intrinsic Bioprobes; Ciphergen, Fremont, Calif.),
resonance light scattering (Genicon Sciences, San Diego, Calif.)
and atomic force microscopy [BioForce Laboratories].
[0149] Capture arrays form the basis of diagnostic chips and arrays
for expression profiling. They employ high affinity capture
reagents, such as conventional antibodies, single domains,
engineered scaffolds, peptides or nucleic acid aptamers, to bind
and detect specific target ligands in high throughput manner.
[0150] Antibody arrays have the required properties of specificity
and acceptable background, and some are available commercially (BD
Biosciences, San Jose, Calif.; Clontech, Mountain View, Calif.;
BioRad; Sigma, St. Louis, Mo.). Antibodies for capture arrays are
made either by conventional immunization (polyclonal sera and
hybridomas), or as recombinant fragments, usually expressed in E.
coli, after selection from phage or ribosome display libraries
(Cambridge Antibody Technology, Cambridge, UK; Biolnvent, Lund,
Sweden; Affitech, Walnut Creek, Calif.; Biosite, San Diego,
Calif.). In addition to the conventional antibodies, Fab and scFv
fragments, single V-domains from camelids or engineered human
equivalents (Domantis, Waltham, Mass.) may also be useful in
arrays.
[0151] The term "scaffold" refers to ligand-binding domains of
proteins, which are engineered into multiple variants capable of
binding diverse target molecules with antibody-like properties of
specificity and affinity. The variants can be produced in a genetic
library format and selected against individual targets by phage,
bacterial or ribosome display. Such ligand-binding scaffolds or
frameworks include `Affibodies` based on Staph. aureus protein A
(Affibody, Bromma, Sweden), `Trinectins` based on fibronectins
(Phylos, Lexington, Mass.) and `Anticalins` based on the lipocalin
structure (Pieris Proteolab, Freising-Weihenstephan, Germany).
These can be used on capture arrays in a similar fashion to
antibodies and may have advantages of robustness and ease of
production.
[0152] Nonprotein capture molecules, notably the single-stranded
nucleic acid aptamers which bind protein ligands with high
specificity and affinity, are also used in arrays (SomaLogic,
Boulder, Colo.). Aptamers are selected from libraries of
oligonucleotides by the Selex.TM. procedure and their interaction
with protein can be enhanced by covalent attachment, through
incorporation of brominated deoxyuridine and UV-activated
crosslinking (photoaptamers). Photocrosslinking to ligand reduces
the crossreactivity of aptamers due to the specific steric
requirements. Aptamers have the advantages of ease of production by
automated oligonucleotide synthesis and the stability and
robustness of DNA; on photoaptamer arrays, universal fluorescent
protein stains can be used to detect binding.
[0153] Protein analytes binding to antibody arrays may be detected
directly or via a secondary antibody in a sandwich assay. Direct
labelling is used for comparison of different samples with
different colours. Where pairs of antibodies directed at the same
protein ligand are available, sandwich immunoassays provide high
specificity and sensitivity and are therefore the method of choice
for low abundance proteins such as cytokines; they also give the
possibility of detection of protein modifications. Label-free
detection methods, including mass spectrometry, surface plasmon
resonance and atomic force microscopy, avoid alteration of ligand.
What is required from any method is optimal sensitivity and
specificity, with low background to give high signal to noise.
Since analyte concentrations cover a wide range, sensitivity has to
be tailored appropriately; serial dilution of the sample or use of
antibodies of different affinities are solutions to this problem.
Proteins of interest are frequently those in low concentration in
body fluids and extracts, requiring detection in the pg range or
lower, such as cytokines or the low expression products in
cells.
[0154] An alternative to an array of capture molecules is one made
through `molecular imprinting` technology, in which peptides (e.g.,
from the C-terminal regions of proteins) are used as templates to
generate structurally complementary, sequence-specific cavities in
a polymerizable matrix; the cavities can then specifically capture
(denatured) proteins that have the appropriate primary amino acid
sequence (ProteinPrint.TM., Aspira Biosystems, Burlingame,
Calif.).
[0155] A multiplexed bead assay, such as, for example, the BD.TM.
Cytometric Bead Array, is a series of spectrally discrete particles
that can be used to capture and quantitate soluble analytes. The
analyte is then measured by detection of a fluorescence-based
emission and flow cytometric analysis. Multiplexed bead assay
generates data that is comparable to ELISA based assays, but in a
"multiplexed" or simultaneous fashion. Concentration of unknowns is
calculated for the cytometric bead array as with any sandwich
format assay, i.e. through the use of known standards and plotting
unknowns against a standard curve. Further, multiplexed bead assay
allows quantification of soluble analytes in samples never
previously considered due to sample volume limitations. In addition
to the quantitative data, powerful visual images can be generated
revealing unique profiles or signatures that provide the user with
additional information at a glance.
[0156] 3. Separation Methods
[0157] Disclosed herein are separation methods that can be used to
detect the number or percentage of cells with detectable Bcl-B
expression for use in the methods disclosed herein. Cells with
detectable Bcl-B expression can be isolated by a fluorescence
activated cell sorting (FACS), protein-conjugated magnetic bead
separation, specific gene expression patterns (using RT-PCR), or
specific antibody staining
[0158] Cells may be selected based on light-scatter properties as
well as their expression of various cell surface antigens. Various
techniques can be employed to separate the cells with detectable
Bcl-B expression. Monoclonal antibodies are particularly useful.
The antibodies can be attached to a solid support to allow for
crude separation. The separation techniques employed should
maximize the retention of viability of the fraction to be
collected.
[0159] Procedures for separation can include magnetic separation,
using antibody-coated magnetic beads, affinity chromatography,
cytotoxic agents joined to a monoclonal antibody or used in
conjunction with a monoclonal antibody, e.g., complement and
cytotoxins, and "panning" with antibody attached to a solid matrix,
e.g., plate, or other convenient technique.
[0160] The antibodies may be conjugated with markers, such as
magnetic beads, which allow for direct separation, biotin, which
can be removed with avidin or streptavidin bound to a support,
fluorochromes, which can be used with a fluorescence activated cell
sorter, or the like, to allow for ease of separation of the
particular cell type. Any technique may be employed which is not
unduly detrimental to the viability of the remaining cells.
[0161] 4. Antibodies
[0162] Disclosed herein are antibodies that specifically bind Bcl-B
that can be used to detect Bcl-B in cancer cells. For example,
disclosed is a polyclonal antibody specific for Bcl-B raised in a
mammal using Bcl-B protein, or an immunogenic fragment thereof as
the immunogen. For example, disclosed is a polyclonal antibody
specific for Bcl-B (BR-49) that was raised in rabbits using the
affinity purified recombinant GST-Bcl-B protein as the immunogen.
Also disclosed is anti-Bcl-B serum (AR-77) generated in rabbits
using a synthetic peptide (NH.sub.2-REPGTPEPAPSTPEAAVLR-amide; SEQ
ID NO:1) corresponding to residues 32 to 50 of human Bcl-B.
[0163] Thus, the immunogenic fragment of Bcl-B can comprise
residues 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46
to 50 of human Bcl-B. Thus, the immunogenic fragment of Bcl-B can
comprise residues 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45 to 49 of human Bcl-B. Thus, the immunogenic fragment of
Bcl-B can comprise residues 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44 to 48 of human Bcl-B. Thus, the immunogenic fragment of
Bcl-B can comprise residues 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43 to 47 of human Bcl-B. Thus, the immunogenic fragment of
Bcl-B can comprise residues 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42 to 46 of human Bcl-B. Thus, the immunogenic fragment of Bcl-B
can comprise residues 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 to 45
of human Bcl-B. Thus, the immunogenic fragment of Bcl-B can
comprise residues 32, 33, 34, 35, 36, 37, 38, 39, 40 to 44 of human
Bcl-B. Thus, the immunogenic fragment of Bcl-B can comprise
residues 32, 33, 34, 35, 36, 37, 38, 39 to 43 of human Bcl-B. Thus,
the immunogenic fragment of Bcl-B can comprise residues 32, 33, 34,
35, 36, 37, 38 to 42 of human Bcl-B. Thus, the immunogenic fragment
of Bcl-B can comprise residues 32, 33, 34, 35, 36, 37 to 41 of
human Bcl-B. Thus, the immunogenic fragment of Bcl-B can comprise
residues 32, 33, 34, 35, 36 to 40 of human Bcl-B. Thus, the
immunogenic fragment of Bcl-B can comprise residues 32, 33, 34, 35
to 39 of human Bcl-B. Thus, the immunogenic fragment of Bcl-B can
comprise residues 32, 33, 34 to 38 of human Bcl-B. Thus, the
immunogenic fragment of Bcl-B can comprise residues 32, 33 to 37 of
human Bcl-B. Thus, the immunogenic fragment of Bcl-B can comprise
residues 32 to 36 of human Bcl-B.
[0164] Thus, the immunogenic fragment of Bcl-B can comprise
residues 32 to 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, or 50 of human Bcl-B. Thus, the immunogenic fragment of Bcl-B
can comprise residues 33 to 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, or 50 of human Bcl-B. Thus, the immunogenic fragment of
Bcl-B can comprise residues 34 to 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, or 50of human Bcl-B. Thus, the immunogenic fragment
of Bcl-B can comprise residues 35 to 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, or 50 of human Bcl-B. Thus, the immunogenic
fragment of Bcl-B can comprise residues 36 to 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, or 50 of human Bcl-B. Thus, the immunogenic
fragment of Bcl-B can comprise residues 37 to 41, 42, 43, 44, 45,
46, 47, 48, 49, or 50 of human Bcl-B. Thus, the immunogenic
fragment of Bcl-B can comprise residues 38 to 42, 43, 44, 45, 46,
47, 48, 49, or 50 of human Bcl-B. Thus, the immunogenic fragment of
Bcl-B can comprise residues 39 to 43, 44, 45, 46, 47, 48, 49, or 50
of human Bcl-B. Thus, the immunogenic fragment of Bcl-B can
comprise residues 40 to 44, 45, 46, 47, 48, 49, or 50 of human
Bcl-B. Thus, the immunogenic fragment of Bcl-B can comprise
residues 41 to 45, 46, 47, 48, 49, or 50 of human Bcl-B. Thus, the
immunogenic fragment of Bcl-B can comprise residues 42 to 46, 47,
48, 49, or 50 of human Bcl-B. Thus, the immunogenic fragment of
Bcl-B can comprise residues 43 to 47, 48, 49, or 50 of human Bcl-B.
Thus, the immunogenic fragment of Bcl-B can comprise residues 44 to
48, 49, or 50 of human Bcl-B. Thus, the immunogenic fragment of
Bcl-B can comprise residues 45 to 49, or 50 of human Bcl-B. Thus,
the immunogenic fragment of Bcl-B can comprise residues 46 to 50 of
human Bcl-B.
[0165] The term "antibodies" is used herein in a broad sense and
includes both polyclonal and monoclonal antibodies. In addition to
intact immunoglobulin molecules, also included in the term
"antibodies" are fragments or polymers of those immunoglobulin
molecules, and human or humanized versions of immunoglobulin
molecules or fragments thereof, as long as they are chosen for
their ability to interact with Bcl-B. The antibodies can be tested
for their desired activity using the in vitro assays described
herein, or by analogous methods, after which their in vivo
therapeutic and/or prophylactic activities are tested according to
known clinical testing methods.
[0166] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a substantially homogeneous population of
antibodies, i.e., the individual antibodies within the population
are identical except for possible naturally occurring mutations
that may be present in a small subset of the antibody molecules.
The monoclonal antibodies herein specifically include "chimeric"
antibodies in which a portion of the heavy and/or light chain is
identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, as long as they exhibit the desired antagonistic
activity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc.
Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
[0167] The disclosed monoclonal antibodies can be made using any
procedure which produces mono clonal antibodies. For example,
disclosed monoclonal antibodies can be prepared using hybridoma
methods, such as those described by Kohler and Milstein, Nature,
256:495 (1975). In a hybridoma method, a mouse or other appropriate
host animal is typically immunized with an immunizing agent to
elicit lymphocytes that produce or are capable of producing
antibodies that will specifically bind to the immunizing agent.
Alternatively, the lymphocytes may be immunized in vitro.
[0168] If these approaches do not produce neutralizing antibodies,
cells expressing cell surface localized versions of these proteins
will be used to immunize mice, rats or other species.
Traditionally, the generation of monoclonal antibodies has depended
on the availability of purified protein or peptides for use as the
immunogen. More recently DNA based immunizations have shown promise
as a way to elicit strong immune responses and generate monoclonal
antibodies. In this approach, DNA-based immunization can be used,
wherein DNA encoding extracellular fragments of Bcl-B expressed as
a fusion protein with human IgG1 or an epitope tag is injected into
the host animal according to methods known in the art (e.g.,
Kilpatrick K E, et al. Hybridoma. 1998 December; 17(6):569-76;
Kilpatrick K E et al. Hybridoma. 2000 August; 19(4):297-302, which
are incorporated herein by referenced in full for the the methods
of antibody production) and as described in the examples.
[0169] An alternate approach to immunizations with either purified
protein or DNA is to use antigen expressed in baculovirus. The
advantages to this system include ease of generation, high levels
of expression, and post-translational modifications that are highly
similar to those seen in mammalian systems. Use of this system
involves expressing the immunogenic fragment of Bcl-B as fusion
proteins with a signal sequence fragment. The antigen is produced
by inserting a gene fragment in-frame between the signal sequence
and the mature protein domain of Bcl-B nucleotide sequence. This
results in the display of the foreign proteins on the surface of
the virion. This method allows immunization with whole virus,
eliminating the need for purification of target antigens.
[0170] Generally, either peripheral blood lymphocytes ("PBLs") are
used in methods of producing monoclonal antibodies if cells of
human origin are desired, or spleen cells or lymph node cells are
used if non-human mammalian sources are desired. The lymphocytes
are then fused with an immortalized cell line using a suitable
fusing agent, such as polyethylene glycol, to form a hybridoma cell
(Goding, "Monoclonal Antibodies: Principles and Practice" Academic
Press, (1986) pp. 59-103). Immortalized cell lines are usually
transformed mammalian cells, including myeloma cells of rodent,
bovine, equine, and human origin. Usually, rat or mouse myeloma
cell lines are employed. The hybridoma cells may be cultured in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
immortalized cells. For example, if the parental cells lack the
enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or
HPRT), the culture medium for the hybridomas typically will include
hypoxanthine, aminopterin, and thymidine ("HAT medium"), which
substances prevent the growth of HGPRT-deficient cells. Preferred
immortalized cell lines are those that fuse efficiently, support
stable high level expression of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. More preferred immortalized cell lines are murine myeloma
lines, which can be obtained, for instance, from the Salk Institute
Cell Distribution Center, San Diego, Calif. and the American Type
Culture Collection, Rockville, Md. Human myeloma and mouse-human
heteromyeloma cell lines also have been described for the
production of human monoclonal antibodies (Kozbor, J. Immunol.,
133:3001 (1984); Brodeur et al., "Monoclonal Antibody Production
Techniques and Applications" Marcel Dekker, Inc., New York, (1987)
pp. 51-63). The culture medium in which the hybridoma cells are
cultured can then be assayed for the presence of monoclonal
antibodies directed against Bcl-B. Preferably, the binding
specificity of monoclonal antibodies produced by the hybridoma
cells is determined by immunoprecipitation or by an in vitro
binding assay, such as radioimmunoassay (RIA) or enzyme-linked
immunoabsorbent assay (ELISA). Such techniques and assays are known
in the art, and are described further in the Examples below or in
Harlow and Lane "Antibodies, A Laboratory Manual" Cold Spring
Harbor Publications, New York, (1988).
[0171] After the desired hybridoma cells are identified, the clones
may be subcloned by limiting dilution or FACS sorting procedures
and grown by standard methods. Suitable culture media for this
purpose include, for example, Dulbecco's Modified Eagle's Medium
and RPMI-1640 medium. Alternatively, the hybridoma cells may be
grown in vivo as ascites in a mammal.
[0172] The monoclonal antibodies secreted by the subclones can be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, protein G, hydroxylapatite
chromatography, gel electrophoresis, dialysis, or affinity
chromatography.
[0173] The monoclonal antibodies can also be made by recombinant
DNA methods, such as those described in U.S. Pat. No. 4,816,567
(Cabilly et al.). DNA encoding the disclosed monoclonal antibodies
can be readily isolated and sequenced using conventional procedures
(e.g., by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). Libraries of antibodies or active antibody fragments
can also be generated and screened using phage display techniques,
e.g., as described in U.S. Pat. No. 5,804,440 to Burton et al. and
U.S. Pat. No. 6,096,441 to Barbas et al.
[0174] In vitro methods are also suitable for preparing monovalent
antibodies. Digestion of antibodies to produce fragments thereof,
particularly, Fab fragments, can be accomplished using routine
techniques known in the art. For instance, digestion can be
performed using papain. Examples of papain digestion are described
in WO 94/29348 published Dec. 22, 1994 and U.S. Pat. No. 4,342,566.
Papain digestion of antibodies typically produces two identical
antigen binding fragments, called Fab fragments, each with a single
antigen binding site, and a residual Fc fragment. Pepsin treatment
yields a fragment that has two antigen combining sites and is still
capable of cross-linking antigen.
[0175] The fragments, whether attached to other sequences or not,
can also include insertions, deletions, substitutions, or other
selected modifications of particular regions or specific amino
acids residues, provided the activity of the antibody or antibody
fragment is not significantly altered or impaired compared to the
non-modified antibody or antibody fragment. These modifications can
provide for some additional property, such as to remove/add amino
acids capable of disulfide bonding, to increase its bio-longevity,
to alter its secretory characteristics, etc. In any case, the
antibody or antibody fragment must possess a bioactive property,
such as specific binding to its cognate antigen. Functional or
active regions of the antibody or antibody fragment may be
identified by mutagenesis of a specific region of the protein,
followed by expression and testing of the expressed polypeptide.
Such methods are readily apparent to a skilled practitioner in the
art and can include site-specific mutagenesis of the nucleic acid
encoding the antibody or antibody fragment. (Zoller, M. J. Curr.
Opin. Biotechnol. 3:348-354, 1992).
[0176] As used herein, the term "antibody" or "antibodies" can also
refer to a human antibody and/or a humanized antibody. Many
non-human antibodies (e.g., those derived from mice, rats, or
rabbits) are naturally antigenic in humans, and thus can give rise
to undesirable immune responses when administered to humans.
Therefore, the use of human or humanized antibodies in the methods
serves to lessen the chance that an antibody administered to a
human will evoke an undesirable immune response.
[0177] As used herein, the term "antibody" encompasses, but is not
limited to, whole immunoglobulin (i.e., an intact antibody) of any
class. Native antibodies are usually heterotetrameric
glycoproteins, composed of two identical light (L) chains and two
identical heavy (H) chains. Typically, each light chain is linked
to a heavy chain by one covalent disulfide bond, while the number
of disulfide linkages varies between the heavy chains of different
immunoglobulin isotypes. Each heavy and light chain also has
regularly spaced intrachain disulfide bridges. Each heavy chain has
at one end a variable domain (V(H)) followed by a number of
constant domains. Each light chain has a variable domain at one end
(V(L)) and a constant domain at its other end; the constant domain
of the light chain is aligned with the first constant domain of the
heavy chain, and the light chain variable domain is aligned with
the variable domain of the heavy chain. Particular amino acid
residues are believed to form an interface between the light and
heavy chain variable domains. The light chains of antibodies from
any vertebrate species can be assigned to one of two clearly
distinct types, called kappa (k) and lambda (l), based on the amino
acid sequences of their constant domains. Depending on the amino
acid sequence of the constant domain of their heavy chains,
immunoglobulins can be assigned to different classes. There are
five major classes of human immunoglobulins: IgA, IgD, IgE, IgG and
IgM, and several of these may be further divided into subclasses
(isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2.
One skilled in the art would recognize the comparable classes for
mouse. The heavy chain constant domains that correspond to the
different classes of immunoglobulins are called alpha, delta,
epsilon, gamma, and mu, respectively.
[0178] The term "variable" is used herein to describe certain
portions of the variable domains that differ in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not usually evenly distributed through the variable
domains of antibodies. It is typically concentrated in three
segments called complementarity determining regions (CDRs) or
hypervariable regions both in the light chain and the heavy chain
variable domains. The more highly conserved portions of the
variable domains are called the framework (FR). The variable
domains of native heavy and light chains each comprise four FR
regions, largely adopting a b-sheet configuration, connected by
three CDRs, which form loops connecting, and in some cases forming
part of, the b-sheet structure. The CDRs in each chain are held
together in close proximity by the FR regions and, with the CDRs
from the other chain, contribute to the formation of the antigen
binding site of antibodies (see Kabat E. A. et al., "Sequences of
Proteins of Immunological Interest," National Institutes of Health,
Bethesda, Md. (1987)). The constant domains are not involved
directly in binding an antibody to an antigen, but exhibit various
effector functions, such as participation of the antibody in
antibody-dependent cellular toxicity.
[0179] The term "antibody" as used herein is meant to include
intact molecules as well as fragments thereof, such as, for
example, Fab and F(ab').sub.2, which are capable of binding the
epitopic determinant.
[0180] As used herein, the term "antibody or fragments thereof"
encompasses chimeric antibodies and hybrid antibodies, with dual or
multiple antigen or epitope specificities, and fragments, such as
F(ab')2, Fab', Fab and the like, including hybrid fragments. Thus,
fragments of the antibodies that retain the ability to bind their
specific antigens are provided. For example, fragments of
antibodies which maintain Bcl-B binding activity are included
within the meaning of the term "antibody or fragment thereof." Such
antibodies and fragments can be made by techniques known in the art
and can be screened for specificity and activity according to the
methods set forth in the Examples and in general methods for
producing antibodies and screening antibodies for specificity and
activity (See Harlow and Lane. Antibodies, A Laboratory Manual.
Cold Spring Harbor Publications, New York, (1988)).
[0181] Also included within the meaning of "antibody or fragments
thereof" are conjugates of antibody fragments and antigen binding
proteins (single chain antibodies) as described, for example, in
U.S. Pat. No. 4,704,692, the contents of which are hereby
incorporated by reference.
[0182] Alternatively, unprotected peptide segments are chemically
linked where the bond formed between the peptide segments as a
result of the chemical ligation is an unnatural (non-peptide) bond
(Schnolzer, M et al. Science, 256:221 (1992)). This technique has
been used to synthesize analogs of protein domains as well as large
amounts of relatively pure proteins with full biological activity
(deLisle Milton R C et al., Techniques in Protein Chemistry IV.
Academic Press, New York, pp. 257-267 (1992)).
[0183] Also disclosed are fragments of antibodies which have
bioactivity. The polypeptide fragments can be recombinant proteins
obtained by cloning nucleic acids encoding the polypeptide in an
expression system capable of producing the polypeptide fragments
thereof, such as an adenovirus or baculovirus expression system.
For example, one can determine the active domain of an antibody
from a specific hybridoma that can cause a biological effect
associated with the interaction of the antibody with Bcl-B. For
example, amino acids found to not contribute to either the activity
or the binding specificity or affinity of the antibody can be
deleted without a loss in the respective activity. For example, in
various embodiments, amino or carboxy-terminal amino acids are
sequentially removed from either the native or the modified
non-immunoglobulin molecule or the immunoglobulin molecule and the
respective activity assayed in one of many available assays. In
another example, a fragment of an antibody comprises a modified
antibody wherein at least one amino acid has been substituted for
the naturally occurring amino acid at a specific position, and a
portion of either amino terminal or carboxy terminal amino acids,
or even an internal region of the antibody, has been replaced with
a polypeptide fragment or other moiety, such as biotin, which can
facilitate in the purification of the modified antibody. For
example, a modified antibody can be fused to a maltose binding
protein, through either peptide chemistry or cloning the respective
nucleic acids encoding the two polypeptide fragments into an
expression vector such that the expression of the coding region
results in a hybrid polypeptide. The hybrid polypeptide can be
affinity purified by passing it over an amylose affinity column,
and the modified antibody receptor can then be separated from the
maltose binding region by cleaving the hybrid polypeptide with the
specific protease factor Xa. (See, for example, New England Biolabs
Product Catalog, 1996, pg. 164.). Similar purification procedures
are available for isolating hybrid proteins from eukaryotic cells
as well.
[0184] The fragments, whether attached to other sequences or not,
include insertions, deletions, substitutions, or other selected
modifications of particular regions or specific amino acids
residues, provided the activity of the fragment is not
significantly altered or impaired compared to the nonmodified
antibody or antibody fragment. These modifications can provide for
some additional property, such as to remove or add amino acids
capable of disulfide bonding, to increase its bio-longevity, to
alter its secretory characteristics, etc. In any case, the fragment
must possess a bioactive property, such as binding activity,
regulation of binding at the binding domain, etc. Functional or
active regions of the antibody may be identified by mutagenesis of
a specific region of the protein, followed by expression and
testing of the expressed polypeptide. Such methods are readily
apparent to a skilled practitioner in the art and can include
site-specific mutagenesis of the nucleic acid encoding the antigen.
(Zoller M J et al. Nucl. Acids Res. 10:6487-500 (1982).
[0185] Techniques can also be adapted for the production of
single-chain antibodies specific to an antigenic protein of the
present disclosure (see e.g., U.S. Pat. No. 4,946,778). In
addition, methods can be adapted for the construction of F (ab)
expression libraries (see e.g., Huse, et al., 1989 Science 246:
1275-1281) to allow rapid and effective identification of
monoclonal F (ab)fragments with the desired specificity for a
protein or derivatives, fragments, analogs or homologs thereof
Antibody fragments that contain the idiotypes to a protein antigen
may be produced by techniques known in the art including, but not
limited to: (i) an F ((ab'))(2)fragment produced by pepsin
digestion of an antibody molecule; (ii) an Fab fragment generated
by reducing the disulfide bridges of an F ((ab'))(2)fragment; (iii)
an F (ab)fragment generated by the treatment of the antibody
molecule with papain and a reducing agent and (iv) F (v),
fragments.
[0186] Methods for the production of single-chain antibodies are
well known to those of skill in the art. The skilled artisan is
referred to U.S. Pat. No. 5,359,046, (incorporated herein by
reference) for such methods. A single chain antibody is created by
fusing together the variable domains of the heavy and light chains
using a short peptide linker, thereby reconstituting an antigen
binding site on a single molecule. Single-chain antibody variable
fragments (scFvs) in which the C-terminus of one variable domain is
tethered to the N-terminus of the other variable domain via a 15 to
25 amino acid peptide or linker have been developed without
significantly disrupting antigen binding or specificity of the
binding (Bedzyk et al., 1990; Chaudhary et al., 1990). The linker
is chosen to permit the heavy chain and light chain to bind
together in their proper conformational orientation. See, for
example, Huston, J. S., et al., Methods in Enzym. 203:46-121
(1991), which is incorporated herein by reference. These Fvs lack
the constant regions (Fc) present in the heavy and light chains of
the native antibody.
[0187] In vitro methods are also suitable for preparing monovalent
antibodies. Digestion of antibodies to produce fragments thereof,
particularly, Fab fragments, can be accomplished using routine
techniques known in the art. For instance, digestion can be
performed using papain. Examples of papain digestion are described
in WO 94/29348 published Dec. 22, 1994, U.S. Pat. No. 4,342,566,
and Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring
Harbor Publications, New York, (1988). Papain digestion of
antibodies typically produces two identical antigen binding
fragments, called Fab fragments, each with a single antigen binding
site, and a residual Fc fragment. Pepsin treatment yields a
fragment, called the F(ab')2 fragment, that has two antigen
combining sites and is still capable of cross-linking antigen.
[0188] The Fab fragments produced in the antibody digestion also
contain the constant domains of the light chain and the first
constant domain of the heavy chain. Fab' fragments differ from Fab
fragments by the addition of a few residues at the carboxy terminus
of the heavy chain domain including one or more cysteines from the
antibody hinge region. The F(ab')2 fragment is a bivalent fragment
comprising two Fab' fragments linked by a disulfide bridge at the
hinge region. Fab'-SH is the designation herein for Fab' in which
the cysteine residue(s) of the constant domains bear a free thiol
group. Antibody fragments originally were produced as pairs of Fab'
fragments which have hinge cysteines between them. Other chemical
couplings of antibody fragments are also known.
[0189] In hybrid antibodies, one heavy and light chain pair is
homologous to that found in an antibody raised against one antigen
recognition feature, e.g., epitope, while the other heavy and light
chain pair is homologous to a pair found in an antibody raised
against another epitope. This results in the property of
multi-functional valency, i.e., ability to bind at least two
different epitopes simultaneously. As used herein, the term "hybrid
antibody" refers to an antibody wherein each chain is separately
homologous with reference to a mammalian antibody chain, but the
combination represents a novel assembly so that two different
antigens are recognized by the antibody. Such hybrids can be formed
by fusion of hybridomas producing the respective component
antibodies, or by recombinant techniques. Such hybrids may, of
course, also be formed using chimeric chains.
[0190] The encoded antibodies can be anti-idiotypic antibodies
(antibodies that bind other antibodies) as described, for example,
in U.S. Pat. No. 4,699,880. Such anti-idiotypic antibodies could
bind endogenous or foreign antibodies in a treated individual,
thereby to ameliorate or prevent pathological conditions associated
with an immune response, e.g., in the context of an autoimmune
disease.
[0191] The targeting function of the antibody can be used
therapeutically by coupling the antibody or a fragment thereof with
a therapeutic agent. Such coupling of the antibody or fragment
(e.g., at least a portion of an immunoglobulin constant region
(Fc)) with the therapeutic agent can be achieved by making an
immunoconjugate or by making a fusion protein, comprising the
antibody or antibody fragment and the therapeutic agent.
[0192] Also included within the meaning of "antibody or fragments
thereof" are conjugates of antibody fragments and antigen binding
proteins (single chain antibodies) as described, for example, in
U.S. Pat. No. 4,704,692, the contents of which are hereby
incorporated by reference.
[0193] One method of producing proteins comprising the antibodies
is to link two or more peptides or polypeptides together by protein
chemistry techniques. For example, peptides or polypeptides can be
chemically synthesized using currently available laboratory
equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or
Boc(tert-butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc.,
Foster City, Calif.). One skilled in the art can readily appreciate
that a peptide or polypeptide corresponding to the antibody, for
example, can be synthesized by standard chemical reactions. For
example, a peptide or polypeptide can be synthesized and not
cleaved from its synthesis resin whereas the other fragment of an
antibody can be synthesized and subsequently cleaved from the
resin, thereby exposing a terminal group which is functionally
blocked on the other fragment. By peptide condensation reactions,
these two fragments can be covalently joined via a peptide bond at
their carboxyl and amino termini, respectively, to form an
antibody, or fragment thereof. (Grant G A (1992) Synthetic
Peptides: A User Guide. W.H. Freeman and Co., N.Y. (1992); Bodansky
M and Trost B., Ed. (1993) Principles of Peptide Synthesis.
Springer-Verlag Inc., NY. Alternatively, the peptide or polypeptide
is independently synthesized in vivo as described above. Once
isolated, these independent peptides or polypeptides may be linked
to form an antibody or fragment thereof via similar peptide
condensation reactions.
[0194] For example, enzymatic ligation of cloned or synthetic
peptide segments allow relatively short peptide fragments to be
joined to produce larger peptide fragments, polypeptides or whole
protein domains (Abrahmsen L et al., Biochemistry, 30:4151 (1991)).
Alternatively, native chemical ligation of synthetic peptides can
be utilized to synthetically construct large peptides or
polypeptides from shorter peptide fragments. This method consists
of a two step chemical reaction (Dawson et al. Synthesis of
Proteins by Native Chemical Ligation. Science, 266:776-779 (1994)).
The first step is the chemoselective reaction of an unprotected
synthetic peptide-alpha-thioester with another unprotected peptide
segment containing an amino-terminal Cys residue to give a
thioester-linked intermediate as the initial covalent product.
Without a change in the reaction conditions, this intermediate
undergoes spontaneous, rapid intramolecular reaction to form a
native peptide bond at the ligation site. Application of this
native chemical ligation method to the total synthesis of a protein
molecule is illustrated by the preparation of human interleukin 8
(IL-8) (Baggiolini M et al. (1992) FEBS Lett. 307:97-101;
Clark-Lewis I et al., J. Biol. Chem., 269:16075 (1994); Clark-Lewis
I et al., Biochemistry, 30:3128 (1991); Rajarathnam K et al.,
Biochemistry 33:6623-30 (1994)).
[0195] The antibody can be bound to a substrate or labeled with a
detectable moiety or both bound and labeled. The detectable
moieties contemplated with the present compositions include
fluorescent, enzymatic and radioactive markers.
[0196] 5. Nucleic Acid Detection
[0197] Disclosed herein are methods for detecting and determining
the abundance of Bcl-B nucleic acid, such as mRNA, in a total or
poly(A) RNA sample from cancer cells for use in the methods
disclosed herein. For example, specific mRNAs can be detected using
Northern blot analysis, nuclease protection assays (NPA), in situ
hybridization, or reverse transcription-polymerase chain reaction
(RT-PCR).
[0198] In theory, each of these techniques can be used to detect
specific RNAs and to precisely determine their expression level. In
general, Northern analysis is the only method that provides
information about transcript size, whereas NPAs are the easiest way
to simultaneously examine multiple messages. In situ hybridization
is used to localize expression of a particular gene within a tissue
or cell type, and RT-PCR is the most sensitive method for detecting
and quantitating gene expression.
[0199] Northern analysis presents several advantages over the other
techniques. The most compelling of these is that it is the easiest
method for determining transcript size, and for identifying
alternatively spliced transcripts and multigene family members. It
can also be used to directly compare the relative abundance of a
given message between all the samples on a blot. The Northern
blotting procedure is straightforward and provides opportunities to
evaluate progress at various points (e.g., intactness of the RNA
sample and how efficiently it has transferred to the membrane). RNA
samples are first separated by size via electrophoresis in an
agarose gel under denaturing conditions. The RNA is then
transferred to a membrane, crosslinked and hybridized with a
labeled probe. Nonisotopic or high specific activity radiolabeled
probes can be used including random-primed, nick-translated, or
PCR-generated DNA probes, in vitro transcribed RNA probes, and
oligonucleotides. Additionally, sequences with only partial
homology (e.g., cDNA from a different species or genomic DNA
fragments that might contain an exon) may be used as probes.
[0200] The Nuclease Protection Assay (NPA) (including both
ribonuclease protection assays and S1 nuclease assays) is an
extremely sensitive method for the detection and quantitation of
specific mRNAs. The basis of the NPA is solution hybridization of
an antisense probe (radiolabeled or nonisotopic) to an RNA sample.
After hybridization, single-stranded, unhybridized probe and RNA
are degraded by nucleases. The remaining protected fragments are
separated on an acrylamide gel. Solution hybridization is typically
more efficient than membrane-based hybridization, and it can
accommodate up to 100 .mu.g of sample RNA, compared with the 20-30
.mu.g maximum of blot hybridizations. NPAs are also less sensitive
to RNA sample degradation than Northern analysis since cleavage is
only detected in the region of overlap with the probe (probes are
usually about 100-400 bases in length).
[0201] NPAs are the method of choice for the simultaneous detection
of several RNA species. During solution hybridization and
subsequent analysis, individual probe/target interactions are
completely independent of one another. Thus, several RNA targets
and appropriate controls can be assayed simultaneously (up to
twelve have been used in the same reaction), provided that the
individual probes are of different lengths. NPAs are also commonly
used to precisely map mRNA termini and intron/exon junctions.
[0202] In situ hybridization (ISH) is a powerful and versatile tool
for the localization of specific mRNAs in cells or tissues. Unlike
Northern analysis and nuclease protection assays, ISH does not
require the isolation or electrophoretic separation of RNA.
Hybridization of the probe takes place within the cell or tissue.
Since cellular structure is maintained throughout the procedure,
ISH provides information about the location of mRNA within the
tissue sample.
[0203] The procedure begins by fixing samples in neutral-buffered
formalin, and embedding the tissue in paraffin. The samples are
then sliced into thin sections and mounted onto microscope slides.
(Alternatively, tissue can be sectioned frozen and post-fixed in
paraformaldehyde.) After a series of washes to dewax and rehydrate
the sections, a Proteinase K digestion is performed to increase
probe accessibility, and a labeled probe is then hybridized to the
sample sections. Radiolabeled probes are visualized with liquid
film dried onto the slides, while nonisotopically labeled probes
are conveniently detected with colorimetric or fluorescent
reagents.
[0204] RT-PCR has revolutionized the study of gene expression. It
is now theoretically possible to detect the RNA transcript of any
gene, regardless of the scarcity of the starting material or
relative abundance of the specific mRNA. In RT-PCR, an RNA template
is copied into a complementary DNA (cDNA) using a retroviral
reverse transcriptase. The cDNA is then amplified exponentially by
PCR. As with NPAs, RT-PCR is somewhat tolerant of degraded RNA. As
long as the RNA is intact within the region spanned by the primers,
the target will be amplified.
[0205] Relative quantitative RT-PCR involves amplifying an internal
control simultaneously with the gene of interest. The internal
control is used to normalize the samples. Once normalized, direct
comparisons of relative abundance of a specific mRNA can be made
across the samples. It is crucial to choose an internal control
with a constant level of expression across all experimental samples
(i.e., not affected by experimental treatment). Commonly used
internal controls (e.g., GAPDH, .beta.-actin, cyclophilin) can vary
in expression. Additionally, most common internal controls are
expressed at much higher levels than the mRNA being studied.
Preferably, all products of the PCR reaction can be analyzed in the
linear range of amplification, which becomes difficult for
transcripts of widely different levels of abundance.
[0206] Competitive RT-PCR is used for absolute quantitation. This
technique involves designing, synthesizing, and accurately
quantitating a competitor RNA that can be distinguished from the
endogenous target by a small difference in size or sequence. Known
amounts of the competitor RNA are added to experimental samples and
RT-PCR is performed. Signals from the endogenous target are
compared with signals from the competitor to determine the amount
of target present in the sample.
[0207] 6. Primers and Probers
[0208] Disclosed herein are nucleic acids that can be used to
detect Bcl-B in cancer cells for use in the methods disclosed
herein. For example, disclosed are compositions including primers
and probes, which are capable of interacting with Bcl-B
transcript.
[0209] The nucleic acid sequence for human Bcl-B is set forth in
Accession No. AF285092 (SEQ ID NO:2), and the amino acid sequence
is set forth in Accession No. AAG00503 (SEQ ID NO:3).
[0210] In certain embodiments the primers are used to support DNA
(e.g., cDNA) amplification reactions. Typically the primers will be
capable of being extended in a sequence specific manner. Extension
of a primer in a sequence specific manner includes any methods
wherein the sequence and/or composition of the nucleic acid
molecule to which the primer is hybridized or otherwise associated
directs or influences the composition or sequence of the product
produced by the extension of the primer. Extension of the primer in
a sequence specific manner therefore includes, but is not limited
to, PCR, DNA sequencing, DNA extension, DNA polymerization, RNA
transcription, or reverse transcription. Techniques and conditions
that amplify the primer in a sequence specific manner are
preferred. In certain embodiments the primers are used for the DNA
amplification reactions, such as PCR or direct sequencing. It is
understood that in certain embodiments the primers can also be
extended using non-enzymatic techniques, where for example, the
nucleotides or oligonucleotides used to extend the primer are
modified such that they will chemically react to extend the primer
in a sequence specific manner. Typically the disclosed primers
hybridize with the disclosed nucleic acids or region of the nucleic
acids or they hybridize with the complement of the nucleic acids or
complement of a region of the nucleic acids.
[0211] The size of the primers or probes for interaction with the
nucleic acids in certain embodiments can be any size that supports
the desired enzymatic manipulation of the primer, such as DNA
amplification or the simple hybridization of the probe or primer. A
typical primer or probe would be at least 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375,
400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900,
950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or
4000 nucleotides long.
[0212] In other embodiments a primer or probe can be less than or
equal to 6, 7, 8, 9, 10, 11, 12 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,
56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200,
225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550,
600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750,
2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides long.
[0213] The primers for the Bcl-B gene or transcript typically will
be used to produce an amplified DNA product that contains a region
of the Bcl-B gene or transcript or the complete gene or transcript.
In general, typically the size of the product will be such that the
size can be accurately determined to within 3, or 2 or 1
nucleotides.
[0214] In certain embodiments this product is at least 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225,
250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600,
650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000,
2250, 2500, 2750, 3000, 3500, or 4000 nucleotides long.
[0215] In other embodiments the product is less than or equal to
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175,
200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500,
550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500,
1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides
long.
[0216] The disclosed nucleic acids can be made up of for example,
nucleotides, nucleotide analogs, or nucleotide substitutes.
Non-limiting examples of these and other molecules are discussed
herein. It is understood that for example, when a vector is
expressed in a cell, the expressed mRNA will typically be made up
of A, C, G, and U. Likewise, it is understood that if, for example,
an antisense molecule is introduced into a cell or cell environment
through for example exogenous delivery, it is advantagous that the
antisense molecule be made up of nucleotide analogs that reduce the
degradation of the antisense molecule in the cellular
environment.
[0217] A nucleotide is a molecule that contains a base moiety, a
sugar moiety and a phosphate moiety. Nucleotides can be linked
together through their phosphate moieties and sugar moieties
creating an internucleoside linkage. The base moiety of a
nucleotide can be adenin-9-yl (A), cytosin-1-yl (C), guanin-9-yl
(G), uracil-1-yl (U), and thymin-1-yl (T). The sugar moiety of a
nucleotide is a ribose or a deoxyribose. The phosphate moiety of a
nucleotide is pentavalent phosphate. An non-limiting example of a
nucleotide would be 3'-AMP (3'-adenosine monophosphate) or 5'-GMP
(5'-guanosine monophosphate). There are many varieties of these
types of molecules available in the art and available herein.
[0218] A nucleotide analog is a nucleotide which contains some type
of modification to either the base, sugar, or phosphate moieties.
Modifications to nucleotides are well known in the art and would
include for example, 5-methylcytosine (5-me-C), 5-hydroxymethyl
cytosine, xanthine, hypoxanthine, and 2-aminoadenine as well as
modifications at the sugar or phosphate moieties. There are many
varieties of these types of molecules available in the art and
available herein.
[0219] Nucleotide substitutes are molecules having similar
functional properties to nucleotides, but which do not contain a
phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide
substitutes are molecules that will recognize nucleic acids in a
Watson-Crick or Hoogsteen manner, but which are linked together
through a moiety other than a phosphate moiety. Nucleotide
substitutes are able to conform to a double helix type structure
when interacting with the appropriate target nucleic acid. There
are many varieties of these types of molecules available in the art
and available herein.
[0220] It is also possible to link other types of molecules
(conjugates) to nucleotides or nucleotide analogs to enhance for
example, cellular uptake. Conjugates can be chemically linked to
the nucleotide or nucleotide analogs. Such conjugates include but
are not limited to lipid moieties such as a cholesterol moiety.
(Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989,86, 6553-6556).
There are many varieties of these types of molecules available in
the art and available herein.
[0221] A Watson-Crick interaction is at least one interaction with
the Watson-Crick face of a nucleotide, nucleotide analog, or
nucleotide substitute. The Watson-Crick face of a nucleotide,
nucleotide analog, or nucleotide substitute includes the C2, N1,
and C6 positions of a purine based nucleotide, nucleotide analog,
or nucleotide substitute and the C2, N3, C4 positions of a
pyrimidine based nucleotide, nucleotide analog, or nucleotide
substitute.
[0222] A Hoogsteen interaction is the interaction that takes place
on the Hoogsteen face of a nucleotide or nucleotide analog, which
is exposed in the major groove of duplex DNA. The Hoogsteen face
includes the N7 position and reactive groups (NH2 or O) at the C6
position of purine nucleotides.
C. DEFINITIONS
[0223] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
skill in the art to which the disclosed method and compositions
belong. Although any methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present method and compositions, the particularly useful
methods, devices, and materials are as described. Publications
cited herein and the material for which they are cited are hereby
specifically incorporated by reference. Nothing herein is to be
construed as an admission that the present invention is not
entitled to antedate such disclosure by virtue of prior invention.
No admission is made that any reference constitutes prior art. The
discussion of references states what their authors assert, and
applicants reserve the right to challenge the accuracy and
pertinency of the cited documents.
[0224] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, reference to "a peptide" includes a plurality of such
peptides, reference to "the peptide" is a reference to one or more
peptides and equivalents thereof known to those skilled in the art,
and so forth.
[0225] "Optional" or "optionally" means that the subsequently
described event, circumstance, or material may or may not occur or
be present, and that the description includes instances where the
event, circumstance, or material occurs or is present and instances
where it does not occur or is not present.
[0226] Ranges can be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another embodiment includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another embodiment. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint. It is
also understood that there are a number of values disclosed herein,
and that each value is also herein disclosed as "about" that
particular value in addition to the value itself For example, if
the value "10" is disclosed, then "about 10" is also disclosed. It
is also understood that when a value is disclosed that "less than
or equal to" the value, "greater than or equal to the value" and
possible ranges between values are also disclosed, as appropriately
understood by the skilled artisan. For example, if the value "10"
is disclosed the "less than or equal to 10''as well as "greater
than or equal to 10" is also disclosed. It is also understood that
the throughout the application, data is provided in a number of
different formats, and that this data, represents endpoints and
starting points, and ranges for any combination of the data points.
For example, if a particular data point "10" and a particular data
point 15 are disclosed, it is understood that greater than, greater
than or equal to, less than, less than or equal to, and equal to 10
and 15 are considered disclosed as well as between 10 and 15. It is
also understood that each unit between two particular units are
also disclosed. For example, if 10 and 15 are disclosed, then 11,
12, 13, and 14 are also disclosed.
[0227] Throughout the description and claims of this specification,
the word "comprise" and variations of the word, such as
"comprising" and "comprises," means "including but not limited to,"
and is not intended to exclude, for example, other additives,
components, integers or steps.
[0228] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this pertains. The references disclosed are also individually
and specifically incorporated by reference herein for the material
contained in them that is discussed in the sentence in which the
reference is relied upon.
[0229] As used herein, the term "epitope" is meant to include any
determinant capable of specific interaction with the anti-Bcl-B
antibodies disclosed. Epitopic determinants usually consist of
chemically active surface groupings of molecules such as amino
acids or sugar side chains and usually have specific three
dimensional structural characteristics, as well as specific charge
characteristics.
[0230] An "epitope tag" denotes a short peptide sequence unrelated
to the function of the antibody or molecule that can be used for
purification or crosslinking of the molecule with anti-epitope tag
antibodies or other reagents.
[0231] By "specifically binds" is meant that an antibody recognizes
and physically interacts with its cognate antigen and does not
significantly recognize and interact with other antigens; such an
antibody may be a polyclonal antibody or a monoclonal antibody,
which are generated by techniques that are well known in the
art.
D. EXAMPLES
[0232] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the compounds, compositions, articles, devices
and/or methods claimed herein are made and evaluated, and are
intended to be purely exemplary and are not intended to limit the
disclosure. Efforts have been made to ensure accuracy with respect
to numbers (e.g., amounts, temperature, etc.), but some errors and
deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, temperature is in .degree. C. or is at
ambient temperature, and pressure is at or near atmospheric.
1. Example 1
Bcl-B Expression in Human Epithelial and Non-Epithelial
Malignancies
[0233] i. Materials and Methods
[0234] a. Patient Specimens
[0235] Bone marrow biopsies from 165 patients, 114 with symptomatic
multiple myeloma (MM), 19 with indolent MM, 13 with monoclonal
gammopathy of undetermined significance (MGUS), and 19 with
reactive plasmacytosis, were obtained from VA Hospital of Los
Angeles. Patients were categorized according to WHO criteria,
assessing the plasma count in bone marrow (group 1: 0-10%, group 2:
11-30%, group 3: >30%) (Went P, et al. 2006).
[0236] Tissue microarrays (TMAs) comprising paraffin-embedded lymph
node specimens from 48 patients diagnosed with DLBCL and from 57
patients with FL were obtained. Tumor specimens were obtained from
79 SCLC patients with limited disease who were treated by surgery
followed by chemotherapy using various multidrug regimens between
1984 and 2001. In addition, thoracic radiation was administered to
4% and prophylactic cranial irradiation to 8% of the patients.
Patients ranged from clinical stage I-IIIA and were of good
performance status (Karnofsky score 80-100). Clinical data
represent a median follow up of 1.3 years. TMAs containing
specimens from 82 NSCLC patients were obtained. The specimens
represented 22 adenocarcinomas, 32 squamous cell carcinomas, and 16
large cell carcinomas (12 unspecified tumors).
[0237] Clinicopathological characteristics related to
paraffin-embedded tissue specimens containing cervical, colorectal,
gastric, breast, prostate and ovarian cancers were described
(Krajewska M, et al. 2005a; Krajewska M, et al. 2005b; Krajewska M,
et al. 2007; Meinhold-Heerlein I, et al. 2001). In addition, a TMA
was produced for 26 cases of Crohn's disease.
[0238] b. Tissue Preparation
[0239] The tissues were prepared for paraffin embedding, as
described (Krajewska M, et al. 2003). TMAs were produced for all
investigated tumors and tissues, as described previously (Krajewska
M, et al. 2005b).
[0240] c. Antibodies
[0241] Glutatione S-transferase (GST)-Bcl-B fusion protein was
produced in bacteria and purified by affinity chromatography as
described (Zhai D, et al. 2006). A polyclonal antibody specific for
Bcl-B (BR-49) was raised in rabbits using the affinity purified
recombinant GST-Bcl-B protein as the immunogen. An additional
anti-Bcl-B serum (AR-77) was generated in rabbits using a synthetic
peptide (NH.sub.2-REPGTPEPAPSTPEAAVLR-amide; SEQ ID NO:1)
corresponding to residues 32 to 50 of human Bcl-B. Commercial mouse
monoclonal antibodies included anti-CD138 (Serotec, Raleigh, N.C.),
anti-CD68 (DakoCytomation, Carpinteria, Calif.), anti-CD10
(Novocastra, Newcastle Upon Tyne, England), anti-Bcl-6
(Novocastra), anti-MUM1 (DakoCytomation), anti-Bcl-2
(DakoCytomation), anti-Hsp60 (Nventa, Victoria, BC, Canada),
anti-.beta.-actin (Sigma-Aldrich, St Louis, Mo.), and anti-GST (BD
Pharmingen, San Diego, Calif.).
[0242] d. Immunohistochemistry (IHC)
[0243] Dewaxed tissue sections were immunostained as reported
(Krajewski S, et al. 1999). To determine specificity, the
immunostaining procedure was performed in parallel using preimmune
Bcl-B serum and immune serum (1:1000) preabsorbed with 10 .mu.g of
GST-Bcl-B, GST-Bcl-X.sub.L recombinant protein or synthetic peptide
immunogens. The immunostaining scoring system was described
(Krajewska M, et al. 2002).
[0244] For double-labeling procedure, tissue sections were stained
as above using Bcl-B rabbit polyclonal antiserum (DAB chromagen,
DAKOCytomation) followed by mouse monoclonal CD138 antibody
(Serotec)(SG chromagen, Vector Lab. Inc). Nuclear Red
(DAKOCytomation) was used for counterstaining of the double-labeled
slides. Automated image analysis system (Aperio Technology Inc,
Vista Calif.) was employed to visualize Bcl-B and CD138 staining
separately, applying a color deconvolution algorithm (Ruifrok A C,
et al. 2001). Quantification of immunostaining was performed using
color translation and an automated thresholding algorithm (Aperio
Technology Inc).
[0245] IHC results for CD10, Bcl-6, and MUM1 were used to
subclassify DLBCL cases into GCB and non-GCB categories (Hans CP,
et al. 2004): cases immunopositive for CD10 alone or for both CD10
and Bcl-6 were assigned to the GCB group, while cases that were
CD10.sup.-/Bcl-6.sup.- or that were
CD10.sup.-/Bcl-6.sup.+/MUM1.sup.+ were considered non-GCB.
[0246] e. Expression Plasmids
[0247] Bcl-B encoding cDNA in pcDNA3-Bcl-B plasmid was digested
with BamHI and XhoI (Promega, USA), purified (Qiagen) from a 1%
agarose gel, and then ligated with modified pTRE2hyg plasmid
(Clontech, USA) previously digested with the same restriction
enzymes. Proper plasmid construction was confirmed by
restriction-enzyme digestion and DNA sequencing.
[0248] f Stable Transfection
[0249] Stable transfection was conducted using the HeLa Tet-on cell
line (Clontech, USA), which was derived from the HeLa cells. This
cell line had been stably transfected with the pTet-On plasmid,
which encoded the tetracycline repressor and allowed the inserted
sequence to be inducibly expressed by tetracycline or doxycycline.
The HeLa
[0250] Tet-on cells were seeded at 50% confluency and cultured
overnight. Transfection was conducted for 3 hours using
LipofectAMINE PLUS. Transfected cells were cultured in complete
media for 24 hours and then split into fresh media. The split cells
were seeded to 10% confluence and cultured in media containing G418
(100 .mu.g/ml) to maintain the integration of the pTet-On construct
and hygromycin B (300 .mu.g/ml) to select stable transfectants of
pTRE2hyg/Bcl-B. Positive foci resistant to both antibiotics were
isolated and expanded. The transfected cells were cultured in the
presence or absence of doxycycline (1 .mu.g/ml) for 16 hours for
immunoblot studies.
[0251] g. Immunoblotting
[0252] Specimens derived from normal and malignant human tissues
with high ratios of cancer cells relative to stroma (>70%) were
provided by M.D. Anderson Cancer Center Orlando for immunoblotting
analysis. The protein lysate preparations, immunoblotting
procedures, and antigen detection were described previously
(Krajewski S, et al. 1996). Blots were probed with rabbit
anti-Bcl-2 antisera (1:2000-1:3000 v/v), mouse anti-Hsp60 or
.beta.-actin antibodies. Expression and purification of recombinant
Bcl-2-family proteins are described elsewhere (Luciano F, et al.
2007).
[0253] h. Microsatellite Instability (MSI)
[0254] Specimens were analyzed for MSI as described (Krajewska M,
et al. 2005b).
[0255] i. Statistical Analysis
[0256] Data were analyzed using the STATISTICA software package
(StatSoft) as described elsewhere (Krajewska M, et al. 2005b).
Median Bcl-B immunopercentage and immunoscore were applied as
cut-offs for Kaplan-Meier survival analyses.
[0257] ii. Results
[0258] a. Characterization of Bcl-B Antibodies and Immunoblot
analysis of Normal and Malignant Human Tissues
[0259] The specificity of the BR-49 antibody was documented,
showing reactivity with Bcl-B but not Bcl-2, Bcl-X.sub.L, Mcl-1,
Bcl-W or Bfl-1 (Luciano F, et al. 2007). To characterize the
specificity of the AR-77 antibody, immunoblot analysis was
performed using recombinant Bcl-2 family proteins generated in
bacteria (FIG. 1A-B). The AR-77 antibody was determined to be
specific for Bcl-B, detecting GST fusion protein containing Bcl-B
and lacking cross-reactivity with other Bcl-2-family members (FIG.
1A-B). Note that the two bands seen with GST-Bcl-B likely
correspond to intact fusion protein (.about.45-50 kD) and
proteolyzed protein separating Bcl-B (.about.23 kD) from GST.
[0260] Probing tissue lysates with the AR-77 or BR-49 antibodies
showed reactivity with a protein at .about.23 kD, corresponding to
the predicted molecular mass of Bcl-B, as well as .about.45-50 kD
band that appears to be an SDS-resistant dimer, based on studies
that have detected this species even when using epitope-tagged
Bcl-B protein that was detected using antibodies directed against
the tag. Also, a dimeric form of Bcl-B was demonstrated by SDS-PAGE
analyses of purified recombinant Bcl-B produced without GST tag. In
some tumor lysates, bands were detected that may correspond to
post-translationally modified forms of Bcl-B, which migrate at a
few kilo-Daltons larger apparent molecular mass than monomeric
Bcl-B in SDS-PAGE (see for example DLCL, MM and BPH samples). The
specificity of the anti-Bcl-B antibodies was further confirmed by
analysis of lysates from HeLa cells containing a
tetracycline-inducible Bcl-B construct, revealing the presence of
the expected .about.23 kD Bcl-B band only when the tetracycline
analog doxycyclin was added to cultures (FIG. 1).
[0261] Levels of endogenous Bcl-B varied among tissues and tumor
specimens analyzed by immunoblotting. Bcl-B was elevated in NSCLC
compared to normal lung in 2 of 2 paired specimens and also higher
in a colorectal cancer compared to normal colonic tissue from the
same patient (FIG. 1C). In contrast, Bcl-B protein levels were
higher in BPH specimen compared to a prostate cancer. Bcl-B protein
was also present in 3 of 4 DLCL and 2 of 3 MM specimens tested.
Reprobing blot with anti-Hsp60 or anti-.beta.-actin antibodies
confirmed equivalent loading of tissue lysates.
[0262] b. Immunohistochemical Analysis of Bcl-B Protein Expression
in Normal Human Tissues
[0263] To lay a foundation for assessing Bcl-B expression in
cancers, the in vivo patterns of Bcl-B protein expression were
first ascertained in normal human tissues by immunohistochemical
analysis. The most intense Bcl-B immunoreactivity was found in
plasma cells. Strongly stained plasma cells were found in bone
marrow, lymphoid tissues, at sites of inflammation, and
infiltrating some tumors (FIG. 2). In contrast, erythroid cells,
myeloid cells, and megakaryocytes in bone marrow, as well as
macrophages, dendritic cells, and most lymphocytes in nodes and
extranodular sites of inflammation were Bcl-B negative.
Immunoblotting analysis of plasma cells isolated from bone marrow
also confirmed the presence of Bcl-B protein (FIG. 6). Moreover,
comparisons of immune (FIG. 2A) and preimmune serum (FIG. 2B), as
well as preabsorption experiments using GST-Bcl-B (FIG. 2C) or
GST-Bcl-X.sub.L fusion proteins (FIG. 2D), confirmed the
specificity of plasma cell immunostaining (FIG. 7). The plasma cell
phenotype of the Bcl-B-positive cells was also confirmed by
two-color immunohistochemical analysis, showing co-expression of
Bcl-B with CD138 (syndecan-1) expressing cells (FIG. 2I-L). In
contrast, the Bcl-B-positive cells were negative for the macrophage
marker CD68 in two-color immunostainings
[0264] In addition to plasma cells, Bcl-B also immunolocalized to
centroblasts and centrocytes in germinal centers, but not to other
types of cells in lymphoid and hematopoietic tissues (FIG. 2E, H),
including bone marrow, spleen, nodes, and thymus. The intensity of
Bcl-B immunostaining in these lymphocytes however was substantially
less than plasma cells (FIG. 2F-G). Among other normal human
tissues, Bcl-B expression was detected in hepatocytes, renal tubule
epithelium, bronchial and nasopharyngeal epithelium, and type II
pneumocytes, as well as cytotrophoblasts in the placenta and some
neuronal cells, again with immunointensity much less than observed
in plasma cells. In the prostate gland, Bcl-B immunoreactivity was
strong in the luminal secretory cells but was absent in basal cells
(FIG. 8), thus constituting a pattern of expression opposite of
Bcl-2 (Krajewski S, et al. 1995). In all cells examined, the Bcl-B
staining pattern was predominantly cytosolic, with a punctate or
granular organellar distribution.
[0265] c. Bcl-B Protein Expression in Hematopoietic
Malignancies
[0266] Due to the predominant expression of Bcl-B protein in plasma
cells, expression of Bcl-B was investigated in plasma cell
dyscrasias. Bone marrow biopsies were immunohistochemically
evaluated from 165 patients, 114 with symptomatic multiple myeloma
(MM), 19 with indolent MM, 13 with monoclonal gammopathy of
undetermined significance (MGUS), and 19 with reactive
plasmacytosis. Unlike normal plasma cells which appeared to be
uniformly Bcl-B-positive, Bcl-B protein was immunolocalized only to
a proportion of plasma cells in the 165 specimens of plasma cell
dyscrasia, with an average prevalence of 29.+-.2.1% immunopositive
cells. Only 16% (26/165) of all tumors showed at least 50%
immunopositivity for Bcl-B, with 21 of these high Bcl-B expressors
belonging to symptomatic MM (21/114; 18%), 3 to the indolent MM
group (3/19; 16%), and 1 each to the MGUS (1/13; 8%) and the
reactive plasmacytosis (1/19; 5%) groups. Using 5% cut-off, 30%
(49/165) of the plasma cell dyscrasia cases were negative for
Bcl-B. FIG. 3(A-D) illustrates examples of Bcl-B immunostaining in
malignant plasma cells. No significant differences in Bcl-B
immunoscore values were observed when specimens from MM, MGUS and
reactive plasmacytosis cases were compared. Similarly, Bcl-B
immunopositivity and immunoscore were comparable in grade 1-3,
categorized according to WHO criteria regarding plasma cell
content. Bcl-B expression was not significantly associated with
patient age, gender, overall survival, or response to therapy in
MM.
[0267] Lymph node specimens from 48 DLBCL patients were
investigated by IHC for Bcl-B expression (FIG. 3E-F). Cases were
considered positive if .gtoreq.30% of tumor cells were Bcl-B
immunoreactive. Of 48 DLBCL cases, 25 (52%) were Bcl-B
immunopositive, using the 30% cut-off that corresponded to the
median percentage of Bcl-B positive cells in this cohort. CD10,
Bcl-6, and MUM1 immunostainings were applied to classify the DLBCL
cases into GCB and non-GCB groups Hans CP, et al. 2004, using the
same cut-off of .gtoreq.30% Hans C P, et al. 2004. In the
investigated patient cohort, 19/48 (40%) were considered GCB and 29
(60%) were classified as non-GCB cases. Both groups contained
almost identical proportions of Bcl-B negative and positive cases
(47% and 53%, respectively, in the GCB group; 48% and 52% in the
non-GCB category). Thus, analysis of the expression of Bcl-B
protein in the GCB and non-GCB lymphomas did not reveal significant
differences. The Bcl-B immunostaining data also did not correlate
with Bcl-2 staining in these DLBCL specimens.
[0268] None of the specimens derived from 57 patients with FL
contained detectable Bcl-B immunostaining in malignant B-cells
(FIG. 3G-H). Positive Bcl-B immunoreactivity in plasma cells
observed in these specimens provided an internal staining
control.
[0269] d. Bcl-B Protein Expression in Solid Tumors
[0270] In addition to hematopoietic malignancies, Bcl-B protein
expression was investigated in breast, cervical, ovarian, prostate,
lung, gastric, and colorectal cancers. The findings provide
evidence of alterations in Bcl-B protein expression in several
types of solid tumors.
[0271] (A) Breast Cancer
[0272] TMAs containing specimens derived from 119 stage I-III
breast cancer patients were immunostained for Bcl-B. The tissue
samples comprised 28 cases of DCIS and 104 ductal, 12 lobular, and
3 mucinous invasive carcinomas. In addition, 12 normal mammary
epithelium specimens, excised from surgical margins, were included
on the arrays, as well as 4 independent samples of normal mammary
gland tissue. Whereas expression of Bcl-B was below the level of
detection in normal mammary epithelium, Bcl-B immunostaining was
prevalent in 64% of in situ carcinomas and in 89% of invasive
cancers (cut-off 10% immunopositive cells), indicating increasing
expression with breast cancer progression. Comparison of Bcl-B
immunostaining results for transformed vs normal mammary epithelium
was highly significant (p<0.0001), using either immunopositivity
or immunoscore data (FIGS. 4A-C, 5A).
[0273] Significantly higher Bcl-B immunostaining was observed in
invasive ductal compared to invasive lobular carcinoma (70% vs 47%
mean immunopercentage, p=0.03; 108 vs 59 mean immunoscore [IS],
p=0.02). Higher Bcl-B immunostaining was associated with more
advanced stage of disease (p=0.01 for immunopercentage; p=0.004 for
IS), more involved lymph nodes (p=0.04 for IS), and higher
histological grade of tumors, with high-grade tumors containing
significantly higher levels of Bcl-B protein as determined by
immunostaining (70.+-.3.2% vs 42.+-.14.1% immunopositive [p=0.02]);
108.+-.6.4 vs 48.+-.17.6 IS [p=0.009]) (FIG. 5B). Bcl-B elevation
in invasive tumors was correlated with higher ER-.beta. expression
(r=0.42, p<0.0001 for immunopercentage; r=0.37, p<0.0001 for
immunoscore) and with lower PR expression (r=-0.22, p=0.02 for
immunoscore), but not with ER-.alpha. expression. Breast tumors
that demonstrated lymphatic vessel invasion (LVI) contained higher
levels of Bcl-B protein (p=0.005 for immunopositivity; p=0.0006 for
IS).
[0274] No correlations were found between Bcl-B expression and
patient age, tumor size, or patient survival. In this particular
cohort of breast cancer patients, only PR was an independent
prognostic factor for overall and disease-free survival in uni- and
multivariate analysis (p=0.008; p=0.01, respectively, Cox
regression), among all variables assessed (patient age, clinical
stage, tumor grade, LVI, ER-.alpha., ER-.beta., PR, Bcl-B % or
IS).
[0275] (B) Cervical Cancer
[0276] TMAs containing cervical specimens derived from Asian women
diagnosed with cervical intraepithelial neoplasia 1 (CIN1;
low-grade squamous intraepithelial lesions; mild dysplasia) (n=47),
CIN2 (high-grade squamous intraepithelial lesion; moderate
dysplasia) (n=46), CIN3 (high-grade squamous intraepithelial
lesion; severe dysplasia--carcinoma in situ) (n=137), and invasive
squamous cell carcinoma (n=109) were stained for Bcl-B. Normal
cervical epithelium adjacent to the transformed cells was available
for each histological entity (n=328) for all patients in the
precancerous groups, and 30 of 109 in women diagnosed with invasive
cancer. Barely detectable Bcl-B immunostaining was observed in
normal epithelium of the exocervix (FIG. 4D) and all stages of the
malignant progression (CIN1 to CIN3), indicating that Bcl-B
expression is not a characteristic of cervical cancer in this Asian
cohort. Strongly stained plasma cells were found infiltrating many
cervical tumors, serving as a positive control (FIG. 4E).
[0277] (C) Ovarian Cancer
[0278] Bcl-B expression was investigated using TMAs containing
tissue specimens from 91 ovarian carcinoma patients, and 6 normal
ovarian surface epithelium or fallopian tube specimens. The patient
cohort comprised 62 individuals with serous carcinomas and 29 cases
of non-serous tumors, including mucinous (n=13), endometrioid
(n=11), clear cell (n=2), granulose (n=1), dysgerminoma (n=1), and
carcinosarcoma (n=1) types.
[0279] Low intensity Bcl-B immunostaining was detected in ovarian
surface epithelium and normal tubal epithelium, with similar levels
of Bcl-B expression found in ovarian cancers. No significant
differences in Bcl-B protein levels were noted between the two
broad histological categories of ovarian cancer--serous and
non-serous. Among these patients, Bcl-B immunostaining did not
correlate with patient age, histological grade of tumor, CA125
serum marker, overall or disease-free survival, or response to
therapy. FIGO stages II-IV showed elevated levels of Bcl-B compared
to stage I tumors (87.+-.7.6 vs 49.+-.18.4, for IS) but the
difference was statistically insignificant. Statistical comparisons
performed for a more homogenous cohort of ovarian cancers, namely
the 64 serous carcinoma cases, failed to reveal significant
associations between Bcl-B expression and the clinical parameters.
Thus, Bcl-B over-expression is not a common trait of ovarian
cancers.
[0280] (D) Prostate Cancer
[0281] To characterize the expression of Bcl-B in prostate cancers,
TMAs containing patient specimens representing the full range of
prostate malignant transformation, including specimens of benign
prostatic hyperplasia (BPH; n=38), prostatic intraepithelial
neoplasia (PIN; n=11), and prostate adenocarcinoma (n=41) derived
from 66 patients were utilized. Gleason score data were available
for all tumors, while clinical stage information (T2-T3) (according
to International Union against Cancer criteria) was known for 48%
of patients. In addition, non-neoplastic prostate epithelium from
14% of cases was available for comparison of protein expression in
non-transformed vs neoplastic epithelium.
[0282] Low expression of Bcl-B was found in the normal prostatic
epithelium (mean immunopercentage 18.+-.11.6). Bcl-B expression was
markedly increased in BPH (63.+-.5%) and PIN (80.+-.7%) lesions,
but less so in the invasive cancers (46.+-.6%) (p=0.0003 for
immunopercentage; p=0.01 for IS) (FIG. 5C). Immunohistochemical
analysis of specimens revealed higher Bcl-B levels in high-grade
tumors (Gleason grade 4) as compared to tumors with Gleason grade
3, however, the difference did not reach the statistical
significance. Higher Bcl-B immunoscores correlated with poor
clinical outcome, with Bcl-B significantly upregulated in tumors
from patients who died from prostate cancer compared to those who
survived without relapse during the follow-up period (median
follow-up=2.7 years) (65.+-.14% vs 33.+-.9% for immunopositivity
[p=0.05]; 126.+-.30 vs 43.+-.18 for IS [p=0.005]) (FIG. 5D).
Comparison of preoperational PSA levels suggested that intratumoral
Bcl-B is higher in patients with higher PSA levels, but the results
did not reach statistical significance.
[0283] (E) Gastric Cancer
[0284] Archival gastric specimens from 169 Asian patients who
underwent surgical resection for localized gastric cancer were
analyzed immunohistochemically for Bcl-B protein expression. Bcl-B
was expressed in normal gastric surface epithelium, but glands deep
within the gastric mucosa were negative for Bcl-B or contained only
trace amounts of this protein (FIG. 4F).
[0285] Expression of Bcl-B was prevalent in gastric cancers, with
89% of tumors showing .gtoreq.10% immunopositive cells. Bcl-B
protein was significantly associated with histological architecture
and cellular differentiation of gastric adenocarcinomas, with
higher levels of Bcl-B expression in well-differentiated tumors
compared to poorly differentiated tumors (90.+-.2.4% vs 52.+-.3%
[p<0.0001], and 155.+-.8 vs 69.+-.5 IS [p<0.0001]) (FIG. 5E)
and higher Bcl-B levels in intestinal-type (FIG. 4G) compared to
diffuse-type cancers (FIG. 4H) (p<0.0001). Similarly, Bcl-B
levels in tumors containing signet ring cells were lower than those
in non-signet ring cell tumors (43.+-.10.0% vs 75.+-.3%
[p<0.0001], and 59.+-.17 vs 118.+-.6 IS [p<0.0001]).
Interestingly, tumors with prominent lymphoid infiltration
demonstrated higher Bcl-B protein content compared to those that
were not infiltrated by lymphocytes (p=0.01 for immunopositivity;
p=0.009 for IS).
[0286] Although tumors from patients who died from cancer contained
slightly lower levels of Bcl-B protein compared to tumors from
those who survived (p=0.01 for IS), no significant association with
OS or DFS was observed for this patient cohort using Kaplan-Meier
survival analysis and log-rank test analysis. Also, Bcl-B
expression did not correlate with the clinical stage or mucin
content in tumors.
[0287] (F) Colorectal Cancer
[0288] TMAs were constructed using primary tumor specimens derived
from a cohort of 106 Asian patients with stage II CRC, who were
treated by surgical resection with curative intent. Of the 106
selected cases, 63 patients survived without recurrence, 7 patients
had recurrent disease, and 36 patients died from CRC. Thus, while
not an unbiased sequential case series, the survival profile of
this cohort closely resembles that of a random population of stage
II CRC patients, with 72.5% of individuals alive at 5 yrs. Adjacent
normal colonic mucosa was present in 65% of the 106 tumor specimens
on the array, permitting side-by-side comparisons of immunostaining
results for morphologically normal vs malignant epithelium. In
addition, 4 specimens of normal colon derived from individuals who
were not diagnosed with colon cancer were stained separately.
[0289] Significantly higher Bcl-B protein expression was found in
colorectal cancers (FIG. 4J) compared to normal colonic epithelium
(FIG. 4I), as assessed by percentage of immunopositive cells
(64.+-.5% vs 92.+-.1%) and by immunoscore (82.+-.7 vs 181.+-.6)
(p<0.0001; p=0.004, respectively). To explore, if differences in
Bcl-B protein expression may correlate with previously identified
prognostic features (Elsaleh H. 2001), Bcl-B immunostaining was
compared with MSI status, anatomical location of tumors, patient
gender, and age. In the investigated cohort, higher Bcl-B levels
were found in microsatellite stable compared to MSI tumors
(p=0.0002 for immunopositivity; p=0.004 for IS), and in the
left-sided as compared to right-sided adenocarcinomas (p=0.02 for
immunopositivity; p=0.04 for IS). Age and gender were not
associated with Bcl-B expression. In univariate analysis, no
correlations were observed between OS or DFS and Bcl-B
immunostaining data.
[0290] Because sections from this same TMA had previously been
analyzed by immunohistochemistry for expression of some other
Bcl-2-family proteins (Krajewska M, et al. 2005a), Bcl-B
immunostaining data was compared with Bcl-2, Bcl-XL, Bax, and Bid.
Bcl-B immunostaining in CRCs correlated with Bcl-X.sub.L (r=0.43,
p<0.0001 for IS) and Bax (r=0.24, p=0.03 for IS), but not with
Bcl-2 or Bid.
[0291] (G) SCLC
[0292] Immunostaining for Bcl-B was performed on SCLC primary
tumors obtained by hemithoracotomy from 79 patients with
limited-stage disease. The cohort comprised 76% men and 24% women
with median age of 57 years, among whom 53% were diagnosed with
non-specified SCLC type, 27% with intermediate, 11% mixed
intermediate, 5% fusiform/spindle, 3% oat cell, and 1% with
polygonal carcinoma. In addition to primary tumors, matching
metastatic mediastinal lymph nodes derived from 24 patients in this
cohort, were available for investigation.
[0293] No significant differences in Bcl-B levels were observed
between primary (FIG. 4K) and metastatic tumors (FIG. 4L), as
determined by paired t-test. Higher Bcl-B content was found in
primary tumors from men (p=0.004 for immunopositivity; p=0.02 for
IS) and from older patients (p=0.003 for immunopositivity; p=0.01
for IS). No association was noted between performance status,
clinical stage (TNM and UICC stage), tumor size or site (left/right
lung) and Bcl-B protein levels in tumors.
[0294] To investigate possible association of Bcl-B expression with
clinical outcome, Bcl-B immunopercentage and immunoscore data were
dichotomized at the median values into "low" vs "high" expression
groups. High Bcl-B prevalence correlated with shorter OS (log-rank
test p=0.009; FIG. 5F) and increased relative risk of death due to
SCLC (univariate Cox proportional hazards analysis HR 2.4; 95%
confidence interval [CI 1.3-4.7]; .p=0.008). However, in
multivariate Cox analysis, which included patient age, gender,
clinical stage, tumor size and size, Bcl-B did not show independent
prognostic significance. Bcl-B immunostaining data also did not
significantly correlate with time to recurrence after surgery and
chemotherapy, though a trend towards higher Bcl-B and shorter time
was noted (median 4.9 vs 3.5 years).
[0295] (H) NSCLC
[0296] TMAs containing specimens from 82 non-small cell lung cancer
(NSCLC) patients were immunohistochemically analyzed for Bcl-B
protein expression, including 22 adenocarcinomas, 32 squamous cell
carcinomas, and 16 large cell carcinomas, and 12 unspecified
tumors. Large cell tumors contained relatively low Bcl-B expression
(81.+-.15 IS), adenocarcinomas showed intermediate expression
(126.+-.20 IS) and squamous cell carcinomas revealed higher Bcl-B
content (154.+-.14 IS) (p=0.005). No follow-up data were available
for these NSCLC patients.
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F. SEQUENCES
TABLE-US-00001 [0333] 1. SEQ ID NO: 1 NH2-REPGTPEPAPSTPEAAVLR-amide
2. (Bcl-B-Accesssion AF285092) SEQ ID NO: 2 atggttgacc agttgcggga
gcgcaccacc atggccgacc cgctgcggga gcgcaccgag ctgttgctgg ccgactacct
ggggtactgc gcccgggaac ccggcacccc cgagccggcg ccatccacgc ccgaggccgc
cgtgctgcgc tccgcggccg ccaggttacg gcagattcac cggtcctttt tctccgccta
cctcggctac cccgggaacc gcttcgagct ggtggcgctg atggcggatt ccgtgctctc
cgacagcccc ggccccacct ggggcagagt ggtgacgctc gtgaccttcg cagggacgct
gctggagaga gggccgctgg tgaccgcccg gtggaagaag tggggcttcc agccgcggct
aaaggagcag gagggcgacg tcgcccggga ctgccagcgc ctggtggcct tgctgagctc
gcggctcatg gggcagcacc gcgcctggct gcaggctcag ggcggctggg atggcttttg
tcacttcttc aggaccccct ttccactggc tttttggaga aaacagctgg tccaggcttt
tctgtcatgc ttgttaacaa cagccttcat ttatctctgg acacgattat tatga
3.(Bcl-B-Accesssion AAG00503) SEQ ID NO: 3 mvdqlrertt madplrerte
llladylgyc arepgtpepa pstpeaavlr saaarlrqih rsffsaylgy pgnrfelval
madsvlsdsp gptwgrvvtl vtfagtller gplvtarwkk wgfqprlkeq egdvardcqr
lvallssrlm gqhrawlqaq ggwdgfchff rtpfplafwr kqlvqaflsc llttafiylw
trll
Sequence CWU 1
1
3119PRTArtificial SequenceSynthetic peptide corresponding to
residues 32-50 of human Bcl-B 1Arg Glu Pro Gly Thr Pro Glu Pro Ala
Pro Ser Thr Pro Glu Ala Ala1 5 10 15Val Leu Arg2615DNAHomo
sapiensgene(1)..(615) 2atggttgacc agttgcggga gcgcaccacc atggccgacc
cgctgcggga gcgcaccgag 60ctgttgctgg ccgactacct ggggtactgc gcccgggaac
ccggcacccc cgagccggcg 120ccatccacgc ccgaggccgc cgtgctgcgc
tccgcggccg ccaggttacg gcagattcac 180cggtcctttt tctccgccta
cctcggctac cccgggaacc gcttcgagct ggtggcgctg 240atggcggatt
ccgtgctctc cgacagcccc ggccccacct ggggcagagt ggtgacgctc
300gtgaccttcg cagggacgct gctggagaga gggccgctgg tgaccgcccg
gtggaagaag 360tggggcttcc agccgcggct aaaggagcag gagggcgacg
tcgcccggga ctgccagcgc 420ctggtggcct tgctgagctc gcggctcatg
gggcagcacc gcgcctggct gcaggctcag 480ggcggctggg atggcttttg
tcacttcttc aggaccccct ttccactggc tttttggaga 540aaacagctgg
tccaggcttt tctgtcatgc ttgttaacaa cagccttcat ttatctctgg
600acacgattat tatga 6153204PRTHOMO SAPIENUNSURE(1)..(204) 3Met Val
Asp Gln Leu Arg Glu Arg Thr Thr Met Ala Asp Pro Leu Arg1 5 10 15Glu
Arg Thr Glu Leu Leu Leu Ala Asp Tyr Leu Gly Tyr Cys Ala Arg 20 25
30Glu Pro Gly Thr Pro Glu Pro Ala Pro Ser Thr Pro Glu Ala Ala Val
35 40 45Leu Arg Ser Ala Ala Ala Arg Leu Arg Gln Ile His Arg Ser Phe
Phe 50 55 60Ser Ala Tyr Leu Gly Tyr Pro Gly Asn Arg Phe Glu Leu Val
Ala Leu65 70 75 80Met Ala Asp Ser Val Leu Ser Asp Ser Pro Gly Pro
Thr Trp Gly Arg 85 90 95Val Val Thr Leu Val Thr Phe Ala Gly Thr Leu
Leu Glu Arg Gly Pro 100 105 110Leu Val Thr Ala Arg Trp Lys Lys Trp
Gly Phe Gln Pro Arg Leu Lys 115 120 125Glu Gln Glu Gly Asp Val Ala
Arg Asp Cys Gln Arg Leu Val Ala Leu 130 135 140Leu Ser Ser Arg Leu
Met Gly Gln His Arg Ala Trp Leu Gln Ala Gln145 150 155 160Gly Gly
Trp Asp Gly Phe Cys His Phe Phe Arg Thr Pro Phe Pro Leu 165 170
175Ala Phe Trp Arg Lys Gln Leu Val Gln Ala Phe Leu Ser Cys Leu Leu
180 185 190Thr Thr Ala Phe Ile Tyr Leu Trp Thr Arg Leu Leu 195
200
* * * * *