U.S. patent application number 17/346630 was filed with the patent office on 2021-11-18 for methods to eliminate cancer stem cells by targeting cd47.
This patent application is currently assigned to The U.S.A, as represented by the Secretary, Department of Health and Human Services. The applicant listed for this patent is The U.S.A, as represented by the Secretary, Department of Health and Human Services, The U.S.A, as represented by the Secretary, Department of Health and Human Services. Invention is credited to Sukhbir Kaur, Chengyu Liu, David D. Roberts.
Application Number | 20210355495 17/346630 |
Document ID | / |
Family ID | 1000005680486 |
Filed Date | 2021-11-18 |
United States Patent
Application |
20210355495 |
Kind Code |
A1 |
Roberts; David D. ; et
al. |
November 18, 2021 |
METHODS TO ELIMINATE CANCER STEM CELLS BY TARGETING CD47
Abstract
Described herein is the discovery that cancer stem cells (CSCs)
can be induced to differentiate by altering CD47 signaling.
Provided herein are methods and compositions for inducing
differentiation of cancer stem cells, for instance irreversible
differentiation, including methods of treating subjects with cancer
such as breast cancer, colon cancer, lung cancer, ovarian cancer,
or melanoma, and including metastatic as well as primary cancer.
Also provided are methods for treating subjects with triple
negative breast cancers involving forcing differentiation of bCSCs
of the subjects through targeting of CD47.
Inventors: |
Roberts; David D.;
(Bethesda, MD) ; Kaur; Sukhbir; (Bethesda, MD)
; Liu; Chengyu; (Boyds, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The U.S.A, as represented by the Secretary, Department of Health
and Human Services |
Bethesda |
MD |
US |
|
|
Assignee: |
The U.S.A, as represented by the
Secretary, Department of Health and Human Services
Bethesda
MD
|
Family ID: |
1000005680486 |
Appl. No.: |
17/346630 |
Filed: |
June 14, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15517345 |
Apr 6, 2017 |
11053314 |
|
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PCT/US2015/055029 |
Oct 9, 2015 |
|
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17346630 |
|
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|
62062675 |
Oct 10, 2014 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2803 20130101;
C12N 2310/3233 20130101; C12N 2501/999 20130101; C12N 2310/11
20130101; A61K 45/06 20130101; C12N 2320/32 20130101; C12N 5/0695
20130101; C12N 15/113 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; A61K 45/06 20060101 A61K045/06; C12N 5/095 20060101
C12N005/095; C07K 16/28 20060101 C07K016/28 |
Claims
1-16. (canceled)
17. A method for inducing differentiation of a cancer stem cell
(CSC), comprising contacting the CSC with an antisense morpholino
oligonucleotide complementary to CD47, thereby inducing
differentiation of the CSC.
18. The method of claim 17, wherein the differentiation of the CSC
is SIRP.alpha.-independent.
19. The method of claim 17, wherein the CSC expresses CD47.
20. The method of claim 17, wherein the antisense morpholino
oligonucleotide comprises the nucleic acid sequence of SEQ ID NO:
70.
21. The method of claim 20, wherein the antisense morpholino
oligonucleotide consists of the nucleic acid sequence of SEQ ID NO:
70.
22. The method of claim 17, wherein the CSC comprises a breast
cancer stem cell, a lung cancer stem cell, a prostate cancer stem
cell, a colon carcinoma stem cell, or a melanoma stem cell.
23. The method of claim 17, wherein the CSC is in a subject.
24. The method of claim 23, wherein the contacting comprises
administering the antisense morpholino oligonucleotide
complementary to CD47 to a subject with cancer.
25. The method of claim 24, wherein the subject has a primary
tumor, has a tumor in regression, has or is suspected of having a
metastatic tumor, or a combination thereof.
26. The method of claim 23, further comprising administering a
therapeutically effective amount of an anti-cancer treatment to the
subject.
27. The method of claim 17, wherein contacting the CSC is in vitro.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/517,345, filed Apr. 6, 2017, which is the
.sctn. 371 U.S. National Stage of International Application No.
PCT/US2015/055029, filed Oct. 9, 2015, published in English under
PCT Article 21(2), which claims the benefit of the earlier filing
date of U.S. Provisional Application No. 62/062,675, filed Oct. 10,
2014; that earlier application is incorporated herein in its
entirety.
FIELD
[0002] This disclosure relates to methods and compositions for
selectively eliminating cancer stem cells, particularly through
perturbing CD47 signaling.
BACKGROUND
[0003] Cancer stem cells (CSC) can be defined by three general
properties: 1) their capability for self-renewal, 2) asymmetric
cell division, and 3) capability to divide and differentiate
(Sugiarto et al., Cancer Cell. 20(3):328-340, 2011). Initially the
concept of cancer stem cells was introduced based on the properties
of CD34.sup.+ and CD38.sup.- leukemia cells isolated from patients
(Lapidot et al., Nature. 367:645-648, 1994). Subsequently, cancer
stem cells have been isolated from many types of solid tumors
including brain and breast tumors (Visvader & Lindeman, Nat Rev
Cancer. 8:755-768, 2008). Studies using cancer cells fractioned for
stem cell markers demonstrate that CSC isolated from various cancer
cell lines have the ability to initiate tumor formation in rodent
models, but the same cell lines depleted of CSC are unable to
initiate tumor formation. Consequently, CSC are also referred to as
tumor initiating cells. Cancer stem cells may originate from
malignant transformation of normal stem cells or tissue-resident
committed progenitor cells. The growing research interest in cancer
stem cells is based on evidence that current cancer therapies
selectively kill fast growing differentiated tumor cells but are
unable to kill tumor initiating cancer stem cells (Dean et al., Nat
Rev Cancer. 5:275-284, 2005). These refractory tumor cells can be
responsible for recurrence of tumors following treatment. Cancer
stem cells may also migrate to distal sites and be responsible for
initiating cancer metastasis.
[0004] Targeting new cancer therapies to cancer stem cells is very
challenging because many of the tumor suppressor and polycomb genes
(Hedgehog and WNT signaling pathway) that are important for normal
stem cell regulation are dysregulated during carcinogenesis
(Karamboulas & Ailles, Biochim Biophys Acta. 1830(2):2481-2495,
2013). The ubiquitous cell surface protein CD47 is up-regulated in
many cancers, especially during metastasis, and high expression is
a negative prognostic indicator for several cancers. Additional
studies have indicated that CD47 expression is elevated in leukemic
cancer stem cells. The current conventional wisdom is that the
function of this elevated CD47 expression on CSC is to serve as a
"don't eat me" signal that protects the CSC from phagocytic
clearance by macrophages (reported online at
news.sciencemag.org/health/2012/03/one-drug-shrink-all-tumors).
Consequently, antibody and ligand therapeutics that engage CD47
have been developed to stimulate the destruction of CSC by
macrophages, and these are now entering human clinical trials. The
CD47 antibody B6H12 is known to block the recognition of CD47 by
its counter-receptor SIRP.alpha. on macrophages. Human tumor
xenografts grown in immunodeficient mice that express a mutant form
of SIRP.alpha. that is capable of binding human CD47 (Nod-SCID)
have been used to test the ability of B6H12 to enhance
macrophage-mediated clearance of human tumor xenografts. Inhibition
of tumor growth by B6H12 in these models provided evidence to
support the humanization of such CD47 antibodies for treating human
cancer patients.
SUMMARY
[0005] However, CD47 is more than a passive SIRP.alpha.
counter-receptor, and B6H12 has effects on CD47 signaling that are
independent of blocking SIRP.alpha. binding. Signal transduction
through CD47 regulates important cell functions and gene
expression. Described herein is the discovery of an unanticipated
activity of B6H12 and other agents that perturb CD47 signaling in
cancer stem cells (CSCs), and methods of using this to inhibit or
treat cancer, or prevent or reduce metastasis, by inducing
differentiation of CSCs.
[0006] Provided herein are methods for inducing differentiation
(for instance, irreversible differentiation) of a cancer stem cell,
involving contacting the cancer stem cell with an agent that alters
CD47 signaling to induce cancer stem cell (CSC) differentiation.
Optionally, the CSC may be in a subject. The agent used in the
method comprises, in various embodiments, an anti-CD47 antibody or
binding fragment thereof, peptide 7N3, or a CD47-targeted CRISPR
construct. Where the agent comprises an antibody or fragment
thereof, it is specifically contemplated that the agent can be the
monoclonal antibody B6H12, humanized B6H12, a binding fragment of
B6H12 or humanized B6H12, or a monoclonal antibody that binds
competitively with B6H12.
[0007] Also provided herein are methods of treating a subject by
inducing differentiation of a cancer stem cell in the subject. It
is contemplated that the subject may have a tumor, such as a
primary tumor, has a tumor in regression, has or is suspected of
having a metastatic tumor, or a combination thereof. Such treatment
methods involve administering to the subject a therapeutically
effective amount of agent that alters CD47 signaling to induce
cancer stem cell (CSC) differentiation, thereby treating the
subject with the tumor.
[0008] Also provided are treatment methods which further involve
administering a therapeutically effective amount of an anti-cancer
treatment to the subject. This can occur before, concurrent with,
or after treatment with the agent that alters CD47 signaling to
induce cancer stem cell (CSC) differentiation. In particular
examples, the anti-cancer treatment is administered first, followed
(for instance 1-4 days, or 1-7 days later) by administration with
the agent that alters CD47 signaling.
[0009] Also provided herein are methods for inducing
differentiation (such as irreversible differentiation) of cancer
stem cells, substantially as described herein.
[0010] The foregoing and other objects, features, and advantages of
the invention will become more apparent from the following detailed
description, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] At least some of the figures submitted herewith contain
color.
[0012] FIG. 1A-1E illustrates characterization of breast cancer
stem cells (bCSCs) derived from suspension cell-enriched MDA-MB-231
triple negative breast carcinoma cells. FIG. 1A) MDA-MB-231 cells
were cultured using RPMI media with 10% FBS+P/S+ Glutamine at 37
C.degree.. Inset showing the presence of bCSCs in the form of small
round suspension cells. FIG. 1B) With gentle agitation of the
flask, loosely bound bCSCs were separated from adherent MDA-MB-231
cells. FIG. 1C) bCSCs form loose aggregates after incubation at
37.degree. C. FIG. 1D) MDA-MB-231 bCSCs exhibit high asymmetric
cell division. Adherent MDA-MB-231 and enriched bCSCs cells were
labeled with BrdU for 10 days and then chased in BrdU free media
for 3-4 days and followed by 2 .mu.M Cytochalasin. The cells were
immunostained using BrdU antibody and mounted with Vectashield
DAPI. The confocal images were taken using a Zeiss 780 microscope
at 63.times.. FIG. 1E) Quantification of asymmetric cell division
ratio between cells negative for BrdU and positive for DAPI counted
manually at 20.times..
[0013] FIG. 2A-2I illustrates differentiation of bCSCs into neural
cells. MDA-MB-231 bCSCs induced to form embryoid bodies can
differentiate along a neural lineage. MDA-MB-231 cells were
cultured in complete RPMI media, and bCSCs were cultured with
neural media containing EBM medium supplemented with FGF2 and EGF
(-5-20 ng/ml), heparin and gentamycin sulfate for 36 h. FIG. 2A-2C)
MDA-MB-231 cells were immunostained with Tuj 1, Neuron-specific
class III beta-tubulin. FIG. 2D) Loosely formed embryoid body-like
cluster of bCSCs. FIGS. 2E & 2F) Differentiation of bCSC
clusters into neuron like cells. FIG. 2G-2I) Neuron like
differentiated cells from bCSCs were immunostained with Tuj 1. The
images were captured using a Zeiss 780 confocal microscope.
[0014] FIG. 3A-3F illustrate differentiation of MDA-MB-231 bCSCs
into endothelial-mimic cells using endothelial growth medium
(EGM2). FIG. 3A-3C) Control adherent MDA-MB-231 cells were cultured
in complete RPMI medium for 36 h and were immunostained for
expression of the endothelial markers CD31 and VEGFR2. FIG. 3D-3F)
Immunostaining of differentiated bCSC cells cultured in EGM2 media
for expression of CD31 and VEGFR2.
[0015] FIG. 4A-4F illustrate differentiation of MDA-MB-231 bCSCs
into smooth muscle cells using Smooth Muscle Basal Medium (Lonza)
supplemented with PDGF (10 ng/ml) and TGF-61 (5 ng/ml). FIG. 4A-4C)
Control MDA-MB-231 cells were cultured in complete RPMI media for
36 h and were immunostained with smooth muscle actin (SMA)
antibody. FIG. 4D-4F) SMA Immunostaining of smooth muscle cells
differentiated from bCSC cells.
[0016] FIG. 5A-5E is a table showing the Ct values of real time PCR
of MDA-MB-231 and bCSCs. MDA-MB-231 bCSCs have up-regulated mRNA
expression of a subset of embryonic and cancer stem cell markers.
FIGS. 5A & 5B) MDA-MB-231 and bCSCs cells were separated, and
total RNA was extracted for real time PCR (q-PCR) analysis using
the indicated primers for VEGF, OCT4, Nanog, Sox2, Nestin, PTPBP1,
CD44 and CD24 with B2M control. FIG. 5C) Real time Ct value of gene
expression of above listed genes after Re-plating of bCSCs. FIGS.
5D & 5E) Real time Ct value of differentiated using EGM2 and
Neural media.
[0017] FIG. 6A-6B show that bCSCs have high cell surface expression
of CD44 and low CD24 than MDA-MB-231 cells. FIGS. 6A & 6B)
bCSCs have higher cell surface expression of CD44 and lower CD24
expression than control MDA-MB-231 cells. Representative flow
cytometry analysis is shown.
[0018] FIG. 7A-7B show global analysis of differential gene
expression in MDA-MB-231 bCSC versus unfractionated MDA-MB-231
cells. FIG. 7A) Hierarchical clustering of differentially expressed
genes based on microarray analysis of MDA-MB-231 and bCSCs. FIG.
7B) List of differential expression of genes between MDA-MB-231 and
bCSCs.
[0019] FIG. 8. Relative gene expression between CD47, IL2, IL10,
CCL3, c-MYC, ALDI, ITG4, TSP1,7a-1,7a-3, 7d, 7f, 7f-2, AGO1, DGCR8,
DROSHA, AGO2, DICER were analyzed using control actin primer. The
Ct value of actin for MDA-MB-231 (12) and for bCSCs (14)
respectively.
[0020] FIG. 9A-9B shows that the CD47 blocking antibody (B6H12 Ab)
exhibits morphological change only in bCSCs not in MDA-MB-231.
FIGS. 9A & 9B) MDA-MB-231 and bCSCs are treated in the presence
or absence of 1 ug/ml of B6H12 for 72 h. The images of live cells
were captured using Olympus microscope at 20.times. and
4.times..
[0021] FIG. 10A-10B show that B6H12 Ab alters gene expression of
bCSCs. FIG. 10A) Microarray analysis clustering of bCSCs treated
with Control IgG antibody or B6H12 Ab for 36 h. FIG. 10B) List of
Differential expression of genes between Control antibody vs B6H12
Ab. Microarray analysis of CD47 Blocking antibody Treated Cancer
Stem Cells.
[0022] FIG. 11A-11H show that CD47 blocking antibody increase mRNA
expression of tumor suppressor and apoptotic genes. Validation of
microarray gene expression results of differentially expressed
genes between bCSCs treated with control IgG antibody or B6H12 Ab
for 36 h. Up-regulation of tumor suppressor (TXNIP in FIG. 11A, LOX
in FIG. 11B and PLS3 in FIG. 11C), activation of apoptotic and
caspases genes (PMAIP1 in FIG. 11E and CRISPLDI in FIG. 11F) and
cell surface markers such as CD14 (FIG. 11D), TIE1 (FIG. 11H) and
TGFBR3 (FIG. 11G) was demonstrated.
[0023] FIG. 12A-12F shows that CD47 blocking antibody globally and
predominantly targets mitochondrial energetics, asymmetric cell
division and cell proliferation in bCSCs. The control MDA-MB-231
and bCSCs were plated using XF24 well plates for 1-4 days.
Mitochondrial energetics was measured using extracellular flux
assay kits. FIG. 12A) B6H12 (1 .mu.g/ml) alters mitochondrial
energetics in bCSCs but not in control MDA-MB-231 cells. FIGS. 12B
& 12C) Long term treatment of bCSC with B6H12 (1 .mu.g/ml)
leads to decrease in basal oxygen consumption rate (OCR) due to
decreased ATP associated OCR and decreases reserve mitochondrial
metabolic capacity. The CD47 ligand TSP1 (1 .mu.g/ml) modestly
changes mitochondrial energetics but stays consistent from 1-4
days. FIG. 12D) B6H12 Ab shows decreased in asymmetric cell
division of bCSCs cells. bCSCs were labeled with BrdU for 10 days,
and then chased, treated with Control Ab (1 .mu.g/ml) or B6H12 Ab
(1 .mu.g/ml) in BrdU-free medium for 3-4 days and followed by 2
.mu.M Cytochalasin. The cells were immunostained using BrdU
antibody and mounted with Vectashield DAPI. The confocal images
were taken using a Zeiss 710 microscope at 63.times.. FIGS. 12E
& 12F) Control MDA-MB-231 cells and bCSCs were treated with
control or B6H12 Ab at 0.1, 1, 2 and 5 (1 .mu.g/ml) for 72 h. The
cell proliferation was measured using CellTiter 96.RTM. Aqueous
Non-Radioactive Cell Proliferation Assay (MTS) according to
manufacturers' instructions.
[0024] FIG. 13A-13C illustrate that EGF and EGFR mRNA expression is
up-regulated in bCSCs, and B6H12-antibody down-regulates mRNA
expression of EGF and EGFR. FIGS. 13A & 13B) Total RNA was
extracted from bCSCs and the gene expression of EGF and EGFR2 was
measured using real time PCR. FIG. 13C) bCSCs were treated with
B6H12 or control Ab (1 .mu.g/ml) in the presence or absence of EGF
(30 ng/ml) for 36 h. EGF and EGFR mRNA expressions were
undetectable in bCSCs treated with B6H12 Ab (1 .mu.g/ml) however
control Ab (1 .mu.g/ml) does not show any effect on bCSCs.
[0025] FIG. 14A-14D show that B6H12-antibody disrupts the
association between EGFR and CD47 and inhibits EGFR phosphorylation
at Y.sup.1068 in bCSCs. FIG. 14A) Unfractionated MDA-MB-231 cells
were treated with B6H12 Ab (1 .mu.g/ml) for 20 minutes and further
stimulated with EGF (30 ng/ml) for 5 minutes. The cells were fixed
immediately using PFA and immunostained with CD47 and EGFR
antibody. The green fluorescence indicates protein expression of
CD47 and red fluorescence shows protein expression of EGFR.
[0026] FIG. 14B) bCSCs were pretreated with B6H12 Ab (1 .mu.g/ml)
for 15 minutes followed by EGF (30 ng/ml) for 5 minutes. The total
lysates were made using NP-40 lysis buffer and immunoprecipitation
was performed using phospho-Tyrosine antibody. Global tyrosine
phosphorylation of proteins was analyzed by SDS-PAGE performed
using Bis-Tris gels 4-12% and stained with Coomassie blue. FIG.
14C) Control MDA-MB-231 and bCSCs were treated with B6H12 (1
.mu.g/ml) and EGF (5 ng/ml) as above, and IP-western blot was
performed using phospho-EGFR antibodies. B6H12 Ab down regulated
EGFR Y.sup.1068 phosphorylation only in bCSCs. FIG. 14D) bCSCs were
further sorted to isolate CD44.sup.high and CD24.sup.low cells
using FACS. The IP-western blot of EGFR was performed using similar
conditions as above. B6H12 Ab (1 .mu.g/ml) treatment reduces EGFR
Y.sup.1068 phosphorylation.
[0027] FIG. 15A-15D show that B6H12-Ab inhibits cell proliferation
but not HER2 phosphorylation in ER+ breast cancer cells. FIGS. 15A
& 15B) MCF7 and T47D1 cells were pretreated with B6H12 Ab (1
.mu.g/ml) for 15 minutes followed by EGF (5 ng/ml) for 5 minutes.
The total lysates were made using NP-40 lysis buffer and
immunoprecipitation was performed using HER2 antibody. IP-western
blots were performed using phospho-HER2 antibodies. FIGS. 15C &
15D) MCF7 and T47D1 cells were labeled with BrdU for 24 h. BrdU
treated cells were further treated with B6H12 Ab (1 .mu.g/ml) for
24 h. The BrdU incorporation was measured using a BrdU cell
proliferation assay kit according to manufactures'
instructions.
[0028] FIG. 16A-16F illustrate that B6H12-Ab decreases cell surface
expression of EGFR via release of Exosomes and K63 Ubiquitination.
FIGS. 16A, 16B and 16C) MCF7, T47D1 and bCSCs were pretreated with
B6H12 Ab (1 .mu.g/ml) for 15 minutes and immediately fixed with 4%
PFA. Immunostaining of HER2 and EGFR was performed and images were
captured using a Zeiss 780 microscope at 63.times.. FIG. 16D)
B6H12-Ab Disrupts the Interaction between CD47 and EGFR in bCSCs.
FIG. 16E) B6H12-Ab (1 .mu.g/ml) blocks the release of Exosomes in
T47D1 and MCF7 Cells but not in bCSCs. FIG. 16F) B6H12-Ab (1
.mu.g/ml) disrupts the Interaction between CD47 and EGFR by
Inducing K63 Ubiquitination of EGFR FIG. 17A-17C illustrate that
B6H12 Ab signal may be independent of SIRP ligand. FIGS. 17A, 17B
& 17C) B6H12-Ab (1 .mu.g/ml) does not affect mRNA expression of
SIRP.alpha., and .gamma. in bCSCs FIG. 18A-18D show that bCSCs
differentiated into neural linage exhibit mammospheres and EMT gene
enrichment. FIG. 18A) MDA-MB-231 cells were cultured in complete
RPMI media, and bCSCs were cultured with neural media containing
EBM medium supplemented with FGF2 and EGF (.about.5-20 ng/ml),
heparin and gentamycin sulfate for 36 h. The total RNA was
extracted and global Microarray was performed. The differential
gene expression of neural linage cancer cells were compared to
MDA-MB-231 derived differentiated and cancer stem cells. FIG. 18B)
Gene enrichment analysis (GSEA) of differentiated neural lineage
MDA-MB231 cells with parental MDA-MB-231 cells and seeder cells
(MDA-231-S1a and S1b). FIGS. 18C & 18D) GSEA of mammospheres
and EMT formation.
[0029] FIG. 19A-19B illustrates that CD47-B6H12 antibody does not
show similar enrichment to MDA231 differentiated or cancer stem
cells. FIG. 19A) Gene enrichment analysis (GSEA) of B6H12 antibody
treated cells vs. differentiated neural lineage MDA-MB-231 cells
and parental MDA-MB-231 cells. FIG. 19B) Principal component
analysis of B6H12 treated cells.
[0030] FIG. 20A-20D illustrates that the anti-CD47 antibody B6H12
inhibits cell proliferation of certain types of breast cancer cell
lines. FIG. 20A) B6H12 antibody Inhibits Cell Proliferation in
CD44.sup.+CD24 breast cancer stem cells (bCSCs) isolated from the
triple negative breast cancer MDA-MB-231 cell line. FIG. 20B)
B6H12-Ab inhibits cell proliferation of T47D1 breast carcinoma
cells, and bCSCs derived from parental T47D1 cells. FIGS. 20C &
20D) B6H12-Ab does not inhibit cell proliferation of normal mammary
epithelial cells (MCF10A) or the ER+ breast carcinoma cell line
MCF7 cells.
[0031] FIG. 21A-21D FIG. 21A) OH-1 (small cell lung cancer cells),
H-128 (small cell lung cancer carcinoma) and PC3 (Prostatic
adenocarcinoma) cells were plated for 0-72 h. The cell
proliferation was performed using CellTiter 96.RTM. AQueous
Non-Radioactive Cell Proliferation kit from Promega after 72 h.
FIG. 21B) A2058 (Human Melanoma cancer cell line) was treated with
B6H12 Ab (1 .mu.g/ml) in the presence or absence of EGF (30 ng/ml)
for 48 h. A2058 cells were labeled with BrdU for 4 h, and BrdU
incorporation was measured using a BrdU cell proliferation assay
kit according to manufacturer's instructions. FIG. 21C) SW480
(human colon adenocarcinoma cell line) and SW620 (colon carcinoma;
derived from metastatic site) was treated with B6H12 Ab (1
.mu.g/ml) for 48 h. SW480 and 620 cell lines were labeled and BrdU
incorporation is measured using above indicated method (FIG. 21B).
FIG. 21D) SW480 and 620 cells were plated overnight using L-15
media. After 24 h, cells were pretreated with B6H12 Ab (1 .mu.g/ml)
for 15 minutes followed by EGF (5 ng/ml) for 5 minutes. The total
lysates were made using NP-40 lysis buffer and immunoprecipitation
was performed using EGF antibody. IP-western blots were performed
using phospho-EGFR Y1068 antibody. B6H12-Ab does not inhibit cell
proliferation of SW480 and SW602 cells. B6H12-Ab inhibits EGFR
phosphorylation only in SW620 cells but not in SW480.
[0032] FIG. 22A-22C illustrates that CD47 targeted CRISPR inhibits
cell proliferation of bCSCs derived from MDA-MB-231.
[0033] FIG. 23 is a bar graph showing that 7N3 peptide increase
mRNA expression of E-cadherin.
[0034] FIG. 24A-24F illustrates characterization of breast cancer
stem cells (bCSCs) derived from suspension cell-enriched MDA-MB-231
triple negative breast carcinoma cells. (FIG. 1A) Routinely
cultured MDA-MB-231 cells. Inset showing loosely attached small
round cells. (FIG. 1B) With gentle agitation, loosely bound bCSCs
were separated from adherent MDA-MB-231 cells. (FIG. 24a) bCSCs
form loose aggregates after incubation at 37.degree. C. for 10
days. (FIG. 6A, 6B) Cell surface protein expression of CD44 and
CD24 determined by flow, (FIG. 24B, FIG. 24C) Replated bCSCs have
higher CD44 and lower CD24 mRNA expression than control MDA-MB-231
cells. Real time Ct values for the indicated genes and reference
B2M are listed. (FIG. 7A) Hierarchical clustering of differentially
expressed genes based on microarray analysis of MDA-MB-231 bCSCs
versus unfractionated MDA-MB-231 cells. (FIG. 24D) Top
differentially expressed genes between MDA-MB-231 and bCSCs. (FIG.
24E) Representative image showing asymmetric division of
BrdU-labeled MDA-MB-231 bCSCs after chasing with unlabeled BrdU and
counterstaining with DAPI. (FIG. 24F) Microscopic quantification of
asymmetric cell division ratios for bCSCs and differentiated
MDA-MB-231 cells (*p<0.05).
[0035] FIG. 25A-25G is a series of bar graphs showing the relative
level of expression of the noted proteins between differentiated
and bCSCs. FIG. 25A-25C shows relative expression of CD47, c-MYC
and TSP1 in Differentiated and bCSCs isolated from the MDA-MB-231
cell line. FIG. 25D-25G show relative Expression of OCT4, NANOG,
SOX2 and NESTIN in Differentiated and bCSCs.
[0036] FIG. 26A-26C is a series of images illustrating that CD47
blocking antibody (B6H12) induces morphological change and reduce
number of aggregates only in bCSCs but not in CSC-depleted
MDA-MB-231 cells. FIGS. 9A & 9B) MDA-MB-231 and bCSCs were
treated in the presence or absence of 1 .mu.g/ml of B6H12 for 72 h.
The images of live cells were captured using an Olympus microscope
at 20.times. and 4.times.. (FIG. 26A). bCSCs were treated with
control or 1 .mu.g/ml of B6H12 for 10 days using cancer stem cell
media form aggregates. FIGS. 26B & 26C are graphs showing
quantification of mammosphere size
[0037] FIG. 27A-27G illustrates that B6H12 alters gene expression
of bCSCs. FIG. 10A) Hierarchical clustering of microarray data
comparing bCSCs treated with control IgG or B6H12 for 36 h. FIG.
27A) List of top differentially expressed genes between control
antibody and B6H12 treatments. Principal component analysis of
parental MDA-MB-231, bCSCs, neural differentiated bCSCs, bCSCs
treated with anti-CD47 B6H12 and control IgG is shown in FIG. 19B.
FIG. 27B-27G are a series of graphs showing Real time PCR
validation of differentially expressed genes between bCSCs treated
with control IgG or B6H12 for 36 h.
[0038] FIG. 28A-28H illustrates that EGF and EGFR mRNA expression
is up-regulated in bCSCs, and down-regulated by B6H12 antibody
treatment. (FIG. 28A & FIG. 28B) EGF and EGFR mRNA expressions
were undetectable in bCSCs treated with B6H12 Ab however control Ab
does not show any effect on bCSCs for 3 days. (FIG. 28C & FIG.
28D) The differentiated and bCSCs cells were treated with B6H12 Ab
for 24 h. The total miRNA was extracted and let-7 family miRNAs
were analyzed using real time PCR. Relative expression of
let-7a-2miRNA in differentiated and bCSCs cells was determined
using U6 as control. (FIG. 28E) EGFR mRNA expression on EVs derived
from MDA-MB-231, bCSCs, T47D1 and MCF7 cells. The cells were
treated with control or B6H12 antibody for 6 h using AB serum
media. (FIG. 28F) Protein expression of EGFR in the presence or
absence of B6H12 for 24 h using FACS sorted CD44.sup.+/CD24-bCSCs
from MDA-MB-231, T47D1 and MCF7 cell lines. FIG. 28G) Relative
expression of let-7a-2miRNA from EVs. FIG. 28H) Amplification and
expression of let-7a-3 miRNA from EV (I don't see a FIG. 28H).
[0039] FIG. 29 is a graph illustrating that Dicer was also
upregulated in Control Vs B6H12 treated bCSCs in microarray. We
asked if B6H12 has alter expression of RNA-induced silencing
complex (RISC) genes. We found that B6H12 increased mRNA expression
of Ago2, DICER, DGCR8 and Drosha, but not Ago1.
[0040] FIG. 30A-30B shows: FIG. 14A is an image of a blot,
illustrating that B6H12 does not inhibit EGHFR.sup.Y992 B6H12.
FIGS. 30A & 30B are graphs showing quantification of EGFRY1068
using Image J program with respect to total Tubulin and EGFR
control. (n=2)
[0041] FIG. 31A-31F is a series of images: FIG. 31A)
EGFR-Immunoprecipitation followed by western blotting shows that
B6H12 treatment for 15 min disrupts the association between EGFR
and CD47 and inhibits EGFRY1068 phosphorylation. FIG. 31B)
CD47-immunoprecipitation showed that small fraction of EGFR
co-immunoprecipitates with EGFR. B6H12 treatment for 15 min leads
to further reduced interaction between CD47 and EGFR in MDA-MB-231
cells. FIGS. 31C & 31D) MDA-MB-231 were pretreated with B6H12
for 15 minutes followed by EGF for 5 minutes, and IP-western blot
was performed using phospho-EGFR antibody (FIG. 31D) quantification
and t-test used for spastically significant value. FIG. 31E)
Differentiated and bCSCs were separated EGFR Immunoprecipitation
was performed using FIG. 31A conditions. One representative of 3
independent experiments is shown (FIG. 31D). FIG. 31F) IP-western
blot of EGFR was performed using CD44high and CD24.sup.low sorted
cells under similar conditions as above.
[0042] FIG. 32A-32G illustrates B6H12-Ab inhibits Asymmetric cell
division and cell proliferation and increase caspase 7. FIG. 32A)
The B6H12-Ab and control antibody treated cells were immunostained
using BrdU antibody (Red) and mounted with Vectashield DAPI at
63.times., and the asymmetric cell division ratio was calculated
(*p<0.05). FIG. 32B) MDA-MB-231 and bCSCs cells were treated
with B6H12 or isotype control antibody for 24 h and immunostained
using KLF4 antibody. Total mean intensity of KLF4 positive cells
was calculated, and t-test was performed. FIG. 32C) Flow cytometry
analysis of KLF4. FIG. 32D) Quantification of BrdU staining using
MFI of BrdU positive cells. FIG. 32E) CD44.sup.high/CD24.sup.low
sorted bCSCs derived from MDA-MB-231 were labeled with BrdU for 24
h and further treated with B6H12 (1 .mu.g/ml) for 24 h. BrdU
incorporation was measured using a BrdU cell proliferation assay
kit. FIG. 32F) Flow cytometry analysis of caspase 7. FIG. 32G)
B6H12 increases caspase 7 expression in MDA-MB-231 cells.
[0043] FIG. 33A-33B are a series of fluorescence microscopy images
showing that MDA-MB-231 cells transfected with EGFR-GFP in the
presence of B6H12 Ab exhibit increase in exosomes secretion as
compared to untreated cells.
[0044] FIG. 34A-34C show that B6H12 treatment of bCSC reduces
expression of KLF4. FIG. 34A and FIG. 34B). MDAMB-231 cells and
bCSCs were plated on LabTek 8 well chamber slides using medium
containing 2% FBS for 24 h at 37.degree. C. The cells were
pre-treated with B6H12 for 24 h. The cells were immunostained with
KLF4 antibody, and images were captured using a Zeiss 710 confocal
microscope. FIG. 34C) Total intensity of KLF4 positive cells was
calculated and t-test was performed.
[0045] FIG. 35A-35E illustrate that B6H12 specifically targets
breast carcinoma cells that have high number of bCSCs/tumor
initiating cells. The following cell lines were treated with 1
.mu.g/ml B6H12, and DNA synthesis was quantified by BrdU
incorporation: (FIG. 35A) MCF10A (FIG. 35B) MCF7 (FIG. 35C) T47D1
and (FIG. 35D) bCSCs sorted for CD44.sup.high CD24.sup.low sorted
bCSCs derived from T47D1 FIG. 35C). The cells were labeled with
BrdU for 24 hours. BrdU treated cells were further treated with
B6H12 Ab (1 .mu.g/ml) for 24 h. The BrdU incorporation was measured
using a BrdU cell proliferation assay kit. (FIG. 35E) MDA-MB-231
cells have a higher percentage of CD44high/CD24.sup.low cells than
MCF7 and T47D1 cells.
[0046] FIG. 36A-36E is a series of images illustrating: FIG. 36A is
a chart of TCGA Kaplan-Meier survival curves for breast invasive
carcinoma patients comparing those with CD47 mRNA expression
determined by RNAseq >1 SD higher than the mean (16% of cases,
lower line) and those without increased CD47 expression (upper
line). FIG. 36B-36E are series of plots showing TCGA expression
data for the indicated proteins in breast tumors determined by
reverse phase protein arrays (RPPA) stratified by CD47 mRNA
expression. Caspase 7 activation was detected using an antibody
specific for the D.sup.198 cleavage. P-values in panels FIG.
36B-36E were based on 2-sided 2 sample t-tests.
[0047] FIG. 37A-37C are a series of plots illustrating: FIG. 37A)
Reverse phase protein array for breast invasive carcinomas
comparing cKit protein expression in those with CD47 mRNA
expression determined by RNAseq >1 SD higher than the mean (16%
of cases, altered) and those without increased CD47 expression
(unaltered). FIG. 37B) TCGA expression data for PDK1
phosphorylation at serine-241 in breast tumors determined by
reverse phase protein arrays (RPPA) stratified by CD47 mRNA
expression. P-values in panels FIG. 37A-37B were based on 2-sided 2
sample t-tests. FIG. 37C) Scatter plot for TCGA data comparing
PATZ1 mRNA expression and CD47 mRNA expression in beast
carcinomas.
SEQUENCE LISTING
[0048] The nucleic and amino acid sequences listed in the
accompanying sequence listing are shown using standard letter
abbreviations for nucleotide bases, and three letter code for amino
acids, as defined in 37 C.F.R. 1.822. Only one strand of each
nucleic acid sequence is shown, but the complementary strand is
understood as included by any reference to the displayed strand.
The Sequence Listing is submitted as an ASCII text file, created on
Jun. 14, 2021, 14,597 bytes, which is incorporated by reference
herein.
[0049] SEQ ID NO: 1 is the peptide p7N3 (FIRVVMYEGKK).
[0050] SEQ ID NO: 2 is gRNA PC200.Hcd47.g3a (CTACTGAAGTATACGTAAG
ngg), a targeting sequence for CRISPR knockout of CD47.
[0051] SEQ ID NOs: 3-52 are primers used as controls to normalize
mRNA expression.
[0052] SEQ ID NOs: 53-69 are additional targeting sequences for
CRISPR knockout of CD47.
[0053] SEQ ID NO: 70 is morpholino CGTCACAGGCAGGACCCACTGCCCA.
DETAILED DESCRIPTION
I. Terms
[0054] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes V, published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
[0055] In order to facilitate review of the various embodiments of
the invention, the following explanations of specific terms are
provided:
[0056] Animal: Living multi-cellular vertebrate organisms, a
category that includes, for example, mammals and birds. The term
mammal includes both human and non-human mammals. Similarly, the
term subject includes both human and veterinary subjects, for
example, humans, non-human primates, rodents, dogs, cats, horses,
and cows.
[0057] Alters CD47 signaling: CD47 is a cell surface receptor that
can impact cellular signal transduction through several known and
undefined mechanisms, and consequently CD47 signaling can be
altered through several signal transduction pathways. One mechanism
involves the lateral association of CD47 with other well
characterized signaling receptors belonging to the integrin family,
with CD14, with VEGF receptor-2, or with Fas. Agents that decrease
the expression of CD47 and pharmacological agents that directly
bind to CD47 or inhibit the binding of thrombospondin-1 (TSP1) or
SIRPs to CD47 may disrupt the association of CD47 with these
receptors and/or induce conformation changes in the respective
receptors that activate or inactivate their signaling. In addition,
CD47 associates with cytoplasmic proteins including PLIC1, BNIP3,
and some heterotrimeric G proteins.
[0058] Agents that change the expression of CD47 or the binding of
natural or pharmacological ligands to CD47 can also alter CD47
signaling. The specific pathways involved are cell type-specific
and depend on which CD47 binding partners are expressed in a given
cell. These upstream perturbations of CD47 signaling lead to
cell-specific alterations in cyclic nucleotide signaling, calcium
signaling, and patterns of gene expression. One of the ultimate
effects of altering CD47 signaling is regulation of stem cell
self-renewal. In non-transformed differentiated cells, blocking
CD47 signaling increases their self-renewal and stem cell
characteristics (see, e.g., International Patent Publication No. WO
2013/155109, incorporated herein by reference), whereas in cancer
cell lines or isolated cancer stem cells (as described herein)
blockade decreases their stem cell markers and results in
differentiation.
[0059] Altered expression: Expression of a biological molecule (for
example, mRNA or protein) in a subject or biological sample from a
subject that deviates from expression if the same biological
molecule in a subject or biological sample from a subject having
normal or unaltered characteristics for the biological condition
associated with the molecule. Normal expression can be found in a
control, a standard for a population, etc. Altered expression of a
biological molecule may be associated with a disease. The term
associated with, in this context, includes an increased risk of
developing the disease as well as the disease itself. Expression
may be altered in such a manner as to be increased or decreased.
The directed alteration in expression of mRNA or protein may be
associated with therapeutic benefits.
[0060] Altered protein expression refers to expression of a protein
that is in some manner different from expression of the protein in
a normal (wild type) situation. This includes but is not
necessarily limited to any one or more of: (1) a mutation in the
protein such that one or more of the amino acid residues is
different; (2) a short deletion or addition of one or a few amino
acid residues to the sequence of the protein; (3) a longer deletion
or addition of amino acid residues, such that an entire protein
domain or sub-domain is removed or added; (4) expression of an
increased amount of the protein, compared to a control or standard
amount; (5) expression of an decreased amount of the protein,
compared to a control or standard amount; (6) alteration of the
subcellular localization or targeting of the protein; (7)
alteration of the temporally regulated expression of the protein
(such that the protein is expressed when it normally would not be,
or alternatively is not expressed when it normally would be); and
(8) alteration of the localized (for example, organ or tissue
specific) expression of the protein (such that the protein is not
expressed where it would normally be expressed or is expressed
where it normally would not be expressed), each compared to a
control or standard.
[0061] Controls or standards appropriate for comparison to a
sample, for the determination of altered expression, include
samples believed to express normally as well as laboratory values,
even though possibly arbitrarily set, keeping in mind that such
values may vary from laboratory to laboratory. Laboratory standards
and values may be set based on a known or determined population
value and may be supplied in the format of a graph or table that
permits easy comparison of measured, experimentally determined
values.
[0062] Analog, derivative or mimetic: An analog is a molecule that
differs in chemical structure from a parent compound, for example a
homolog (differing by an increment in the chemical structure, such
as a difference in the length of an alkyl chain), a molecular
fragment, a structure that differs by one or more functional
groups, a change in ionization. Structural analogs are often found
using quantitative structure activity relationships (QSAR), with
techniques such as those disclosed in Remington (The Science and
Practice of Pharmacology, 19th Edition (1995), chapter 28). A
derivative is a biologically active molecule derived from the base
structure. A mimetic is a molecule that mimics the activity of
another molecule, such as a biologically active molecule.
Biologically active molecules can include chemical structures that
mimic the biological activities of a compound. It is acknowledged
that these terms may overlap in some circumstances.
[0063] Antibody: A protein (or protein complex) that includes one
or more polypeptides substantially encoded by immunoglobulin genes
or fragments of immunoglobulin genes. The recognized immunoglobulin
genes include the kappa, lambda, alpha, gamma, delta, epsilon and
mu constant region genes, as well as the myriad immunoglobulin
variable region genes. Light chains are classified as either kappa
or lambda. Heavy chains are classified as gamma, mu, alpha, delta,
or epsilon, which in turn define the immunoglobulin classes, IgG,
IgM, IgA, IgD and IgE, respectively.
[0064] The basic immunoglobulin (antibody) structural unit is
generally a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one light (about 25
kD) and one heavy chain (about 50-70 kD). The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms
variable light chain (V.sub.L) and variable heavy chain (V.sub.H)
refer, respectively, to these light and heavy chains.
[0065] As used herein, the term antibody includes intact
immunoglobulins as well as a number of well-characterized fragments
produced by digestion with various peptidases, or genetically
engineered artificial antibodies. Thus, for example, pepsin digests
an antibody below the disulfide linkages in the hinge region to
produce F(ab)'.sub.2, a dimer of Fab which itself is a light chain
joined to V.sub.H-C.sub.H 1 by a disulfide bond. The F(ab)'.sub.2
may be reduced under mild conditions to break the disulfide linkage
in the hinge region thereby converting the F(ab)'.sub.2 dimer into
an Fab' monomer. The Fab' monomer is essentially a Fab with part of
the hinge region (see, Fundamental Immunology, W. E. Paul, ed.,
Raven Press, N.Y., 1993). While various antibody fragments are
defined in terms of the digestion of an intact antibody, it will be
appreciated that Fab' fragments may be synthesized de novo either
chemically or by utilizing recombinant DNA methodology. Thus, the
term antibody as used herein also includes antibody fragments
either produced by the modification of whole antibodies or
synthesized de novo using recombinant DNA methodologies.
[0066] Antibodies for use in the methods, compositions, and systems
of this disclosure can be monoclonal or polyclonal. Merely by way
of example, monoclonal antibodies can be prepared from murine
hybridomas according to the classical method of Kohler and Milstein
(Nature 256:495-497, 1975) or derivative methods thereof. Detailed
procedures for monoclonal antibody production are described in
Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York,
1988).
[0067] The terms bind specifically and specific binding refer to
the ability of a specific binding agent (such as, an antibody) to
bind to a target molecular species in preference to binding to
other molecular species with which the specific binding agent and
target molecular species are admixed. A specific binding agent is
said specifically to recognize a target molecular species when it
can bind specifically to that target.
[0068] A single-chain antibody (scFv) is a genetically engineered
molecule containing the V.sub.H and V.sub.L domains of one or more
antibody(ies) linked by a suitable polypeptide linker as a
genetically fused single chain molecule (see, for example, Bird et
al., Science, 242:423-426, 1988; Huston et al., Proc. Natl. Acad.
Sci., 85:5879-5883, 1988). Diabodies are bivalent, bispecific
antibodies in which V.sub.H and V.sub.L domains are expressed on a
single polypeptide chain, but using a linker that is too short to
allow for pairing between the two domains on the same chain,
thereby forcing the domains to pair with complementary domains of
another chain and creating two antigen binding sites (see, for
example, Holliger et al., Proc. Natl. Acad. Sci., 90:6444-6448,
1993; Poljak et al., Structure, 2:1121-1123, 1994). One or more
CDRs may be incorporated into a molecule either covalently or
noncovalently to make the resultant molecule an immunoadhesin. An
immunoadhesin may incorporate the CDR(s) as part of a larger
polypeptide chain, may covalently link the CDR(s) to another
polypeptide chain, or may incorporate the CDR(s) noncovalently. The
CDRs permit the immunoadhesin to specifically bind to a particular
antigen of interest. A chimeric antibody is an antibody that
contains one or more regions from one antibody and one or more
regions from one or more other antibodies.
[0069] An antibody may have one or more binding sites. If there is
more than one binding site, the binding sites may be identical to
one another or may be different. For instance, a
naturally-occurring immunoglobulin has two identical binding sites,
a single-chain antibody or Fab fragment has one binding site, while
a bispecific or bifunctional antibody has two different binding
sites.
[0070] A neutralizing antibody or an inhibitory antibody is an
antibody that inhibits at least one activity of a target--usually a
polypeptide--such as by blocking the binding of the polypeptide to
a ligand to which it normally binds, or by disrupting or otherwise
interfering with a protein-protein interaction of the polypeptide
with a second polypeptide. An activating antibody is an antibody
that increases an activity of a polypeptide. Antibodies may
function as mimics of a target protein activity, or as blockers of
the target protein activity, with therapeutic effect derived
therein.
[0071] Antisense, Sense, and Antigene: Double-stranded DNA (dsDNA)
has two strands, a 5'->3' strand, referred to as the plus
strand, and a 3'->5' strand (the reverse complement), referred
to as the minus strand. Because RNA polymerase adds nucleic acids
in a 5'->3' direction, the minus strand of the DNA serves as the
template for the RNA during transcription. Thus, the RNA formed
will have a sequence complementary to the minus strand and
identical to the plus strand (except that U is substituted for
T).
[0072] Antisense molecules are molecules that are specifically
hybridizable or specifically complementary to either RNA or plus
strand DNA. Sense molecules are molecules that are specifically
hybridizable or specifically complementary to the minus strand of
DNA. Antigene molecules are either antisense or sense molecules
complimentary to a dsDNA target. In one embodiment, an antisense
molecule specifically hybridizes to a target mRNA and inhibits
transcription of the target mRNA.
[0073] Aptamer: A single-stranded nucleic acid molecule (such as
DNA or RNA) that assumes a specific, sequence-dependent shape and
binds to a target protein with high affinity and specificity.
Aptamers generally comprise fewer than 100 nucleotides, fewer than
75 nucleotides, or fewer than 50 nucleotides. Mirror-image
aptamer(s) (also called Spiegelmers.TM.) are high-affinity
L-enantiomeric nucleic acids (for example, L-ribose or
L-2'-deoxyribose units) that display high resistance to enzymatic
degradation compared with D-oligonucleotides (such as aptamers).
The target binding properties of mirror-image aptamers are designed
by an in vitro-selection process starting from a random pool of
oligonucleotides, as described for example, in Wlotzka et al.,
Proc. Natl. Acad. Sci. 99(13):8898-8902, 2002. Applying this
method, high affinity mirror-image aptamers specific for a
polypeptide can be generated.
[0074] Binding affinity: A term that refers to the strength of
binding of one molecule to another at a site on the molecule. If a
particular molecule will bind to or specifically associate with
another particular molecule, these two molecules are said to
exhibit binding affinity for each other. Binding affinity is
related to the association constant and dissociation constant for a
pair of molecules, but it is not critical to the methods herein
that these constants be measured or determined. Rather, affinities
as used herein to describe interactions between molecules of the
described methods are generally apparent affinities (unless
otherwise specified) observed in empirical studies, which can be
used to compare the relative strength with which one molecule
(e.g., an antibody or other specific binding partner) will bind two
other molecules (e.g., two versions or variants of a peptide). The
concepts of binding affinity, association constant, and
dissociation constant are well known.
[0075] Binding domain: The molecular structure associated with that
portion of a receptor that binds ligand. More particularly, the
binding domain may refer to a polypeptide, natural or synthetic, or
nucleic acid encoding such a polypeptide, the amino acid sequence
of which represents a specific region (binding domain) of a
protein, which either alone or in combination with other domains,
exhibits binding characteristics. Neither the specific sequences
nor the specific boundaries of such domains are critical, so long
as binding activity is exhibited. Likewise, used in this context,
binding characteristics necessarily includes a range of affinities,
avidities and specificities, and combinations thereof, so long as
binding activity is exhibited.
[0076] Binding partner: Any molecule or composition capable of
recognizing and binding to a specific structural aspect of another
molecule or composition. Examples of such binding partners and
corresponding molecule or composition include antigen/antibody,
hapten/antibody, lectin/carbohydrate, apoprotein/cofactor and
biotin/(strept)avidin.
[0077] Breast cancer: A neoplastic condition of breast tissue that
can be benign or malignant. The most common type of breast cancer
is ductal carcinoma. Ductal carcinoma in situ is a non-invasive
neoplastic condition of the ducts. Lobular carcinoma is not an
invasive disease but is an indicator that a carcinoma may develop.
Infiltrating (malignant) carcinoma of the breast can be divided
into stages (I, IIA, IIB, IIIA, IIIB, and IV).
[0078] Breast carcinomas lose the typical histology and
architecture of normal breast glands. Generally, carcinoma cells
overgrow the normal cells and lose their ability to differentiate
into glandular like structures. The degree of loss of
differentiation in general is related to the aggressiveness of the
tumor. For example, "in situ" carcinoma by definition retains the
basement membrane intact, whereas as it progresses to "invasive",
the tumor shows breakout of basement membranes. Thus one would not
expect to see, within breast carcinomas, staining of a discrete
layer of basal cells as seen in normal breast tissue. For a
discussion of the physiology and histology of normal breast and
breast carcinoma, see Ronnov-Jessen (Physiol Rev 76, 69-125,
1996).
[0079] Breast cancers can be divided into groups based on their
expression profiles. Basal-type carcinomas usually are negative for
expression of estrogen receptor (ER) and negative for expression of
HER2 (erbB2) and progesterone receptor (PR), and thus are referred
to as "triple-negative breast cancers" or "TNBC." This type of
breast cancer is also denoted ER.sup.-/HER2.sup.-/PR.sup.- and
represents about 15-20% of all breast cancer, and generally cannot
be treated using Her2 targeted or estrogen targeted therapies. It
is believed that the aggressive nature of this cancer is correlated
with an enrichment for cancer stem cells (CSC) with a
CD44.sup.+CD24.sup.-/lo phenotype. In some embodiments, basal
carcinomas are negative for expression of progesterone receptor
(PR), positive for expression of epidermal growth factor receptor
(EGFR), and positive for expression of cytokeratin 5 (CK5). This
phenotype is denoted as follows:
ER.sup.-/PR.sup.-/HER2.sup.-/CK5.sup.+/EGFR.sup.+.
[0080] A breast cancer sample is a sample that includes tissue or
cells from a breast tumor. In some examples, a breast cancer sample
is a breast tumor biopsy, lymph node tissue from a subject with
breast cancer, or a metastasis from a breast tumor.
[0081] Cancer: A malignant neoplasm that has undergone
characteristic anaplasia with loss of differentiation, increase
rate of growth, invasion of surrounding tissue, and is capable of
metastasis. For example, thyroid cancer is a malignant neoplasm
that arises in or from thyroid tissue, and breast cancer is a
malignant neoplasm that arises in or from breast tissue (such as a
ductal carcinoma). Residual cancer is cancer that remains in a
subject after any form of treatment given to the subject to reduce
or eradicate thyroid cancer. Metastatic cancer is a cancer at one
or more sites in the body other than the site of origin of the
original (primary) cancer from which the metastatic cancer is
derived. Cancer includes, but is not limited to, solid tumors.
[0082] Cancer Stem Cells (CSCs): The cancer stem cell hypothesis
suggests that tumors contain a small subpopulation of cells which
are exclusively responsible for cancer initiation and maintenance
and contribute to therapeutic failure, called cancer stem cells
(CSC). Although, CSC were first identified in hematologic
malignancies, recent studies have suggested the presence of CSC in
solid tumors of various organs including breast, brain, pancreas,
colon, and liver. CSC have been identified using non-specific
markers such as CD133, CD24, CD44, CD90, although these markers are
present on other cells. The ability to efflux the Hoechst dye,
defined as the "side-population," has also been used to identify
CSC. Collectively, these methods resulted in heterogenic
populations enriched with putative CSC. However, cells identified
using these methods generally were not able to initiate tumors with
less than 100 cells.
[0083] Cancer cells found within solid tumors or hematological
cancers that possess characteristics associated with normal stem
cells, specifically the ability to give rise to all cell types
found in a particular cancer, and also have the ability to
self-renew. Cancer stem cells are tumorigenic (tumor-forming). CSCs
can generate tumors through the stem cell processes of self-renewal
and differentiation into multiple cell types. Such cells are
proposed to persist in tumors as a distinct population and cause
relapse and metastasis by giving rise to new tumors at distant
sites. CSCs have recently been identified in several solid tumors,
including cancers of the brain, breast, colon, ovary, pancreas and
prostate, as well as in hematologic cancers. A standard for testing
CSC is xenogeneic transplantation into immunosuppressed mice. It
tests CSC capacity to initiate tumors in a
non-human-environment.
[0084] cDNA (complementary DNA): A piece of DNA lacking internal,
non-coding segments (introns) and transcriptional regulatory
sequences. cDNA can also contain untranslated regions (UTRs) that
are responsible for translational control in the corresponding RNA
molecule. cDNA is synthesized in the laboratory by reverse
transcription from messenger RNA extracted from cells.
[0085] Cell Culture: Cell culture or culturing cells refers to
placing cells in a dish, flask, or other container with an
appropriate medium (such as a growth medium or differentiation
medium) for the type of cells utilized (such as a medium including
glucose, essential amino acids, vitamins, trace elements, salts, a
buffer to maintain pH, and/or other components for particular
applications).
[0086] Chemotherapy; chemotherapeutic agents: As used herein, these
terms include any chemical agent with therapeutic usefulness in the
treatment of diseases characterized by abnormal cell growth. Such
diseases include tumors, neoplasms, and cancer as well as diseases
characterized by hyperplastic growth such as psoriasis. In one
embodiment, a chemotherapeutic agent is an agent of use in treating
neoplasms such as solid tumors. In one embodiment, a
chemotherapeutic agent is radioactive molecule. One of skill in the
art can readily identify a chemotherapeutic agent of use (e.g., see
Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in
Harrison's Principles of Internal Medicine, 14th edition; Perry et
al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2.sup.nd
ed., .COPYRGT. 2000 Churchill Livingstone, Inc.; Baltzer L.,
Berkery R. (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed.
St. Louis, Mosby-Year Book, 1995; Fischer D S, Knobf M F, Durivage
H J (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis,
Mosby-Year Book, 1993). Chemotherapeutic agents include those known
by those skilled in the art, including but not limited to:
5-fluorouracil (5-FU), azathioprine, cyclophosphamide,
antimetabolites (such as Fludarabine), antineoplastics (such as
Etoposide, Doxorubicin, methotrexate, and Vincristine),
carboplatin, cis-platinum and the taxanes, such as taxol. Rapamycin
has also been used as a chemotherapeutic. Additional
chemotherapeutic agents are described herein, and are known in the
art.
[0087] Colon cancer: Colorectal cancer, also called large bowel
cancer, includes cancerous growths in the colon, rectum and
appendix. With 655,000 deaths worldwide per year, it is the third
most common form of cancer and the second leading cause of
cancer-related death in the Western world. Many colorectal cancers
are thought to arise from adenomatous polyps in the colon. These
mushroom-like growths are usually benign, but some may develop into
cancer over time. The majority of the time, the diagnosis of
localized colon cancer is through colonoscopy. Therapy is usually
through surgery, which in many cases is followed by chemotherapy.
The first symptoms of colon cancer are usually vague, such as
bleeding, weight loss, and fatigue (tiredness). Local (bowel)
symptoms are rare until the tumor has grown to a large size.
Generally, the nearer the tumor is to the anus, the more bowel
symptoms there will be.
[0088] Control or control sample: Samples believed to be normal (in
that they are not altered for the desired characteristic, for
example non-tumor cell or tissue) as well as laboratory values,
even though possibly arbitrarily set, keeping in mind that such
values can vary from laboratory to laboratory. In one example, a
control sample is non-tumor cell or tissue from an individual
without a particular cancer, such as without breast cancer. In
another example, a control sample is non-tumor cell or tissue from
an individual with cancer.
[0089] Conjugated (Linked): Two entities are conjugated when under
physiological conditions of pH, ionic strength and osmotic
potential, the majority of the entities are associated with each
other at equilibrium, such as due to the presence of a covalent
bond. Covalent linkage may be by any of a variety of chemical
linking and cross-linking agents including, for example,
homobifunctional or heterobifunctional crosslinking reagents, many
of which are commercially available (see, e.g., Pierce Chemical Co.
or Sigma Chemical Co.). Linking or crosslinking can be achieved by
any of a variety of chemistries well known in the art including,
for example, activated polyethylene glycols, aldehydes,
isocyanates, maleimides and the like. Linking or cross-linking can
also be achieved using physical methods, such as irradiation, for
example gamma irradiation or ultraviolet (UV) irradiation.
[0090] DNA (deoxyribonucleic acid): A long chain polymer that
comprises the genetic material of most living organisms (some
viruses have genes comprising ribonucleic acid (RNA)). The
repeating units in DNA polymers are four different nucleotides,
each of which comprises one of the four bases, adenine, guanine,
cytosine and thymine bound to a deoxyribose sugar to which a
phosphate group is attached. Triplets of nucleotides (referred to
as codons) code for each amino acid in a polypeptide. The term
codon is also used for the corresponding (and complementary)
sequences of three nucleotides in the mRNA into which the DNA
sequence is transcribed.
[0091] Unless otherwise specified, any reference to a DNA molecule
is intended to include the reverse complement of that DNA molecule.
Except where single-strandedness is required by the text herein,
DNA molecules, though written to depict only a single strand,
encompass both strands of a double-stranded DNA molecule. Thus, a
reference to the nucleic acid molecule that encodes a specific
protein, or a fragment thereof, encompasses both the sense strand
and its reverse complement. Thus, for instance, it is appropriate
to generate probes or primers from the reverse complement sequence
of the disclosed nucleic acid molecules.
[0092] Deletion: The removal of a sequence of DNA (which may be as
short as a single nucleotide), the regions on either side being
joined together.
[0093] Encode: A polynucleotide is said to encode a polypeptide if,
in its native state or when manipulated by methods known to those
skilled in the art, it can be transcribed and/or translated to
produce the mRNA for and/or the polypeptide or a fragment
thereof.
[0094] Epitope: An antigenic determinant. These are particular
chemical groups or peptide sequences on a molecule that are
antigenic, for instance, that elicit a specific immune response. An
antibody binds a particular antigenic epitope, based on a 3D
structure of the antibody and the matching or cognate epitope.
Epitopes can be formed both from contiguous amino acids or
noncontiguous amino acids juxtaposed by tertiary folding of a
protein. Epitopes formed from contiguous amino acids are typically
retained on exposure to denaturing solvents whereas epitopes formed
by tertiary folding are typically lost on treatment with denaturing
solvents. An epitope typically includes at least 3, and more
usually, at least 5, about 9, or 8 to 10 amino acids in a unique
spatial conformation. Methods of determining spatial conformation
of epitopes include, for example, x-ray crystallography and
2-dimensional nuclear magnetic resonance. See, e.g., "Epitope
Mapping Protocols" in Methods in Molecular Biology, Vol. 66, Glenn
E. Morris, Ed (1996). In one embodiment, an epitope binds an MHC
molecule, such an HLA molecule or a DR molecule. These molecules
bind polypeptides having the correct anchor amino acids separated
by about eight to about ten amino acids, such as nine amino
acids.
[0095] Expand: A process by which the number or amount of cells in
a cell culture is increased due to cell division. Similarly, the
terms "expansion" or "expanded" refers to this process. The terms
"proliferate," "proliferation" or "proliferated" may be used
interchangeably with the words "expand," "expansion," or
"expanded." Typically, during an expansion phase, the cells do not
differentiate to form mature cells, but divide to form more
cells.
[0096] Fluorescence Activated Cell Sorting (FACS): A technique for
counting, examining and separating microscopic particles, such as
cells and chromosomes, by suspending them in a stream of fluid and
passing them by an electronic detection apparatus. It allows
simultaneous multiparametric analysis of the physical and/or
chemical characteristics of up to thousands of particles per
second. Flow cytometry is routinely used in the diagnosis of cancer
and the isolation of populations of lymphocytes, but has many other
applications in both research and clinical practice.
[0097] A beam of light (usually laser light) of a single wavelength
is directed onto a hydrodynamically-focused stream of fluid. A
number of detectors are aimed at the point where the stream passes
through the light beam: one in line with the light beam (Forward
Scatter or FSC) and several perpendicular to it (Side Scatter (SSC)
and one or more fluorescent detectors). Each suspended particle
from 0.2 to 150 micrometers passing through the beam scatters the
light in some way, and fluorescent chemicals found in the particle
or attached to the particle may be excited into emitting light at a
longer wavelength than the light source. This combination of
scattered and fluorescent light is picked up by the detectors, and,
by analyzing fluctuations in brightness at each detector (one for
each fluorescent emission peak), it is then possible to derive
various types of information about the physical and chemical
structure of each individual particle.
[0098] A flow cytometer generally has several main components: (1)
a flow cell--liquid stream (sheath fluid), which carries and aligns
the cells so that they pass single file through the light beam for
sensing; (2) a measuring system, such as those that allow
measurement of impedance (or conductivity) and optical
systems--lamps (mercury, xenon); high-power water-cooled lasers
(argon, krypton, dye laser); low-power air-cooled lasers (argon
(488 nm), red-HeNe (633 nm), green-HeNe, HeCd (UV)); diode lasers
(blue, green, red, violet) resulting in light signals; (3) a
detector and Analogue-to-Digital Conversion (ADC) system which
generates FSC and SSC as well as fluorescence signals from light
into electrical signals that can be processed by a computer; (4) an
amplification system (either linear or logarithmic); and (5) a
computer for analysis of the signals. FACS instruments usually have
multiple lasers and fluorescence detectors (such as up to 4 lasers
and 18 fluorescence detectors). Increasing the number of lasers and
detectors allows for multiple antibody labeling, and can more
precisely identify a target population by their phenotypic
markers.
[0099] Using fluorescent activated cell sorting (FACS), a
heterogeneous mixture of biological cells can be separated into two
or more containers, one cell at a time, based upon the specific
light scattering and fluorescent characteristics of each cell. The
cell suspension is entrained in the center of a narrow, rapidly
flowing stream of liquid. The flow is arranged so that there is a
large separation between cells relative to their diameter. A
vibrating mechanism causes the stream of cells to break into
individual droplets. The system is adjusted so that there is a low
probability of more than one cell per droplet. Just before the
stream breaks into droplets, the flow passes through a fluorescence
measuring station where the fluorescent character of interest of
each cell is measured. An electrical charging ring is placed just
at the point where the stream breaks into droplets. A charge is
placed on the ring based on the immediately-prior fluorescence
intensity measurement, and the opposite charge is trapped on the
droplet as it breaks from the stream. The charged droplets then
fall through an electrostatic deflection system that diverts
droplets into containers based upon their charge. In some systems,
the charge is applied directly to the stream, and the droplet
breaking off retains a charge of the same sign as the stream. The
stream is then returned to neutral after the droplet breaks off.
FACS can be used to identify and isolate cancer stem cells, as
described herein.
[0100] Gating: The data generated by FACS can be plotted in a
single dimension, to produce a histogram, or in two-dimensional dot
plots or even in three dimensions. The regions on these plots can
be sequentially separated, based on fluorescence intensity, by
creating a series of subset extractions, termed "gates." Specific
gating protocols exist for diagnostic and clinical purposes
especially in relation to hematology and the identification of stem
cells.
[0101] The plots are often made on logarithmic scales. Because
different fluorescent dyes' emission spectra overlap, signals at
the detectors have to be compensated electronically as well as
computationally. Data accumulated using the flow cytometer can be
analyzed using software, e.g., FLOJO.TM., or CELLQUEST PRO.TM..
[0102] Gene expression: The process by which the coded information
of a nucleic acid transcriptional unit (including, for example,
genomic DNA or cDNA) is converted into an operational,
non-operational, or structural part of a cell, often including the
synthesis of a protein. Gene expression can be influenced by
external signals; for instance, exposure of a subject to an agent
that inhibits gene expression. Expression of a gene also may be
regulated anywhere in the pathway from DNA to RNA to protein.
Regulation of gene expression occurs, for instance, through
controls acting on transcription, translation, RNA transport and
processing, degradation of intermediary molecules such as mRNA, or
through activation, inactivation, compartmentalization or
degradation of specific protein molecules after they have been
made, or by combinations thereof. Gene expression may be measured
at the RNA level or the protein level and by any method known in
the art, including Northern blot, RT-PCR, Western blot, or in
vitro, in situ, or in vivo protein activity assay(s).
[0103] The expression of a nucleic acid may be modulated compared
to a control state, such as at a control time (for example, prior
to administration of a substance or agent that affects regulation
of the nucleic acid under observation) or in a control cell or
subject, or as compared to another nucleic acid. Such modulation
includes but is not necessarily limited to overexpression,
underexpression, or suppression of expression. In addition, it is
understood that modulation of nucleic acid expression may be
associated with, and in fact may result in, a modulation in the
expression of an encoded protein or even a protein that is not
encoded by that nucleic acid.
[0104] Interfering with or inhibiting gene expression refers to the
ability of an agent to measurably reduce the expression of a target
gene. Expression of a target gene may be measured by any method
known to those of ordinary skill in the art, including for example
measuring mRNA or protein levels. It is understood that interfering
with or inhibiting gene expression is relative, and does not
require absolute suppression of the gene. Thus, in certain
embodiments, interfering with or inhibiting gene expression of a
target gene requires that, following application of an agent, the
gene is expressed at least 5% less than prior to application, at
least 10% less, at least 15% less, at least 20% less, at least 25%
less, or even more reduced. Thus, in some particular embodiments,
application of an agent reduces expression of the target gene by
about 30%, about 40%, about 50%, about 60%, or more. In specific
examples, where the agent is particularly effective, expression is
reduced by 70%, 80%, 85%, 90%, 95%, or even more. Gene expression
is substantially eliminated when expression of the gene is reduced
by 90%, 95%, 98%, 99% or even 100%.
[0105] Growth factor: A substance that promotes cell growth,
survival, and/or differentiation. Growth factors include molecules
that function as growth stimulators (mitogens), factors that
stimulate cell migration, factors that function as chemotactic
agents or inhibit cell migration or invasion of tumor cells,
factors that modulate differentiated functions of cells, factors
involved in apoptosis, or factors that promote survival of cells
without influencing growth and differentiation. Examples of growth
factors are a fibroblast growth factor (such as FGF-2), epidermal
growth factor (EGF), cilliary neurotrophic factor (CNTF), nerve
growth factor (NGF), activin-A, and insulin.
[0106] Heterologous: A type of sequence (nucleic acid or protein)
that is not normally (for example, in the wild-type sequence) found
adjacent to a second sequence. In one embodiment, the sequence is
from a different genetic source, such as a virus or other organism,
than the second sequence.
[0107] Hybridization: Oligonucleotides and their analogs hybridize
by hydrogen bonding, which includes Watson-Crick, Hoogsteen or
reversed Hoogsteen hydrogen bonding, between complementary bases.
Generally, nucleic acid consists of nitrogenous bases that are
either pyrimidines (cytosine (C), uracil (U), and thymine (T)) or
purines (adenine (A) and guanine (G)). These nitrogenous bases form
hydrogen bonds between a pyrimidine and a purine, and the bonding
of the pyrimidine to the purine is referred to as base pairing.
More specifically, A will hydrogen bond to T or U, and G will bond
to C. Complementary refers to the base pairing that occurs between
to distinct nucleic acid sequences or two distinct regions of the
same nucleic acid sequence.
[0108] Inhibiting protein activity: To decrease, limit, or block an
action, function, association, or expression of a protein. The
phrase "inhibit protein activity" is not intended to be an absolute
term. Instead, the phrase is intended to convey a wide range of
inhibitory effects that various agents may have on the normal (for
example, uninhibited or control) protein activity. Inhibition of
protein activity may, but need not, result in a change (increase or
decrease) in the level or activity of an indicator of the protein's
activity. By way of example, this can happen when the protein of
interest is acting as an inhibitor or suppressor of a downstream
indicator. Thus, protein activity may be inhibited when the level
or activity of any direct or indirect indicator of the protein's
activity is changed (for example, increased or decreased) by at
least 10%, at least 20%, at least 30%, at least 50%, at least 80%,
at least 100% or at least 250% or more as compared to control
measurements of the same indicator.
[0109] A signaling protein (such as CD47) may control several
activities in parallel, and inhibition may occur when one or more
downstream indicators is altered while downstream indicators of
other signaling responses controlled by the same protein remain
unchanged. Conversely, a given inhibitor may alter additional
downstream indicators that are irrelevant to a specific application
of that protein inhibitor.
[0110] Inhibition of protein activity may also be effected, for
example, by inhibiting expression of the gene encoding the protein
or by decreasing the half-life of the mRNA encoding the
protein.
[0111] Injectable composition: A pharmaceutically acceptable fluid
composition comprising at least one active ingredient, for example,
a protein, peptide, or antibody. The active ingredient is usually
dissolved or suspended in a physiologically acceptable carrier, and
the composition can additionally comprise minor amounts of one or
more non-toxic auxiliary substances, such as emulsifying agents,
preservatives, pH buffering agents and the like. Such injectable
compositions that are useful for use with the compositions of this
disclosure are conventional; appropriate formulations are well
known in the art.
[0112] Isolated: An isolated biological component (such as a
nucleic acid, peptide or protein) has been substantially separated,
produced apart from, or purified away from other biological
components in the cell of the organism in which the component
naturally occurs, for instance, other chromosomal and
extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides
and proteins that have been isolated thus include nucleic acids and
proteins purified by standard purification methods. The term also
embraces nucleic acids, peptides and proteins prepared by
recombinant expression in a host cell as well as chemically
synthesized nucleic acids. The terms isolated and purified do not
require absolute purity; rather, it is intended as a relative term.
Thus, for example, an isolated peptide preparation is one in which
the peptide or protein is more enriched than the peptide or protein
is in its natural environment within a cell. In certain
embodiments, a preparation is purified such that the protein or
peptide represents at least 50% of the total peptide or protein
content of the preparation.
[0113] Label: An agent capable of detection, for example by ELISA,
spectrophotometry, flow cytometry, or microscopy. In some
instances, the detectable compound or composition is conjugated
directly or indirectly to another molecule to facilitate detection
of that molecule. For example, a label can be attached to a nucleic
acid molecule or protein, thereby permitting detection of the
nucleic acid molecule or protein. Examples of labels include, but
are not limited to, radioactive isotopes, enzyme substrates,
co-factors, ligands, chemiluminescent agents, fluorophores,
fluorescent tags, haptens, enzymes, and combinations thereof.
Methods for labeling and guidance in the choice of labels
appropriate for various purposes are discussed for example in
Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor, N.Y., 1989) and Ausubel et al. (In Current Protocols
in Molecular Biology, John Wiley & Sons, New York, 1998).
[0114] Lung cancer: The main type of lung cancer is carcinoma of
the lung, which includes small cell lung carcinoma and non-small
cell lung carcinoma. Non-small cell lung carcinoma (NSCLC) is
sometimes treated with surgery, while small cell lung carcinoma
(SCLC) usually responds to chemotherapy and radiation. The most
common cause of lung cancer is long-term exposure to tobacco
smoke.
[0115] 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. 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, adenocarcinoma is the most common form of
lung cancer. A subtype of adenocarcinoma, the bronchioalveolar
carcinoma, is more common in females.
[0116] Small cell lung cancers (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 also is strongly associated with smoking.
[0117] Lymphoma: A cancer that begins in the lymphocytes and
presents as a solid tumor of lymphoid cells. Lymphomas are
generally treatable with chemotherapy, and in some cases
radiotherapy and/or bone marrow transplantation, and can be
curable, depending on the histology, type, and stage of the
disease. The WHO classification is a generally accepted system for
the classification of lymphoma and is based upon the foundations
laid within the "Revised European-American Lymphoma classification"
(REAL). This system attempts to group lymphomas by cell type (i.e.
the normal cell type that most resembles the tumor) and defining
phenotypic, molecular or cytogenetic characteristics. There are
three large groups of lymphoma: the B cell, T cell, and natural
killer cell tumors. Hodgkin's lymphoma, although considered
separately within the WHO classification, is now recognized as
being a tumor of lymphocytes of the mature B cell lineage.
[0118] Lymphomas include mature B cell lymphomas such as chronic
lymphocytic leukemia/Small lymphocytic lymphoma, B-cell
prolymphocytic leukemia, lymphoplasmacytic lymphoma (such as
Waldenstrom macroglobulinemia), splenic marginal zone lymphoma,
plasma cell neoplasms: plasma cell myeloma, plasmacytoma,
monoclonal immunoglobulin deposition diseases, heavy chain diseases
extranodal marginal zone B cell lymphoma, nodal marginal zone B
cell lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma,
diffuse large B cell lymphoma, mediastinal (thymic) large B cell
lymphoma, intravascular large B cell lymphoma, primary effusion
lymphoma, and Burkitt lymphoma/leukemia. Lymphomas also include
mature T cell and natural killer cell neoplasms, such as T cell
prolymphocytic leukemia, T cell large granular lymphocytic
leukemia, aggressive NK cell leukemia, adult T cell
leukemia/lymphoma, extranodal NK/T cell lymphoma (nasal type),
enteropathy-type T cell lymphoma, hepatosplenic T cell lymphoma,
blastic NK cell lymphoma, mycosis fungoides/Sezary syndrome,
primary cutaneous CD30-positive T cell lymphoproliferative
disorders, primary cutaneous anaplastic large cell lymphoma,
lymphomatoid papulosis, angioimmunoblastic T cell lymphoma,
peripheral T cell lymphoma, and anaplastic large cell lymphoma.
[0119] Modulator: An agent that increases or decreases (modulates)
the activity of a protein or other bio-active compound, as measured
by the change in an experimentally measured biological parameter. A
modulator can be essentially any compound or mixture (for example,
two or more proteins or a protein and another compound), such as an
antibody molecule or other polypeptide, a hormone, a nucleic acid,
a sugar, a lipid and the like.
[0120] Morpholino: A morpholino oligo is structurally different
from natural nucleic acids, with morpholino rings replacing the
ribose or deoxyribose sugar moieties and non-ionic
phosphorodiamidate linkages replacing the anionic phosphates of DNA
and RNA. Each morpholino ring suitably positions one of the
standard bases (A, G, C, T/U), so that a 25-base morpholino oligo
strongly and specifically binds to its complementary 25-base target
site in a strand of RNA via Watson-Crick pairing. Because the
backbone of the morpholino oligo is not recognized by cellular
enzymes of signaling proteins, it is stable to nucleases and does
not trigger an innate immune response through the toll-like
receptors. This avoids loss of oligo, inflammation or interferon
induction. Morpholinos can be delivered by a number of techniques,
including direct injection to tissues or via infusion pump and
intravenous bolus. A morpholino is one example of a stabilized
nucleic acid molecule.
[0121] Neoplasm: An abnormal cellular proliferation, which includes
benign and malignant tumors, as well as other proliferative
disorders.
[0122] Non-viable cells: Cells that are in the process of dying or
are dead. These cells do not divide. Non-viable cells include
necrotic and apoptotic cells.
[0123] Nucleic acid molecule: A polymeric form of nucleotides,
which may include both sense and antisense strands of RNA, cDNA,
genomic DNA, and synthetic forms and mixed polymers thereof. A
nucleotide refers to a ribonucleotide, deoxynucleotide or a
modified form of either type of nucleotide. A nucleic acid molecule
as used herein is synonymous with nucleic acid and polynucleotide.
A nucleic acid molecule is usually at least 10 bases in length,
unless otherwise specified. The term includes single- and
double-stranded forms. A polynucleotide may include either or both
naturally occurring and modified nucleotides linked together by
naturally occurring and/or non-naturally occurring nucleotide
linkages.
[0124] Nucleic acid molecules may be modified chemically or
biochemically or may contain non-natural or derivatized nucleotide
bases, as will be readily appreciated by those of ordinary skill in
the art. Such modifications include, for example, labels,
methylation, substitution of one or more of the naturally occurring
nucleotides with an analog, internucleotide modifications, such as
uncharged linkages (for example, methyl phosphonates,
phosphotriesters, phosphoramidates, carbamates, etc.), charged
linkages (for example, phosphorothioates, phosphorodithioates,
etc.), pendent moieties (for example, polypeptides), intercalators
(for example, acridine, psoralen, etc.), chelators, alkylators, and
modified linkages (for example, alpha anomeric nucleic acids,
etc.). The term nucleic acid molecule also includes any topological
conformation, including single-stranded, double-stranded, partially
duplexed, triplexed, hairpinned, circular and padlocked
conformations. Also included are synthetic molecules that mimic
polynucleotides in their ability to bind to a designated sequence
via hydrogen bonding and other chemical interactions. Such
molecules are known in the art and include, for example, those in
which peptide linkages substitute for phosphate linkages in the
backbone of the molecule.
[0125] Unless specified otherwise, the left hand end of a
polynucleotide sequence written in the sense orientation is the 5'
end and the right hand end of the sequence is the 3' end. In
addition, the left hand direction of a polynucleotide sequence
written in the sense orientation is referred to as the 5'
direction, while the right hand direction of the polynucleotide
sequence is referred to as the 3' direction. Further, unless
otherwise indicated, each nucleotide sequence is set forth herein
as a sequence of deoxyribonucleotides. It is intended, however,
that the given sequence be interpreted as would be appropriate to
the polynucleotide composition: for example, if the isolated
nucleic acid is composed of RNA, the given sequence intends
ribonucleotides, with uridine substituted for thymidine.
[0126] An antisense nucleic acid is a nucleic acid (such as, an RNA
or DNA oligonucleotide) that has a sequence complementary to a
second nucleic acid molecule (for example, an mRNA molecule). An
antisense nucleic acid will specifically bind with high affinity to
the second nucleic acid sequence. If the second nucleic acid
sequence is an mRNA molecule, for example, the specific binding of
an antisense nucleic acid to the mRNA molecule can prevent or
reduce translation of the mRNA into the encoded protein or decrease
the half-life of the mRNA, and thereby inhibit the expression of
the encoded protein.
[0127] Oligonucleotide: A plurality of joined nucleotides joined by
native phosphodiester bonds, between about 6 and about 300
nucleotides in length. An oligonucleotide analog refers to moieties
that function similarly to oligonucleotides but have non-naturally
occurring portions. For example, oligonucleotide analogs can
contain non-naturally occurring portions, such as altered sugar
moieties or inter-sugar linkages, such as a phosphorothioate
oligodeoxynucleotide. Functional analogs of naturally occurring
polynucleotides can bind to RNA or DNA, and include stabilized
oligonucleotides, such as peptide nucleic acid (PNA) molecules and
morpholinos.
[0128] Particular oligonucleotides and oligonucleotide analogs can
include linear sequences up to about 200 nucleotides in length, for
example a sequence (such as DNA or RNA) that is at least 6 bases,
for example at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or
even 200 bases long, or from about 6 to about 50 bases, for example
about 10-25 bases, such as 12, 15 or 20 bases.
[0129] Operably linked: A first nucleic acid sequence is operably
linked with a second nucleic acid sequence when the first nucleic
acid sequence is placed in a functional relationship with the
second nucleic acid sequence. For instance, a promoter is operably
linked to a coding sequence if the promoter affects the
transcription or expression of the coding sequence. Generally,
operably linked DNA sequences are contiguous and, where necessary
to join two protein-coding regions, in the same reading frame.
[0130] Open reading frame: A series of nucleotide triplets (codons)
coding for amino acids without any internal termination codons.
These sequences are usually translatable into a peptide.
[0131] Ortholog: Two nucleic acid or amino acid sequences are
orthologs of each other if they share a common ancestral sequence
and diverged when a species carrying that ancestral sequence split
into two species. Orthologous sequences are also homologous
sequences.
[0132] Parenteral: Administered outside of the intestine, for
example, not via the alimentary tract. Generally, parenteral
formulations are those that will be administered through any
possible mode except ingestion. This term especially refers to
injections, whether administered intravenously, intrathecally,
intramuscularly, intraperitoneally, or subcutaneously, and various
surface applications including intranasal, intradermal, and topical
application, for instance.
[0133] Pharmaceutically acceptable carriers: The pharmaceutically
acceptable carriers useful in this disclosure are conventional.
Remington: The Science and Practice of Pharmacy, The University of
the Sciences in Philadelphia, Editor, Lippincott, Williams, &
Wilkins, Philadelphia, Pa., 21.sup.st Edition (2005), describes
compositions and formulations suitable for pharmaceutical delivery
of the compounds herein disclosed.
[0134] In general, the nature of the carrier will depend on the
particular mode of administration being employed. For instance,
parenteral formulations usually comprise injectable fluids that
include pharmaceutically and physiologically acceptable fluids such
as water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(for example, powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically-neutral carriers, pharmaceutical
compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
[0135] Pharmaceutical agent or drug: A chemical compound or
composition capable of inducing a desired therapeutic or
prophylactic effect when properly administered to a subject.
Incubating includes exposing a target to an agent for a sufficient
period of time for the agent to interact with a cell. Contacting
includes incubating an agent in solid or in liquid form with a
cell. Pharmaceutical agents include, but are not limited to,
chemotherapeutic agents and anti-infective agents.
[0136] Polypeptide: A polymer in which the monomers are amino acid
residues that are joined together through amide bonds. When the
amino acids are alpha-amino acids, either the L-optical isomer or
the D-optical isomer can be used, the L-isomers usually being
preferred. The term polypeptide or protein as used herein
encompasses any amino acid sequence and includes modified sequences
such as glycoproteins. The term polypeptide is specifically
intended to cover naturally occurring proteins, as well as those
that are recombinantly or synthetically produced.
[0137] The term polypeptide fragment refers to a portion of a
polypeptide that exhibits at least one useful epitope. The phrase
"functional fragment(s) of a polypeptide" refers to all fragments
of a polypeptide that retain an activity, or a measurable portion
of an activity, of the polypeptide from which the fragment is
derived. Fragments, for example, can vary in size from a
polypeptide fragment as small as an epitope capable of binding an
antibody molecule to a large polypeptide capable of participating
in the characteristic induction or programming of phenotypic
changes within a cell. An epitope is a region of a polypeptide
capable of binding an immunoglobulin generated in response to
contact with an antigen.
[0138] Conservative amino acid substitution tables providing
functionally similar amino acids are well known to one of ordinary
skill in the art. The following six groups are examples of amino
acids that are considered to be conservative substitutions for one
another:
[0139] 1) Alanine (A), Serine (S), Threonine (T);
[0140] 2) Aspartic acid (D), Glutamic acid (E);
[0141] 3) Asparagine (N), Glutamine (Q);
[0142] 4) Arginine (R), Lysine (K);
[0143] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
and
[0144] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0145] In some circumstances, variations in the cDNA sequence that
result in amino acid changes, whether conservative or not, are
minimized in order to preserve the functional and immunologic
identity of the encoded protein. The immunologic identity of the
protein may be assessed by determining whether it is recognized by
an antibody; a variant that is recognized by such an antibody is
immunologically conserved. Any cDNA sequence variant will
preferably introduce no more than twenty, and preferably fewer than
ten amino acid substitutions into the encoded polypeptide. Variant
amino acid sequences may, for example, be 80%, 90%, or even 95% or
98% identical to the native amino acid sequence. Programs and
algorithms for determining percentage identity are well known, and
representative programs and algorithms can be found at the NCBI
website.
[0146] Preventing or treating a disease: Preventing a disease
refers to inhibiting the full development of a disease, for example
inhibiting the development of myocardial infarction in a person who
has coronary artery disease or inhibiting the progression or
metastasis of a tumor in a subject with a neoplasm. Treatment
refers to a therapeutic intervention that ameliorates at least one
sign or symptom of a disease or pathological condition, or
interferes with a pathophysiological process after it has begun to
develop. Treatment includes inhibiting or preventing the partial or
full development or progression of a disease, for example in a
person who is known to have a predisposition to a disease.
[0147] Primary cells: Cells directly obtained or isolated from
tissue. Primary cells are not transformed and are not immortalized.
These cells generally do not proliferate indefinitely when placed
in cell culture unless they undergo spontaneous immortalization or
malignant transformation. Primary cells obtained from a tissue may
include a population of multiple cell types, including multiple
types of differentiated cells, lineage-committed cells, and/or stem
cells (such as adult stem cells, for example hematopoietic stem
cells, mesenchymal stem cells, or neural stem cells). Primary cells
obtained from a tissue may also include primarily a single cell
type (or a single cell type may be isolated or selected from a
population of primary cells), such as human umbilical vein
endothelial cells (HUVEC).
[0148] Purified: In a more pure form than is found in nature. The
term purified does not require absolute purity; rather, it is
intended as a relative term. Thus, for example, a purified protein
preparation is one in which the protein referred to is more pure
than the protein in its natural environment within a cell.
[0149] The term substantially purified as used herein refers to a
molecule (for example, a nucleic acid, polypeptide,
oligonucleotide, etc.) that is substantially free of other
proteins, lipids, carbohydrates, or other materials with which it
is naturally associated. In one embodiment, a substantially
purified molecule is a polypeptide that is at least 50% free of
other proteins, lipids, carbohydrates, or other materials with
which it is naturally associated. In another embodiment, the
polypeptide is at least at least 80% free of other proteins,
lipids, carbohydrates, or other materials with which it is
naturally associated. In yet other embodiments, the polypeptide is
at least 90% or at least 95% free of other proteins, lipids,
carbohydrates, or other materials with which it is naturally
associated.
[0150] Recombinant: A nucleic acid that has a sequence that is not
naturally occurring or has a sequence that is made by an artificial
combination of two otherwise separated segments of sequence. This
artificial combination can be accomplished by chemical synthesis
or, more commonly, by the artificial manipulation of isolated
segments of nucleic acids, for example, by genetic engineering
techniques.
[0151] RNA interference (RNA silencing; RNAi): A gene-silencing
mechanism whereby specific double-stranded RNA (dsRNA) trigger the
degradation of homologous mRNA (also called target RNA).
Double-stranded RNA is processed into small interfering RNAs
(siRNA), which serve as a guide for cleavage of the homologous mRNA
in the RNA-induced silencing complex (RISC). The remnants of the
target RNA may then also act as siRNA; thus resulting in a cascade
effect.
[0152] Senescence: The biological process(es) of aging and showing
the effects of increased age. In one embodiment, a senescent cell
does not divide and/or has a reduced capacity to divide.
[0153] Separating: In reference to the separation of a stem cell,
such as a cancer stem cell, from other cells, such as a
non-cancerous stem cell. More generally, the term refers to the
purification of one cell type from another cell type, as used
herein, refers to spatially segregating cells of the different cell
types from each other so as to yield a fraction that is relatively
enriched in a first cell type, with respect to a second cell type,
and another fraction that is relatively enriched in a second cell
type, with respect to a first cell type. In certain embodiments,
cell types (e.g., cancer stem cells and/or progenitor cells and
non-cancerous stem cells and/or progenitor cells) can be separated
from each other such that the segregated fractions of the
respective cell types are enriched in the desired cells by at least
a factor of about 5, in some embodiments by at least a factor of
about 10, in some embodiments by at least a factor of about 100, in
some embodiments by at least a factor of about 1000, in some
embodiments by at least a factor of about 104, in some embodiments
by at least a factor of about 105, in some embodiments by at least
a factor of about 10.sup.6, and in yet other embodiments the
desired cells in the segregated fraction are free of cells of the
undesired type.
[0154] Sequence identity: The similarity between two nucleic acid
sequences, or two amino acid sequences, is expressed in terms of
the similarity between the sequences, otherwise referred to as
sequence identity. Sequence identity is frequently measured in
terms of percentage identity (or similarity or homology); the
higher the percentage, the more similar the two sequences are.
[0155] Methods of alignment of sequences for comparison are well
known in the art. Various programs and alignment algorithms are
described in: Smith and Waterman (Adv. Appl. Math. 2: 482, 1981);
Needleman and Wunsch (J. Mol. Biol. 48: 443, 1970); Pearson and
Lipman (PNAS. USA 85: 2444, 1988); Higgins and Sharp (Gene, 73:
237-244, 1988); Higgins and Sharp (CABIOS 5: 151-153, 1989); Corpet
et al. (Nuc. Acids Res. 16: 10881-10890, 1988); Huang et al. (Comp.
Appls Biosci. 8: 155-165, 1992); and Pearson et al. (Meth. Mol.
Biol. 24: 307-31, 1994). Altschul et al. (Nature Genet., 6:
119-129, 1994) presents a detailed consideration of sequence
alignment methods and homology calculations.
[0156] The alignment tools ALIGN (Myers and Miller, CABIOS 4:11-17,
1989) or LFASTA (Pearson and Lipman, 1988) may be used to perform
sequence comparisons (Internet Program .COPYRGT. 1996, W. R.
Pearson and the University of Virginia, fasta20u63 version 2.0u63,
release date December 1996). ALIGN compares entire sequences
against one another, while LFASTA compares regions of local
similarity. These alignment tools and their respective tutorials
are available on the Internet at the NCSA Website. Alternatively,
for comparisons of amino acid sequences of greater than about 30
amino acids, the Blast 2 sequences function can be employed using
the default BLOSUM62 matrix set to default parameters, (gap
existence cost of 11, and a per residue gap cost of 1). When
aligning short peptides (fewer than around 30 amino acids), the
alignment should be performed using the Blast 2 sequences function,
employing the PAM30 matrix set to default parameters (open gap 9,
extension gap 1 penalties). The BLAST sequence comparison system is
available, for instance, from the NCBI web site; see also Altschul
et al., J. Mol. Biol. 215:403-410, 1990; Gish. & States, Nature
Genet. 3:266-272, 1993; Madden et al. Meth. Enzymol. 266:131-141,
1996; Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; and
Zhang & Madden, Genome Res. 7:649-656, 1997.
[0157] Orthologs of proteins are typically characterized by
possession of greater than 75% sequence identity counted over the
full-length alignment with the amino acid sequence of specific
protein using ALIGN set to default parameters. Proteins with even
greater similarity to a reference sequence will show increasing
percentage identities when assessed by this method, such as at
least 80%, at least 85%, at least 90%, at least 92%, at least 95%,
or at least 98% sequence identity. In addition, sequence identity
can be compared over the full length of particular domains of the
disclosed peptides.
[0158] When significantly less than the entire sequence is being
compared for sequence identity, homologous sequences will typically
possess at least 80% sequence identity over short windows of 10-20
amino acids, and may possess sequence identities of at least 85%,
at least 90%, at least 95%, or at least 99% depending on their
similarity to the reference sequence. Sequence identity over such
short windows can be determined using LFASTA; methods are described
at the NCSA Website. One of skill in the art will appreciate that
these sequence identity ranges are provided for guidance only; it
is entirely possible that strongly significant homologs could be
obtained that fall outside of the ranges provided.
[0159] An alternative indication that two nucleic acid molecules
are closely related is that the two molecules hybridize to each
other under stringent conditions. Stringent conditions are
sequence-dependent and are different under different environmental
parameters. Generally, stringent conditions are selected to be
about 5.degree. C. to 20.degree. C. lower than the thermal melting
point (T.sub.m) for the specific sequence at a defined ionic
strength and pH. The T.sub.m is the temperature (under defined
ionic strength and pH) at which 50% of the target sequence
hybridizes to a perfectly matched probe. Conditions for nucleic
acid hybridization and calculation of stringencies can be found in
Sambrook et al. (In Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor, N.Y., 1989) and Tijssen (Laboratory Techniques in
Biochemistry and Molecular Biology Part I, Ch. 2, Elsevier, New
York, 1993).
[0160] Hybridization conditions resulting in particular degrees of
stringency will vary depending upon the nature of the hybridization
method of choice and the composition and length of the hybridizing
nucleic acid sequences. Generally, the temperature of hybridization
and the ionic strength (especially the Na+ concentration) of the
hybridization buffer will determine the stringency of
hybridization, though waste times also influence stringency.
Calculations regarding hybridization conditions required for
attaining particular degrees of stringency are discussed by
Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd
ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989, chapters 9 and 11, herein incorporated by
reference. The following is an exemplary set of hybridization
conditions:
Very High Stringency (detects sequences that share 90% identity)
[0161] Hybridization: 5.times.SSC at 65.degree. C. for 16 hours
[0162] Wash twice: 2.times.SSC at room temperature (RT) for 15
minutes each [0163] Wash twice: 0.5.times.SSC at 65.degree. C. for
20 minutes each High Stringency (detects sequences that share 80%
identity or greater) [0164] Hybridization: 5.times.-6.times.SSC at
65.degree. C.-70.degree. C. for 16-20 hours [0165] Wash twice:
2.times.SSC at RT for 5-20 minutes each [0166] Wash twice:
1.times.SSC at 55.degree. C.-70.degree. C. for 30 minutes each Low
Stringency (detects sequences that share greater than 50% identity)
[0167] Hybridization: 6.times.SSC at RT to 55.degree. C. for 16-20
hours [0168] Wash at least twice: 2.times.-3.times.SSC at RT to
55.degree. C. for 20-30 minutes each.
[0169] Nucleic acid sequences that do not show a high degree of
identity may nevertheless encode similar amino acid sequences, due
to the degeneracy of the genetic code. It is understood that
changes in nucleic acid sequence can be made using this degeneracy
to produce multiple nucleic acid sequences that each encode
substantially the same protein.
[0170] Specifically hybridizable and specifically complementary are
terms that indicate a sufficient degree of complementarity such
that stable and specific binding occurs between the oligonucleotide
(or its analog) and the DNA or RNA target. The oligonucleotide or
oligonucleotide analog need not be 100% complementary to its target
sequence to be specifically hybridizable. An oligonucleotide or
analog is specifically hybridizable when binding of the
oligonucleotide or analog to the target DNA or RNA molecule
interferes with the normal function of the target DNA or RNA, and
there is a sufficient degree of complementarity to avoid
non-specific binding of the oligonucleotide or analog to non-target
sequences under conditions where specific binding is desired, for
example under physiological conditions in the case of in vivo
assays or systems. Such binding is referred to as specific
hybridization.
[0171] Small interfering RNAs: Synthetic or naturally-produced
small double stranded RNAs (dsRNAs) that can induce gene-specific
inhibition of expression in invertebrate and vertebrate species are
provided. These RNAs are suitable for interference or inhibition of
expression of a target gene and comprise double stranded RNAs of
about 15 to about 40 nucleotides containing a 3' and/or 5' overhang
on each strand having a length of 0- to about 5-nucleotides,
wherein the sequence of the double stranded RNAs is essentially
identical to a portion of a coding region of the target gene for
which interference or inhibition of expression is desired. The
double stranded RNAs can be formed from complementary ssRNAs or
from a single stranded RNA that forms a hairpin or from expression
from a DNA vector.
[0172] Small molecule inhibitor: A molecule, typically with a
molecular weight less than 1000, or in some embodiments, less than
about 500 Daltons, wherein the molecule is capable of inhibiting,
to some measurable extent, an activity of some target molecule.
[0173] Stabilized nucleic acid molecules: A variety of synthetic
nucleic acid derivatives with increased stability as compared to
native (e.g., non-modified) nucleic acids. Stabilized nucleic acid
molecules include nucleic acids where the labile phosphodiester
bonds in nucleic acids are replaced with more stable
phosphoramidates or peptide amide backbones, or oligonucleotides
including one or more such nucleic acid derivatives. Also included
are nucleic acids having a substitution of the deoxyribosyl moiety
with a more stable morpholine derivative (e.g., morpholinos) or
oligonucleotides including one or more morpholino nucleic acids. In
other examples, stabilized nucleic acid molecules include "locked"
nucleic acids where the ribose moiety is modified with a bridge
connecting the 2' oxygen and the 4' carbon, or oligonucleotides
including one or more locked nucleic acid.
[0174] Stem cell: A cell that can generate a fully differentiated
functional cell of a more than one given cell type. The role of
stem cells in vivo is to replace cells that are destroyed during
the normal life of an animal. Generally, stem cells can divide
asymmetrically without limit and may be lineage-committed,
totipotent, or pluripotent. After division, the stem cell may
remain as a stem cell, become a precursor cell, or proceed to
terminal differentiation. A nervous system stem cell is, for
example, a cell of the central nervous system that can self-renew
and can generate astrocytes, neurons and oligodendrocytes.
[0175] A "somatic precursor cell" is a cell that can generate a
fully differentiated functional cell of at least one given cell
type from the body of an animal, such as a human. A neuronal
precursor cell can generate of fully differentiated neuronal cell,
such as, but not limited to, and adrenergic or a cholinergic
neuron. A glial precursor cell can generate fully differentiated
glial cells, such as but not limited to astrocytes, microglia and
oligodendroglia. Generally, precursor cells can divide and are
pluripotent. After division, a precursor cell can remain a
precursor cell, or may proceed to terminal differentiation. A
neuronal precursor cell can give rise to one or more types of
neurons, such as dopaminergic, adrenergic, or serotonergic cells,
but is more limited in its ability to differentiate than a stem
cell. In one example, a neuronal stem cell gives rise to all of the
types of neuronal cells (such as dopaminergic, adrenergic, and
serotonergic neurons) but does not give rise to other cells, such
as glial cells.
[0176] Suspension of cells; Cellular suspension: A mixture of cells
suspended in a carrier liquid. The carrier liquid may be naturally
part of the biological sample from which the cells derive, for
example blood is a suspension of blood cells suspended in plasma,
or, for cells which are not normally present in a suspension, the
carrier liquid can be any suitable diluent or medium. A cellular
suspension can include a plurality of stem cells of one or more
specific and desired types, for example cancer stem cells,
lympho-hematopoietic stem cells. For example, for such an
embodiment in the context of cancer treatment, diagnostics, or
research, the methods described herein can be used to generate
cellular suspension including a plurality of cancer stem cells.
[0177] Target sequence: A target sequence is a portion of ssDNA,
dsDNA, or RNA that, upon hybridization to a therapeutically
effective oligonucleotide or oligonucleotide analog (e.g., a
morpholino), results in the inhibition of expression of the target.
Either an antisense or a sense molecule can be used to target a
portion of dsDNA, as both will interfere with the expression of
that portion of the dsDNA. The antisense molecule can bind to the
plus strand, and the sense molecule can bind to the minus strand.
Thus, target sequences can be ssDNA, dsDNA, and RNA.
[0178] Therapeutically effective amount: A quantity of compound
sufficient to achieve a desired effect in a subject being treated.
An effective amount of a compound may be administered in a single
dose, or in several doses, for example daily, during a course of
treatment. However, the effective amount will be dependent on the
compound applied, the subject being treated, the severity and type
of the affliction, and the manner of administration of the
compound. For example, a therapeutically effective amount of an
active ingredient can be measured as the concentration (moles per
liter or molar-M) of the active ingredient (such as a small
molecule, peptide, protein, oligonucleotide, or antibody) in blood
(in vivo) or a buffer (in vitro) that produces an effect. The
effective amount of the compound usually will be dependent on the
compound applied, the subject being treated, the severity and type
of the affliction, and the manner of administration of the
compound.
[0179] Treating a disease: Includes inhibiting or preventing the
partial or full development or progression of a disease, for
example in a person who is known to have a predisposition to a
disease. Furthermore, treating a disease refers to a therapeutic
intervention that ameliorates at least one sign or symptom of a
disease or pathological condition, or interferes with a
pathophysiological process, after the disease or pathological
condition has begun to develop.
[0180] Tumor: An abnormal growth of cells, which can be benign or
malignant. Cancer is a malignant tumor, which is characterized by
abnormal or uncontrolled cell growth. Other features often
associated with malignancy include metastasis, interference with
the normal functioning of neighboring cells, release of cytokines
or other secretory products at abnormal levels and suppression or
aggravation of inflammatory or immunological response, invasion of
surrounding or distant tissues or organs, such as lymph nodes, etc.
"Metastatic disease" refers to cancer cells that have left the
original tumor site and migrate to other parts of the body for
example via the bloodstream or lymph system.
[0181] Tumors of the same tissue type may be divided into tumor of
different sub-types (a classic example being bronchogenic
carcinomas (lung tumors) which can be an adenocarcinoma, small
cell, squamous cell, or large cell tumor). Breast cancers can be
divided histologically into scirrhous, infiltrative, papillary,
ductal, medullary and lobular.
[0182] Tumors include original (primary) tumors, recurrent tumors,
and metastases (secondary) tumors. A tumor recurrence is the return
of a tumor, at the same site as the original (primary) tumor, after
the tumor has been removed surgically, by drug or other treatment,
or has otherwise disappeared. A metastasis is the spread of a tumor
from one part of the body to another. Tumors formed from cells that
have spread are called secondary tumors and contain cells that are
like those in the original (primary) tumor. There can be a
recurrence of either a primary tumor or a metastasis.
[0183] The amount of a tumor in an individual is the "tumor burden"
which can be measured as the number, volume, or weight of the
tumor. A tumor that does not metastasize is referred to as
"benign." A tumor that invades the surrounding tissue and/or can
metastasize is referred to as "malignant." Examples of
hematological tumors include leukemias, including acute leukemias
(such as 11q23-positive acute leukemia, acute lymphocytic leukemia,
acute myelocytic leukemia, acute myelogenous leukemia and
myeloblastic, promyelocytic, myelomonocytic, monocytic and
erythroleukemia), chronic leukemias (such as chronic myelocytic
(granulocytic) leukemia, chronic myelogenous leukemia, and chronic
lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's
disease, non-Hodgkin's lymphoma (indolent and high grade forms),
multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain
disease, myelodysplastic syndrome, hairy cell leukemia and
myelodysplasia.
[0184] Examples of solid tumors, such as sarcomas and carcinomas,
include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,
osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma,
Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
lymphoid malignancy, pancreatic cancer, breast cancer (including
basal breast carcinoma, ductal carcinoma and lobular breast
carcinoma), lung cancers, ovarian cancer, prostate cancer,
hepatocellular carcinoma, squamous cell carcinoma, basal cell
carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid
carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous
gland carcinoma, papillary carcinoma, papillary adenocarcinomas,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor,
cervical cancer, testicular tumor, seminoma, bladder carcinoma, and
CNS tumors (such as a glioma, astrocytoma, medulloblastoma,
craniopharyrgioma, ependymoma, pinealoma, hemangioblastoma,
acoustic neuroma, oligodendroglioma, meningioma, melanoma,
neuroblastoma and retinoblastoma). In several examples, a tumor is
melanoma, lung cancer, lymphoma breast cancer or colon cancer.
[0185] Under conditions sufficient for: A phrase that is used to
describe any environment that permits the desired activity. In one
example, includes administering a therapeutically effective amount
of a composition that includes a peptide, antibody, or
oligonucleotide (e.g., morpholino), sufficient to enable the
desired activity.
[0186] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this invention
belongs.
[0187] The singular terms "a," "an," and "the" include plural
referents unless context clearly indicates otherwise. Similarly,
the word or is intended to include and unless the context clearly
indicates otherwise. Hence "comprising A or B" means "including A,
or including B, or including A and B." It is further to be
understood that all base sizes or amino acid sizes, and all
molecular weight or molecular mass values, given for nucleic acids
or polypeptides are approximate, and are provided for description.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, suitable methods and materials are described
below. All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety.
[0188] In case of conflict, the present specification, including
explanations of terms, will control. In addition, the materials,
methods, and examples are illustrative only and not intended to be
limiting.
II. Overview of Several Embodiments
[0189] Provided herein in a first embodiment is a method for
inducing differentiation of a cancer stem cell, wherein the method
includes contacting the cancer stem cell with an agent that alters
CD47 signaling to induce cancer stem cell (CSC) differentiation,
thereby inducing differentiation of CSCs. In examples of this
embodiment, the induced differentiation is irreversible.
Optionally, the agent used in the method comprises an anti-CD47
antibody or binding fragment thereof, peptide 7N3, or a
CD47-targeted CRISPR construct. By way of example, the agent is the
monoclonal antibody B6H12, humanized B6H12, a binding fragment of
B6H12 or humanized B6H12, or a monoclonal antibody that binds
competitively with B6H12.
[0190] In embodiments of the disclosed methods, the CSC comprises a
breast cancer stem cell, a lung cancer stem cell, a prostate cancer
stem cell, a colon carcinoma stem cell, or a melanoma stem
cell.
[0191] By way of examples, in embodiment of the disclosed methods
for inducing differentiation of CSCs, the agent that alters CD47
signalling induces in the CSC down-regulation of EGFR and
up-regulation of one or more of the molecules listed in Table 1 as
having increased expression.
[0192] It is contemplated herein that the methods of inducing
cancer stem cell (CSC) differentiation disclosed herein can be
practiced on CSCs that are in a subject. In examples of such
treatment methods, the subject has a primary tumor, has a tumor in
regression, has or is suspected of having a metastatic tumor, or a
combination thereof
[0193] Also provided herein are methods of treating a subject with
a tumor, which methods involve administering a therapeutically
effective amount of agent that alters CD47 signaling to induce
cancer stem cell (CSC) differentiation to the subject, thereby
treating the subject with the tumor. In examples of this method,
the agent comprises an anti-CD47 antibody or binding fragment
thereof, peptide 7N3, or a CD47-targeted CRISPR construct. By way
of example, the agent is the monoclonal antibody B6H12, humanized
B6H12, a binding fragment of B6H12 or humanized B6H12, or a
monoclonal antibody that binds competitively with B6H12.
[0194] Also provided are treatment methods which further involve
administering a therapeutically effective amount of an anti-cancer
treatment to the subject. This can occur before, concurrent with,
or after treatment with the agent that alters CD47 signaling to
induce cancer stem cell (CSC) differentiation. In particular
examples, the anti-cancer treatment is administered first, followed
(for instance 1-4 days, or 1-7 days later) by administration with
the agent that alters CD47 signaling.
[0195] In embodiments of treatment methods, the subject has a
primary tumor, has a tumor in regression, has or is suspected of
having a metastatic tumor, or a combination thereof.
[0196] Also provided herein are methods for inducing
differentiation (such as irreversible differentiation) of cancer
stem cells, substantially as described herein.
III. Methods to Eliminate Cancer Stem Cells by Targeting CD47
[0197] CD47 is a ubiquitously expressed cell surface receptor for
thrombospondin-1 and the counter-receptor for signal-regulatory
protein-.alpha. (SIRP.alpha.). High expression of CD47 on several
types of cancer cells has been identified as a `don't eat me
signal` that inhibits their killing by macrophages or NK cells.
Conversely, the CD47 antibody B6H12 that blocks SIRP.alpha. binding
enhances macrophage-dependent clearance of tumors in several mouse
models, although others have shown that such clearance can be
independent of SIRP.alpha. signaling. We recently found that the
absence of CD47 enhances stem cells in vitro and in vivo by
increasing expression of four stem cell transcription factors (see,
e.g., International Patent Publication WO 2013/155109, incorporated
herein by reference in its entirety).
[0198] Stem cells also play an important role in the pathogenesis
of cancer. Cancer stem cells have been reported to express elevated
CD47 levels, but the role of CD47 in cancer stem cell function,
apart from protecting CSC from phagocytosis by engaging SIRP.alpha.
on macrophages, has not been examined. Breast cancer stem cells
(bCSCs) isolated from the triple negative MDA-MB-231 cell line have
up-regulation of embryonic as well as cancer stem cell markers and
exhibit high asymmetric cell division as compared to differentiated
MDA-MB-231 cells. bCSCs can form mammospheres and can differentiate
into endothelial, smooth muscle, and neural phenotypes, but they
preserve the gene expression patterns of differentiated MDA-MB-231
cells. Relative to differentiated MDA-MB-231 cells, bCSCs have low
expression of genes associated with tumor suppression, the RNAi
silencing complex, and metabolism.
[0199] Global analysis of gene expression of bCSCs treated with
B6H12 antibody showed up-regulation of tumor suppressor genes
(TXNIP, LOX and PLS3), the RNAi silencing gene DICER 1, and TOP1,
but decreased expression of epidermal growth factor receptor
(EGFR). We further found that CD47 and EGFR are co-localized and
interact with each other in MDA-MB-231 breast cancer cells.
Treatment with B6H12 antibody inhibits EGF-induced EGFR-tyrosine
phosphorylation and cell proliferation. Data presented herein show
that B6H12 specifically targets bCSCs but not differentiated cancer
cells, and this CD47 signaling is independent of SIRP.alpha..
Treatment of the estrogen receptor-positive breast cancer cell line
MCF7 with B6H12 antibody did not alter expression of the above
indicated genes. This suggests that therapeutic antibodies
targeting CD47 may selectively benefit patients with triple
negative breast cancers that are associated with high rates of
metastasis by forcing differentiation of bCSCs.
[0200] Breast epithelial stem cells are important for tissue self
renewal (Villadsen et al., J Cell Biol. 177:87 101, 2007). The
presences of breast progenitor cells has been reported and
characterized in normal adults (Stingl et al., Breast Canc Res
Treat. 67:93 109, 2001). Normal stem and progenitor cells play
active role in cyclic changes that takes place during pregnancy and
ovulation in women (Petersen & Polyak, CSH Persps in Biol.
2:a003160, 2010). Human breast epithelial cells with stem cell like
phenotype showing increased telomerase activity and led to
immortalization (Gudjonsson et al., Genes & Deve. 16:693 706,
2002; Ince et al., Cancer Cell. 12:160 170, 2007; Sun et al.,
Cancer Res. 59:6118 6123, 1999). This suggested that breast cancer
initiation might involve cells having stem cell like properties and
defined as cancer stem cells. These cancer stem cells have distinct
properties from differentiated tumor cells. It is thought that
restoration, progression and resistance of cancer to therapy is due
to small pool of cancer stem cells. These may account for the
frequency of breast cancer recurrence in 5 7 years (Rosen et al., J
Clin Oncol 7:1239 1251, 1989). It has been speculated that cancer
stem cells are in a dormant stage and become active during
epithelial mesenchymal transition (Giancotti, Cell. 155:750 764,
2013).
[0201] Reports have shown that cancer stem cells are lin.sup.-- and
express the CD133.sup.+ marker. CD133.sup.+ expressing cells are
tumorigenic in Nod SCID mice, but CD133 engrafted cells do not form
tumors (Singh et al., Nature. 432:396 401, 2004). Other flow
cytometric analysis has shown that a CD44.sup.+(high) and CD24 low
population represents cancer stem cells (Fillmore &
Kuperwasser, Breast Cancer Res: BCR. 9:303, 2007). ALD1, a
metabolic dehydrogenase, has been associated with cancer stem cells
(Charafe Jauffret et al., Cancer Res. 73:7290 7300, 2013).
[0202] CD47 is also known as hematopoietic stem cell marker that
inhibits phagocytosis via SIRP alpha (SIRP-.alpha.) (Jaiswal et
al., Cell. 138(2):271-285-2009). Inhibition of CD47 using the
antibody B6H12 has been in pre-clinical trials where human tumor
xenografts are implanted into Nod-SCID mice that express a mutant
form of mouse SIRP.alpha. that can recognize human CD47 (Majeti,
Cell. 2009 Jul. 23; 138(2):286-99; Chan et al., Proc Natl Acad Sci
USA. 2009 Aug. 18; 106(33):14016-21; Chao et al., Cell. 2010 Sep.
3; 142(5):699-713; Cancer Res. 2011 Feb. 15; 71(4):1374-84; Chao et
al., Blood. 2011 Nov. 3; 118(18):4890-901). In these mice, B6H12
inhibits the interaction between human CD47 on the tumor cells and
SIRP.alpha. on mouse macrophages and thereby enhances clearance of
the tumor xenograft by the mouse innate immune system. Humanized
CD47 antibodies are potential candidates for treating many cancers
(Willingham et al., PNAS USA. 109:6662 6667, 2012). Many labs have
argued that data in the above publications is not fully supportive
of the CD47-SIRP-.alpha. hypothesis (Zhao et al., Blood. 119:4333
4334; author reply 4334 4335, 2012; Zhao et al., PNAS USA.
109:E2843; author reply E2844 2845, 2012; Soto-Pantoja et al., Proc
Natl Acad Sci USA. 109(42): E2842, 2012; author reply E2844-5).
However, none of these publications has identified a cancer
cell-autonomous function of CD47 that is blocked by B6H12 and could
lead to tumor ablation. On the contrary, previous publications that
have identified other CD47 antibodies that directly induce death of
certain leukemic and breast cancer cell lines, but B6H12 was
generally used as a negative control in those studies. The CD47
antibody Ad22 induced apoptosis of Jurkat cells and
CD3epsilon-stimulated PBMC, but the CD47 mAbs 2D3 and B6H12 were
inactive (Pettersen et al., J Immunol. 162(12):7031-40, 1999)
Another study reported that immobilized but not soluble B6H12
induced caspase-independent cell death of human B cell chronic
lymphocytic leukemia cells (Mateo et al., Nat Med. 5(11):1277-84,
1999; Mateo et al., Blood. 100(8):2882-90, 2002). However, in the
same study CD34.sup.+ hematopoietic progenitor stem cells were not
killed by immobilized B6H12, despite their high level of CD47
expression.
[0203] Recently we discovered that CD47 null primary cells derived
from mouse lungs exhibit enhanced self renewal, which is mediated
by up-regulation of the four stem cell transcription factors c-MYC,
KLF4, OCT4 and SOX2. Global transcriptome analysis confirmed a
close resemblance with ES and iPS cells (Kaur et al., Scientific
Rep. 3:1673, 2013).
[0204] Breast cancer stem cells (bCSCs) isolated from the triple
negative MDA-MB-231 cell line have up regulation of embryonic as
well as cancer stem cell markers and exhibit high asymmetric cell
division as compared to differentiated MDA-MB-231 cells. These
bCSCs can form mammospheres and can differentiate into endothelial,
smooth muscle, and neural phenotypes, but they preserve the gene
expression patterns of differentiated MDA-MB-231 cells. Relative to
differentiated MDA-MB-231 cells, the bCSCs have low expression of
genes associated with tumor suppression, the RNAi silencing
complex, and metabolism. Remarkably, treating the bCSCs with the
CD47 function-blocking antibody B6H12 induces irreversible
differentiation of the bCSC and inhibits their proliferation.
Global analysis of gene expression of bCSCs treated with B6H12
showed up-regulation of tumor suppressor genes (TXNIP, LOX and
PLS3), the RNAi silencing gene DICER 1, and TOP1, but decreased
expression of EGFR.
[0205] Further, CD47 and EGFR are co-localized and interact with
each other in MDA-MB-231 breast cancer cells. Treatment with B6H12
antibody inhibits EGF induced EGFR tyrosine phosphorylation and
cell proliferation. Our preliminary data show that B6H12
specifically targets bCSCs but not differentiated cancer cells, and
this CD47 signaling is independent of SIRP-.alpha.. Treatment of
estrogen receptor positive breast cancer cell lines with B6H12
antibody did not alter expression of the above indicated genes.
This suggests that therapeutic antibodies targeting CD47 may
selectively benefit patients with triple negative breast cancers
that are associated with high rates of metastasis by forcing
differentiation of bCSCs. These experiments reveal an unexpected
direct activity of CD47 blockade to target breast cancer by
suppressing bCSC (by inducing terminal differentiation, thereby
eliminating the cancer stem cells). This challenges the current
dogma that CD47 antibodies have therapeutic benefit exclusively
through blocking clearance of tumor cells by macrophage
phagocytosis.
[0206] Though exemplified herein with cancer stem cells (including
particularly triple negative breast cancer), it is to be understood
that the methods provided herein are application in other cancer
types, including but not limited to colon cancer, lung cancer,
kidney cancer, bone cancer, brain cancer, leukemia, lymphoma,
prostate cancer, bladder cancer, or pancreatic cancer.
[0207] Evidence is emerging that CSC also play a major role in the
metastatic spread of cancer, which is the major cause of treatment
failure and death from malignancies (Li et al., Cell Res.
17(1):3-14, 2007; Baccelli and Trumpp, J Cell Biol. 198(3):281-93,
2012; Shiozawa et al., Pharmacol Ther. 2013 May; 138(2):285-93).
Metastatic cells share many molecular features with CSC, and in
common with CSC: the cells that initiate distant metastases have a
self-renewal capability and can give rise to various differentiated
tumor cell types in any given metastasis. Elevated expression of
CD47 is emerging as another characteristic shared between CSC and
metastatic cancer cells. Luminal-type breast cancers with the
highest expression of CD47 were associated with lymph node
metastasis and poor survival (Baccelli et al., Oncotarget. 2014
Sep. 2. [Epub ahead of print]). Circulating cancer cells in these
patients also had elevated CD47 expression. In mouse xenograft
studies, circulating tumor cells selected for high expression of
CD47 and other CSC markers, but not bulk circulating tumor cells
efficiently gave rise to bone, lung and liver metastases in mice
(Baccelli et al., Nat Biotechnol. 31(6):539-44, 2013). Elevated
CD47 expression was also reported on circulating colorectal cancer
cells (Steinert et al., Cancer Res. 74(6):1694-704, 2014). In the
context of the present findings, these studies indicate that the
high CD47 expression on circulating tumor cells serves not merely
to prevent their clearance by phagocytes, but rather is important
for maintaining their stem cell character. Conversely, one
application of the agents and methods described herein as useful to
alter CD47 signaling in CSC is to differentiate circulating tumor
cells and cancer cells in nascent micrometastases, thereby
preventing the establishment and growth of metastatic lesions.
IV. Production of Antibodies
[0208] Examples of the therapeutic methods described herein employ
an anti-CD47 antibody or derivative thereof; one specific example
is the monoclonal antibody B6H12. However, additional antibodies
can be used in these methods, including for instance humanized
versions of B6H12, binding fragments of B6H12, and monoclonal
antibodies that bind competitively with B6H12. Additional specific
antibodies include BRIC 126, 6H9, Clkm1, OVTL16, OX101, mIAP410, or
mIAP301. Some of these antibodies, e.g. B6H12, are known to inhibit
both TSP1 and SIRP.alpha. binding to CD47. mIAP301 blocks binding
of murine CD47 to murine SIRP.alpha.. Given the large size of
antibodies, some CD47 antibodies may bind to epitopes distinct from
the SIRP or TSP1 binding sites but still be capable of sterically
blocking the binding of one or both of these ligands. Evidence
provided herein suggests that the activity of B6H12 to
differentiate CSC is independent of blocking SIRP binding. Blocking
of EGFR or TSP1 binding may be important, but B6H12 and related
CD47 antibodies may induce CSC differentiation without inhibiting
EGFR association or TSP1 binding to CD47. Thus some CD47 antibodies
that do not inhibit SIRP or TSP1 binding but similarly alter the
conformation of CD47 would be expected to share the ability to
differentiate CSC. Tests are provided herein for evaluating the
usefulness of any particular antibody for inducing differentiation
of CSCs.
[0209] Optimally, antibodies raised against a target protein (such
as CD47) would specifically detect that peptide/protein, and
optimally would inhibit the functional interaction between CD47 and
a protein with which it normally interacts, such as EGFR or TSP1 or
SIRP.alpha.. Antibodies that specifically detect a target protein
would recognize and bind that protein (and peptides derived
therefrom) and would not substantially recognize or bind to other
proteins or peptides found in a biological sample. The
determination that an antibody specifically detects its target
protein is made by any one of a number of standard immunoassay
methods; for instance, the Western blotting technique (Sambrook et
al., In Molecular Cloning: A Laboratory Manual, CSHL, New York,
1989). Antibodies that do not inhibit binding of these ligands but
similarly alter the conformation of CD47 or its lateral
associations in the cell membrane are another preferred
embodiment.
[0210] To determine by Western blotting that a given antibody
preparation (such as one produced in a mouse or rabbit)
specifically detects the target peptide, the peptide of interest is
synthesized and transferred to a membrane (for example,
nitrocellulose) by Western blotting, and the test antibody
preparation is incubated with the membrane. After washing the
membrane to remove non-specifically bound antibodies, the presence
of specifically bound antibodies is detected by the use of an
anti-mouse or anti-rabbit antibody conjugated to an enzyme such as
alkaline phosphatase.
[0211] Application of an alkaline phosphatase substrate
5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium results
in the production of a dense blue compound by immunolocalized
alkaline phosphatase. Antibodies that specifically detect the
target peptide will, by this technique, be shown to bind to the
target peptide band (which will be localized at a given position on
the gel determined by its molecular weight). Non-specific binding
of the antibody to other proteins may occur and may be detectable
as a weak signal on the Western blot. The non-specific nature of
this binding will be recognized by one skilled in the art by the
weak signal obtained on the Western blot relative to the strong
primary signal arising from the specific antibody-target peptide
binding.
[0212] The determination that an antibody inhibits the association
between CD47 and EGFR, or induces differentiation of CSCs through
blockade of CD47, may be made, for example, using any one of the
assays described herein. For instance, the determination that an
antibody inhibits EGFR association with purified or recombinant
CD47 can be made by comparing the binding (or functional) activity
alone with the binding (or functional activity in the presence of
the antibody using a solid phase ligand binding assay.
[0213] A. Monoclonal Antibody Production by Hybridoma Fusion
[0214] Monoclonal antibody to epitopes of a target peptide (e.g.,
from CD47) can be prepared from murine hybridomas according to the
classical method of Kohler and Milstein (Nature 256:495, 1975) or
derivative methods thereof. Briefly, a mouse is repetitively
inoculated with a few micrograms of the selected protein over a
period of a few weeks. The mouse is then sacrificed, and the
antibody-producing cells of the spleen are isolated. The spleen
cells are fused by means of polyethylene glycol with mouse myeloma
cells, and the excess un-fused cells destroyed by growth of the
system on selective media comprising aminopterin (HAT media). The
successfully fused cells are diluted and aliquots of the dilution
placed in wells of a microtiter plate where growth of the culture
is continued. Antibody-producing clones are identified by detection
of antibody in the supernatant fluid of the wells by immunoassay
procedures, such as ELISA, as originally described by Engvall
(Enzymol. 70:419, 1980), and derivative methods thereof. Selected
positive clones can be expanded and their monoclonal antibody
product harvested for use. Detailed procedures for monoclonal
antibody production are described in Harlow and Lane (Antibodies, A
Laboratory Manual, CSHL, New York, 1988).
[0215] B. Polyclonal Antibody Production by Immunization
[0216] Polyclonal antiserum containing antibodies to heterogeneous
epitopes of a single protein can be prepared by immunizing suitable
animals with the expressed protein, which can be unmodified or
modified to enhance immunogenicity. Effective polyclonal antibody
production is affected by many factors related both to the antigen
and the host species. For example, small molecules tend to be less
immunogenic than others and may require the use of carriers and
adjuvant. Also, host animals vary in response to site of
inoculations and dose, with either inadequate or excessive doses of
antigen resulting in low titer antisera. Small doses (ng level) of
antigen administered at multiple intradermal sites appear to be
most reliable. An effective immunization protocol for rabbits can
be found in Vaitukaitis et al. (J. Clin. Endocrinol. Metab.
33:988-991, 1971).
[0217] Booster injections can be given at regular intervals, and
antiserum harvested when antibody titer thereof, as determined
semi-quantitatively, for example, by double immunodiffusion in agar
against known concentrations of the antigen, begins to fall. See,
for example, Ouchterlony et al. (In Handbook of Experimental
Immunology, Wier, D. (ed.) chapter 19. Blackwell, 1973). Plateau
concentration of antibody is usually in the range of about 0.1 to
0.2 mg/ml of serum (about 12 .mu.M). Affinity of the antisera for
the antigen is determined by preparing competitive binding curves,
as described, for example, by Fisher (Manual of Clinical
Immunology, Ch. 42, 1980).
[0218] C. Antibodies Raised Against Synthetic Peptides
[0219] A third approach to raising antibodies against a target
peptide is to use synthetic peptides synthesized on a commercially
available peptide synthesizer based upon the amino acid sequence of
the native protein (e.g., CD47).
[0220] By way of example only, polyclonal antibodies to a CD47
peptide can be generated through well-known techniques by injecting
rabbits or another mammal with chemically synthesized peptide.
[0221] D. Antibodies Raised by Injection of a Peptide-Encoding
Sequence
[0222] Antibodies may be raised against a target peptide by
subcutaneous injection of a DNA vector that expresses that peptide
into laboratory animals, such as mice. Delivery of the recombinant
vector into the animals may be achieved using a hand-held form of
the Biolistic system (Sanford et al., Particulate Sci. Technol.
5:27-37, 1987) as described by Tang et al. (Nature 356:152-154,
1992). Expression vectors suitable for this purpose may include
those that express the desired peptide-encoding sequence under the
transcriptional control of either the human .beta.-actin promoter
or the cytomegalovirus (CMV) promoter.
[0223] E. Humanized Antibodies
[0224] Also contemplated are humanized antibodies, for instance
humanized equivalents of the described murine monoclonal
antibodies. A "humanized" immunoglobulin is an immunoglobulin
including a human framework region and one or more CDRs from a
non-human (for example a mouse, rat, or synthetic) immunoglobulin.
The non-human immunoglobulin providing the CDRs is termed a
"donor," and the human immunoglobulin providing the framework is
termed an "acceptor." In one embodiment, all the CDRs are from the
donor immunoglobulin in a humanized immunoglobulin. Constant
regions need not be present, but if they are, they must be
substantially identical to human immunoglobulin constant regions,
such as at least about 85-90%, such as about 95% or more identical.
Hence, all parts of a humanized immunoglobulin, except possibly the
CDRs, are substantially identical to corresponding parts of natural
human immunoglobulin sequences. A "humanized antibody" is an
antibody comprising a humanized light chain and a humanized heavy
chain immunoglobulin. A humanized antibody binds to the same
antigen as the donor antibody that provides the CDRs. The acceptor
framework of a humanized immunoglobulin or antibody may have a
limited number of substitutions by amino acids taken from the donor
framework. Humanized or other monoclonal antibodies can have
additional conservative amino acid substitutions which have
substantially no effect on antigen binding or other immunoglobulin
functions. Humanized immunoglobulins can be constructed by means of
genetic engineering (see for example, U.S. Pat. No. 5,585,089).
[0225] The use of antibody components derived from humanized
monoclonal antibodies obviates potential problems associated with
the immunogenicity of the constant regions of the donor antibody.
Techniques for producing humanized monoclonal antibodies are
described, for example, by Jones et al., Nature 321:522, 1986;
Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science
239:1534, 1988; Carter et al., Proc. Natl. Acad. Sci. U.S.A.
89:4285, 1992; Sandhu, Crit. Rev. Biotech. 12:437, 1992; and Singer
et al., J. Immunol. 150:2844, 1993. The antibody may be of any
isotype, but in several embodiments the antibody is an IgM or an
IgG, including but not limited to, IgG.sub.1, IgG.sub.2, IgG.sub.3
and IgG.sub.4.
[0226] Humanized monoclonal antibodies can be produced by
transferring donor complementarity determining regions (CDRs) from
heavy and light variable chains of the donor mouse (or other
animal) immunoglobulin. The production of chimeric antibodies,
which include a framework region from one antibody and the CDRs
from a different antibody, is well known in the art. For example,
humanized antibodies can be routinely produced. The antibody or
antibody fragment can be a humanized immunoglobulin having
complementarity determining regions (CDRs) from a donor monoclonal
antibody that binds a cell surface antigen of pancreatic cells
(such as endocrine, exocrine or ductal cells) and immunoglobulin
and heavy and light chain variable region frameworks from human
acceptor immunoglobulin heavy and light chain frameworks.
Generally, the humanized immunoglobulin specifically binds to the
cell surface antigen (or cells expressing the antigen) with an
affinity constant of at least 10.sup.7 M.sub.-1, such as at least
10.sup.8 M.sup.-1 at least 5.times.10.sup.8 M.sup.-1 or at least
10.sup.9 M.sup.-1.
[0227] In one embodiment, the sequence of the humanized
immunoglobulin heavy chain variable region framework can be at
least about 65% identical to the sequence of the donor
immunoglobulin heavy chain variable region framework. Thus, the
sequence of the humanized immunoglobulin heavy chain variable
region framework can be at least about 75%, at least about 85%, at
least about 95%, or at least about 99% identical to the sequence of
the donor murine immunoglobulin heavy chain variable region
framework. Human framework regions, and mutations that can be made
in a humanized antibody framework regions, are known in the art
(see, for example, U.S. Pat. No. 5,585,089, which is incorporated
herein by reference). One of skill in the art can readily select a
human framework region of use.
[0228] Also contemplated are fully human antibodies. Mice have been
generated that express only human immunoglobulin genes, instead of
mouse genes. These mice are immunized with the antigen, such as
CD47, and resultant antibodies that are raised are selected for the
activity desired. In the current instance, it is contemplated that
this technique can be used to generate antibodies (including
monoclonal antibodies) useful for blocking CD47 interactions. These
procedures are substantially similar just those used to select a
mouse anti-human Ab, but result in a fully human antibody since the
mouse only has human Ig genes.
V. Peptides and Peptide Variants
[0229] The peptides useful in methods disclosed herein (e.g.,
peptide p7N3, FIRVVMYEGKK; SEQ ID NO: 1) can be chemically
synthesized by standard methods, or can be produced recombinantly.
The synthesis of the presently disclosed peptide compounds can be
accomplished using standard chemical reactions known to be useful
for preparing a variety of analogous compounds. Indeed, exemplary
techniques known to those of ordinary skill in the art of peptide
synthesis are taught by Bodanszky & Bodanszky (The Practice of
Peptide Synthesis; Springer Verlag, New York, 1994) and by Jones
(Amino Acid and Peptide Synthesis; 2nd ed.; Oxford University
Press, 2002), both of which are incorporated herein by reference.
The Bodanszky and Jones references detail the parameters and
techniques for activating and coupling amino acids and amino acid
derivatives. Moreover, the references teach how to select, use and
remove various useful protecting groups. An exemplary specific
process for (poly)peptide production is described in Lu et al.
(Fed. Europ Biochem Societies Lett. 429:31-35, 1998).
[0230] Polynucleotides encoding the peptides disclosed herein are
also provided. These polynucleotides include DNA, cDNA and RNA
sequences that encode the peptide of interest. Silent mutations in
the coding sequence result from the degeneracy (i.e., redundancy)
of the genetic code, whereby more than one codon can encode the
same amino acid residue. Thus, for example, leucine can be encoded
by CTT, CTC, CTA, CTG, TTA, or TTG; serine can be encoded by TCT,
TCC, TCA, TCG, AGT, or AGC; asparagine can be encoded by AAT or
AAC; aspartic acid can be encoded by GAT or GAC; cysteine can be
encoded by TGT or TGC; alanine can be encoded by GCT, GCC, GCA, or
GCG; glutamine can be encoded by CAA or CAG; tyrosine can be
encoded by TAT or TAC; and isoleucine can be encoded by ATT, ATC,
or ATA. Tables showing the standard genetic code can be found in
various sources (e.g., L. Stryer, 1988, Biochemistry, 3.sup.rd
Edition, W.H. 5 Freeman and Co., NY).
[0231] A nucleic acid encoding a peptide can be cloned or amplified
by in vitro methods, such as the polymerase chain reaction (PCR),
the ligase chain reaction (LCR), the transcription-based
amplification system (TAS), the self-sustained sequence replication
system (3SR) and the Q.beta. replicase amplification system (QB).
For example, a polynucleotide encoding the peptide (or a longer
polypeptide, such as an expression fusion polypeptide, containing
the peptide) can be isolated by polymerase chain reaction of cDNA
using primers based on the DNA sequence of the molecule. A wide
variety of cloning and in vitro amplification methodologies are
well known to persons skilled in the art. PCR methods are described
in, for example, U.S. Pat. No. 4,683,195; Mullis et al., Cold
Spring Harbor Symp. Quant. Biol. 51:263, 1987; and Erlich, ed., PCR
Technology, (Stockton Press, N Y, 1989). Polynucleotides also can
be isolated by screening genomic or cDNA libraries with probes
selected from the sequences of the desired polynucleotide under
stringent hybridization conditions.
[0232] The polynucleotides encoding a peptide (e.g., a peptide from
or derived from TSP1 or CD47) include a recombinant DNA which is
incorporated into a vector into an autonomously replicating plasmid
or virus or into the genomic DNA of a prokaryote or eukaryote, or
which exists as a separate molecule (such as a cDNA) independent of
other sequences. The nucleotides of the invention can be
ribonucleotides, deoxyribonucleotides, or modified forms of either
nucleotide. The term includes single and double forms of DNA.
[0233] In one embodiment, vectors are used for expression in yeast
such as S. cerevisiae or Kluyveromyces lactis. Several promoters
are known to be of use in yeast expression systems such as the
constitutive promoters plasma membrane H.sup.+-ATPase (PMA1),
glyceraldehyde-3-phosphate dehydrogenase (GPD), phosphoglycerate
kinase-1 (PGK1), alcohol dehydrogenase-1 (ADH1), and pleiotropic
drug-resistant pump (PDR5). In addition, many inducible promoters
are of use, such as GAL1-10 (induced by galactose), PHOS (induced
by low extracellular inorganic phosphate), and tandem heat shock
HSE elements (induced by temperature elevation to 37.degree. C.).
Promoters that direct variable expression in response to a
titratable inducer include the methionine-responsive MET3 and MET25
promoters and copper-dependent CUP1 promoters. Any of these
promoters may be cloned into multicopy (2.mu.) or single copy (CEN)
plasmids to give an additional level of control in expression
level. The plasmids can include nutritional markers (such as URA3,
ADE3, HIS1, and others) for selection in yeast and antibiotic
resistance (AMP) for propagation in bacteria. Plasmids for
expression on K. lactis are known, such as pKLAC1. Thus, in one
example, after amplification in bacteria, plasmids can be
introduced into the corresponding yeast auxotrophs by methods
similar to bacterial transformation.
[0234] The peptides can be expressed in a variety of yeast strains.
For example, seven pleiotropic drug-resistant transporters, YOR1,
SNQ2, PDR5, YCF1, PDR10, PDR11, and PDR15, together with their
activating transcription factors, PDR1 and PDR3, have been
simultaneously deleted in yeast host cells, rendering the resultant
strain sensitive to drugs. Yeast strains with altered lipid
composition of the plasma membrane, such as the erg6 mutant
defective in ergosterol biosynthesis, can also be utilized.
Proteins that are highly sensitive to proteolysis can be expressed
in a yeast lacking the master vacuolar endopeptidase Pep4, which
controls the activation of other vacuolar hydrolases. Heterologous
expression in strains carrying temperature-sensitive (ts) alleles
of genes can be employed if the corresponding null mutant is
inviable.
[0235] Viral vectors can also be prepared encoding the peptides
disclosed herein. Myriad viral vectors have been constructed and
are known to those of skill in the art, including but not limited
to polyoma, SV40 (Madzak et al., J. Gen. Virol. 73:1533-1536,
1992), adenovirus (Berkner, Cur. Top. Microbiol. Immunol.,
158:39-36, 1992; Berliner et al., BioTechniques, 6:616-629, 1988;
Gorziglia et al., J. Virol. 66:4407-4412, 1992; Quantin et al.,
Proc. Nad. Acad. Sci. USA 89:2581-2584, 1992; Rosenfeld et al.,
Cell, 68:143-155, 1992; Wilkinson et al., Nucl. Acids Res.
20:2233-2239, 1992; Stratford-Perricaudet et al., Hum. Gene Ther.,
1:241-256), vaccinia virus (Mackett et al., Biotechnology,
24:495-499, 1992), adeno-associated virus (Muzyczka, Curr. Top.
Microbiol. Immunol. 158:91-123, 1992; On et al., 1990, Gene,
89:279-282), herpes viruses including HSV and EBV (Margolskee,
Curr. Top. Microbiol. Immunol., 158:67-90, 1992; Johnson et al., J.
Virol. 66:2952-2965, 1992; Fink et al., Hum. Gene Ther. 3:11-19,
1992; Breakfield et al., Mol. Neurobiol., 1:337-371, 1987; Fresse
et al., Biochem. Pharmacol. 40:2189-2199, 1990), Sindbis viruses
(Herweijer et al., Human Gene Therapy 6:1161-1167, 1995; U.S. Pat.
No. 5,091,309), alphaviruses (Schlesinger, Trends Biotechnol.
11:18-22, 1993; Frolov et al., Proc. Natl. Acad. Sci. USA
93:11371-11377, 1996) and retroviruses of avian (Brandyopadhyay et
al., Mol. Cell Biol. 4:749-754, 1984; Petropouplos et al., J.
Virol. 66:3391-3397, 1992), murine (Miller, Curr. Top. Microbiol.
Immunol., 158:1-24, 1992; Miller et al., 1985, Mol. Cell Biol.,
5:431-437; Sorge et al., Mol. Cell Biol. 4:1730-1737, 1984; Mann et
al., J. Virol. 54:401-407, 1985), and human origin (Page et al., J.
Virol. 64:5370-5276, 1990; Buchschalcher et al., J. Virol.
66:2731-2739, 1992). Baculovirus (Autographa californica
multinuclear polyhedrosis virus; AcMNPV) vectors are also known in
the art, and may be obtained from commercial sources (such as
PharMingen, San Diego, Calif.; Protein Sciences Corp., Meriden,
Conn.; Stratagene, La Jolla, Calif.).
[0236] The characteristics of the peptides disclosed herein lie not
in their precise and entire amino acid sequence, but rather in the
three-dimensional structure inherent in the amino acid sequences
encoded by the DNA sequences. It is possible to recreate the
binding characteristics of any of these peptides, for instance the
binding characteristics of any one of the specific peptides
described herein, by recreating the three-dimensional structure,
without necessarily recreating the exact amino acid sequence.
Production of variations is enabled particularly in view of the
guidance provided for the tolerance of variations at various
positions within the core peptide. Such modifications and
variations can be achieved for instance by designing a nucleic acid
sequence that encodes for the three-dimensional structure, but
which differs, for instance by reason of the redundancy of the
genetic code or the substitution of one or more specific amino
acids. Similarly, the DNA sequence may also be varied, while still
producing a functional peptide.
[0237] Variant therapeutic peptides include peptides that differ in
amino acid sequence from the disclosed sequence, but that share
structurally significant sequence homology with any of the provided
peptides. Such variants may be produced by manipulating the
nucleotide sequence of the encoding sequence, using standard
procedures, including site-directed mutagenesis or PCR. The
simplest modifications involve the substitution of one or more
amino acids for amino acids having similar biochemical properties.
These so-called conservative substitutions are likely to have
minimal impact on the activity of the resultant peptide, especially
when made outside of the binding site of the peptide. One of
ordinary skill in the art will be able to predict or empirically
determine (particularly in view of the provided teachings) amino
acids that may be substituted for an original amino acid in a
peptide.
[0238] More substantial changes in peptide structure may be
obtained by selecting amino acid substitutions that are less
conservative. Such changes include changing residues that differ
more significantly in their effect on maintaining polypeptide
backbone structure (for example, sheet or helical conformation)
near the substitution, charge or hydrophobicity of the molecule at
the target site, or bulk of a specific side chain. The following
substitutions are generally expected to produce the greatest
changes in protein properties: (a) a hydrophilic residue (for
example, seryl or threonyl) is substituted for (or by) a
hydrophobic residue (for example, leucyl, isoleucyl, phenylalanyl,
valyl or alanyl); (b) a cysteine or proline is substituted for (or
by) any other residue; (c) a residue having an electropositive side
chain (for example, lysyl, arginyl, or histadyl) is substituted for
(or by) an electronegative residue (for example, glutamyl or
aspartyl); or (d) a residue having a bulky side chain (for example,
phenylalanine) is substituted for (or by) one lacking a side chain
(for example, glycine).
[0239] Variant peptide-encoding sequences may be produced by
standard DNA mutagenesis techniques, for example, M13 primer
mutagenesis. Details of these techniques are provided in Sambrook
(In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,
N.Y., 1989), Ch. 15. By the use of such techniques, variants may be
created which differ in minor ways from the angiogenic and
anti-angiogenic-encoding sequences disclosed. DNA molecules and
nucleotide sequences which are derivatives of those specifically
disclosed herein and that differ from those disclosed by the
deletion, addition, or substitution of nucleotides while still
encoding a peptide that promotes or inhibits angiogenesis, are
comprehended by this disclosure. In their most simple form, such
variants may differ from the disclosed sequences by alteration of
the coding region to fit the codon usage bias of the particular
organism into which the molecule is to be introduced.
[0240] Alternatively, the coding region may be altered by taking
advantage of the degeneracy of the genetic code to alter the coding
sequence such that, while the nucleotide sequence is substantially
altered, it nevertheless encodes a peptide having an amino acid
sequence substantially similar to the disclosed peptide sequences.
For example, one nucleotide codon triplet GCT encodes alanine.
Because of the degeneracy of the genetic code, three other
nucleotide codon triplets--(GCG, GCC and GCA)--also code for
alanine. Thus, a nucleotide sequence containing GCT for alanine
could be changed at the same position to any of the three
alternative codons without affecting the amino acid composition or
characteristics of the encoded peptide. Based upon the degeneracy
of the genetic code, variant DNA molecules may be derived from the
cDNA and gene sequences disclosed herein using standard DNA
mutagenesis techniques as described above, or by synthesis of DNA
sequences. Thus, this disclosure also encompasses nucleic acid
sequences which encode the subject peptides, but which vary from
the disclosed nucleic acid sequences by virtue of the degeneracy of
the genetic code.
[0241] The present disclosure includes biologically active
molecules that mimic the action of the inhibitor/blockade peptides
of the present disclosure. The peptides of the disclosure include
synthetic embodiments of naturally-occurring peptides described
herein, as well as analogues (non-peptide organic molecules),
derivatives (chemically functionalized protein molecules obtained
starting with the disclosed peptide sequences) and variants
(homologs) of these peptides that specifically bind CD47, or that
block one or more signalling functions of CD47. Each peptide of the
disclosure is comprised of a sequence of amino acids, which may be
either L- and/or D-amino acids, naturally occurring and
otherwise.
[0242] Peptides may be modified by a variety of chemical techniques
to produce derivatives having essentially the same activity as the
unmodified peptides, and optionally having other desirable
properties. For example, carboxylic acid groups of the peptides,
whether carboxyl-terminal or side chain, may be provided in the
form of a salt of a pharmaceutically-acceptable cation or
esterified to form a C.sub.1-C.sub.16 ester, or converted to an
amide of formula NR.sub.1R.sub.2 wherein R.sub.1 and R.sub.2 are
each independently H or C.sub.1-C.sub.16 alkyl, or combined to form
a heterocyclic ring, such as a 5- or 6-membered ring. Amino groups
of the peptides, whether amino-terminal or side chain, may be in
the form of a pharmaceutically-acceptable acid addition salt, such
as the HCl, HBr, acetic, benzoic, toluene sulfonic, maleic,
tartaric and other organic salts, or may be modified to
C.sub.1-C.sub.16 alkyl or dialkyl amino or further converted to an
amide.
[0243] Hydroxyl groups of the peptide side chains may be converted
to C.sub.1-C.sub.16 alkoxy or to a C.sub.1-C.sub.16 ester using
well-recognized techniques. Phenyl and phenolic rings of the
peptide side chains may be substituted with one or more halogen
atoms, such as fluorine, chlorine, bromine or iodine, or with
C.sub.1-C.sub.16 alkyl, C.sub.1-C.sub.16 alkoxy, carboxylic acids
and esters thereof, or amides of such carboxylic acids. Methylene
groups of the peptide side chains can be extended to homologous
C.sub.2-C.sub.4 alkylenes. Thiols can be protected with any one of
a number of well-recognized protecting groups, such as acetamide
groups. Those skilled in the art will also recognize methods for
introducing cyclic structures into the peptides of this disclosure
to select and provide conformational constraints to the structure
that result in enhanced stability.
[0244] Peptidomimetic and organomimetic embodiments are also within
the scope of the present disclosure, whereby the three-dimensional
arrangement of the chemical constituents of such peptido- and
organomimetics mimic the three-dimensional arrangement of the
peptide backbone and component amino acid side chains in the
described inhibitor peptides, resulting in such peptido- and
organomimetics of the peptides of this disclosure having measurable
or enhanced angiogenic or anti-angiogenic activity. For computer
modeling applications, a pharmacophore is an idealized,
three-dimensional definition of the structural requirements for
biological activity. Peptido- and organomimetics can be designed to
fit each pharmacophore with current computer modeling software
(using computer assisted drug design or CADD). See Walters,
Computer-Assisted Modeling of Drugs, in Klegerman & Groves,
eds., 1993, Pharmaceutical Biotechnology, Interpharm Press: Buffalo
Grove, Ill., pp. 165-174 and Principles of Pharmacology Munson
(ed.) 1995, Ch. 102, for descriptions of techniques used in CADD.
Also included within the scope of the disclosure are mimetics
prepared using such techniques that produce angiogenic or
anti-angiogenic peptides.
VI. Compositions and Methods for Inducing CSC Differentiation
[0245] The disclosed methods include inhibiting or blocking CD47
signaling, for example to induce differentiation of cancer stem
cells, and particularly to induce irreversible differentiation of
cancer stem cells. In various embodiments, inhibiting CD47
signaling includes one or more of inhibiting the expression of
CD47, or blockading or inhibiting the interaction between CD47 and
EGFR.
[0246] Agents that alter CD47 signaling to increase or induce CSC
differentiation include but are not limited to peptides,
antibodies, antisense oligonucleotides, morpholinos, or small
molecule inhibitors. The agents include, in various embodiments,
synthetic peptides having specific binding affinity for CD47;
oligonucleotide comprising at least about 15 contiguous bases and
that hybridizes to the mature or unprocessed nuclear mRNA of CD47
under high stringency conditions; isolated or recombinant CD47
molecules or soluble fragments thereof, or molecule that binds
thereto; agents that decrease the expression of CD47; agents that
enhance the proteolysis of CD47; agents that enhance removal of
CD47 from the cell surface; CD47 antagonists; antibodies that
specifically bind CD47; or a mixture of two or more thereof.
Exemplary agents that influence CD47 signaling include those
described in U.S. Pat. No. 8,236,313 and International Pat. Publ.
No. WO 2010/017332, both of which are incorporated herein by
reference in their entirety.
[0247] A. Suppression of Protein Expression
[0248] In some embodiments, inhibition or blockade of CD47
signaling is achieved by reducing or suppressing CD47 protein
expression, for example in methods of inducing differentiation
(such as irreversible differentiation) of cancer stem cells, such
as exemplified herein.
[0249] Although the mechanism by which antisense RNA molecules
interfere with gene expression has not been fully elucidated, it is
believed that antisense RNA molecules (or fragments thereof) bind
to the endogenous mRNA molecules and thereby inhibit translation of
the endogenous mRNA, splicing of the nuclear mRNA precursor, or
result in its degradation. A reduction of protein expression in a
cell may be obtained by introducing into cells an antisense
construct based on CD47 encoding sequences, including the human (or
other mammalian) CD47 cDNA or gene sequence or flanking regions
thereof. For antisense suppression, a nucleotide sequence from a
CD47-encoding sequence, for example all or a portion of a CD47 cDNA
or gene, is arranged in reverse orientation relative to the
promoter sequence in the transformation vector. One of ordinary
skill in the art will understand how other aspects of the vector
may be chosen.
[0250] The introduced sequence need not be the full length of the
cDNA or gene, or reverse complement thereof, and need not be
exactly homologous to the equivalent sequence found in the cell
type to be transformed. Generally, however, where the introduced
sequence is of shorter length, a higher degree of homology to the
native target sequence will be needed for effective antisense
suppression. The introduced antisense sequence in the vector may be
at least 15 nucleotides in length, and improved antisense
suppression will typically be observed as the length of the
antisense sequence increases. The length of the antisense sequence
in the vector advantageously may be greater than about 20
nucleotides, greater than about 30 nucleotides, or greater than
about 100 nucleotides. For suppression of the CD47 gene itself,
transcription of an antisense construct results in the production
of RNA molecules that are the reverse complement of mRNA molecules
transcribed from the endogenous CD47 gene in the cell.
[0251] Suppression of CD47 expression can also be achieved using
ribozymes. Ribozymes are synthetic molecules that possess highly
specific endoribonuclease activity. The production and use of
ribozymes are disclosed in U.S. Pat. Nos. 4,987,071 and 5,543,508.
The inclusion of ribozyme sequences within antisense RNAs may be
used to confer RNA cleaving activity on the antisense RNA, such
that endogenous mRNA molecules that bind to the antisense RNA are
cleaved, which in turn leads to an enhanced antisense inhibition of
endogenous gene expression.
[0252] Suppression can also be achieved using RNA interference,
using known and previously disclosed methods. Several models have
been put forward to explain RNAi, in particular the mechanisms by
which the cleavage derived small dsRNAs or siRNAs interact with the
target mRNA and thus facilitate its degradation (Hamilton et al.,
Science 286:950, 1999; Zamore et al., Cell 101:25, 2000; Hammond et
al., Nature 404:293, 2000; Yang et al., Curr. Biol. 10:1191, 2000;
Elbashir et al., Genes Dev. 15:188, 2001; Bass Cell 101:235, 2000).
It has been proposed that the cleavage derived small dsRNAs or
siRNAs act as a guide for the enzymatic complex required for the
sequence specific cleavage of the target mRNA. Evidence for this
includes cleavage of the target mRNA at regular intervals of about
21-23 nucleotides in the region corresponding to the input dsRNA
(Zamore et al., Cell 101, 25, 2000), with the exact cleavage sites
corresponding to the middle of sequences covered by individual 21
or 22 nucleotide small dsRNAs or siRNAs (Elbashir et al., Genes
Dev. 15:188, 2001). Although mammals and lower organisms appear to
share dsRNA-triggered responses that involve a related intermediate
(small dsRNAs), it is likely that there will be differences as well
as similarities in the underlying mechanism. dsRNAs can be formed
from RNA oligomers produced synthetically (for technical details
see material from the companies Xeragon and Dharmacon, both
available on the internet). Small dsRNAs and siRNAs can also be
manufactured using standard methods of in vitro RNA production. In
addition, the Silencer.TM. siRNA Construction kit (and components
thereof) available from Ambion (Catalog #1620; Austin, Tex.), which
employs a T7 promoter and other well-known genetic engineering
techniques to produce dsRNAs. Double stranded RNA triggers could
also be expressed from DNA based vector systems.
[0253] Inhibition also can be accomplished using morpholino
oligonucleotides, for instance as described herein. The morpholino
can be delivered directly to cells (for example, in vitro) or can
be administered to a subject as herein described. In particular
embodiments, the morpholino is an antisense morpholino
oligonucleotide complementary to CD47 (such as human and/or murine
CD47). Example CD47 morpholino are provided, for instance, in
International Patent Publication No. WO 2008/060785, which is
incorporated herein by reference in its entirety for all that it
teaches. For instance, in some embodiments, the methods include the
use of a morpholino comprising an antisense oligonucleotide to CD47
having the sequence CGTCACAGGCAGGACCCACTGCCCA (SEQ ID NO: 70), or
another sequence from CD47 or a sequence from TSP1.
[0254] The nucleic acids and nucleic acid analogs that are used to
suppress CD47 expression may be modified chemically or
biochemically or may contain one or more non-natural or derivatized
nucleotide bases, as will be readily appreciated by those of
ordinary skill in the art. Such modifications include, for example,
labels, methylation, substitution of one or more of the naturally
occurring nucleotides with an analog, internucleotide
modifications, such as uncharged linkages (for example, methyl
phosphonates, phosphotriesters, phosphoramidates, carbamates,
etc.), charged linkages (for example, phosphorothioates,
phosphorodithioates, etc.), pendent moieties (for example,
polypeptides), intercalators (for example, acridine, psoralen,
etc.), chelators, alkylators, and/or modified linkages (for
example, alpha anomeric nucleic acids, etc.). The term nucleic acid
molecule also includes any topological conformation, including
single-stranded, double-stranded, partially duplexed, triplexed,
hair-pinned, circular and padlocked conformations. Also included
are synthetic molecules that mimic polynucleotides in their ability
to bind to a designated sequence via hydrogen bonding and other
chemical interactions. Such molecules are known in the art and
include, for example, those in which peptide linkages substitute
for phosphate linkages in the backbone of the molecule.
[0255] Additionally, although particular exemplary sequences are
disclosed herein, one of ordinary skill in the art will appreciate
that the present methods also encompass sequence alterations of the
disclosed agents that yield the same results as described herein.
Such sequence alterations can include, but are not limited to,
deletions, base modifications, mutations, labeling, and
insertions.
[0256] Suppression of protein expression may also be achieved
through agents that enhance proteolysis of CD47 (Allen et al.,
Endocrinology 150:1321-1329, 2009). In other particular examples,
the suppression of CD47 expression involves an agent that enhances
the removal of CD47 from the cell surface or decreases the
transcription, mRNA processing, or translation of CD47.
[0257] It is also contemplated that suppression of expression can
be achieved through the engineered, directed editing of the genomic
sequence encoding CD47, for instance through the clustered,
regularly interspaced, short palindromic repeat (CRISPR) technology
(Sander & Joung. Nature Biotechnol 32:347-355, 2014, doi:
10.1038/nbt.2842, which is incorporated by reference herein in its
entirety); this approach generates RNA-guided nucleases, such as
Cas9, with customizable specificities. CRISPRs are DNA loci
containing short repetitions of base sequences. Each repetition is
followed by short segments of "spacer DNA" from previous exposures
to a virus (Marraffini & Sontheimer. Nature Rev Genetics
11:181-190, 2010, doi: 10.1038/nrg2749, which is incorporated by
reference herein in its entirety). CRISPRs are often associated
with Cas genes that code for proteins related to CRISPRs. The
CRISPR/Cas system is a prokaryotic immune system that confers
resistance and acquired immunity against invading foreign genetic
elements such as plasmids and via RNA-guided DNA cleavage
(Wiedenheft et al., Nature 482:331-338, 2012, which is incorporated
by reference herein in its entirety). In the type II CRISPR/Cas
system, short segments of foreign DNA, termed `spacers` are
integrated within the CRISPR genomic loci and transcribed and
processed into short CRISPR RNA (crRNA). These crRNAs anneal to
transactivating crRNAs (tracrRNAs) and direct sequence-specific
cleavage and silencing of pathogenic DNA by Cas proteins. Target
recognition by the Cas9 protein requires a `seed` sequence within
the crRNA and a conserved dinucleotide-containing protospacer
adjacent motif (PAM) sequence upstream of the crRNAbinding region
(Jinek et al. Science 337:816-821, 2012, which is incorporated by
reference herein in its entirety). The CRISPR/Cas system can
thereby be retargeted to cleave virtually any DNA sequence by
redesigning the crRNA. Significantly, the CRISPR/Cas system has
been shown to be directly portable to human cells by co-delivery of
plasmids expressing the Cas9 endonuclease and the necessary crRNA
components (Cho et al., Nature Biotechnol 31:230-232, 2013; Cong et
al., Science 339:819-823, 2013; Mali et al., Science 339:823-826,
2013, each of which is incorporated by reference herein in its
entirety). These programmable RNA-guided DNA endonucleases have
demonstrated multiplexed gene disruption capabilities (Cong et al.,
Science 339:819-823, 2013, which is incorporated by reference
herein in its entirety) and targeted integration in iPS cells (Mali
et al., Science 339:823-826, 2013, which is incorporated by
reference herein in its entirety). Cas9 endonucleases have also
been converted into nickases (Cong et al., Science 339:819-823,
2013, which is incorporated by reference herein in its entirety),
enabling an additional level of control over the mechanism of DNA
repair. The CRISPR/Cas system can be used for gene editing (adding,
disrupting or changing the sequence of specific genes) and gene
regulation in species throughout the tree of life (Mali et al.,
Nature Methods 10:957-963, 2013; doi: 10.1038/nmeth.2649, which is
incorporated by reference herein in its entirety). By delivering
the Cas9 protein and appropriate guide RNAs into a cell, the
organism's genome can be cut at any desired location. It may be
possible to use CRISPR to build RNA-guided gene drives capable of
altering the genomes of entire populations (Esvelt et al., eLife
doi:10.7554/eLife.03401, which is incorporated by reference herein
in its entirety).
[0258] The following table lists representative gRNA sequences
useful for CRISPR disruption of CD47:
TABLE-US-00001 SEQ ID NO: Name gRNA 53 hCD47.g1
TCCATGCTTTGTTACTAATANGG 54 hCD47.g10 ATCGAGCTAAAATATCGTGTNGG 55
hCD47.g11 GATGATCGTTTCACCTTCTCNGG 56 hCD47.g12
ACTCTTATCCATCTTCAAACNGG 57 hCD47.g13 TTTTGCACTACTAAAGTCAGNGG 58
hCD47.g14 TTTGCACTACTAAAGTCAGTNGG 59 hCD47.g15
TTGCACTACTAAAGTCAGTGNGG 60 hCD47.g16 ACTAAAGTCAGTGGGGACAGNGG 61
hCD47.g17 CTTGTTTAGAGCTCCATCAANGG 62 hCD47.g18
TCCATATTAGTAACAAAGCANGG 63 hCD47.g2 ATGCTTTGTTACTAATATGGNGG 2
hCD47.g3 CTACTGAAGTATACGTAAAGNGG 64 hCD47.g4
TACGTAAAGTGGAAATTTAANGG 65 hCD47.g5 AGAGATATTTACACCTTTGANGG 66
hCD47.g6 GAAGTCTCACAATTACTAAANGG 67 hCD47.g7
AGGAGATGCCTCTTTGAAGANGG 68 hCD47.g8 AGTGATGCTGTCTCACACACNGG 69
hCD47.g9 GTAACAGAATTAACCAGAGANGG
[0259] There is provided herein an example CRISPR-mediated
disruption of CD47 expression. One of ordinary skill will recognize
that there are hundreds of possible CRISPR binding sites in the
CD47 gene (for instance, most cites with a "GG" or CC" sequence can
be used to target Cas9-mediated gene inactivation), and thus the
example provided herein is intended to be illustrative rather than
limiting. Likewise, CRISPR is not the only art-recognized way to
knock out or knock down expression of a garget gene, such as CD4'7.
In addition to the many CRISPR systems (including systems that use
Cpf1) that have been harnessed for use in the laboratory, Zinc
Finger Nucleases (ZFN) and TALEN are alternative methods for
knocking out the CD47 gene. Other art-recognized methods and
systems are also contemplated.
[0260] B. Suppression of Protein Activity
[0261] In some embodiments, inhibition or blockade of CD47
signaling is achieved by reducing or suppressing CD47 protein
activity, for example in methods of inducing differentiation of
cancer stem cells, such as exemplified herein.
[0262] In some examples, an inhibitor of CD47 signaling includes an
agent that decreases or blocks binding of a ligand (such as EGFR or
TSP1) to CD47. The determination that an agent (such as an antibody
or a peptide) inhibits an association with or functional signalling
from CD47 may be made, for example, using assays known to one of
ordinary skill in the art. For instance, the determination that an
agent inhibits EGFR binding to purified or recombinant CD47 can be
made by comparing the binding activity alone with the binding
activity in the presence of the agent using a solid phase ligand
binding assay, an EGFR tyrosine phosphorylation assay, or an assay
of a known downstream signaling target of EGFR. An agent that
inhibits a signaling pathway through CD47 on cells may reduce the
activity of a cGMP-dependent reporter in a suitable transfected
cell assay by a certain amount, for example, by 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, or even by 100%. In addition, an agent that
inhibits the activity or CD47 can be identified using any one of
the assays described herein, including, but not limited to,
determining TXNIP expression in a cell, or the change in expression
of any of the other genes listed in Table 1 (where an agent useful
in methods described herein will influence the gene expression
level in a manner similar to that seen with the antibody B6H12).
For instance, an agent that inhibits CD47 signaling (such that the
agent will induce differentiation of CSCs) will increase TXNIP
expression (such as an increase in TXNIP mRNA or TXNIP protein) in
a cell or population of cells by a certain amount, for example by
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, or
more as compared to a suitable control. Altered expression of genes
that are known markers of CSC such as CD24 can also be measured by
changes in mRNA expression or in surface expression by flow
cytometry.
[0263] Thus, in various embodiments an agent that alters CD47
signaling to induce CSC differentiations includes antibodies (such
as monoclonal antibodies or humanized antibodies) that specifically
bind to CD47. In some examples, an antibody that specifically binds
CD47 is of use in the methods disclosed herein. Antibodies that
specifically bind to CD47 include polyclonal antibodies, monoclonal
antibodies, or humanized monoclonal antibodies, or fragments
thereof. Methods of constructing such antibodies are known in the
art (see, for example, Green et al., "Production of Polyclonal
Antisera," in: Immunochemical Protocols, pages 1-5, Manson, ed.,
Humana Press, 1992; Coligan et al., "Production of Polyclonal
Antisera in Rabbits, Rats, Mice and Hamsters," in: Current
Protocols in Immunology, section 2.4.1, 1992; Kohler &
Milstein, Nature 256:495, 1975; Coligan et al., sections
2.5.1-2.6.7; and Harlow et al. in: Antibodies: a Laboratory Manual,
page 726, Cold Spring Harbor Pub., 1988). In addition, such
antibodies may be commercially available.
[0264] In some examples, an agent that alters CD47 signaling
includes an anti-CD47 antibody, such as anti-CD47 antibodies B6H12,
1F7, BRIC 126, 6H9, Clkm1, OVTL16, OX101, mIAP410, or mIAP301 (also
referred to as ab301), a binding fragment of any one of these, or a
humanized version of any one of these, or an antibody or fragment
thereof that competes with B6H12, BRIC 126, 6H9, Clkm1, OVTL16,
OX101, mIAP410, or mIAP301 for binding. It is to be understood that
agents that alter CD47 signaling for use in the present disclosure
also include novel CD47 antibodies developed in the future.
[0265] In other embodiments, an inhibitor of CD47 signaling
includes a peptide or recombinant protein derived from TSP1 that
specifically binds to CD47. In some examples an inhibitor of CD47
signaling is a CD47-binding peptide, such as a TSP1-derived
CD47-binding peptide. An exemplary CD47-binding peptides is 7N3
(FIRVVMYEGKK; SEQ ID NO: 1). Additional CD47-binding peptides
include those described in U.S. Pat. No. 8,236,313, which is
incorporated herein by reference in its entirety. It is to be
understood that CD47 signaling inhibitors for use in the present
disclosure also include novel CD47 binding peptides developed in
the future.
[0266] In additional embodiments, an inhibitor of CD47 signaling
includes a small molecule (such as a small organic molecule). Some
small molecule inhibitors may inhibit CD47 expression or activity.
It is to be understood that CD47 signaling inhibitors for use in
the present disclosure also include novel CD47 small molecule
inhibitors developed in the future.
VII. Administration of Compounds that Induce Differentiation of
CSCs
[0267] The disclosed methods include selectively inducing terminal
and irreversible differentiation of cancer stem cells by
administering to a CSC, or to CSCs in a subject in need of
treatment for cancer, a compound that alters CD47 signaling. In
particular examples, the subject in need of treatment has a cancer
such as colon cancer, breast cancer (including particularly triple
negative breast cancer), lung cancer, kidney cancer, bone cancer,
or brain cancer. In additional examples, the subject in need of
treatment may have a leukemia, lymphoma, prostate cancer, bladder
cancer, or pancreatic cancer.
[0268] Agents that influence CD47 signalling are administered in
any suitable manner, preferably with pharmaceutically acceptable
carriers. Pharmaceutically acceptable carriers are determined in
part by the particular composition being administered, as well as
by the particular method used to administer the composition.
Accordingly, there is a wide variety of suitable formulations of
pharmaceutical compositions of the present disclosure. The
pharmaceutically acceptable carriers useful in this disclosure are
conventional. Remington's Pharmaceutical Sciences, by E. W. Martin,
Mack Publishing Co., Easton, Pa., 15th Edition (1975), describes
compositions and formulations suitable for pharmaceutical delivery
of the compounds herein disclosed.
[0269] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's, or fixed oils. Intravenous vehicles include
fluid and nutrient replenishers, electrolyte replenishers (such as
those based on Ringer's dextrose), and the like. Preservatives and
other additives may also be present such as, for example,
antimicrobials, anti-oxidants, chelating agents, and inert gases
and the like.
[0270] Formulations for topical administration may include
ointments, lotions, creams, gels, drops, suppositories, sprays,
liquids and powders. Conventional pharmaceutical carriers, aqueous,
powder or oily bases, thickeners and the like may be necessary or
desirable.
[0271] Compositions for oral administration include powders or
granules, suspensions or solutions in water or non-aqueous media,
capsules, sachets, or tablets. Thickeners, flavorings, diluents,
emulsifiers, dispersing aids or binders may be desirable.
[0272] In some embodiments, the CD47 blocking agent is administered
to a subject in a single dose. In other embodiments, the CD47
blocking agent is administered to a subject in multiple doses. When
administered in multiple doses, the time period between each
administration can vary and will depend in part on the subject
being treated and the type of cancer being treated. In some
examples, an agent that alters CD47 signaling to increase CSC
differentiation is administered daily, bi-weekly, weekly,
bi-monthly or monthly. When administered in multiple doses, the
time period between each administration can vary and will depend in
part on the subject being treated and the type of cancer being
treated. One of skill in the art can determine an appropriate
dosing schedule for each subject.
[0273] It is particularly contemplated that an agent that alters
CD47 signaling to increase CSC differentiation can be administered
in conjunction with standard of care chemotherapy or radiation
therapy. Dosing with the agent (such as B6H12) 1-4 days before
administering chemotherapy or radiation is expected to sensitize
the CSC to respond to these therapies, thereby making the otherwise
conventional therapy more effective.
[0274] In one example, administration of an agent that alters CD47
signaling to increase CSC differentiation decreases the volume of a
tumor or a metastatic tumor, or both, or initiation of metastatic
tumor(s). Decreasing the volume of a tumor or a metastatic tumor
does not require a 100% reduction in the volume, and in some
examples includes decreasing the volume by at least 10%, for
example by at least 20% or more as compared to a volume in the
absence of the therapeutic agent. In one example, such
administration enhances apoptosis of the cancer cells, or
metastatic tumor cells, or both. Increasing the apoptosis of cancer
cells, or metastatic tumor cells, does not require a 100%
apoptosis, and in some examples includes increasing the apoptosis
by at least 10%, for example by at least 20% or more as compared to
an amount of apoptosis in the absence of the therapeutic agent. In
addition, the disclosed methods can result in a decrease in the
symptoms associated with a tumor or a metastatic tumor.
[0275] In further embodiments, the methods include administering a
second anti-cancer therapeutic to the subject in addition to the
CD47 blocking agent, such as a cell-cycle checkpoint inhibitor.
Anti-cancer therapeutics include, but are not limited to,
chemotherapeutic drug treatment, radiation, gene therapy, hormonal
manipulation, immunotherapy and antisense oligonucleotide therapy.
Chemotherapeutic agents include, but are not limited to alkylating
agents, such as nitrogen mustards (for example, chlorambucil,
chlormethine, cyclophosphamide, ifosfamide, and melphalan),
nitrosoureas (for example, carmustine, fotemustine, lomustine, and
streptozocin), platinum compounds (for example, carboplatin,
cisplatin, oxaliplatin, and bbr3464), busulfan, dacarbazine,
mechlorethamine, procarbazine, temozolomide, thiotepa, and
uramustine; antimetabolites, such as folic acid (for example,
methotrexate, pemetrexed, and raltitrexed), purine (for example,
cladribine, clofarabine, fludarabine, mercaptopurine, and
tioguanine), pyrimidine (for example, capecitabine), cytarabine,
fluorouracil, and gemcitabine; plant alkaloids, such as podophyllum
(for example, etoposide, and teniposide), taxane (for example,
docetaxel and paclitaxel), vinca (for example, vinblastine,
vincristine, vindesine, and vinorelbine); cytotoxic/antitumor
antibiotics, such as anthracycline family members (for example,
daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone,
and valrubicin), bleomycin, hydroxyurea, and mitomycin;
topoisomerase inhibitors, such as topotecan and irinotecan;
monoclonal antibodies, such as alemtuzumab, bevacizumab, cetuximab,
gemtuzumab, rituximab, and trastuzumab; photosensitizers, such as
aminolevulinic acid, methyl aminolevulinate, porfimer sodium, and
verteporfin; and other agents, such as alitretinoin, altretamine,
amsacrine, anagrelide, arsenic trioxide, asparaginase, bexarotene,
bortezomib, celecoxib, denileukin diftitox, erlotinib,
estramustine, gefitinib, hydroxycarbamide, imatinib, pentostatin,
masoprocol, mitotane, pegaspargase, and tretinoin.
[0276] Co-administration of the disclosed agents with radiation
therapy is also contemplated. Methods for treating cancers using
radiation therapy are well known in the art. In addition, methods
of radioprotection are taught, for instance, in International
Patent Publication No. WO 2010/017332, which is incorporated herein
by reference.
[0277] The CD47 blocking agent and the second anti-cancer
therapeutic can be delivered at the same time (such as part of the
same composition or as separate compositions), or can be
administered at different times. When administered at different
times, the second anti-cancer therapeutic can either be
administered before the agent that alters CD47 signaling to induce
cancer stem cell (CSC) differentiation or after that agent. The
time between administration of the CD47 blocking agent and the
second anti-cancer therapeutic can vary and will depend on the type
of second anti-cancer therapy selected, the cancer being treated
and the subject being treated. Similarly, the second anti-cancer
therapeutic can be administered in a single dose or in multiple
doses. One of skill in the art can determine an appropriate dosing
schedule for each subject.
[0278] Administration to cells of inhibitors of CD47 signaling
(such as the monoclonal antibody B6H12, a humanized version of
B6H12, a binding fragment of B6H12, or a monoclonal antibody that
binds competitively with B6H12) can be local or systemic. Examples
of local administration include, but are not limited to, topical
administration, subcutaneous administration, transdermal
administration, intramuscular administration, intrathecal
administration, intrapericardial administration, intra-ocular
administration, topical ophthalmic administration, or
administration to the nasal mucosa or lungs by inhalational
administration. In addition, local administration includes routes
of administration typically used for systemic administration, for
example by directing intravascular administration to the arterial
supply for a particular organ. Thus, in particular embodiments,
local administration includes intra-arterial administration and
intravenous administration when such administration is targeted to
the vasculature supplying a particular organ. Local administration
also includes the incorporation of active compounds and agents into
implantable devices or constructs, such as vascular stents or other
reservoirs, which release the active agents and compounds over
extended time intervals for sustained treatment effects.
[0279] Systemic administration includes any route of administration
intended to distribute an active compound or composition widely
throughout the body, for example, via the circulatory system. Thus,
systemic administration includes, but is not limited to
intra-arterial and intravenous administration. Systemic
administration also includes, but is not limited to, topical
administration, subcutaneous administration, transdermal
administration, intramuscular administration, or administration by
inhalation, when such administration is directed at absorption and
distribution throughout the body by the circulatory system.
Systemic administration also includes oral administration, in some
examples.
[0280] In other embodiments, the methods include administering an
agent that alters CD47 signaling to induce CSC differentiation to a
subject, for example, to induce differentiation of CSCs in vivo
[0281] In some embodiments, an agent that alters CD47 signaling to
induce CSC differentiation (such as a peptide, antibody or antibody
fragment, nucleic acid, or inhibitory oligonucleotide (e.g.,
morpholino)) is administered locally to an affected area, for
example by direct administration to a tumor, or is incorporated
into an implant device and placed directly at an affected area,
such as at or near a tumor, or in contact with bone marrow. In some
embodiments, administration is, for example, by direct topical
administration, or by intra-arterial, intravenous, subcutaneous, or
intramuscular injection into the affected area. Efficacy of the
treatment is shown, for example, by a regression of symptoms, for
example reduction of tumor size, reduction of metastasis, reduction
of recurrence of cancer, or any other measurable return to or
towards a system more characteristic of health (for instance,
changes in gene expression to be more like a healthy profile and
less like a cancerous profile).
[0282] Liposomes containing a therapeutic compound, including
cationic and anionic liposomes, can be made using standard
procedures as known to one skilled in the art. Liposomes can be
applied topically, either in the form of drops or as an aqueous
based cream, or can be injected intraocularly. The cells or an
agent that alters CD47 signaling to induce CSC differentiation can
also be included in a delivery system that can be implanted at
various sites, depending on the size, shape and formulation of the
implant, and the type of transplant procedure. The delivery system
is then introduced into the subject, for instance at or near a
tumor or the site from which a tumor has been removed.
[0283] An effective amount of an agent that alters CD47 signaling
to induce CSC differentiation (such as a peptide, antibody,
inhibitor peptide-encoding DNA, or oligonucleotide (e.g.,
morpholino)) can be administered in a single dose, or in multiple
doses, for example daily, weekly, every two weeks, or monthly
during a course of treatment. Additionally, the therapeutic agents
may be incorporated into or on implantable constructs or devices,
such as vascular stents, for sustained regional or local
release.
[0284] In some examples, the methods include identifying or
selecting a subject for administration of an agent that alters CD47
signaling to induce CSC differentiation. For example, the methods
include selecting a subject with cancer. In some instances, the
method involves selecting a subject at risk for metastasis, or
already showing evidence of metastatic disease.
VIII. Pharmaceutical Compositions
[0285] The therapeutic compounds described herein may be formulated
in a variety of ways depending on the location and type of disease
to be treated or prevented in the subject. Pharmaceutical
compositions are thus provided for both local use at or near an
affected area and for systemic use (in which the agent is
administered in a manner that is widely disseminated via the
cardiovascular system).
[0286] Pharmaceutical compositions that include at least one
peptide or other inhibitor or therapeutic compound as described
herein as an active ingredient, or that include both a therapeutic
peptide or inhibitor/blockade agent and an additional agent as
active ingredients, or that include both an ischemia-influencing
peptide or inhibitor and an additional therapeutic agent, may be
formulated with an appropriate solid or liquid carrier, depending
upon the particular mode of administration chosen. Additional
active ingredients include, for example, nitric oxide donors,
nitrovasodilators, activators of the enzyme soluble guanylyl
cyclase, or cGMP phosphodiesterase inhibitors.
[0287] A suitable administration format may best be determined by a
medical practitioner for each subject individually. Various
pharmaceutically acceptable carriers and their formulation are
described in standard formulation treatises, for example,
Remington's Pharmaceutical Sciences by E. W. Martin. See also Wang
and Hanson, J. Parenteral Sci. Technol., 10(Supp. 42): 2S,
1988.
[0288] The dosage form of the pharmaceutical composition will be
determined by the mode of administration chosen. For instance, in
addition to injectable fluids, inhalational, topical, ophthalmic,
peritoneal, and oral formulations can be employed. Inhalational
preparations can include aerosols, particulates, and the like. In
general, the goal for particle size for inhalation is about 1 .mu.m
or less in order that the pharmaceutical reach the alveolar region
of the lung for absorption. Oral formulations may be liquid (for
example, syrups, solutions, or suspensions), or solid (for example,
powders, pills, tablets, or capsules). For solid compositions,
conventional non-toxic solid carriers can include pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. Actual
methods of preparing such dosage forms are known, or will be
apparent, to those of ordinary skill in the art.
[0289] The compositions or pharmaceutical compositions can be
administered by any route, including parenteral administration, for
example, intravenous, intramuscular, intraperitoneal, intrasternal,
or intra-articular injection or infusion, or by sublingual, oral,
topical, intra-nasal, ophthalmic, or transmucosal administration,
or by pulmonary inhalation. When the active compounds are provided
as parenteral compositions, for example, for injection or infusion,
they are generally suspended in an aqueous carrier, for example, in
an isotonic buffer solution at a pH of about 3.0 to about 8.0,
preferably at a pH of about 3.5 to about 7.4, 3.5 to 6.0, or 3.5 to
about 5.0. Useful buffers include sodium citrate-citric acid and
sodium phosphate-phosphoric acid, and sodium acetate/acetic acid
buffers. A form of repository or depot slow release preparation may
be used so that therapeutically effective amounts of the
preparation are delivered into the bloodstream over many hours or
days following transdermal injection or delivery.
[0290] Active compounds (e.g., peptides, proteins, antibodies or
fragments thereof, oligos, and so forth) are also suitably
administered by sustained-release systems. Suitable examples of
sustained-release formulations include suitable polymeric materials
(such as, for example, semi-permeable polymer matrices in the form
of shaped articles, for example, films, or mirocapsules), suitable
hydrophobic materials (for example as an emulsion in an acceptable
oil) or ion exchange resins, and sparingly soluble derivatives
(such as, for example, a sparingly soluble salt). Sustained-release
compounds may be administered by intravascular, intravenous,
intra-arterial, intramuscular, subcutaneous, intra-pericardial, or
intra-coronary injection. Administration can also be oral, rectal,
parenteral, intracisternal, intravaginal, intraperitoneal, topical
(as by powders, ointments, gels, drops or transdermal patch),
buccal, or as an oral or nasal spray.
[0291] Preparations for administration can be suitably formulated
to give controlled release of the therapeutic agent(s) (e.g.,
peptides, antibodies, oligonucleotides or other compounds that
block CD47 and/or TSP1 activity or interaction). For example, the
pharmaceutical compositions may be in the form of particles
comprising a biodegradable polymer and/or a polysaccharide
jellifying and/or bioadhesive polymer, an amphiphilic polymer, an
agent modifying the interface properties of the particles and a
pharmacologically active substance. These compositions exhibit
certain biocompatibility features that allow a controlled release
of the active substance. See, for example, U.S. Pat. No.
5,700,486.
[0292] In some embodiments, therapeutic agent(s) are delivered by
way of a pump (see Sefton, CRC Crit. Ref. Biomed. Eng. 14:201,
1987; Buchwald et al., Surgery 88:507, 1980; Saudek et al., N.
Engl. J. Med. 321:574, 1989) or by continuous subcutaneous
infusions, for example, using a mini-pump. An intravenous bag
solution may also be employed. The key factor in selecting an
appropriate dose is the result obtained, as measured by increases
or decreases in angiogenesis, or by other criteria for measuring
control or prevention of disease, as are deemed appropriate by the
practitioner. Other controlled release systems are discussed in the
review by Langer (Science 249:1527-1533, 1990).
[0293] In another aspect of the disclosure, therapeutic agent(s)
are delivered by way of an implanted pump, described, for example,
in U.S. Pat. Nos. 6,436,091; 5,939,380; and 5,993,414. Implantable
drug infusion devices are used to provide subjects with a constant
and long term dosage or infusion of a drug or any other therapeutic
agent. Essentially, such device may be categorized as either active
or passive.
[0294] Active drug or programmable infusion devices feature a pump
or a metering system to deliver the drug into the patient's system.
An example of such an active drug infusion device currently
available is the Medtronic SynchroMed.TM. programmable pump. Such
pumps typically include a drug reservoir, a peristaltic pump to
pump the drug out from the reservoir, and a catheter port to
transport the pumped out drug from the reservoir via the pump to a
patient's anatomy. Such devices also typically include a battery to
power the pump, as well as an electronic module to control the flow
rate of the pump. The Medtronic SynchroMed.TM. pump further
includes an antenna to permit the remote programming of the
pump.
[0295] Passive drug infusion devices, in contrast, do not feature a
pump, but rather rely upon a pressurized drug reservoir to deliver
the drug. Thus, such devices tend to be both smaller as well as
cheaper as compared to active devices. An example of such a device
includes the Medtronic IsoMed.TM.. This device delivers the drug
into the patient through the force provided by a pressurized
reservoir applied across a flow control unit.
[0296] The implanted pump can be completely implanted under the
skin of a subject, thereby negating the need for a percutaneous
catheter. These implanted pumps can provide the patient with
therapeutic agent(s) at a constant or a programmed delivery rate.
Constant rate or programmable rate pumps are based on either
phase-change or peristaltic technology. When a constant, unchanging
delivery rate is required, a constant-rate pump is well suited for
long-term implanted drug delivery. If changes to the infusion rate
are expected, a programmable pump may be used in place of the
constant rate pump system. Osmotic pumps may be much smaller than
other constant rate or programmable pumps, because their infusion
rate can be very low. An example of such a pump is described listed
in U.S. Pat. No. 5,728,396.
[0297] The therapeutic agents may also be delivered passively and
in sustained fashion as part of and incorporated into implantable
devices, such as vascular stents which can be placed directly into
diseased blood vessels through several standard approaches,
including direct surgical insertion or percutaneoulsy with
angiographic control.
[0298] For oral administration, the pharmaceutical compositions can
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (for example, pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(for example, lactose, microcrystalline cellulose or calcium
hydrogen phosphate); lubricants (for example, magnesium stearate,
talc or silica); disintegrants (for example, potato starch or
sodium starch glycolate); or wetting agents (for example, sodium
lauryl sulphate). The tablets can be coated by methods well known
in the art. Liquid preparations for oral administration can take
the form of, for example, solutions, syrups or suspensions, or they
can be presented as a dry product for constitution with water or
other suitable vehicle before use. Such liquid preparations can be
prepared by conventional means with pharmaceutically acceptable
additives such as suspending agents (for example, sorbitol syrup,
cellulose derivatives or hydrogenated edible fats); emulsifying
agents (for example, lecithin or acacia); non-aqueous vehicles (for
example, almond oil, oily esters, ethyl alcohol or fractionated
vegetable oils); and preservatives (for example, methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations can
also contain buffer salts, flavoring, coloring, and sweetening
agents as appropriate.
[0299] For administration by inhalation, the compounds for use
according to the present disclosure are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebulizer, with the use of a suitable propellant, for example,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol, the dosage unit can be
determined by providing a valve to deliver a metered amount.
Capsules and cartridges for use in an inhaler or insufflator can be
formulated containing a powder mix of the compound and a suitable
powder base such as lactose or starch.
[0300] For topical administration, the compounds can be, for
example, mixed with a liquid delivery agent for administration
locally. The agents used therapeutically (such as peptides,
antibodies and morpholinos) are readily soluble or suspendable in
water and saline, and as such these would be useful for delivery
since water or saline do not cause adverse biological tissue
effects. This allows sufficiently high doses to be administered
locally or systemically, without secondary toxicity from the
delivery vehicle.
[0301] By way of example, in the treatment of burns agents can be
given by direct injection into the wound bed or topically dissolved
in saline as a spray to the burn area, to skin grafts and/or to
graft wound beds. They may also be mixed directly into antibiotic
creams used to treat burns, such as bacitracin or silver sulfadine,
or incorporated in a manner allowing release into dressing and
bandaging materials applied to wounds, grafts or burns.
[0302] Pharmaceutical compositions that comprise at least one
therapeutic agent as described herein as an active ingredient will
normally be formulated with an appropriate solid or liquid carrier,
depending upon the particular mode of administration chosen. The
pharmaceutically acceptable carriers and excipients useful in this
disclosure are conventional. For instance, parenteral formulations
usually comprise injectable fluids that are pharmaceutically and
physiologically acceptable fluid vehicles such as water,
physiological saline, other balanced salt solutions, aqueous
dextrose, glycerol or the like. Excipients that can be included
are, for instance, proteins, such as human serum albumin or plasma
preparations. If desired, the pharmaceutical composition to be
administered may also contain minor amounts of non-toxic auxiliary
substances, such as wetting or emulsifying agents, preservatives,
and pH buffering agents and the like, for example sodium acetate or
sorbitan monolaurate. Actual methods of preparing such dosage forms
are known, or will be apparent, to those skilled in the art.
[0303] For example, for parenteral administration, therapeutic
agent(s) can be formulated generally by mixing them at the desired
degree of purity, in a unit dosage injectable form (solution,
suspension, or emulsion), with a pharmaceutically acceptable
carrier, for instance, one that is non-toxic to recipients at the
dosages and concentrations employed and is compatible with other
ingredients of the formulation. A pharmaceutically acceptable
carrier is a non-toxic solid, semisolid or liquid filler, diluent,
encapsulating material or formulation auxiliary of any type.
[0304] Generally, the formulations are prepared by contacting the
therapeutic agent(s) each uniformly and intimately with liquid
carriers or finely divided solid carriers or both. Then, if
necessary, the product is shaped into the desired formulation.
Optionally, the carrier is a parenteral carrier, and in some
embodiments it is a solution that is isotonic with the blood of the
recipient. Examples of such carrier vehicles include water, saline,
Ringer's solution, and dextrose solution. Non-aqueous vehicles such
as fixed oils and ethyl oleate are also useful herein, as well as
liposomes.
[0305] The pharmaceutical compositions that comprise at least one
therapeutic agent, in some embodiments, will be formulated in unit
dosage form, suitable for individual administration of precise
dosages. The amount of active compound(s) administered will be
dependent on the subject being treated, the severity of the
affliction, and the manner of administration, and is best left to
the judgment of the prescribing clinician. Within these bounds, the
formulation to be administered will contain a quantity of the
active component(s) in amounts effective to achieve the desired
effect in the subject being treated.
[0306] The therapeutically effective amount of therapeutic agent,
such as a peptide, antibody, or oligonucleotide (e.g., morpholino
or other antisense molecule) will be dependent on the peptide or
inhibitor utilized, the subject being treated, the severity and
type of the affliction, and the manner of administration. The exact
dose is readily determined by one of skill in the art based on the
potency of the specific compound, the age, weight, sex and
physiological condition of the subject.
[0307] The peptides/proteins of the present disclosure (for
example, CD47 or TSP1 peptides, or a peptide that inhibits or
alters binding between TSP1 and CD47) also can be administered as
naked DNA encoding the peptide. To simplify the manipulation and
handling of the nucleic acid encoding the peptide, the nucleic acid
is generally inserted into a cassette, where it is operably linked
to a promoter. Preferably, the promoter is capable of driving
expression of the protein in cells of the desired target tissue.
The selection of appropriate promoters can readily be accomplished.
Preferably, the promoter is a high expression promoter, for example
the 763-base-pair cytomegalovirus (CMV) promoter, the Rous sarcoma
virus (RSV) promoter (Davis et al., Hum. Gene. Ther. 4:151, 1993),
or the MMT promoter.
[0308] Other elements that enhance expression also can be included,
such as an enhancer or a system that results in high levels of
expression, such as a tat gene or tar element. This cassette is
inserted into a vector, for example, a plasmid vector such as
pUC118, pBR322, or other known plasmid vector, that includes, for
example, an E. coli origin of replication. See, Sambrook, et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press (1989). The plasmid vector may also include a
selectable marker such as the -lactamase gene for ampicillin
resistance, provided that the marker polypeptide does not adversely
affect the metabolism of the organism being treated. The cassette
also can be bound to a nucleic acid binding moiety in a synthetic
delivery system, such as the system disclosed in PCT publication WO
95/22618.
[0309] Optionally, the DNA may be used with a microdelivery vehicle
such as cationic liposomes and adenoviral vectors. (For a review of
the procedures for liposome preparation, targeting and delivery of
contents, see Mannino & Gould-Fogerite, BioTechniques, 6:682,
1988; Feigner & Holm, Bethesda Res. Lab. Focus, 11(2):21,
1989); and Maurer, Bethesda Res. Lab. Focus, 11(2):25, 1989.)
Replication-defective recombinant adenoviral vectors can be
produced in accordance with known techniques. (See Quantin et al.,
Proc. Natl. Acad. Sci. USA, 89:2581-2584, 1992;
Stratford-Perricadet et al., J. Clin. Invest., 90:626-630, 1992;
and Rosenfeld et al., Cell, 68:143-155, 1992).
[0310] In order to facilitate injection, the nucleic acid is
formulated with a pharmaceutically acceptable carrier. Examples of
suitable carriers include, but are not limited to, saline, albumin,
dextrose and sterile water. The nucleic acid is injected into the
ischemic tissue using standard injection techniques by use of, for
example, a hypodermic needle, for example a hypodermic needle size
between No. 29 and No. 16. The nucleic acid also may be injected by
an externally applied local injection apparatus, such as that used
to inject antigens for allergy testing; or a transcutaneous patch
capable of delivery to subcutaneous muscle. The nucleic acid is
injected at one site, or at multiple sites throughout the ischemic
tissue.
[0311] Once injected, the nucleic acid capable of expressing the
desired angiogenic protein is taken up and expressed by the cells
of the tissue. Because the vectors containing the nucleic acid of
interest are not normally incorporated into the genome of the
cells, expression of the protein of interest takes place for only a
limited time. Typically, the angiogenic protein is only expressed
in therapeutic levels for about two days to several weeks,
preferably for about one to two weeks. Reinjection of the DNA can
be utilized to provide additional periods of expression of the
angiogenic protein. If desired, use of a retrovirus vector to
incorporate the heterologous DNA into the genome of the cells will
increase the length of time during which the therapeutic
polypeptide is expressed, from several weeks to indefinitely.
[0312] The therapeutic agents can also be administered directly as
part of a surgical procedure, or at the bedside by a treating
physician. Drug quality product (e.g., peptide, antibody or
morpholino) can be diluted for instance in sterile saline and given
by injection using sterile 1 cc syringes and small bore needles (25
gauge and less) to ischemic soft tissue units. Alternatively, a
wound bed can be irrigated for instance with a saline or other
therapeutically effective solution containing a known concentration
(dosage) of drug or compound, or a combination thereof. Precise
control and localization of therapeutic effects can thus be
obtained.
[0313] Controlled release parenteral formulations can be made as
implants, oily injections, or as particulate systems. For a broad
overview of protein delivery systems, see Banga, Therapeutic
Peptides and Proteins: Formulation, Processing, and Delivery
Systems, Technomic Publishing Company, Inc., Lancaster, Pa., 1995.
Particulate systems include microspheres, microparticles,
microcapsules, nanocapsules, nanospheres, and nanoparticles.
Microcapsules contain the therapeutic peptide as a central core. In
microspheres, the therapeutic agent is dispersed throughout the
particle. Particles, microspheres, and microcapsules smaller than
about 1 .mu.m are generally referred to as nanoparticles,
nanospheres, and nanocapsules, respectively. Capillaries have a
diameter of approximately 5 .mu.m so that only nanoparticles are
administered intravenously. Microparticles are typically around 100
.mu.m in diameter and are administered subcutaneously or
intramuscularly (see Kreuter, Colloidal Drug Delivery Systems, J.
Kreuter, ed., Marcel Dekker, Inc., New York, N.Y., pp. 219-342,
1994; Tice & Tabibi, Treatise on Controlled Drug Delivery, A.
Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp. 315-339,
1992).
[0314] Also contemplated is the use of nanoparticles as delivery
agents, which can be targeted to specific cells, tissues or organ
for instance by incorporation on their surface ligands of receptors
specific in their expression to the targeted cells, tissues or
organs, The targeting entity can be the same or different than the
therapeutically active agent carried by the nanoparticle. Further,
distribution of nanoparticles to certain tissues spaces (e.g. the
blood versus the central nervous system protected by the
blood-brain barrier) can be determined by altering the size of the
nanoparticles thereby allowing or preventing their transit of such
barriers between tissue compartments.
[0315] Polymers can be used for ion-controlled release. Various
degradable and non-degradable polymeric matrices for use in
controlled drug delivery are known in the art (Langer, Accounts
Chem. Res. 26:537, 1993). For example, the block copolymer,
polaxamer 407 exists as a viscous yet mobile liquid at low
temperatures but forms a semisolid gel at body temperature. It has
shown to be an effective vehicle for formulation and sustained
delivery of recombinant interleukin-2 and urease (Johnston et al.,
Pharm. Res. 9:425, 1992; Pec, J. Parent. Sci. Tech. 44(2):58,
1990). Alternatively, hydroxyapatite has been used as a
microcarrier for controlled release of proteins (Ijntema et al.,
Int. J. Pharm. 112:215, 1994). In yet another aspect, liposomes are
used for controlled release as well as drug targeting of
lipid-capsulated compounds (Betageri et al., Liposome Drug Delivery
Systems, Technomic Publishing Co., Inc., Lancaster, Pa., 1993).
Numerous additional systems for controlled delivery of therapeutic
proteins are known (e.g., U.S. Pat. Nos. 5,055,303; 5,188,837;
4,235,871; 4,501,728; 4,837,028; 4,957,735; and 5,019,369;
5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505;
5,506,206; 5,271,961; 5,254,342; and 5,534,496).
IX. Kits
[0316] Also disclosed herein are kits that can be used to induce
differentiation in CSCs in vivo or in cells in culture. In some
embodiments, the kit includes one or more agent that alters CD47
signaling to induce CSC differentiation, such as one or more of an
anti-CD47 antibody or fragment thereof, a CD47-binding peptide, a
CD47 antisense oligonucleotide, a CD47 morpholino. In other
embodiments, the kit includes a small molecule capable of binding
to CD47.
[0317] In another example, the kit includes an anti-CD47 antibody
or fragment thereof, such as monoclonal antibody B6H12 or a
humanized B6H12. By way of example, humanized versions of B6H12 are
taught in International Patent Publication No. WO 2011/143624,
which is incorporated by reference in its entirety.
[0318] The kits may further include additional components such as
instructional materials and additional reagents, for example cell
culture medium (such as growth medium or differentiation medium)
for one or more cell types. The kits may also include additional
components to facilitate the particular application for which the
kit is designed (for example tissue culture plates). The
instructional materials may be written, in an electronic form (such
as a computer diskette or compact disk), or may be visual (such as
video files).
[0319] The following examples are provided to illustrate certain
particular features and/or embodiments. These examples should not
be construed to limit the invention to the particular features or
embodiments described.
EXAMPLES
Example 1: Characterization of bCSC Phenotype
[0320] This example illustrates characterization of the stem cell
phenotype of breast cancer stem cells (bCSC) isolated from cultures
of the human triple negative breast carcinoma cell line
MDA-MB-231.
[0321] The bCSC are enriched in nonadherent fractions of parental
MDA-MB-231 cells and were further purified for some experiments by
flow sorting for CD44hi/CD24lo cells. The bCSC exhibit increased
asymmetric cell division characteristic of stem cells (FIG. 1). The
bCSCs also exhibit abilities to differentiate along several
lineages including neuronal and vascular (FIGS. 2, 3 and 4).
[0322] The bCSC exhibit elevated mRNA and protein expression of the
bCSC marker CD44 and characteristic decreased expression of CD24
(FIGS. 5, 6). They also exhibit elevated expression of the stem
cell transcription factor Sox2 and the stem cell markers nestin and
PTP-BP1 (FIG. 5), but not the pluripotency marker Nanog. Upon
addition of differentiating growth factors, the elevated stem cell
marker expression is reversed. Global transcriptome analysis and
qRT-PCR confirmed altered CD24 expression and identified additional
changes in gene expression in MDA-MB-231 bCSC (FIGS. 7, 8). CD47
expression is elevated in the bCSC, consistent with published
reports of elevated CD47 expression in other CSC, but expression of
the CD47 ligand thrombospondin-1 (TSP1) is decreased.
Example 2: Differentiation of bCSCs Through CD47 Blockade Using
B6H12
[0323] The CD47-blocking antibody B6H12 is known to inhibit binding
of CD47 to its counter-receptor SIRP.alpha. on phagocytic cells,
including macrophages. Consequently, this blocking antibody is
known to increase in vitro killing of tumor cells by macrophages or
NK cells. Based on these observations, others have proposed that
the ability of B6H12 to inhibit the growth of various human tumor
xenografts grown in Nod.SCID mice, which express a mutant murine
SIRP.alpha. that can recognize human CD47, results from blocking
the "don't eat me" signal of the human tumor cell CD47 engaging
Nod.SIRP.alpha. on the mouse macrophages. However, we have
discovered a potent cell-autonomous activity of the same CD47
antibody that directly suppresses the viability of human breast
cancer stem cells.
[0324] Treatment of unfractionated MDA-MB-231 cells with B6H12
results in rapid loss of rounded non-adherent cells from the
culture (FIG. 6A). Isolated bCSC from the same cell line also
rapidly adopt a flattened morphology and stop proliferating when
treated with B6H1, but not with an isotype-matched control antibody
(FIG. 6B). Microarray analysis and qRT-PCR demonstrated reversal by
B6H12 of many of the gene expression changes in bCSCs but not by
the control antibody (FIGS. 10, 11).
[0325] To define the mechanism by which the CD47 antibody B6H12
directly inhibits bCSC, we examined effects on energy metabolism
using the Seahorse Analyzer (FIGS. 12A-12C). B6H12 increased the
basal oxygen consumption rate (OCR) at day 1 and day 3 but this was
lost at day 4, when both the basal and spare metabolic capacity
were decreased. The CD47 ligand TSP1 had less effect than the CD47
antibody. B6H12 decreased asymmetric division and proliferation
measured by the MTS assay (FIGS. 12D-12F).
[0326] Based on our recently published discovery that CD47
associates with and regulates the tyrosine kinase receptor VEGFR2
in endothelial cells and T lymphocytes, and our previously
published finding that TSP1 synergized with EGF to inhibit growth
of small cell lung carcinoma cells, we considered the possibility
that CD47 may also regulate EGFR signaling in bCSC, and this could
be a mechanism through which the CD47 antibody B6H12 inhibits bCSC.
EGF and EGFR both showed moderately elevated mRNA expression in
bCSC relative to control MDA-MB-231 cells (FIGS. 13A, 13B).
Remarkably, treatment of the bCSC with B6H12 alone or in the
presence of EGF dramatically reduced expression of both EGF and
EGFR (FIG. 13C). The control IgG was inactive, and B6H12 did not
alter the same mRNAs on differentiated MDA-MB-231 cells or in the
ER+ breast carcinoma cell lines MCF7 or T47D (FIG. 17). At the
protein level, B6H12 suppressed global tyrosine phosphorylation of
proteins either in the absence or presence of RGF (FIG. 14B) and
preferentially inhibited EGFR phosphorylation at Y1068 but not Y998
in bCSC but not in control MDA-MB-231 cells (FIG. 14C, 14D).
[0327] The EGFR family member HER2 is not expressed well in the
triple negative cell line MDA-MB-231, so we used the Her2+ cell
lines MCF7 and T47D to examine the specificity of B6H12 effects on
EGFR signaling (FIG. 15). B6H12 also inhibited proliferation of
these cell lines, but this was not accompanied by inhibition of
HER2 phosphorylation. Unlike EGFR, Her2 showed no colocalization
with CD47 in these cell lines (FIG. 16). In the MDA-MB-231 bCSCs,
CD47 co-immunoprecipitated with EGFR, and preincubation with B6H12
decreased the cell surface expression of EGFR (FIG. 16D). Reduced
expression of EGFR after B6H12 treatment is associated with exosome
shedding of EGFR (FIG. 16E) and K63 ubiquitinylation of EGFR (FIG.
16F).
[0328] In contrast to the SIRP.alpha.-dependent role of CD47 in
tumor innate immunity, the three SIRP genes are poorly expressed in
breast cancer cells, and B6H12 did not affect their expression
(FIG. 17).
[0329] Based on these data, we propose a novel mechanism of
anti-tumor action for certain CD47 antibodies, including those
based on B6H12 that have been recently humanized and are expected
to soon enter clinical trials for treating cancer patients (Edris
et al., Proc Natl Acad Sci USA. 109(17):6656-6661, 2012). Our data
demonstrates that these antibodies directly act on human breast
cancer cells to suppress bCSC, and our preliminary results suggest
that this finding will generalize to other cancers that express
elevated levels of CD47. Because elevated CD47 expression is a
negative prognostic factor for many major human cancers, certain
CD47 antibodies may generally exhibit direct tumor suppressive
activity independent of the proposed indirect macrophage-dependent
mechanism. This discovery is an extension of but not predictable
from our recent discovery that CD47 regulates stem cell
self-renewal and reprogramming in healthy tissues (see, e.g., PCT
publication WO 2013/155109, incorporated herein by reference in its
entirety). We demonstrate here a direct signaling function of CD47
on CSC that sustains their stem cell properties. This contrasts
with the signaling by CD47 in normal stem cells, where it limits
their self-renewal. More importantly, we demonstrate that ligation
of CD47 with antibodies has the potential to suppress CSC and
thereby stop cancer growth.
Example 3: Differentiation of Additional CSCs Through CD47
Blockade
[0330] Targeting new cancer therapies to cancer stem cells is very
challenging because many of the tumor suppressor and polycomb genes
(Hedgehog and WNT signaling pathway) that are important for normal
stem cell regulation are dysregulated during carcinogenesis. The
ubiquitous cell surface protein CD47 is up-regulated in many
cancers, especially during metastasis, and high expression is a
negative prognostic indicator for several cancers. Additional
studies have indicated that CD47 expression is elevated in leukemic
cancer stem cells. The conventional wisdom has been that the
function of this elevated CD47 expression on CSC is to serve as a
"don't eat me" signal that protects the CSC from phagocytic
clearance by macrophages (reported online at
news.sciencemag.org/health/2012/03/one-drug-shrink-all-tumors).
Consequently, antibody and ligand therapeutics that engage CD47
have been developed to stimulate the destruction of CSC by
macrophages, and these are now entering human clinical trials.
[0331] The CD47 antibody B6H12 is known to block the recognition of
CD47 by its counter-receptor SIRP.alpha. on macrophages. Human
tumor xenografts grown in immunodeficient mice that express a
mutant form of SIRP.alpha. that is capable of binding human CD47
(Nod-SCID) have been used to test the ability of B6H12 to enhance
macrophage-mediated clearance of human tumor xenografts. Inhibition
of tumor growth by B6H12 in these models provided evidence to
support the humanization of such CD47 antibodies for treating human
cancer patients.
[0332] The central assumption for developing such antibody-based
therapies is that CD47 is a passive cell surface protein that
serves only to protect CSC from host innate immune surveillance.
However, our recent work has revealed that expression of CD47 in
non-transformed cells plays a critical role in regulating stem cell
homeostasis. Specifically, CD47 signaling inhibits the expression
of the Yamanaka transcription factors cMyc, Sox2, Oct3/4 and Klf4.
Decreasing CD47 in non-transformed cells increases their
self-renewal, asymmetric division and ability to reprogram into
other differentiated cell types.
[0333] Because high CD47 expression limits the stem cell character
of non-transformed cells, the high expression of CD47 on cancer
stem cells seemed paradoxical. This suggested that the CD47
expressed on CSC may lack the signaling activity to control stem
cell self-renewal, either due to alterations in the CD47 or
inactivation of genes that mediate its signaling in CSC. However,
to date the signaling function of CD47 in CSC has not been
examined. Therefore, we isolated CSC and cells depleted of CSC from
the triple-negative breast carcinoma cell line MDA-MB-231 and
examined the effects of ligating CD47 on these cells when treated
with the CD47 antibody B6H12.
[0334] bCSCs were isolated and differentiated similar to FIG. 2D-F.
bCSCs were cultured using neural differentiation media for 36 h.
Total RNA was extracted using TriZol method and global microarray
analysis was performed. FIG. 18A shows comparison between
differentiated neural lineage bCSCs vs. differentiated MDA-MB-231
or bCSCs; FIG. 18B-18D show gene enrichment of bCSCs differentiated
into neural lineage using published Geo sets data's
(GSE21475_TUBO_MAMMOSPHERES; GSE18833_MDA231_PARENRAL_VS_S1) and
CHARAFE_BREAST_CANCER_LUMINAL_VS_BASAL_DN (Charafe-Jauffret et al.,
Oncogene. 25(15):2273-2284, 2006). These molecular characteristics
analyses of differentiated neural lineage cancer cells showed
enrichment of genes related with mammosphere formation, epithelial
mesenchymal transition and tumorigenesis. We further compared gene
enrichment analysis to committed neural lineage or neural cells or
parts of brain region, none of these markers show any enrichment of
neural biomarkers. This indicates that bCSCs derived from neural
linage differentiated MDA-MB-231 cells are tumorigenic and
cancerous cells.
[0335] Microarray global expression data showed that treating CSC
isolated from the MDA-MB-231 cell line with the CD47 blocking
antibody B6H12 down regulated EGFR and up regulated Dicer 1 and
many more genes (Table 1). In Table 1, a blank gene ( - - - )
symbol indicates that gene has not been annotated in genome/gene
bank but the probe sequences are hybridizing to an expressed
sequence tag (EST). Dicer 1 cleaves double stranded and pre-micro
RNA into short stranded RNA and miRNA. Decreased dicer level
correlates with advanced tumor stages and poor survival and
outcomes. In addition to this, B6H12 up regulated CDC14 (cell
division cycle 14 homolog B, S. cerevisiae), which regulates CDK1
activity at the G2/M transition. The CD47 blocking antibody also
up-regulated MOB kinase activator 1A, which is known as a tumor
suppressor that controls cell proliferation and apoptosis. Our gene
enrichment analysis (FIG. 19A) also suggested that CD47-B6H12
blocking antibody does not enrich the expression of genes related
with tumorigenesis. These studies clearly establish that CD47 is
functional signaling receptor in breast CSC and suggested that
B6H12 may directly inhibit the tumorigenic potential of breast
CSC.
[0336] To assess the effect of B6H12 on CSC proliferation, bCSC
from MDA-MB-231 cells were treated with B6H12 or an isotype-matched
control antibody, and proliferation was assessed by
bromodeoxyuridine (BrdU) incorporation (FIG. 20A). B6H12
significantly inhibited DNA synthesis, but the control antibody was
inactive. This indicated that ligation of CD47 by B6H12 initiates
an anti-proliferative signal in bCSC, but it remained unclear
whether CD47 expression on bCSC positively regulates their growth
or, as we demonstrated on non-transformed stem cells, is a negative
regulator of stem cell function. We addressed this question by
using a CRISPR vector (based on pX330-U6-Chimeric_BB-CBh-hSpCas9;
Cong et al., Science. 339(6121):819-23, 2013, PMID: 23287718;
sequence and other information available online at
addgene.org/42230/) designed to specifically disrupt the CD47 gene
in MDA-MB-231 cells (FIGS. 22A and 22B), using the following gRNA
targeting sequence: 5' CTACTGAAGTATACGTAAG ngg 3' (PC200.Hcd47.g3a;
SEQ ID NO: 2). PC200.Hcd47.g3a was transfected into bCSCs derived
from MDA-MB-231 using a NUCLEOFECTOR.TM. kit (Lonza, Switzerland).
CD47 negative cells were sorted using FACS analysis. CD47.sup.neg
bCSCs cells were expanded in culture and cell proliferation was
measured using BrdU cell proliferation kit (EMD Millipore). CD47
targeted clone showed diminished BrdU incorporation, indicating
that CD47 has the opposite effect on bCSC proliferation as what we
reported in normal tissue stem cells (FIG. 22C).
[0337] We next examined whether other breast cancer cell lines
(MCF-7 and T47D) and a normal immortalized breast epithelial cell
line (MCF10A) showed similar responses to B6H12 (FIG. 20B-20D).
Consistent with our published studies using lung epithelial cells
and T cells, the CD47 blocking antibody B6H12 increased DNA
synthesis in MCF10A cells (FIG. 20C). MCF7 is an ER+
well-differentiated breast carcinoma cell line with limited
malignant potential and also exhibited a positive response to B6H12
(FIG. 20D). However, the T47D breast carcinoma cell line showed a
similar inhibition of proliferation by B6H12 as we found for
MDA-MB-231 cells (FIGS. 20A &B).
[0338] To determine whether the inhibitory effects of B6H12 were
limited to breast cancer cells, we examined proliferation in cell
lines from other major human cancers (FIG. 21). The classic small
cell lung carcinoma (SCLC) cell line OH-1 showed inhibition by
b6H12, and the variant SCLC cell line showed greater inhibition of
proliferation assessed by the MTS assay (FIG. 21A). Proliferation
of the prostate carcinoma cell line PC-3 was also inhibited by
B6H12. Proliferation of the human melanoma cell line A2058 was also
inhibited by B6H12, but consistent with the regulation of EGF
pathway genes we observed in bCSC, combining B6H12 with EGF
reversed the inhibitor effects of both agents (FIG. 21B).
[0339] We compared the proliferative responses of metastatic
(SW620) and non-metastatic variants of a human colon carcinoma
(SW480) to B6H12 (FIG. 21C). B6H12 had a positive effect on SW480
cells but not on SW620. Correspondingly, EGFR phosphorylation in
SW480 cells was increased following B6H12 treatment, but EGFR
phosphorylation in SW620 cells was inhibited (FIG. 21D). This
suggests that binding of B6H12 to CD47 elicits a signal that
inhibits EGF signaling only in the more metastatic cell line.
[0340] In summary, these data demonstrate that CD47 is an active
signaling receptor in multiple human cancer types including breast,
lung, prostate, and colon carcinomas and human melanoma. B6H12
antibody does not inhibit cell growth in a subset of the cancer
cell lines we have tested, but stimulation of proliferation was
also observed in some cell lines with lower malignant potential,
which is consistent with known CD47 signaling in non-transformed
cells. Our cell proliferation and EGFR phosphorylation data
indicates that B6H12 antibody predominantly inhibits proliferation
of aggressive cancer cells as compared to less malignant tumor cell
lines.
Example 4: Inducing Differentiation of CSCs in an In Vivo Model
[0341] This example describes representative methods to assess the
efficacy of a compound to selectively induce differentiation of
cancer stem cells or prevent tumor growth or metastasis in an in
vivo model.
[0342] Mice having a human cancer xenograft(s) can be generated
using the method of Forsberg et al. (British J. Cancer 85:129-36,
2001). Briefly, the xenograph is established in immune-deficient
mice (such as SCID or Balb/c nu/nu mice). The xenograft can be
allowed to grow for a period of time prior to administration of the
compound (such as 5-20 days). Alternatively, the compound can be
administered at the same time as the cancer cells (and optionally
administration can continue for a period of time after
administration of the cancer cells), and growth of the xenograft
monitored in the absence of further administration of the compound
(to determine if xenograft growth can be prevented). The
development of the xenograft can be compared to mice which are
administered vehicle or a control compound.
[0343] Tumor metastasis can also be measured by intravenously
injecting tumor cells in the tail vein of athymic nude mice. Gross
tumor number in a site such as lung tissue is assessed by gross
observation at necroscopy 21 days after transplantation.
[0344] Administration of a compound that inhibits CD47 functional
interaction with EGFR and induces differentiation of cancer stem
cells should reduce tumor size, tumor cell volume, or number of
tumor metastases or prevent tumor growth or metastasis to a greater
extent than the control tumors (e.g., in untreated or control
treated animals).
[0345] It is believed that administration of CD47 blocking agents
will be useful for selectively inducing differentiation and
senescence of cancer, such as breast tumors (e.g., including triple
negative breast cancer) as well as other cancers.
[0346] By way of example, athymic Nude mice BALB/c can be used for
animal study in vivo. .about.1*10.sup.6/0.2 ml WT and CD47-CRISPR
transfected bCSCs derived from MDA-MB-231 cells are injected into
subaxillary mammary fat pads at both sites of 4-6 week-old female
BALB/c nude mice.
[0347] Tumor size is monitored weekly after inoculation; a tumor
with a diameter 100 mm.sup.3 will be further treated with B6H12 Ab,
or a test agent being evaluated for activity in the methods claimed
herein.
[0348] To measure spontaneous metastasis (using same experimental
conditions as above described), when the mean tumor diameter
reaches 1.0 cm, the primary tumor is surgically removed. Mice are
then maintained for an additional 2 months to allow further growth
of lung metastasis. To produce lung metastasis;
.about.1*10.sup.6/0.2 ml WT and CD47-CRISPR transfected bCSCs
derived from MDA-MB-231 cells are injected into lateral tail veins
of female athymic nude mice using a restraint box. The bCSCs cells
are treated with B6H12 Ab after the growth of tumor (100 mm.sup.3).
After 4 weeks, the mice are euthanized and the lung metastases
examined.
Example 5: Antibody-induced CD47 Signaling Suppresses Stem Cells in
Triple-Negative Breast Cancer
[0349] CD47 is a signaling receptor for thrombospondin-1 and the
counter-receptor for signal-regulatory protein-.alpha.
(SIRP.alpha.). By inducing inhibitory SIRP.alpha. signaling,
elevated CD47 expression by some cancers prevents phagocytosis by
macrophages. In preclinical studies the CD47 blocking antibody
B6H12 inhibited tumor growth in several xenograft models,
presumably by preventing SIRP.alpha. engagement. Evidence that CD47
signaling in non-transformed and some malignant cells regulates
self-renewal suggested that B6H12 and related therapeutic
antibodies may have a second mechanism of action involving cancer
stem cells (CSC).
[0350] This example provides data illustrating that breast CSC
isolated from the triple negative MDA-MB-231 cell line showed
up-regulation of CD47 and CSC markers and more frequent asymmetric
cell division than differentiated MDA-MB-231 cells. Gene expression
analysis in breast CSCs treated with B6H12 showed up-regulation of
tumor suppressor genes and decreased expression of epidermal growth
factor receptor (EGFR) and the stem cell transcription factor KLF4.
B6H12 treatment also inhibited co-immunoprecipitation of EGFR with
CD47. EGFR and CD47 co-immunoprecipitation analysis revealed that
very small fraction of CD47 and EGFR co-immunoprecipitate, and
pretreatment with B6H12 antibody disrupted this interaction.
EGF-induced EGFR tyrosine phosphorylation, asymmetric cell
division, and cell proliferation and up-regulated caspase 3/7
activity. Similarly, B6H12 inhibited proliferation of T47D CSCs but
not of MCF7 breast carcinoma or MCF10A breast epithelial cells.
Caspase-7 cleavage and EGFR expression in human breast cancers
correlated with CD47 expression, suggesting that this CD47
signaling pathway is functional in vivo.
[0351] These data reveal a novel SIRP.alpha.-independent mechanism
by which therapeutic CD47 antibodies can control tumor growth by
directly forcing differentiation of CSC. Therefore, therapeutic
CD47 antibodies intended to enhance macrophage-mediated tumor cell
clearance may also benefit triple negative breast cancer patients
by directly suppressing CSCs.
[0352] The subject matter of this Example overlaps at least in part
with that of Examples 2 and 3.
INTRODUCTION
[0353] Breast progenitor cells play an active role in the cyclic
changes that take place during pregnancy and ovulation in women
(Stingl et al., Breast Cancer Res Treat, 67(2):93-109, 2001;
Petersen & Polyak, "Stem cells in the human breast." Cold
Spring Harb Perspect Biol, 2(5):a003160, 2010). A minor subset of
tumor cells have the capacity to initiate a new tumor upon
transplantation into a healthy host. These tumor initiating cells
have stem cell-like properties and are also known as cancer stem
cells (CSC). In contrast to CSCs, the bulk tumor cells have limited
proliferative capacity and cannot form new tumors.
[0354] Despite advances in the diagnosis and treatment of breast
cancer, these cancers frequently recur with a relapse time of 5-7
years (Rosen et al., J Clin Oncol, 7(9):1239-51, 1989). One
proposed mechanism is that CSCs are more resistant to
chemoradiation therapies and persist in a dormant state during
therapy but later reinitiate tumor regrowth. As few as 100
CD133.sup.+-expressing brain and breast cancer cells were
sufficient to establish a new cancer in non-obese diabetic, severe
combined immunodeficient (NOD-SCID) mice. In contrast, engrafted
CD133.sup.- cells did not form any tumor (Singh et al., Nature,
432(7015):396-401, 2004; Wright et al., Breast Cancer Res,
10(1):R10, 2008). Flow cytometric analysis has shown that a
CD44.sup.high and CD24.sup.low population is enriched in CSCs
(Sheridan et al., Breast Cancer Res, 8(5):R59, 2006). However, most
existing therapies to treat solid tumors do not efficiently target
cancer stem cells.
[0355] Breast cancers comprise four major molecular subtypes:
luminal A, luminal B, triple negative/basal-like, and HER2 type
(Sorlie, Eur J Cancer, 40(18):2667-75, 2004). Triple negative
breast cancers (TNBC) represent approximately 20% of cases and are
defined by their lack of expression of estrogen receptor (ER),
progesterone receptor, and human epidermal growth factor receptor-2
(HER2). TNBC highly express epidermal growth factor receptor-1
(EGFR) and are highly proliferative, aggressive and resistant to
systemic chemotherapies. Consequently, outcome for these patients
is poor compared to ER.sup.+ and HER2.sup.+ cancers. Approximately
30-40% of deaths are caused by recurrence and metastasis of TNBC.
Even though EGFR inhibitors have shown some promise for treating
TNBC (Al-Ejeh et al., J Nucl Med, 54(6):913-21, 2013), no FDA
approved therapies have improved patient outcome for TNBC. Thus,
there is urgency to identify signaling pathways required for TNBC
and develop therapies targeting these pathways.
[0356] The ubiquitous cell surface protein CD47 is up-regulated in
many cancers, especially during metastasis, and high expression is
a negative prognostic indicator for several cancers including
invasive breast cancer (Zhao et al., Proc Natl Acad Sci USA,
108(45):18342-7, 2011; Willingham et al., Proc Natl Acad Sci USA,
109(17):6662-7, 2012). One proposed function of elevated CD47
expression on CSC is to serve as a "don't eat me" signal that
protects the CSC from phagocytic clearance by macrophages (Jaiswal
et al., Trends Immunol, 31(6):212-9, 2010). Consequently, antibody
and recombinant protein therapeutics that engage CD47 and block
SIRP.alpha. binding have been developed to stimulate the
destruction of CSC by macrophages. The CD47 antibody B6H12 blocks
the recognition of CD47 by its counter-receptor SIRP.alpha. on
macrophages. Human tumors grown in immunodeficient NOD-SCID mice
that express a mutant form of SIRP.alpha. capable of binding human
CD47 have been used to test the ability of B6H12 to enhance
macrophage-mediated clearance of human tumor xenografts (Willingham
et al., Proc Natl Acad Sci USA, 109(17):6662-7, 2012).
[0357] Inhibition of tumor growth by B6H12 in these models provided
evidence to support the humanization of related CD47 antibodies for
treating human cancer patients, which humanized antibodies have now
entered human clinical trials (NCT02216409, NCT02367196,
NCT02096770). Based in part on evidence that B6H12 has effects on
CD47 signaling that are independent of blocking SIRP.alpha. binding
(Soto-Pantoja et al., Cancer Res, 74(23):6771-83, 2014;
Soto-Pantoja et al., Proc Natl Acad Sci USA, 109(42):E2842; author
reply E2844-5, 2012; Zhao et al., Proc Natl Acad Sci USA,
108(45):18342-7, 2011), however, others have concluded that the
CD47/SIRP.alpha. hypothesis is not sufficient to explain the
antitumor activity of CD47 blockade and have reported that CD47 is
more than a passive SIRP.alpha. counter-receptor.
[0358] In this Example, we present evidence for an unanticipated
activity of B6H12 and other agents that perturb CD47 signaling in
CSC. We demonstrate a signaling role of the prototypical CD47
blocking antibody B6H12 on breast CSC but not differentiated cancer
cells from the MDA-MB-231 cell line. These data provide evidence
that this blocking antibody can function independently of its known
ligands TSP1 and SIRP.alpha. and should be considered a
pharmacological agonist of CD47 signaling.
Material and Methods
Asymmetric Cell Division
[0359] MDA-MB-231 cells (ATCC) were labeled with
5-bromo-2'-deoxyuridine (BrdU) for two weeks. The cells were then
grown in BrdU-free medium for at least two consecutive cell
divisions. The numbers of asymmetric cells were quantified as
described (Kaur et al., Sci Rep, 3:1673, 2013; Pine et al., Proc
Natl Acad Sci USA, 107(5):2195-200, 2010).
RNA Extraction and Real-Time PCR
[0360] CSC-depleted MDA-MB-231 and bCSCs were plated at
1.times.10.sup.6 cells in 6-well plates and were-treated with B6H12
or Isotype control antibody (1 .mu.g/ml) for 36 h. Total RNA was
extracted using TriPure (Roche). One .mu.g of total RNA was used
for first strand cDNA synthesis using a Maxima kit (2-Step RT PCR,
Thermo Scientific) according to the manufacturer's instructions.
Real time PCR was performed using SYBR Green (Roche) on an MJ
Research Opticon I instrument (Bio-Rad) with the amplification
program as described in (Kaur et al., Matrix Biol, 37:49-59, 2014).
.beta.2-microglobulin (B2M), HPRT1 or 18S rRNA primers were used as
control to normalize mRNA expression (Table 2).
TABLE-US-00002 TABLE 2 SEQ ID NO: Gene Name Primer Sequence 3 EGF-F
TGG TTC CTT CTG TGT CAA TCC 4 EGF-R GTA CTC TCG CAG GAA ATG GG 5
EGFR-F TCC TCT GGA GGC TGA GAA AA 6 EGFR-R GGG CTC TGG AGG AAA AGA
AA 7 18S rRNA-F AGG ACC GCG GTT CTA TTT TGT TGG 8 18S rRNA-R CCC
CCG GCC GTC CCT CTT A 9 hMYC-F CGT CCA AGC AGA GGA GCA AAA GCT 10
hMYC-R CGC ACA AGA GTT CCG TAG CTG 11 B2M-F TCC TGA ATT GCT ATG TGT
CTG GGT 12 B2M-R GAT AGA AAG ACC AGT CCT TGC T 13 TXNIP-F AGG AAG
CTC AAA GCC GAA CT 14 TXNIP-R ACG CTT CTT CTG GAA GAC CA 15
PMAIP1-F AAG TTT CTG CCG GAA GTT CA 16 PMAIP1-R GCA AGA ACG CTC AAC
CGA G 17 PLS3-F TTG CAA AGG CCT CTT TGA GT 18 PLS3-R CCC AGG ACT
CTG CGA CTT TA 19 NRIP1-F ATG GAT GAC TGC ATT CCA CA 20 NRIP1-R GAG
AAA CCA GCC CAA AAT GA 21 LOX-F TGG CAG TCT ATG TCT GCA CC 22 LOX-R
CTA TGG CTA CCA CAG GCG AT 23 CRISPLD1-F CCA TGT TCC CAC AAG CAA CT
24 CRISPLD1-R TCA GGT GTA TCC AAC AGC CTC 25 CD14-F CTC ACA AGG TTC
TGG CGT G 26 CD14-R TGA GCT CAG AGG TTC GGA AG 27 U6-Forward:
CTCGCTTCGGCAGCACA 28 U6-Reverse: AACGCTTCACGAATTTGCGT 29 Drosha
forward TAGGCTGTGGGAAAGGACCAAG 30 Drosha reverse
GTTCGATGAACCGCTTCTGATG 31 DGCR8 forward CAAGCAGGAGACATCGGACAAG 32
DGCR8 reverse CACAATGGACATCTTGGGCTTC 33 Actin forward
TGAAGTGTGACGTGGACATC 34 Actin reverse GGAGGAGCAATGATCTTGAT 35 Ago1
forward CGTAGAGTCAACCGGGAAGT 36 Ago1 reverse CCTCAAAGTCGACCCGTTC 37
Ago2 forward TGCAGGCGTTACACGATGC 38 Ago2 reverse
TACCTCATGGATGGCAAGTGC 39 Dicer forward CAGGTATACTTCTCAGCCATGTGA 40
Dicer reverse GCCCACTTCTGTCAGTAAATGGT 41 let 7a-1-F CAA CGT AAG TGA
ATG AAA ATG GT 42 let 7a-1-R TAA ATT AAT TTA TTT CCA GGC CA 43 let
7a-2-F GAA AAT ACA GCA TGG GTA CAA GGA 44 let 7a-2-R TTA AGA AAT
GGT AGT TTT CCA GCC A 45 let 7a-3-F AGA ATC CCT GTG CCC TTG G 46
let 7a-3-R GGC ACC TAG GCC TGT CAG ACT 47 let 7d-F CAG GTT AAT TTG
AAG TGC ATC TG 48 let 7d-R AGC ACC ATG CTG CAG TTT ATA A 49 let
7f-1-F GGG GAA ACC TTT TGC TTC T 50 let 7f-1-R CTG TGC CTA CTG TAC
TTG AAC A 51 let 7f-2-F TAC ATG AAC ATG CTG TTT CAG AG 52 let
7f-2-R CAT CTA AGT CAA ATG ACT CCA CTG
BrdU Cell Proliferation Assay
[0361] Approximately 8,000 cells were plated per well on 96-well
plates and incubated overnight at 37.degree. C. The cells were
treated with B6H12 or isotype control antibody (1 .mu.g/ml) for 24
h. BrdU was added for 4 or 24 h as indicated in the figures, and
BrdU incorporation was quantified using a BrdU Cell Proliferation
Kit according to the manufacturer's instructions (EMD Millipore).
For flow analysis, MDA-MB-231 cells were labeled with BrdU, and
unlabeled cells were used as a negative control.
Microarray Processing and Analysis
[0362] Samples were prepared according to Affymetrix protocols
(Affymetrix, Santa Clara, Calif.). RNA quality and quantity was
ensured using the Bioanalyzer (Agilent, Santa Clara, Calif.) and
NanoDrop (Thermo Scientific, Waltham, Mass.) respectively. Per RNA
labeling, 500 nanograms of total RNA was used in conjunction with
the Affymetrix recommended protocol for the HG_U133_Plus 2.0 chips.
The hybridization cocktail containing the fragmented and labeled
cDNAs was hybridized to the Affymetrix Human HG_U133_Plus 2.0
GeneChip. The chips were washed and stained by the Affymetrix
Fluidics Station using the standard format and protocols as
described by Affymetrix and the Affymetrix Gene Chip Scanner 3000
was used to scan the probe arrays. Gene expression intensities were
extracted using Affymetrix AGCC software. Partek Genomic Suite was
used to RMA normalize (Robust Multichip Analysis), summarize, log 2
transform the data and run the ANOVA analysis. The raw data is
deposited in NCBI Gene Expression Omnibus (GEO): GSE67966.
Cell Culture and Reagents
[0363] The breast carcinoma cell lines MDA-MB-231, MCF7, MCF10A and
T47D1 cells were purchased from ATCC (Manassas, Va.) and cultured
at 37.degree. C. in 5% CO.sub.2 using Gibco RPMI 1640 medium with
10% FBS, Penicillin Streptomycin and Glutamine (Life Technologies,
Grand Island, N.Y.). The bCSCs were cultured using cancer stem cell
media from (ProMab, Richmond, Calif.). APC-conjugated antibodies
for EGF and human KLF4 were obtained from R&D Systems.
Antibodies specific for EGFR and phospho-Tyr.sup.1175 EGFR, actin,
nanog, OCT4, and SOX2 were obtained from Cell Signaling (Danvers,
Mass.), CD47 antibody B6H12 (Abcam, Cambridge, Mass.), Human
CD47-FITC (BD Biosciences). Functional grade purified anti-human
CD47-B6H12 and isotype-matched control antibody were from
eBioscience, (San Diego, Calif.), Anti-BrdU APC, EGFR-PE,
CD44-FITC, CD24-PE conjugated and Isotype control antibodies were
obtained from Bio Legend. Cytoclasin D and anti-tubulin were
purchased from Sigma Aldrich. For functional studies B6H12 and its
isotype-matched control antibody were used at 1 .mu.g/ml for all
experiment analysis throughout the manuscript using RPMI medium
containing 2% FBS.
Immunoprecipitation and Western Blots
[0364] bCSC-depleted MDA-MB-231 cells and bCSCs were plated at
1.times.10.sup.6 cells in 6-well plates. The cells were
serum-starved for 2 hours using serum-free RPMI medium. The cells
were pre-treated with B6H12 (1 .mu.g/ml) for 20 min. Cell lysates
were made using immunoprecipitation buffer (50 mM Tris-HCl, 150 mM
NaCl, and 1% Nonidet P-40) along with 1.times. Complete
Mini-protease inhibitor mixture (Roche Applied Science). Cell
lysates were centrifuged at 13,000 rpm for 15 min. A BCA assay
(Thermo scientific) was used to quantify total protein.
DYNABEADS.RTM. magnetic separation beads (Invitrogen) were used for
Immunoprecipitation. The DYNABEADS.RTM. magnetic separation beads
were washed three times with activation buffer. The cell lysates
were incubated in DYNABEADS.RTM.-protein G along with anti-EGFR and
CD47 antibodies (1:500) and incubated for 24 hours at 4.degree. C.
on a shaker. The beads were washed three times with lysate buffer
and heated at 95.degree. C. for 5 min. The immunoprecipitated cell
lysates were loaded on 4-12% NuPAGE gels (Life Technologies), and
Western blotting was performed. For immunoprecipitation, primary
antibody against phospho-EGFR (1:1000) was used. Normalization of
protein lysates used for Western blotting was performed by
reprobing with anti-.beta.-actin (1:3000) or tubulin or EGFR
antibodies.
CD47 and EGFR Immunoprecipitation
[0365] MDA-MB-231 cells were pretreated with B6H12 (1 .mu.g/ml) for
15 min. The cells were further treated with EGF for 7 min and total
Lysate were performed using NP-40 lysis buffer as described above.
CD47 and EGFR immunoprecipitation was performed using
DYNABEADS.RTM. Protein G Immunoprecipitation Kit (Life
technologies) according to manufacturer's instructions with slight
modifications in incubation time (3 h) for EGFR and CD47 antibodies
with Dyna beads. The immunoprecipitated cell lysates were loaded on
4-12% NuPAGE gels (Life Technologies) and transferred using
IBLOT.RTM.--Western Blotting System (Life Technologies). The
membrane was blocked with 3% milk with addition of Complete, mini,
Pellet protease inhibitor mix (Roche Life Sciences) for 20 minutes.
IP-western blots were performed using -EGFR.sup.Y1068, EGFR and
CD47 antibodies (1:1000) overnight at 4.degree. C. The membrane was
washed two times with TBST for 10 minutes. Secondary HRP
(Amersham), IRDye 800 or 680 (LI-COR) 1:3000 were used for 1 h at
RT. The membranes were further washed 3 times for 10 minutes
interval. The images were captured by using WesternSure PREMIUM
Chemiluminescent Substrate with ODYSSEY.RTM. Fc(LI-COR). The
membranes were immunoblotted with EGFR and CD47 for total
IP-input.
Transfection MDA-MB-231 cells were plated overnight using Lab-Tek
Chambers. EGFR-GFP (Addgene) was transfected using Fugene 6. Cells
were treated with B6H12 and immunostaining performed as above
described section.
Exosomes Secretion
[0366] MDA-MB-231, bCSCs, T47D1 and MCF7 cells were treated with
B6H12 for 6 hours and condition media was harvested. The exosomes
were isolated using ExoQuick Kit (SBI-System Biosciences) as
previously described (Kaur et al., Matrix Biol, 37:49-59,
2014).
Flow Cytometry
[0367] MDA-MB-231 and bCSCs cells were stained with either isotype
control antibody or anti-CD44-FITC and anti-CD24-PE antibodies for
30 min. at room temperature. bCSCs cells treated with control
antibody or EGF or B6H12 antibodies alone or in combinations were
incubated at 4.degree. C. or 37.degree. C. for 2 hours and stained
with anti-CD47-FITC and anti-EGFR-PE antibodies for 30 min. at room
temperature. For caspase 3/7 staining bCSCs cells treated with
either control antibody or B6H12 antibody were stained using
CellEvent.TM. Caspase-3/7 Green READYPROBES.RTM. Reagent. For
intracellular staining, bCSCs cells were stimulated with Leukocyte
Activation Cocktail, with GOLGIPLUS.TM. reagent (BD Pharmingen) for
6 hours at 37.degree. C. before being stained with anti-KLF4 using
Foxp3 staining buffer set kit (eBiosciences). Cells were analyzed
using a LSR II System flow cytometer (BD Biosciences), and the data
were subsequently analyzed and presented using FlowJO software
(TreeStar).
Extended Asymmetric Cell Division
[0368] With gentle agitation of the flask, loosely bound bCSCs were
separated from adherent MDA-MB-231 cells. bCSCs form loose
aggregates after incubation at 37.degree. C. Adherent MDA-MB-231
and enriched bCSCs cells were labeled with BrdU for 10 days and
then chased in BrdU free medium for 3-4 days and followed by 2
.mu.M Cytochalasin. The cells were immunostained using BrdU
antibody and mounted with Vectashield DAPI. The confocal images
were taken using a Zeiss 780 microscope at 63.times. quantification
of asymmetric cell division ratio between cells negative for BrdU
and positive for DAPI counted manually (Kaur et al., Sci Rep,
3:1673, 2013; Pine et al., Proc Natl Acad Sci USA, 107(5):2195-200,
2010; Sunderaraman et al., Circ Res, 110(9):1169-73, 2012).
[0369] The MDA-MB-231 cells were labeled with BrdU for two weeks.
The cells were grown in BrdU free media at least to two consecutive
cell divisions. BrdU free media at 0 h and rest of them split into
two plates and treated with antiB6H12 or Isotype control antibody
(1 .mu.g/ml) for 5 days. BrdU staining was performed using BrdU
Cell Proliferation Kit 2752 from EMD Millipore. BrdU negative and
positive for DAPI for DNA segregation was counted manually. The
total numbers of DAPI were divided by BrdU negative cells. The
untreated or control asymmetric cells were normalized to 1. The
ratio of B6H12 was determined as compared to 1 (control).
Mammosphere Formation Medium
[0370] Cultured MDA-MB-231 cells were washed with IXPBS and
Suspension cells (bCSCs) were harvested with gentle agitation from
flask. Mammosphere formation medium with slight modification
according to Cioce et al. (Cell Cycle, 9(14):2878-87, 2010) for
FIGS. 1B and 24A only. bCSCs were seeded using Petri dishes
(Corning) in DMEM/F-12 (1:1) Invitrogen media) containing 5
.mu.g/ml insulin, 0.5 .mu.g/ml hydrocortisone, 25 ng/ml EGF, 25
ng/ml bFGF, 4 .mu.g/ml heparin and gentamycin sulfate. In other
experiments the bCSCs was cultured using cancer stem cell medium
from ProMab.
TCGA Invasive Breast Carcinoma Patient Tumor Data
[0371] mRNA (RNA Seq V2 RSEM) and protein expression data from the
TCGA Breast Invasive Carcinoma raw data at the NCI was analyzed
using cBioPortal tools (Gao et al., Sci Signal, 6(269):pl1, 2013;
Cerami et al., Cancer Discov, 2(5):401-404, 2012).
Statistical Analysis
[0372] The p-values for asymmetric cell division, cell
proliferation, cell imaging intensity and flow MFI were measured
using the t-test for two samples assuming equal variances. The
p-value less than .ltoreq.0.05 were used as statistically
significant. *, ** and *** corresponding to .ltoreq.0.05, 0.05 and
0.005 respectively.
Results
[0373] Characterization of Breast CSCs Derived from Suspension
Cell-Enriched MDA-MB-231 Cells
[0374] Routinely cultured MDA-MB-231 cells displayed abundant
loosely attached round cells as well as firmly attached spread
cells (FIG. 1A and inset). The loosely attached cells could be
harvested by gentle shaking (FIG. 1B) and formed aggregates within
10 days at 37.degree. C. in cancer stem cell medium (FIG. 24A). The
presence of CD44.sup.high and CD24.sup.low cancer stem/progenitor
cells is a hallmark of aggressive metastatic TNBC (Idowu et al.,
Hum Pathol, 43(3):364-73, 2012). Based on flow cytometry the
loosely attached cells expressed more surface CD44 than the firmly
attached MDA-MB-231 cells (FIGS. 6A & 6B). Gene expression
analysis of CD44 and CD24 mRNA in suspension and attached cells
indicated that the suspension cells have 257-fold up-regulation of
CD44 as compared to attached cells, and re-plating the suspension
cells in stem cell media further increased CD44 gene expression
(FIG. 24B). On the other hand, the suspension cells expressed
8-fold less CD24 than attached cells, which did not further change
after re-plating. Global microarray gene expression analysis of
these two subsets indicated that loosely bound MDA-MB-231 cells
differentially express many genes characteristic of CSC (FIG. 7A).
Among them, 8 transcripts were significantly upregulated (P=0.005),
and 90 transcripts were down regulated in suspension cells
including four CD24 transcripts. (FIG. 24D and Table 3). Based on
these characteristics, we hereafter refer to the suspension cells
as bCSC and to the firmly attached cells as differentiated
MDA-MB-231 cells. Interestingly, CD47 showed 2.3-fold higher
expressions in bCSCs, whereas TSP1 and c-Myc showed decreased
expression in bCSCs (FIG. 25A-25C).
[0375] CSCs share some characteristics with embryonic stem cells.
Correspondingly, real time PCR analysis of bCSCs revealed
up-regulation of OCT4, Nanog, SOX2, and nestin relative to attached
cells (FIG. 25D-25G). Another defining characteristic of stem cells
is asymmetrical division. MDA-MB-231 cells have been reported to
divide asymmetrically for self-renewal (Liu et al., Mol Cancer,
12(1):139, 2013), and asymmetric division is correlated with the
CD44.sup.high/CD24.sup.low phenotype (Ghiabi et al., PLoS One,
9(11):e112424, 2014). We chased BrdU-labeled bCSCs with unlabeled
BrdU to quantify asymmetric DNA template strand segregation (Pine
et al., Proc Natl Acad Sci USA, 107(5):2195-200, 2010).
Differentiated MDA-MB-231 cells and bCSCs were labeled with BrdU
for two weeks and chased for 2 divisions in BrdU-free complete RPMI
medium. The cells were treated with cytochalasin D, and symmetric
versus asymmetric DNA segregation was counted microscopically.
bCSCs enriched for CD44.sup.highCD24.sup.low showed an increase in
asymmetric cell division (FIG. 24E-24F).
CD47 Blocking Antibody B6H12 Specifically Targets bCSCs
[0376] To investigate if the CD47 blocking antibody B6H12 has
direct effects on bCSCs cells, we cultured differentiated
MDA-MB-231 cells and bCSCs in the presence of B6H12 or an
isotype-matched control IgG for 3 days. The CD47 antibody decreased
the number of round non-adherent bCSCs cells (arrows) but had no
effect on differentiated MDA-MB-231 cells (FIG. 9A-9B). B6H12
reduced size of mammosphere when bCSCs were cultured for 10 days
using cancer stem cell medium (FIG. 26A-26C). Global microarray
assessment of mRNA expression revealed that B6H12 altered the
expression of 225 transcripts as compared to isotype control
antibody (FIG. 10A, 27A). Principle component analysis showed that
B6H12 antibody-treated bCSC showed distinct gene expression
relative to control antibody-treated and differentiated MDA-MB-231
cells (FIG. 19B). Representative genes identified in the arrays
were validated by qPCR (FIG. 27C-G). B6H12-treated bCSCs showed an
up-regulation of thioredoxin-interacting protein (TXNIP) and LOX
(lysyl oxidase), which are known tumor suppressors (Shin et al.,
Biochem Biophys Res Commun, 372(4):880-5, 2008; Min et al., A
Cancer Res, 69(16):6685-93, 2009). Altered TXNIP mRNA expression
was previously reported following CD47 knockdown in hepatocellular
carcinoma stem cells (Lee et al., Hepatology, 60(1):179-91, 2014).
Similarly, B6H12 treatment selectively increased bCSC expression of
plastin-3 (PLS3), which is associated with actin and calcium ion
binding (Lin et al., Mol Cell Biol, 10(4):1818-21, 1990; Lin et
al., J Biol Chem, 268(4):2781-92, 1993), PMAIP1
(phorbol-12-myristate-13-acetate-induced protein-11), which
promotes activation of caspases and apoptosis (Bertin-Ciftci et
al., Cell Death Differ, 20(5):755-64, 2013), cysteine-rich
secretory protein LCCL domain-containing-1 (CRISPLD1), which is
released into extracellular vesicles, and CD14, which activates
cell adhesion and NF.kappa.B (Palma et al., Mol Cancer, 13:79,
2014).
B6H12 Down-Regulates EGFR mRNA Expression and Tyrosine.sup.1068
Phosphorylation in bCSCs
[0377] High expression of EGFR is characteristic of ER-breast
tumors and has been linked to poor prognosis (Foley et al., Semin
Cell Dev Biol, 21(9):951-60, 2010; Wang et al., Lancet,
365(9460):671-9, 2005; Bos et al., Nature, 459(7249):1005-9, 2009).
ER.sup.-/HER2.sup.low cancers with a EGFR.sup.high phenotype were
reported to have a higher number of stem/progenitor cells (Foley et
al., Semin Cell Dev Biol, 21(9):951-60, 2010; Dontu et al., Trends
Endocrinol Metab, 15(5):193-7, 2004; Zhu et al., J Theor Biol,
269(1):138-49, 2011). Treatment of bCSCs with B6H12 for 3 days
down-regulated EGFR at the mRNA level on microarray analysis (FIG.
27A). Real time PCR confirmed that treatment with B6H12 alone or in
the presence of EGF completely blocked detectable EGF and EGFR
transcript expression (FIG. 28A-28B), whereas treatment with
isotype control antibody in the presence or absence of EGF did not
significantly alter mRNA expression of EGF and EGFR.
[0378] CD47 regulates the release of mRNA into exosomes in T cells,
which suggested that the loss of EGFR mRNA induced by B6H12
treatment could result from export of this mRNA. We isolated
exosomes from conditioned medium of MDA-MB-231 cells and derived
bCSCs, T47D1 cells, and MCF7 cells in the presence or absence of
B6H12 for 6 h. Treatment with B6H12 increased EGFR mRNA levels in
EVs 2-fold in MDA-MB-231 cells, 5-fold in bCSCs. HPRT1, actin and
18S have different Ct levels in B6H12 treated samples as compared
to untreated. U6 was used for this analysis, which is more
appropriate for miRNA but less so for mRNA expression; but not in
T47D1 and MCF7 cells (FIG. 28C). In contrast to EGFR mRNA, EGF mRNA
was not detected in the EVs, and levels of other mRNAs were not
significantly changed FACS sorted bCSCs_MDA-MB-231 showed increase
in EGFR protein level expression on EVs but not bCSCs derived from
T47D1 and MCF7 (FIG. 28D). To test exosomes secretion, we
transfected EGFR-GFP in MDA-MB-231 cells were transfected with
EGFR-GFP and confocal microscopy analysis showed that B6H12
stimulated EGFR release in the form of vesicles. We further
isolated a pure population of CD44.sup.high/CD24.sup.low from
MDA-MB-231, T47D1 and MCF7 cell lines and treated with B6H12 for 24
hours. The exosomes were extracted and protein expression of EGFR
was analyzed by western blot. B6H12 treatment specifically
upregulate EGFR level only in bCSCs derived from MDA-MB-231 but not
from T47D1 and MCF7 cells which could play role as decoy receptor
for EGFR (available online at
grantome.com/grant/NIH/R01-CA163563-03). We further found that
B6H12 treatment increase MicroRNA-7a-2 and 7a-3 expression (Table
4). Down-regulation of EGFR has been reported by MicroRNA-7 (miR-7)
in human cancer cells (Webster et al., J Biol. Chem. 284:5731-5741,
2009). MiR-7 has been under-expressed in lung, breast and
glioblastoma cancer cells and an emerging cancer therapeutic target
(Barh et al., Curr Oncol 17:70-80, 2010). This could be a potential
mechanism by which B6H12 decrease EGFR expression in Triple
negative breast cancer (TNBC).
TABLE-US-00003 TABLE 4 Ct value of miR 1et7-family from cellular
RNA of MDA-MB-231 18S RNA 7a-2 7a-3 7d 7f-2 UT 18.81 .+-. 0.46
25.94 .+-. 0.06 28.29 .+-. 0.13 27.12 .+-. 0.16 32.88 .+-. 0.18
B6H12 22.27 .+-. 0.02 32.41 .+-. 0.23 31.66 .+-. 0.44 29.27 .+-.
0.03 38.50 .+-. 0.50 B6H12 + EGF 22.03 .+-. 0.03 29.63 .+-. 0.04
32.99 .+-. 0.05 32.60 .+-. 0.14 34.40 .+-. 0.07 EGF 17.65 .+-. 0.35
26.09 .+-. 0.50 28.88 .+-. 0.71 27.38 .+-. 0.18 33.24 .+-. 0.05 EGF
+ Control Ab 17.60 .+-. 0.02 25.63 .+-. 0.66 28.94 .+-. 0.47 27.18
.+-. 0.44 32.67 .+-. 0.43 Control Ab 17.43 .+-. 0.03 24.10 .+-.
0.01 28.99 .+-. 0.26 26.51 .+-. 0.18 31.78 .+-. 0.07
[0379] Based on our previous finding that CD47 laterally associates
with the tyrosine kinase receptor VEGFR2 (Kaur et al., J Biol Chem,
285(50):38923-32, 2010), we asked whether CD47 similarly interacts
with EGFR. Immunoprecipitation revealed that EGFR and CD47 very
small fraction of CD47 and EGFR co-immunoprecipitate, and
pretreatment with B6H12 antibody disrupted this interaction and
inhibit EGFRY.sup.1061 phosphorylation (FIG. 31A-31B). To determine
whether B6H12 treatment acutely altered EGFR tyrosine
phosphorylation, we pre-treated MDA-MB-231 cells, for 15 min with
B6H12. EGF stimulation for 5 min significantly increased EGFR
Y.sup.1061. B6H12 treatment in the presence or absence of EGF
inhibited EGFR phosphorylation at Y.sup.1068 but not Y.sup.992.
B6H12 treatment alone inhibited basal EGFR Y.sup.1068
phosphorylation as compared to untreated (FIG. 28C-28D). Similarly,
differentiated cells and bCSCs derived from MDA-MB-231 cells were
treated with either EGF alone or in combination with B6H12. B6H12
treatment inhibited basal and EGF-stimulated EGFR phosphorylation
in bCSCs but not in differentiated cells (FIG. 28E). We also
examined Y.sup.998 phosphorylation but did not observe any change
(FIG. 29B) which is consistent with (FIG. 28C). To further validate
these results, we isolated a pure population of
CD44.sup.high/CD24.sup.low MDA-MB-231 cells by cell sorting.
Stimulation of FACS-sorted bCSCs with EGF did not further increase
EGFR Y.sup.1068 phosphorylation, but B6H12 strongly inhibited EGFR
Y.sup.1068 phosphorylation in the absence and presence of
co-stimulation with EGF (FIG. 28F). These data demonstrate that
B6H12 selectively targets EGFR signaling in bCSCs by
down-regulating EGFR phosphorylation.
B6H12 Inhibits Asymmetric Cell Division and Expression of KLF4
[0380] To observe the effect of B6H12 on asymmetric cell division,
we labeled bCSCs with BrdU and chased using BrdU-free medium in the
presence of B6H12 or control antibody. The cells were immunostained
using anti-BrdU and quantified using confocal microscopy imaging
(FIG. 32A). The fraction of cells exhibiting asymmetric division
significantly decreased after B6H12 treatment.
[0381] We further analyzed the effect of B6H12 on the embryonic
stem cell markers OCT4, SOX2, NANOG and KLF4 using differentiated
cells and bCSCs. OCT4, SOX2, and NANOG immunostaining did not
change between isotype control and B6H12 treatments as well as in
microarray analysis, KLF4 decreased moderately in differentiated
cells (FIGS. 33A and 33C), but a statistically significant
reduction of KLF4 was observed in bCSCs (FIG. 32B and FIG. 34B).
Decreased KLF4 expressing was confirmed using flow cytometry (FIG.
32C). Because KLF4 is an essential gene for breast cancer stem cell
maintenance and it's decrease leads to decreased proportion of
stem/progenitor cells (Yu et al., Oncogene, 30(18):2161-72, 2011),
B6H12 treatment may reduce the number of bCSC by down-regulation of
KLF4 and inhibition of asymmetric cell division.
B6H12 Inhibits Proliferation of bCSCs and Induces Caspase 3/7
Activity
[0382] Flow cytometric analysis of equilibrium BrdU-labeled bCSCs
chased with unlabeled BrdU showed that B6H12 treatment
significantly limited the mean dilution of BrdU compared to isotype
control antibody (FIG. 32D). This indicated that ligation of CD47
by B6H12 initiates an anti-proliferative signal in bCSC, we
examined the specificity of B6H12 for TNBC by testing two ER+
breast cancer cell lines (MCF7 and T47D) and a normal immortalized
breast epithelial cell line (MCF10A). Consistent with our published
studies using lung endothelial and T cells, the CD47 blocking
antibody B6H12 increased DNA synthesis in MCF10A cells (FIG. 35B).
MCF7, a well-differentiated ER+ breast carcinoma cell line with
limited malignant potential, also exhibited a positive response to
B6H12 (FIG. 34A). However, the breast carcinoma cell line T47D and
sorted bCSC showed a similar inhibition of proliferation by B6H12
as MDA-MB-231-sorted bCSC (FIG. 35C-35D). To verify the sensitivity
of bSCS derived from MDA-MB-231 and T47D, we sorted
CD44High/CD24.sup.low cells and assessed BrdU incorporation. B6H12
significantly inhibited cell proliferation of bCSCs derived from
MDA-MB-231 and T47D (FIGS. 31E and 35D). This data indicates that
B6H12 specifically targets CD44.sup.high and CD24.sup.low subsets
of cells in triple negative and certain ER+ breast cancers that
have more tumor initiating cells but is not effective on other ER
positive breast cancers or normal breast epithelial cells (FIG.
35E).
[0383] It was previously reported that ligation CD47 on several
breast cancer cell lines by thrombospondin-1, a CD47-binding
peptide, or the CD47 antibody 1F7 induce programmed cell death
(Manna & Frazier, Cancer Res, 64(3):1026-36, 2004). One
potential mechanism for the decrease in cell proliferation
following B6H12 treatment is increased programmed cell death.
Although a CD47-binding peptide induced mitochondrial-independent
death of MDA-MB-231 and three additional breast cancer cell lines
without caspase activation, such effects of the 4N1K peptide may be
independent of CD47 (Barazi et al., J Biol Chem, 277(45):42859-66,
2002; Leclair & Lim, PLoS One, 9(5):e98358, 2014).
[0384] We treated MDA-MB-231 cells with B6H12 or isotype control
for 36 hours and found that levels of the initiator caspases-2, 3,
9 and 8 were upregulated as compared to isotype control and
untreated cells, but cleavage of these caspases were not observed
in any treatments. However, flow cytometry demonstrated that B6H12
increased executioner caspase 3/7 activity (FIGS. 32F and 32G).
Consistent with previous reports (Pettersen et al., J Immunol,
166(8):4931-42, 2001), B6H12 treatment did not induce binding of
the apoptosis indicator annexin V. Thus, B6H12-induced cell death
does not occur by the canonical apoptosis pathway.
Stem Cell and Cell Death Markers Correlate with CD47 Expression in
Human Breast Cancers
[0385] The relevance of this in vitro data to human breast cancers
was examined using cBioPortal tools to analyze The Cancer Genome
Atlas (TCGA) mRNA and protein expression data for invasive breast
carcinoma (Cancer Genome Atlas Network, Nature, 490(7418):61-70,
2012; Gao et al., Sci Signal, 6(269):pl1, 2013; Cerami et al.,
Cancer Discov, 2(5):401-4, 2012). CD47 mRNA expression >1 SD
higher than the mean by RNAseq analysis was associated with
significantly decreased overall survival (median survival 93.7
versus 114.7 months, log rank p-value 0.029, FIG. 36A). Consistent
with the diminished sensitivity of ER.sup.+ breast cancer cells to
B6H12 observed here and a previous report that basal breast
carcinomas express higher CD47 (Zhao et al., Proc Natl Acad Sci
USA, 108(45):18342-7, 2011), CD47 mRNA expression in the TCGA
dataset was negatively correlated with ER and with HER2 protein
expression (p=1.7.times.10.sup.-6 and 2.5.times.10.sup.-5,
respectively, FIG. 34B-34C). In contrast to HER2 but consistent
with our in vitro data, EGFR protein expression in the tumors
positively correlated with CD47 mRNA expression (p=0.009, FIG.
32D)
[0386] CD47 mRNA expression data showed a strong positive
correlation with levels of caspases-7 protein cleaved at
Asp.sup.198 (FIG. 32F, G, p=2.6.times.10.sup.-6, FIG. 36E). This
was the fifth most significant change in protein levels associated
with CD47 mRNA expression in the breast cancer dataset. In
contrast, levels of the anti-apoptotic protein Bcl2 were
significantly lower in breast cancers with elevated CD47 mRNA
(p=0.001). Similar positive correlations between CD47 mRNA and
caspase-7 cleavage were found in TCGA melanoma
(p=1.times.10.sup.-3) and bladder carcinoma (p=9.7.times.10.sup.-5)
datasets. These results are consistent with a recent study showing
that long term blocking of CD47 using B6H12 directly induced
apoptosis of pancreatic cancer stem cells in the absence of
macrophages (Cioffi et al., Clin Cancer Res, 21:2325, 2015). This
suggests that high CD47 expression in cancer stem cells may
generally increase caspase-7 cleavage and be a liability for their
survival.
[0387] Several known stem cell markers correlated with CD47 mRNA
expression in the TCGA breast carcinoma dataset including a
positive correlation with cKit protein expression (p=0.042). In
contrast, phosphorylation of PDK1 at Ser241, which induces a cancer
stem cell gene expression signature (Tan et al., Cancer Discov,
3(10):1156-71, 2013; Casamayor et al., Biochem J, 342(Pt 2):287-92,
1999), was negatively correlated with CD47 mRNA expression and was
the fourth most significant change in protein phosphorylation in
the breast cancer dataset (p=0.0015). The transcription factor
PATZ1 maintains stem cells by its regulation of Pou5f1 and Nanog
(Ow et al., Stem Cells Dev, 23(10):1062-73, 2014). PATZ1 mRNA
expression in breast cancers negatively correlated with CD47 mRNA
expression (Spearman correlation=-0.41, FIG. 37C). Similar negative
correlations between PATZ1 and CD47 mRNA expression were found in
TCGA datasets for melanoma (-0.41), head and neck squamous cell
carcinoma (-0.45), and bladder carcinoma (-0.50). Therefore,
elevated CD47 expression correlates with known markers and
regulators of stem cell maintenance in breast tumors, but CD47
expression may have both positive and negative effects on bCSC
differentiation.
[0388] We examined the specificity of B6H12 for TNBC by testing two
ER+ breast cancer cell lines (MCF7 and T47D) and a normal
immortalized breast epithelial cell line (MCF10A). Consistent with
our published studies using lung endothelial and T cells, the CD47
blocking antibody B6H12 increased DNA synthesis in MCF10A cells.
However, the breast carcinoma cell line T47D and sorted bCSC showed
a similar inhibition of proliferation by B6H12 as MDA-MB-231-sorted
bCSC. This data indicates that B6H12 specifically targets CD44high
and CD24low subsets of cells in triple negative and certain ER+
breast cancers that have more tumor initiating cells but is not
effective on other ER positive breast cancers or normal breast
epithelial cells. Our results indicate that bCSC express both high
levels of CD47 and characteristic stem cell genes. Treatment with
B6H12 down regulates KLF4 and inhibits asymmetric cell division of
bCSCs. The treated cells resemble differentiated MDA-MB-231 cells
and have limited proliferative capacity. This suggests that CD47
expression supports bCSC maintenance.
DISCUSSION
[0389] The premise for developing therapeutic antibodies that
target CD47 was that high expression of this cell surface protein
protects tumor cells from host innate immune surveillance (Jaiswal
et al., Cell, 138(2):271-85, 2009). However, we recently reported
that expression of CD47 in non-transformed cells plays a critical
role in regulating stem cell homeostasis. Specifically, CD47
signaling inhibits the expression of cMyc, SOX2, OCT3/4 and KLF4
(Kaur et al., Sci Rep, 3:1673, 2013). Decreasing CD47 in
non-transformed cells increases their self-renewal, asymmetric
division and ability to reprogram into other differentiated cell
types. Because, high CD47 expression limits the stem cell character
of non-transformed cells, the high expression of CD47 on cancer
stem cells appeared paradoxical, suggesting that the CD47 expressed
on CSC may lack the signaling activity to control stem cell
self-renewal, either due to alterations in the CD47 or inactivation
of pathways that mediate its signaling in CSC. A recent study in
hepatocarcinoma stem cells demonstrated that reducing CD47
expression resulted in loss of stem cell character (Lee et al.,
Hepatology, 60(1):179-91, 2014), suggesting that CD47 signaling
differentially regulates normal versus malignant stem cells.
Conversely, treatment with the CD47 ligand thrombospondin-1 was
recently reported to inhibit proliferation, sphere formation, and
expression of stem cell transcription factors in Lewis lung
carcinoma cells, and CD47 shRNA knockdown blocked this activity
(Zheng et al., J Biol Chem, 290(14):8975-86, 2015). Our results
indicate that bCSC express both high levels of CD47 and
characteristic stem cell genes. Treatment with B6H12 down regulates
KLF4 and inhibits asymmetric cell division of bCSCs. The treated
cells resemble differentiated MDA-MB-231 cells and have limited
proliferative capacity. This suggests that CD47 expression supports
bCSC maintenance. However, increased CD47 expression in human
breast cancers correlates with increased cleavage of the
executioner caspase-7 and loss of Bcl2. Therefore, maintaining high
CD47 expression to evade host immunity and support CSC may have an
associated cost in terms of tumor cell viability.
[0390] From a therapeutic perspective, our data indicates that the
prototypical CD47 "blocking" antibody B6H12 has a second activity
that could provide therapeutic benefit by suppressing stem cell
character in bCSC. B6H12 down regulates EGFR expression at the mRNA
and protein levels and inhibits Tyr.sup.1068 phosphorylation of
EGFR. CD47 and EGFR co-immunoprecipitation and their interaction
are disturbed by B6H12, but only a small fraction of these proteins
interact and co-localize at the cell surface however B6H12 alone
enhance EGFR release on exosomes only in TNBC. Currently EGFR
inhibitors are in phase II clinical trials and show efficacy to
inhibit tumor growth in xenograft models (Gluz et al., Ann Oncol,
20(12):1913-27, 2009; Zhang et al., Clin Cancer Res,
15(21):6639-48, 2009; Friess et al., Clin Cancer Res,
11(14):5300-9, 2005; Sohn et al., J Cancer, 5(9):745-53, 2014). The
correlation between CD47 and EGFR expression in human breast tumors
suggests that therapeutic CD47 antibodies may be effective against
tumors with high EGFR expression alone or in combination with EGFR
inhibitors.
[0391] B6H12 may also limit tumor growth by inhibiting asymmetric
division of CSCs. This activity was observed in a TNBC line, but
not in the less aggressive MCF7 cell line or MCF10 immortalized
mammary epithelial cells, which showed increased proliferation
consistent with our primary endothelial cell data (Kaur et al., Sci
Rep, 3:1673, 2013). Based on our data and a recent report that
shRNA knockdown of CD47 suppressed cancer stem cells of hepatocyte
carcinoma cells (Lee et al., Hepatology, 60(1):179-91, 2014),
suggesting that a direct cell-autonomous effects of therapeutic
CD47 antibodies to suppress CSC may extend to additional
cancers.
[0392] Others have shown that certain CD47 antibodies, but not
B6H12, directly induce apoptosis of B-cell chronic lymphocytic
leukemia associated with cell shrinkage, decreased mitochondrial
transmembrane potential, and phosphatidylserine externalization,
but independent of apoptotic caspase activation (Pettersen et al.,
J Immunol, 166(8):4931-42, 2001). These studies suggest that each
CD47 antibody may have different effects on CD47 signaling, which
may involve direct agonist activities of a given antibody as well
as antagonism of the signaling induced by SIRP.alpha. or TSP1
binding.
[0393] In summary, these data demonstrate that CD47 is an active
signaling receptor in triple negative human breast cancer. The CD47
antibody B6H12 directly inhibits cell growth and CSC maintenance in
an aggressive subset of the breast cancer cell lines we have
tested, whereas stimulation of proliferation was also observed in
cell lines with less malignant potential, which is consistent with
known CD47 signaling in non-transformed cells. The EGFR
phosphorylation data presented here reveals a novel lateral
signaling mechanism through which B6H12 inhibits proliferation of
aggressive cancer cells.
[0394] In view of the many possible embodiments to which the
principles of the disclosed invention may be applied, it should be
recognized that the illustrated embodiments are only preferred
examples of the invention and should not be taken as limiting the
scope of the invention. Rather, the scope of the invention is
defined by the following claims. We therefore claim as our
invention all that comes within the scope and spirit of these
claims.
TABLE-US-00004 TABLE 1 Fold- Change Entrez Gene RefSeq p-value
(B6H12_Ab Col # Column ID Gene Symbol Gene Title Transcript ID
(Attribute) vs. Ctrl_Ab) 7865 1565483_at 1956 EGFR epidermal
NM_005228 /// 0.00758284 -1.9058 growth NM_201282 /// factor
NM_201283 /// receptor NM_201284 7866 1565484_x_at 1956 EGFR
epidermal NM_005228 /// 0.00929699 -1.09678 growth NM_201282 ///
factor NM_201283 /// receptor NM_201284 50111 240854_x_at -- -- --
-- 0.00716955 -1.55596 43862 234605_at 8555 CDC14B CDC14 cell
NM_001077181 /// 0.00145854 1.50173 division NM_003671 /// cycle 14
NM_033331 /// homolog B NM_033332 S. cerevisiae) 49205 239948_at
9972 NUP153 nucleoporin NM_005124 0.00537521 1.50258 153 kDa 13503
204048_s_at 9749 PHACTR2 phosphatase NM_001100164 /// 0.0141831
1.50419 and actin NM_001100165 /// regulator 2 NM_001100166 ///
NM_014721 53002 243745_at -- -- -- -- 0.00180785 1.50472 52905
243648_at 100381270 ZBED6 zinc NM_001174108 0.0219142 1.50648
finger, BED-type containing 6 31907 222620_s_at 64215 DNAJC1 DnaJ
(Hsp40) NM_022365 0.000177968 1.50759 homolog subfamily C, member 1
33266 223984_s_at 9818 NUPL1 nucleoporin NM _001008564 ///
0.0426645 1.50852 like 1 NM_901008565 /// NM_014089 4385 1558733_at
253461 ZBTB38 zinc NM_001080412 /// 0.037835 1.50948 finger,
NM_152535 and BTB domain containing 38 10370 200914_x_at 3895 KTN1
kinectin 1 NM_001079521 /// 0.00536708 1.50981 (kinesin NM
_001079522 /// receptor) NM_004986 /// NM_182926 48863 239606_at --
-- -- -- 0.00109509 1.51 49561 240304_s_at 79838 TMC5 transmembrane
NM_001105248 /// 0.0212464 1.51049 channel-like 5 NM_001105249 ///
NM_001261841 /// NM_024780 / 38782 229520_s_at 55668 C14orf118
chromosome NM_017926 /// 0.000662047 1.51135 14 open NM_017972
reading frame 118 30846 221555_x_at 8555 CDC14B CDC14 cell
NM_001077181 /// 0.0488715 1.5127 division NM_003671 /// cycle 14
NM_033331 /// homolog B NM_033332 S. cerevisiae) 17149 207700_s_att
8202 NCOA3 nuclear NM_001174087 // 0.0104055 1.51282 receptor
NM_001174088 /// coactivator 3 NM_006534 /// NM_181659 44682
235425_at 151246 SGOL2 shugoshin- NM_001160033 /// 0.00247415
1.51305 like 2 NM_001160046 /// S. pombe) NM_152524 28863
219571_s_at 7559 ZNF12 zinc finger NM_006956 /// 0.00148539 1.5166
protein 12 NM_016265 41357 232095_at 100509683 LOC100509683
uncharacterized XR_112004 0.00460052 1.51713 LOC100509683 14770
205315_s_at 6645 SNTB2 syntrophin, NM_006750 /// 0.000695291
1.51732 beta 2, NM_130845 (dystrophin- associated protein A1, 59
kDa, basic component 2) 46009 236752_at -- -- -- -- 0.0108735
1.51862 42339 233078_at 8539 API5 apoptosis NM_001142930 ///
0.0074819 1.5193 inhibitor 5 NM_001142931 /// NM_001243747 ///
NM_006595 /// NR_024625 54032 244774_at 9749 PHACTR2 phosphatase
0.0216076 1.52175 and actin NM_001100164 /// regulator 2
NM_001100165 /// NM_001100165 /// NM_014721 33528 224250_s_at 79048
SECISBP2 SECIS NM_024077 0.000168024 1.52234 binding protein 2
11373 201917_at 55186 SLC25A36 solute NM_002204547 /// 0.00462846
1.52367 carrier NM_018155 family 25 pyrimidine nucleotide carrier),
member 36 49912 240655_at -- -- -- -- 0.0345829 1.52775 21534
212220_at 23198 PSME4 proteasome NM_014614 0.0146262 1.52807
(prosome, macropain) activator subunit 4 2498 1555677_s_at 8243
SMC1A structural NM_006306 0.0006529 1.52887 maintenance of
chromosomes 1A 27814 218521_s_at 55284 UBE2W ubiquitin-
NM_001001481 /// 0.0214975 1.53094 conjugating NM_001001482 ///
enzyme NM_018299 E2W (putative) 1390 1554178_a_at 285172 FAM1268
family with NM_173822 0.00140223 1.53106 sequence similarity 126,
member B 5111 1559993_at 81855 SFXN3 Sideroflexin 3 NM_030971
0.0256685 1.53147 21427 212113_at 552889 ATXN7L3B ataxin
NM_001136262 0.092876 1.53378 7-like 3B 31975 222688_at 55331 ACER3
alkaline NM_018367 0.00568208 1.53322 ceramidase 3 48650 239393_at
-- -- -- -- 0.0333635 1.53379 29867 220575_at 80039 FAM106A family
with NM_024974 /// 0.0473866 1.53439 sequence NM_026809 similarity
106, member A 37002 227740_at 127933 UHMK1 U2AF NM_001184763 ///
0.00366961 1.5347 homology NM_144524 /// motif NM_175866 (UHM)
kinase 1 12428 202971_s_at 8445 DYRK2 dual- NM_003583 /// 0.0135754
1.53583 specificity NM_006482 tyrosine- (Y)- phosphorylation
regulated kinase 2 48649 239392_s_at 57645 POGK pogo NM_017542
0.000501282 1.53627 transposable element with KRAB domain 13502
204047_s_at 9749 PHACTR2 phosphatase NM_001100164 /// 0.00411187
1.53867 and actin NM_001100165 /// regulator 2 NM_001100166 ///
NM_014721 50640 241391_at -- -- -- -- 0.00027805 1.53944 12432
202975_s_at 22836 RHOBTB3 Rho-related NM_14899 0.000538566 1.5403
BTB domain containing 3 35318 226054_at 23476 BRD4 bromodomain
NM_058243 0.00748653 1.5403 containing 4 47598 238341_at -- -- --
-- 0.00349413 1.54212 39892 230630_at 100507855 /// AK4 ///
adenylate NM_001005353 /// 0.0236654 1.54404 205 LOC100507855
kinase 4 /// NM_013410 /// adenylate NM_203464 /// kinase
XM_003119530 isoenzyme 4, mitochondrial- like 36643 227383_att
727820 LOC727820 uncharacterized XR_041980 0.00982574 1.54717
LOC727820 53599 244341_at -- -- -- -- 0.00832358 1.54938 45781
236524_at -- -- -- -- 0.00272043 1.54934 48999 239742_at 56995
TULP4 Tubby like NM_001007466 /// 0.0419099 1.55012 protein 4
NM_020245 38295 229033_s_at 84939 MUM1 melanoma NM_032853 ///
0.0350633 1.55263 associated NR_024247 antigen (mutated) 1 11439
201983_s_at 1956 EGFR epidermal NM_005228 /// 0.0160253 1.55342
growth NM_201282 /// factor NM_201283 /// receptor NM_201284 37193
227931_at 54891 INO8D INO8D complex NM_017759 0.00144066 1.55524
subunit D 30232 220940_at 57730 ANKRD368 ankyrin repeat NM_020970
/// 0.00169712 1.55578 domain 36B NM_025190 9181 1569362_at 214
ALCAM activated NM_001243280 /// 0.00991597 1.55629 leukocyte
NM_001243281 /// cell NM_001243283 /// adhesion NM_001627 molecule
38848 229586_at 80205 CHD9 chromodomain NM _025134 0.000565799
1.55647 helicase DNA binding protein 9 40665 231403_at 7204 TRIO
triple NM_007118 0.000351701 1.55759 functional domain (PTPRF
interacting) 2044 1555058_a_at 9926 LPGAT1 lysophosphatidyl-
NM_014873 0.0246991 1.55787 glycerol acyltransferase 1 19548
210136_at 4155 MBP myelin NM_001025081 /// 0.00513611 1.55807 basic
NM_001025090 /// protein NM_001025092 /// NM_001025094 ///
NM_001025098 /// NM 17407 207966_s_at 2734 GLG1 goigi NM_001145666
/// 0.0150821 1.561042 glycoprotein 1 NM_001145667 /// NM _012201
/// NR_027264 /// NR_027265 44314 235057_at 83737 ITCH itchy E3
NM_001257137 /// 0.0267355 1.56109 ubiquitin NM_001257138 protein
NM_031483 ligase 24526 215224_at 619505 SNORA21 small NR_002576
0.000536919 1.56208 nucleolar RNA, H/ACA box 21 22539 213229_at
23405 DICER1 dicer 1, NM_001195573 /// 0.0200868 1.56391
ribonuclease NM_030621 /// type III NM_177438 11835 202379_s_at
4820 NKTR natural NM_001012654 /// 0.00469482 1.56815 killer-
NM_005385 tumor recognition sequence 15515 206061_s_at 23405 DICER1
dicer 1, NM_001195573 /// 0.0255576 1.5686
ribonuclease NM_030621 /// type III NM_177438 41270 232008_s_at
56987 BBX bobby sox NM_001142568 /// 0.00092687 1.56873 homolog
NM_020235 (Drosophila) 46640 237383_at -- -- -- -- 0.00131189
1.57192 52270 243013_at -- -- -- -- 0.00115308 1.57343 5295
1560318_at 9411 ARHGAP29 Rho NM_004815 0.000103225 1.57462 GTPase
activating protein 29 10755 201299_s_at 55233 MOB1A MOB kinase
NM_018221 0.0102216 1.57694 activator 1A 45257 236000_s_at -- -- --
-- 0.0120607 1.57749 36520 227257_s_at 143384 CACUL1 CDK2-
NM_153810 0.00322839 1.57983 associated cullin domain 1 37391
228129_at 26135 SERBP1 SERPINE1 NM_001018067 /// 0.00444628 1.58016
mRNA NM_001018068 /// binding NM_001018069 /// protein 1 NM_015640
38615 229353_s_at 64710 NUCKS1 nuclear NM_022731 0.00527112 1.58045
casein kinase and cyclin- dependent kinase substrate 1 31827
222540_s_at 51773 RSF1 remodeling NM_016578 0.00134395 1.58096 and
spacing factor 1 48044 238787_at 163486 DENND1B DENN/ NM_001142795
/// 0.003337621 1.58315 MADD NM_001195215 /// containing
NM_001195216 /// 1B NM_019049 /// NM _144977 53443 244185_at -- --
-- -- 0.00109563 1.58432 18679 209257_s_at 9126 5MC3 structural
NM_005445 0.00218202 1.58551 maintenance of chromosomes 3 52846
243589_at 284058 KANSL1 KATB NM_001193465 /// 0.0109478 1.58729
regulatory NM_001193466 /// NSL NM_015443 /// complex XM_003403598
/// subunit 1 XM_003403599 /// XM_00 31831 222544_s_at 54904
WHSC1L1 Wolf- NM_017778 /// 0.0105939 1.59152 Hirschhorn NM_023034
syndrome candidate 1- like 1 4581 1559060_a_at 96459 FNIP1
Folliculin NM_001008738 /// 0.017307 1.59165 interacting NM_133372
protein 1 34354 225089_at 55230 USP40 ubiquitin NM_018218
0.000538702 1.592 specific peptidase 40 16511 207057_at 9194
SLC16A7 solute NM_004731 0.000510397 1.59641 carrier family 16,
member 7 (monocarboxylic acid transporter 2) 9247 1569472_s_at
286495 /// TTC3 /// tetra- NM_001001894 /// 0.00421749 1.59679 7247
TTC3P1 tricopeptide NM_003316 /// repeat NR_030737 domain 3 ///
tetra- tricopeptide repeat domain 3 pseudogene 1 13083 203628_at
3480 IGF1R insulin- NM_000875 /// 0.006082 1.59853 like NM_152452
growth factor 1 receptor 34199 224933_ s_at 221037 JMJD1C jumonji
NM_004241 /// 0.00521599 1.59926 domain NM_032776 containing 1C
13258 203203_at 51449 PCYOX1 prenyicysteine NM_016297 0.00020689
1.59966 oxidase 1 29364 220072_at 79848 CSPP1 centrosome
NM_001077204 /// 0.00731063 1.60277 and NM_024790 spindle pole
associated protein 1 45729 236472_at -- -- -- -- 0.00720358 1.603
44645 235388_at 80205 CHD9 chromodomain NM_025134 0.00343612
1.60332 helicase DNA binding protein 9 30972 221683_s_at 80184
CEP290 centrosomal NM_025114 0.0430594 1.60434 protein 290 kDa
43290 234032_at -- -- -- -- 0.0136508 1.60619 37146 227884_at 8148
TAF15 TAF15 NM_003487 /// 0.015747 1.60873 RNA NM_139215 polymerase
II, TATA box binding protein (TBP)- associated factor, 68 kDa 45613
236356_at 4719 NDUFS1 NADH NM_001199981 /// 0.0240567 1.61047
dehydrogenase NM_001199982 /// (ubiquinone) NM_001199983 /// Fe-S
protein 1 NM_001199984 /// 75 kDa NM_005006 (NADH- coenzyme Q
reductase) 34755 225490_at 196528 ARID2 AT rich NM_152641
0.00224833 1.61096 interactive domain 2 (ARID, RFX-like) 48588
239331_at -- -- -- -- 0.00275564 1.6118 20457 211085_s_at 6789 STK4
serine/ NM_006282 0.0235459 1.61205 threonine kinase 4 30794
221503_s_at 3839 KPNA3 karyopherin NM_02267 0.0205337 1.61663 alpha
3 (importin alpha 4) 38569 229307_at 23243 ANKRD28 ankyrin
NM_001195098 /// 0.00139142 1.61668 repeat NM_001195099 /// domain
28 NM_015199 18353 208930_s_at 3609 ILF3 interleukin NM_001137673
/// 0.0182301 1.61869 enhancer NM_004516 /// binding NM_012218 ///
factor 3, NM_017620 /// 90 kDa NM_153464 45060 235803_at -- -- --
-- 0.0225123 1.62271 48279 239022_at -- -- -- -- 0.0334072 1.62429
22887 213579_s_at 2033 EP300 E1A NM_001429 0.0036532 1.62606
binding protein 300 46098 236841_at 100134445 LOC100134445
uncharacterized XM_001720526 0.00243628 1.62736 LOC1001341445 36717
227454_at 57551 TAOK1 TAO Kinase 1 NM_020791 /// 0.00720828 1.62742
NM_025142 12671 203215_s_at 4646 MYO6 myosin VI NM_004999 0.0126125
1.62761 34836 225571_at 3977 LIFR leukemia NM_001127671 ///
0.0161661 1.62983 inhibitory NM_002310 factor reception alpha 28450
219158_s_at 80155 NAA15 N(alpha)- NM_057175 0.00232713 1.63131
acetyltransferase 15 NatA auxiliary subunit 52940 243683_at 9643
MORF4L2 Mortality NM_001142418 /// 0.0205152 1.63181 factor 4
NM_001142419 /// like 2 NM_001142420 /// NM_001142421///
NM_001142422 /// NM 18680 209258_s_a t 9126 SMC3 structural
NM_005445 0.00172564 1.63185 maintenance of chromosomes 3 7160
1563321_s_at 8028 MLLT10 myeloid/ NM_001009569 /// 0.0176147
1.63255 lymphoid NM_001195626 /// or mixed- NM_001195627 ///
lineage NM_001195628 /// leukemia NM_001195630 /// (trithorax- NM
homolog (Drosophila); translocate 44783 235526_at 55553 SOX6 SRY
(sex NM_001145811 /// 0.0330642 1.63454 determining NM_001145819
/// region Y)- NM_017508 /// box6 NM_033326 36776 227514_at 162073
ITPRIPL2 inositol-1,4,5- NM_001034841 /// 0.0145369 trisphosphate
NM_028028 receptor interacting protein-like 2 38661 229399_at 55088
C10orf118 chromosome 10 NM_018017 0.0413405 1.63704 open reading
frame 118 33223 223940_x_at LOC100507645 /// LOC100507645 ///
uncharacterized NR_02819 /// _ 0.0292591 1.64434 378938 MALAT1
LOC100507645 /// XR_110915 /// metastasis XR_110916 /// associated
XR_110917 /// lung XR_111190 /// adenocarcinoma XR_111191 ///
transcript 1 XR 51077 241820_at 55183 RIF1 RAP1 NM_0011776643 ///
0.0245286 1.64497 interacting NM_001177664 /// factor NM_001177665
/// homolog NM_018151 (yeast) 3442 1557100_s_at 25831 HECTD1 HECT
NM_015382 0.0252237 1.64535 domain containg E3 ubiquitin protein
ligase 1 28513 219221_at 253461 ZBTB38 zinc finger NM_001080412 ///
0.0011571 1.64824 and BTB NM_152535 domain containing 38 10638
201182_s_at 1108 CHD4 chromodomain NM_001273 0.0058932 1.64852
helicase DNA binding protein 4 51967 242710_at -- -- -- --
0.0327547 1.64964 11452 201996_s_at 23013 spen homolog, NM_015001
0.0171906 1.65008 transcriptional regulator (Drosophila) 36647
227384_s_at 727820 /// LOC727820 /// uncharacterized NR_024510 ///
0.0255387 1.65063 728855 /// LOC728855 /// LOC727820 /// NR_024511
/// 728875 LOC728875 uncharacterized NR_024584 /// LOC728855 ///
XR_041980 uncharacterized LOC728875 5267 1560271_at -- -- -- --
0.00391521 1.65177 42186 232925_at -- -- -- -- 0.038837 1.65236
27244 217951_s_at 23469 PHF3 PHD finger NM_015153 0.00244501
1.65357 protein 3 51490 242233_at -- -- -- -- 0.00271001
1.65646
3544 1557267_s_at 348654 GEN1 Gen NM_001130009 /// 0.0228586
1.66227 endonuclease NM_182625 homolog 1 (Drosophila) 41128
231866_at 4012 LNPEP leucyl/ NM_005575 /// 0.00429813 1.66515
cystinyl NM_175920 aminopeptidase 4048 1558080_s_at 5611 DNAIC3
DnaJ (Hsp40) NM_006260 0.00438813 1.66648 homolog subfamily C.
member 3 34685 225420_at 57678 GPAM glycerol-3- NM_001244949 ///
0.0679399 1.65814 phosphate NM_020918 acyltransferase mitochondrial
45525 236268_at 9117 SEC22C SEC22 NM_001201572 /// 0.00208389
1.67104 vesicle NM_001201584 /// trafficking NM_004206 /// protein
NM_032970 homolog (S.cerevisiae) 39120 229858_at -- -- -- --
0.0386334 1.68237 49564 240307_at -- -- -- -- 0.0282523 1.68804
44523 2.35266_at 29028 ATAD2 ATPase family, NM_014109 0.0040998
1.69135 AAA domain containing 2 32861 223577_x_at 378938 MALAT1
metastasis NR_002819 0.0152794 1.69738 associated lung
adenocarcinoma transcript 1 (non-protein coding) 17459 208022_s_at
8555 CDC14B CDC14 cell NM_001077181 /// 0.00142907 1.69967 division
NM_003671 /// cycle 14 NM_0333321 /// homolog B NM_033332
(S.cerevisiae) 10796 201340_at 8507 ENC1 ectodermal- NM_001256574
/// 0.0138129 1.70015 neural NM_001256575 /// cortex 1 NM 001256576
/// (with BTB- NM_003633 /// like domain) NR_046318 44979 235722_at
55333 SYNJ2BP synaptojanin 2 NM_018373 0.0217892 1.70451 binding
protein 37418 228156_at -- -- -- -- 0.00856184 1.70602 24924
215623_x_at 10051 SMC4 structural NM_001002799 /// 0.000631502
1.70906 maintenance of NM_001002800 /// chromosomes 4 NM_005496
31920 222633_at 79718 TBL1XR1 transducin NM_024665 0.00298632
1.71649 (beta)-like 1 X-linked receptor 1 33102 223818_s_at 51773
RSF1 remodeling NM_ 016578 0.00129842 1.72008 and spacing factor 1
27106 217813_s_at 10927 SPIN1 spindlin 1 NM_006717 0.00939252
1.72364 19166 209750_at 9975 NR1D2 nuclear NM_001145425 ///
0.00030303 1.72724 receptor NM_005126 subfamily 1, group D, member
2 18302 208879_x_at 24148 PRP6 PRP6 pre- NM_012469 0.00561885
1.73723 mRNA processing factor 6 homolog (S.cerevisiae) 33898
224631_at 80829 ZFP91 zinc finger NM_001197051 /// 0.00241181
1.73771 protein 91 NM_053023 homolog (mouse) 48018 238761_at 2005
ELK4 ELK4, ETS- NM_001973 /// 0.0162532 1.74057 domain protein
NM_021795 (SRF accessory protein 1) 17440 208003_s_at 10725 NFATS
nuclear NM_001113178 /// 0.00457515 1.74202 factor of NM_006599 ///
activated NM_138713 /// T-cells 5, NM_138714 /// tonicity-
NM_173214 /// responsive NM_173215 256 1552611_a_at 3716 JAK1 Janus
kinase 1 NM_002227 0.0186674 1.74617 33835 224568_ x_at 100507645
/// LOC100507645 /// uncharacterized NR_002819 /// 0.0404992
1.75208 378938 MALAT1 LOC100507645 /// XR_110915 /// metastasis
XR_110916 /// associated XR_110917 /// lung XR_111190 ///
adenocarcinoma XR_111191 /// transcripts 1 XR_ 10541 201085_s_t
6651 SON SON DNA NM_003103 /// 0.00303166 1.75255 binding NM_032195
/// protein NM_058183 /// NM_138925 /// NM_138926 /// NM_138927
21392 212078_s_at 4297 MLL myeloid/ NM_001197104 /// 0.00635272
1.73681 lymphoid NM_005933 /// or mixed NM_024891 lineage leukemia
(trithorax homolog, Drosophila) 43958 234701_at 29123 ANKRD11
ankyrin NM_001256182 /// 0.00023534 1.75952 repeat NM_001256183 ///
domain 11 NM_013275 /// NR_045839 11323 201867_s_at 6907 TBL1X
transducin NM_001139466 /// 0.00368541 1.76902 (beta)-like
NM_001139467 /// 1X linked NM_001139468 /// NM_005647 49947 240690t
-- -- -- -- 0.00198799 1.76926 15954 206500_s_at 55320 MIS18BP1
MIS18 NM_18353 0.0259793 1.7749 binding protein 1 44316 235059_at
201475 RAB12 RAB12, NM_001025300 0.00165136 1.77978 member RAS
oncogene family 44266 235009_at 259282 BOD1L1 biorientation of
NM_148894 0.0241619 1.77982 chromosomes in cell division 1- like 1
10291 200835_s_at 4134 microtubule- NM_001134364 /// 0.00250008
1.78104 associated NM_001134365 /// protein 4 NM_002375 ///
NM_030884 /// NM_030885 44966 235709_at 283431 GAS2L3 growth
NM_174942 0.0256883 1.78185 arrest- specific 2 like 3 4392
1558747_at 23347 SMCHD1 structural NM_015295 0.0330839 1.78271
maintenance of chromosomes flexible hinge domain containing 1 37740
228478_at -- -- -- -- 0.0192989 1.78339 27171 217878_s_at 996 CDC27
cell division NM_0011114091 /// 0.039132 1.78366 cycle 27 NM_001256
homolog (S.cerevisiae) 39756 230494_at 6574 SLC20A1 solute
NM_005415 0.00670771 1.78563 carrier family 20 (phosphate
transporter), member 1 38534 229272_at 23360 FNBP4 formin NM_015308
0.00528746 1.78979 binding protein 4 48303 239046_at -- -- -- --
0.00260028 1.78979 31700 222413_s_at 58508 MLL3 myeloid/ NM_021230
/// 0.0276727 1.79679 lymphoid NM_170606 or mixed lineage leukemia
3 24288 214985_at 2131 EXT1 exostosin 1 NM_000127 0.0112472 1.80175
24640 215338_s_at 4820 NKTR natural NM_001012651 /// 0.00360133
1.80846 killer- NM_005385 tumor recognition sequence 4092
1558173_a_at 7798 LUZP1 leucine NM_001142546 /// 0.0100121 1.81185
zipper NM_033631 protein 1 18808 208663_s_at 286495 /// TTC3 ///
tetra- NM_001001894 /// 0.00674795 1.81703 7267 TTC3P1 tricopeptide
NM_003316 /// repeat NR_030737 domain 3 /// tetra- tricopeptide
repeat domain 3 pseudogene 1 12125 202669_s_at 1948 EFNB2 ephrin-B2
NM_004093 0.0226477 1.82888 18134 208711_s_at 595 CCND1 cyclin D1
NM_053056 0.0115323 1.83042 25237 215936_s_at 23325 KIAA1033
KIAA1033 NM_015275 0.0166419 1.83448 24508 215206_at -- -- -- --
0.00376487 1.8582 7608 1564378_a_at -- -- -- -- 0.0045512 1.85918
25861 216563_at 23253 ANKRD12 ankyrin NM_001083625 /// 0.00786514
1.87083 repeat NM_001204056 /// domain 12 NM_015208 38377 229115_at
1778 DYNC1H1 dynein, NM_001376 0.00311892 1.87432 cytoplasmic 1,
heavy chain 1 20396 211022_s_at 546 ATRX alpha NM_000489 ///
0.0304891 1.87675 thalassemia/ NM_138270 /// mental NM_138271
retardation syndrome X-linked 14291 204840_s_at 8411 EEA1 early
NM_003566 0.00819668 1.88274 endosome antigen 1 21963 212650_at
23301 EHBP1 EH domain NM_0001142614 /// 0.009319 1.88314 binding
NM_001142615 /// protein 1 NM_001142616 /// NM_015252 10297
200841_s_at 2058 EPRS glutamyl- NM_004446 0.00258631 1.93041
prolyl-tRNA synthetase 21781 212468_at 9043 SPAG9 sperm
NM_001130527 /// 0.00362449 1.93517 associated NM_01130528 ///
antigen 9 NM_001251971 /// NM_003971 /// NM_172325 22107
212794_s_at 23325 KIAA1033 KIAA1033 NM_015275 0.00765823 1.94138
22378 213067_at 4628 MYH10 myosin, heavy NM_001256012 ///
0.00750319 1.94171 chain 10, NM_001256095 /// non-muscle NM_005964
2764 1556054_at -- -- -- -- 0.00387442 1.98692 40997 231735_s_at
378938 MALAT1 metastasis NR_002819 0.00182841 1.99074 lung
adenocarcinoma transcript 1 (non-protein coding) 51325 242068_at --
-- -- -- 0.0197402 1.99077 39027 229756_at 7756 ZNF207 zinc finger
NM_001032293 /// 0.000769356 1.99093 protein 207 NM_001098507 ///
NM_003457 51517 242260_at 9782 MATR3 Matrin 3 NM_001194954 ///
0.018593 2.00734 NM_0001194955 /// NM_001194956 /// NM_018834 ///
NM_199189 /// NM_03653 23659 214352_s_at 3845 KRAS v-Ki-ras2
NM_004985 /// 0.0262788 2.03062 Kirsten NM_033360
rat sarcoma viral oncogene homolog 18049 208624_s_at 1981 EIF4G1
eukaryotic NM_001194946 /// 0.0119994 2.03273 translation
NM_001194947 /// intiation NM_004953 /// factor 4 NM_182917 ///
gamma, 1 NM_198241 /// NM_198242 / 29377 220085_at 3070 HELLS
helicase, NM_018063 0.00839666 2.03541 lymphoid- specific 51212
241955_at 25831 HECTD1 HECT NM_015382 0.000788387 2.03696 domain
containing E3 ubiquitin protein ligase 1 50030 240773_at -- -- --
-- 0.0137585 2.04324 31726 222439_s_at 9967 THRAP3 thyroid
NM_005119 0.00504398 2.05116 hormone receptor associated protein 3
3461 1557129_a_at 374393 FAM1116 family with NM_001142703 ///
0.0287632 2.0535 sequence NM_001142704 /// similarity 111,
NM_198947 member B 28679 219387_ at 55704 CCDC88A coiled-coil
NM_001135597 /// 0.00355158 2.0778 domain NM_001254943 ///
containing NM_018084 88A 4502 1558924_s_at 6249 CLIP1 CAP-GLY
NM_001241997 /// 0.0267528 2.09463 domain NM_002956 /// containing
NM_198240 linker protein 1 53114 243857_at 9643 MORF4L2 Mortality
NM_001142418 /// 0.0074179 2.13027 factor 4 NM_001142419 /// like 2
NM_001142420 /// NM_001142421 /// NM_01142422 /// NM 18323
208900_s_at 7150 TOP1 topoisomerase NM_003286 0.0365807 2.13487
(DNA) I 49893 240636_at -- -- -- -- 0.0377752 2.1411 40398
231136_at 407032 MIR30C2 microRNA NR_029598 0.00328686 2.15034
30c-2 309 1552680_s_at 57082 CASC5 cancer NM_144508 /// 0.00799316
2.15456 susceptibilty NM_170589 candidate 5 18036 208610_s_at 23524
SRRM2 serine/ NM_016333 0.00110455 2.16673 arginine repetitive
matrix 2 20375 2110000_s_at 3572 IL65T interleukin NM_001190981 ///
0.0136659 2.2082 6 signal NM_002184 /// transducer NM_175767 (gp130
oncostatin M receptor) 18510 209088_s_at 29855 UBN1 ubinuclein 1
NM_001079514 /// 0.00669916 2.22201 NM_016936 14318 204863_s_at
3572 IL65T interleukin NM_001190981 /// 0.00704479 2.24897 6 signal
NM_002184 /// transducer NM_175767 (gp130, oncostatin M receptor)
21393 212079_s_at 4297 MLL myeloid/ NM _001197104 /// 0.00889717
2.25754 lymphoid or NM_005933 /// mixed-lineage NM_024891 leukemia
(trithorax homolog, Drosophila) 38248 228986_at 114882 OSBPL8
oxysterol NM_001003712 /// 0.0161915 2.26479 binding NM_020841
protein- like 8 23771 214464_at 8476 CDC42BPA CDC42 NM_003607 ///
0.00114942 2.27563 binding NM_014826 protein kinase alpha
(DMPK-like) 4349 1558678_s_at 100507645 /// LOC100507645 ///
uncharacterized NR_002819 /// 0.00592243 2.28297 378938 MALAT1
LOC100507645 /// XR_110915 /// metastasis XR_110916 /// associated
XR_110917 /// lung XR_111190 /// adenocarcinima XR_111191 ///
transcript 1 XR_ 33834 224567_x_at 100507645 /// LOC100507645 ///
uncharacterized NR_002819 /// 0.00410374 2.29298 378938 MALAT1
LOC100507645 /// XR_110915 /// metastasis XR_110916 /// associated
XR_110917 /// lung XR_111190 /// adenocarcinima XR_111191 ///
transcript 1 XR_ 18282 208859_s_at 546 ATRX alpha NM_000489 ///
0.000865449 2.31098 thalassemia/ NM_138270 /// mental NM_138271
retardation syndrome X-linked 33830 224563_at 10163 WASF2 WAS
protein NM_001201404/// 0.00632155 2.36071 family, NM_006990 member
2 45877 236620_at 55183 RIF1 RAP1 NM_001177663 /// 0.00480654
2.46908 interacting NM_001177664 /// factor NM_001177665 ///
homolog NM_018151 (yeast) 3521 1557227_s_at 7175 TPR translocated
NM_003292 0.0185624 2.56223 promoter region, nuclear basket protein
11186 201730_s_at 7175 TPR translocated NM_003292 0.0023957 2.62456
promoter region, nuclear basket protein 35938 226675_s_at 100507645
/// LOC100507645 /// uncharacterized NR_002819 /// 0.0061131
2.63183 378938 MALATI LOC100507645/// XR_110915 /// metastasis
XR_110916 /// associated XR_110917 /// lung XR_111190 ///
adenocarcinima XR_111191 /// transcript 1 XR_ 22755 213446_s_at
8826 IQGAP1 IQ motif NM_003870 0.0118332 2.68542 containing GTPase
activating protein 1 52175 242918_at 4678 NASP Nuclear NM_001195193
/// 0.017061 2.75136 autoantigenic NM_002482 /// sperm NM_152298
/// protein NM_172164 (histone binding) 15264 205809_s_at 8976 WASL
Wiskitt- NM_003941 0.0252923 2.82213 Aldrich syndrome- like 41500
232238_at 259266 ASPM asp (abnormal NM_0012068468/// 0.00155378
2.83547 spindle) NM_018136 homolog, microcephaly associated
(Drosophila) 51609 242352_at 25836 NIPBL Nipped-8 NM_015384 ///
0.0177762 2.92333 homolog NM_133433 (Drosophila)
TABLE-US-00005 TABLE 3 Part 1 Entrez Column # Column ID Gene Gene
Symbol Gene Title 18582 209160_at 8644 AKR1C3 aldo-keto reductase
family 1, member C3 (3-alpha hydroxysteroid dehydrogenase, type II)
20985 211653_x_at 100653286 /// AKR1C2 /// aldo-keto reductase
family 1, member 1646 LOC100653286 C2 (dihydrodiol dehydrogenase 2;
bile acid binding 13606 204151_x_at 1645 AKR1C1 aldo-keto reductase
family 1, member C1 (dihydrodiol dehydrogenase 1; 20- alpha
(3-alpha 13026 203571_s_at 10974 C10orf116 chromosome 10 open
reading frame 116 25892 216594_x_at 1645 AKR1C1 aldo-keto reductase
family 1, member C1 (dihydrodiol dehydrogenase 1; 20- alpha
(3-alpha 13796 204341_at 10626 TRIM16 tripartite motif containing
16 18317 208894_at 3122 HLA-DRA major histocompatibility complex,
class II, DR alpha 19115 209699_x_at 100653286 /// AKR1C2 ///
aldo-keto reductase family 1, member 1646 LOC100653286 C2
(dihydrodiol dehydrogenase 2; bile acid binding 25005 215704_at
2312 FLG filaggrin 13753 204298_s_at 4015 LOX lysyl oxidase 41497
232235_at 92126 DSEL dermatan sulfate epimerase-like 11693
202237_at 4837 NNMT nicotinamide N-methyltransferase 19171
209755_at 23057 NMNAT2 nicotinamide nucleotide adenylyltransferase
2 13743 204288_s_at 8470 SORBS2 sorbin and SH3 domain containing 2
3439 1557094_at 100652762 LOC100652762 uncharacterized LOC100652762
22738 213429_at 80114 BICC1 bicaudal C homolog 1 (Drosophila) 19707
210299_s_at 2273 FHL1 four and a half LIM domains 1 19154
209738_x_at 5675 PSG6 pregnancy specific beta- 1-glycoprotein 6
21468 212154_at 6383 SDC2 syndecan 2 17182 207733_x_at 5678 PSG9
pregnancy specific beta- 1-glycoprotein 9 28341 219049_at 55790
CSGALNACT1 chondroitin sulfate N- acetylgalactosaminyltransferase 1
14713 205258_at 3625 INHBB inhibin, beta B IFIH1 interferon induced
with helicase C 28501 219209_at 64135 domain 1 37497 228235_at
84848 MGC16121 uncharacterized protein MGC16121 12959 203504_s_at
19 ABCA1 ATP-binding cassette, sub-family A (ABC1),member 1 10996
201540_at 2273 FHL1 four and a half LIM domains 1 21802 212489_at
1289 COL5A1 collagen, type V, alpha 1 NEDD9 neural precursor cell
expressed, 11606 202150_s_at 4739 developmentally down-regulated 9
22054 212741_at 4128 MAOA monoamine oxidase A GPRC5B G
protein-coupled receptor, family C, 13087 203632_s_at 51704 group
5, member B 37712 228450_at 144100 PLEKHA7 pleckstrin homology
domain containing, family A member 7 33112 223828_s_at 85329
LGALS12 lectin, galactoside-binding, soluble, 12 RECK
reversion-inducing-cysteine-rich protein 14862 205407_at 8434 with
kazal motifs 11694 202238_s_at 4837 NNMT nicotinamide
N-methyltransferase 13279 203824_at 7103 TSPAN8 tetraspanin 8 36110
226847_at 10468 FST follistatin 10962 201506_at 100652886 ///
LOC100652886 /// uncharacterized LOC100652886 /// 100653157 ///
LOC100653157 /// uncharacterized LOC100653157 /// 7045 TGFBI
transforming growth f 19322 209908_s_at 7042 TGFB2 transforming
growth factor, beta 2 28815 219523_s_at 55714 ODZ3 odz, odd
Oz/ten-m homolog 3 (Drosophila) 32994 223710_at 10344 CCL26
chemokine (C-C motif) ligand 26 46044 236787_at 100507286
LOC100507286 uncharacterized LOC100507286 28959 219667_s_at 55024
BANK1 B-cell scaffold protein with ankyrin repeats 1 14403
204948_s_at 10468 FST follistatin 35482 226218_at 3575 IL7R
interleukin 7 receptor 13844 204389_at 4128 MAOA monoamine oxidase
A 38147 228885_at 256691 MAMDC2 MAM domain containing 2 45055
235798_at 100113407 TMEM170B transmembrane protein 170B 9315
1569582_at 201651 LOC201651 arylacetamide deacetylase (esterase)
pseudogene 12222 202766_s_at 2200 FBN1 fibrillin 1 37233 227971_at
203447 NRK Nik related kinase 28821 219529_at 9022 CLIC3 chloride
intracellular channel 3 12531 203074_at 244 /// ANXA8 /// annexin
A8 /// annexin A8-like 1 /// 653145 /// ANXA8L1 /// annexin A8-like
2 728113 ANXA8L2 15749 206295_at 3606 IL18 interleukin 18
(interferon-gamma- inducing factor) 41330 232068_s_at 7099 TLR4
toll-like receptor 4 caspase 1, apoptosis-related cysteine 20711
211367_s_at 834 CASP1 peptidase 14388 204933_s_at 4982 TNFRSF11B
tumor necrosis factor receptor superfamily, member 11b 11806
202350_s_at 100506558 /// LOC100506558 /// uncharacterized
LOC100506558 /// 4147 MATN2 matrilin 2 20252 210869_s_at 4162 MCAM
melanoma cell adhesion molecule 32680 223395_at 25890 ABI3BP ABI
family, member 3 (NESH) binding protein 33059 223775_at 64399 HHIP
hedgehog interacting protein 46723 237466_s_at 64399 HHIP hedgehog
interacting protein 12221 202765_s_at 2200 FBN1 fibrillin 1 21472
212158_at 6383 SDC2 syndecan 2 14285 204830_x_at 5673 PSG5
pregnancy specific beta- 1-glycoprotein 5 39409 230147_at 2151
F2RL2 coagulation factor II (thrombin) receptor-like 2 21949
212636_at 9444 QKI QKI, KH domain containing, RNA binding 20712
211368_s_at 834 CASP1 caspase 1, apoptosis-related cysteine
peptidase 12595 203139_at 1612 DAPK1 death-associated protein
kinase 1 16378 206924_at 3589 IL11 interleukin 11 19012 209594_x_at
5678 PSG9 pregnancy specific beta- 1-glycoprotein 9 46692 237435_at
-- -- -- 21048 211719_x_at 2335 FN1 fibronectin 1 12144 202688_at
8743 TNFSF10 tumor necrosis factor (ligand) superfamily, member 10
19894 210495_x_at 2335 FN1 fibronectin 1 21777 212464_s_at 2335 FN1
fibronectin 1 25741 216442_x_at 2335 FN1 fibronectin 1 neural
precursor cell expressed, 11605 202149_at 4739 NEDD9
developmentally down-regulated 9 39397 230135_at 64399 HHIP
hedgehog interacting protein 16667 207214_at 27290 SPINK4 serine
peptidase inhibitor, Kazal type 4 29306 220014_at 51334 PRR16
proline rich 16 25678 216379_x_at 100133941 CD24 CD24 molecule
22421 213110_s_at 1287 COL4A5 collagen, type IV, alpha 5 36662
227399_at 389136 VGLL3 vestigial like 3 (Drosophila) 19187
209771_x_at 100133941 CD24 CD24 molecule 324 1552703_s_at 114769
/// CARD16 /// caspase recruitment domain family, 834 CASP1 member
16 /// caspase 1, apoptosis- related cysteine 54150 266_s_at
100133941 CD24 CD24 molecule 18075 208650_s_at 100133941 CD24 CD24
molecule caspase recruitment domain family, 323 1552701_a_at 114769
CARD16 member 16 23636 214329_x_at 8743 TNFSF10 tumor necrosis
factor (ligand) superfamily, member 10 15018 205563_at 3814 KISS1
KiSS-1 metastasis-suppressor 22253 212942_s_at 57214 KIAA1199
KIAA1199 19909 210511_s_at 3624 INHBA inhibin, beta A 12143
202687_s_at 8743 TNFSF10 tumor necrosis factor (ligand)
superfamily, member 10 34993 225728_at 8470 SORBS2 sorbin and SH3
domain containing 2 36403 227140_at 3624 INHBA inhibin, beta A
19363 209949_at 4688 NCF2 neutrophil cytosolic factor 2 14464
205009_at 7031 TFF1 trefoil factor 1
TABLE-US-00006 TABLE 3 Part 2 Ratio p-value (Suspended Fold-Change
RefSeq Transcript p-value (Suspended vs. (Suspended vs. Column # ID
(Attribute) vs. Attached) Attached) Attached) 18582 NM_001253908
/// NM_001253909 /// NM_003739 0.000170367 0.000170367 1.81864
1.81864 20985 NM_001135241 /// NM_001354 /// NM_205845 ///
XM_003403856 0.000143927 0.000143927 1.67318 1.67318 13606
NM_001353 0.000659841 0.000659841 1.65919 1.65919 13026 NM_006829
0.000387207 0.000387207 1.65068 1.65068 25892 NM_001353 0.000756883
0.000756883 1.60532 1.60532 13796 NM_006470 1.72E-05 1.72E-05
1.51663 1.51663 18317 NM_019111 0.00512881 0.00512881 1.51385
1.51385 19115 NM_001135241 /// NM_001354 /// NM_205845 ///
XM_003403856 0.00033636 0.00033636 1.50424 1.50424 25005 NM_002016
0.00218557 0.00218557 0.665474 -1.50269 13753 NM_001178102 ///
NM_002317 6.59E-05 6.59E-05 0.664062 -1.50588 41497 NM_032160
0.00226259 0.00226259 0.66251 -1.50941 11693 NM_006169 0.000462864
0.000462864 0.662281 -1.50993 NM_015039 /// 19171 NM_170706
0.020548 0.020548 0.661458 -1.51181 NM_001145670 /// NM_001145671
/// NM_001145672 /// NM_001145673 /// NM_001145674 /// 13743 NM
0.0112829 0.0112829 0.660839 -1.51323 3439 XM_003403544 0.00309358
0.00309358 0.660605 -1.51376 22738 NM_001080512 0.0062046 0.0062046
0.659525 -1.51624 NM_001159699 /// NM_001159700 /// NM_001159701
/// NM_001159702 /// NM_001159703 /// 19707 NM 0.025324 0.025324
0.659366 -1.51661 NM_001031850 /// 19154 NM_002782 0.000207499
0.000207499 0.657119 -1.52179 21468 NM_002998 0.000566015
0.000566015 0.656899 -1.5223 17182 NM_002784 0.00758296 0.00758296
0.6558 -1.52486 NM_001130518 /// NM_018371 /// 28341 NR_024040
0.000369511 0.000369511 0.654509 -1.52786 14713 NM_002193
0.00235168 0.00235168 0.654085 -1.52885 28501 NM_022168 0.00655627
0.00655627 0.65325 -1.53081 NM_032762 /// 37497 NR_024607
0.00968185 0.00968185 0.652485 -1.5326 12959 NM_005502 0.00392514
0.00392514 0.651694 -1.53446 10996 NM_001159699 /// 0.00223441
0.00223441 0.650122 -1.53817 NM_001159700 /// NM_001159701 ///
NM_001159702 /// NM_001159703 /// NM 21802 NM_000093 0.000341289
0.000341289 0.649968 -1.53854 11606 NM_001142393 /// NM_006403 ///
NM_182966 0.0280295 0.0280295 0.645034 -1.55031 22054 NM_000240
0.00100831 0.00100831 0.643901 -1.55303 13087 NM_016235 0.00133541
0.00133541 0.642282 -1.55695 37712 NM_175058 0.000419231
0.000419231 0.64162 -1.55855 33112 NM_001142535 /// 0.0390828
0.0390828 0.641364 -1.55918 NM_001142536 /// NM_001142537 ///
NM_001142538 /// NM_033101 14862 NM_021111 0.00695804 0.00695804
0.639083 -1.56474 11694 NM_006169 0.000173649 0.000173649 0.638349
-1.56654 13279 NM_004616 0.000799162 0.000799162 0.63719 -1.56939
36110 NM_006350 /// 0.0030227 0.0030227 0.636925 -1.57004 NM_013409
10962 NM_000358 /// 0.000241245 0.000241245 0.635083 -1.5746
XR_132813 /// XR_133407 19322 NM_001135599 /// 0.0300973 0.0300973
0.634683 -1.57559 NM_003238 28815 NM_001080477 0.016908 0.016908
0.633668 -1.57811 32994 NM_006072 0.0445369 0.0445369 0.632818
-1.58023 46044 XR_109951 0.00634762 0.00634762 0.631059 -1.58464
28959 NM_001083907 /// 0.00600733 0.00600733 0.630334 -1.58646
NM_001127507 /// NM_017935 14403 NM_006350 /// 0.000425631
0.000425631 0.629981 -1.58735 NM_013409 35482 NM_002185 0.000443453
0.000443453 0.629004 -1.58981 13844 NM_000240 0.00104301 0.00104301
0.627119 -1.59459 38147 NM_153267 0.000232264 0.000232264 0.626074
-1.59725 45055 NM_001100829 0.00992984 0.00992984 0.622005 -1.6077
9315 NR_026915 0.00258393 0.00258393 0.618288 -1.61737 12222
NM_000138 0.00176066 0.00176066 0.618179 -1.61765 37233 NM_198465
0.0138066 0.0138066 0.61709 -1.62051 28821 NM_004669 0.000316266
0.000316266 0.614298 -1.62787 12531 NM_001039801 /// 0.00261053
0.00261053 0.613273 -1.6306 NM_001040084 /// NM_001098845 ///
NM_001630 15749 NM_001243211 /// 0.00789586 0.00789586 0.612172
-1.63353 NM_001562 41330 NM_003266 /// 0.00707246 0.00707246
0.610647 -1.63761 NM_138554 /// NM_138556 /// NM_138557 ///
NR_024168 /// NR_024169 20711 NM_001223 /// 0.00177852 0.00177852
0.609003 -1.64203 NM_001257118 /// NM_001257119 /// NM_033292 ///
NM_033293 /// NM_033294 / 14388 NM_002546 0.0034562 0.0034562
0.608783 -1.64262 11806 NM_002380 /// 0.00269868 0.00269868
0.603891 -1.65593 NM_030583 /// XR_108869 /// XR_133494 20252
NM_006500 0.00214434 0.00214434 0.603836 -1.65608 32680 NM_015429
0.0154958 0.0154958 0.602208 -1.66056 33059 NM_022475 0.000751578
0.000751578 0.600994 -1.66391 46723 NM_022475 0.000230794
0.000230794 0.5999 -1.66695 12221 NM_000138 0.0112647 0.0112647
0.598516 -1.6708 21472 NM_002998 0.000137552 0.000137552 0.595021
-1.68061 14285 NM_001130014 /// 0.00469336 0.00469336 0.593415
-1.68516 NM_002781 39409 NM_001256566 /// 0.00262479 0.00262479
0.592707 -1.68717 NM_004101 21949 NM_006775 /// 0.0109828 0.0109828
0.588126 -1.70032 NM_206853 /// NM_206854 /// NM_206855 20712
NM_001223 /// 0.00544332 0.00544332 0.586424 -1.70525 NM_001257118
/// NM_001257119 /// NM_033292 /// NM_033293 /// NM_033294 / 12595
NM_004938 0.00070315 0.00070315 0.583547 -1.71366 16378 NM_000641
/// 0.00206342 0.00206342 0.575597 -1.73733 NM_001267718 19012
NM_002784 0.000350804 0.000350804 0.567073 -1.76344 46692 --
0.00486443 0.00486443 0.565559 -1.76816 21048 NM_002026 ///
0.000320581 0.000320581 0.56548 -1.76841 NM_054034 /// NM_212474
/// NM_212475 /// NM_212476 /// NM_212478 /// NM_ 12144
NM_001190942 /// 0.00322745 0.00322745 0.563625 -1.77423
NM_001190943 /// NM_003810 /// NR_033994 19894 NM_002026 ///
0.000274524 0.000274524 0.563315 -1.77521 NM_054034 /// NM_212474
/// NM_212475 /// NM_212476 /// NM_212478 /// NM_ 21777 NM_002026
/// 0.000185237 0.000185237 0.560338 -1.78464 NM_054034 ///
NM_212474 /// NM_212475 /// NM_212476 /// NM_212478 /// NM_ 25741
NM_002026 /// 0.000355233 0.000355233 0.558125 -1.79171 NM_054034
/// NM_212474 /// NM_212475 /// NM_212476 /// NM_212478 /// NM_
11605 NM_001142393 /// 5.75E-05 5.75E-05 0.557276 -1.79444
NM_006403 /// NM_182966 39397 NM_022475 0.00081091 0.00081091
0.556999 -1.79534 16667 NM_014471 0.00379337 0.00379337 0.556208
-1.79789 29306 NM_016644 0.000373382 0.000373382 0.551386 -1.81361
25678 NM_013230 0.000831434 0.000831434 0.550468 -1.81664 22421
NM_000495 /// 0.000153119 0.000153119 0.550254 -1.81734 NM_033380
/// NM_033381 36662 NM_016206 0.00145565 0.00145565 0.545624
-1.83276 19187 NM_013230 0.00103971 0.00103971 0.542121 -1.84461
324 NM_001017534 /// 0.0012893 0.0012893 0.535819 -1.8663 NM_001223
/// NM_001257118 /// NM_001257119 /// NM_033292 /// NM_03329 54150
NM_013230 0.000136369 0.000136369 0.535661 -1.86685 18075 NM_013230
9.53E-05 9.53E-05 0.528598 -1.8918 323 NM_001017534 /// 0.0112715
0.0112715 0.526508 -1.89931 NM_052889 23636 NM_001190942 ///
6.44E-05 6.44E-05 0.52413 -1.90792 NM_001190943 /// NM_003810 ///
NR_033994 15018 NM_002256 3.43E-05 3.43E-05 0.510055 -1.96057 22253
NM_018689 4.12E-05 4.12E-05 0.491527 -2.03448 19909 NM_002192
0.000271854 0.000271854 0.484229 -2.06514 12143 NM_001190942 ///
0.00371971 0.00371971 0.480021 -2.08324 NM_001190943 /// NM_003810
/// NR_033994 NM_001145670 /// NM_001145671 /// NM_001145672 ///
NM_001145673 /// NM_001145674 /// 34993 NM 0.00394697 0.00394697
0.477446 -2.09448 36403 NM_002192 0.00472489 0.00472489 0.469154
-2.13149 19363 NM_000433 /// 0.00011872 0.00011872 0.464326
-2.15366 NM_001127651 /// NM_001190789 /// NM_001190794 14464
NM_003225 9.43E-06 9.43E-06 0.246286 -4.06032
Sequence CWU 1
1
70111PRTartificial sequencePeptide 1Phe Ile Arg Val Val Met Tyr Glu
Gly Lys Lys1 5 10222DNAartificial
sequencePrimermisc_feature(20)..(20)n is a, c, g, or t 2ctactgaagt
atacgtaagn gg 22321DNAartificial sequencePrimer 3tggttccttc
tgtgtcaatc c 21420DNAartificial sequencePrimer 4gtactctcgc
aggaaatggg 20520DNAartificial sequencePrimer 5tcctctggag gctgagaaaa
20620DNAartificial sequencePrimer 6gggctctgga ggaaaagaaa
20724DNAartificial sequencePrimer 7aggaccgcgg ttctattttg ttgg
24819DNAartificial sequencePrimer 8cccccggccg tccctctta
19924DNAartificial sequencePrimer 9cgtccaagca gaggagcaaa agct
241021DNAartificial sequencePrimer 10cgcacaagag ttccgtagct g
211124DNAartificial sequencePrimer 11tcctgaattg ctatgtgtct gggt
241222DNAartificial sequencePrimer 12gatagaaaga ccagtccttg ct
221320DNAartificial sequencePrimer 13aggaagctca aagccgaact
201420DNAartificial sequencePrimer 14acgcttcttc tggaagacca
201520DNAartificial sequencePrimer 15aagtttctgc cggaagttca
201619DNAartificial sequencePrimer 16gcaagaacgc tcaaccgag
191720DNAartificial sequencePrimer 17ttgcaaaggc ctctttgagt
201820DNAartificial sequencePrimer 18cccaggactc tgcgacttta
201920DNAartificial sequencePrimer 19atggatgact gcattccaca
202020DNAartificial sequencePrimer 20gagaaaccag cccaaaatga
202120DNAartificial sequencePrimer 21tggcagtcta tgtctgcacc
202220DNAartificial sequencePrimer 22ctatggctac cacaggcgat
202320DNAartificial sequencePrimer 23ccatgttccc acaagcaact
202421DNAartificial sequencePrimer 24tcaggtgtat ccaacagcct c
212519DNAartificial sequencePrimer 25ctcacaaggt tctggcgtg
192620DNAartificial sequencePrimer 26tgagctcaga ggttcggaag
202717DNAartificial sequencePrimer 27ctcgcttcgg cagcaca
172820DNAartificial sequencePrimer 28aacgcttcac gaatttgcgt
202922DNAartificial sequencePrimer 29taggctgtgg gaaaggacca ag
223022DNAartificial sequencePrimer 30gttcgatgaa ccgcttctga tg
223122DNAartificial sequencePrimer 31caagcaggag acatcggaca ag
223222DNAartificial sequencePrimer 32cacaatggac atcttgggct tc
223320DNAartificial sequencePrimer 33tgaagtgtga cgtggacatc
203420DNAartificial sequencePrimer 34ggaggagcaa tgatcttgat
203520DNAartificial sequencePrimer 35cgtagagtca accgggaagt
203619DNAartificial sequencePrimer 36cctcaaagtc gacccgttc
193719DNAartificial sequencePrimer 37tgcaggcgtt acacgatgc
193821DNAartificial sequencePrimer 38tacctcatgg atggcaagtg c
213924DNAartificial sequencePrimer 39caggtatact tctcagccat gtga
244023DNAartificial sequencePrimer 40gcccacttct gtcagtaaat ggt
234123DNAartificial sequencePrimer 41caacgtaagt gaatgaaaat ggt
234223DNAartificial sequencePrimer 42taaattaatt tatttccagg cca
234324DNAartificial sequencePrimer 43gaaaatacag catgggtaca agga
244425DNAartificial sequencePrimer 44ttaagaaatg gtagttttcc agcca
254519DNAartificial sequencePrimer 45agaatccctg tgcccttgg
194621DNAartificial sequencePrimer 46ggcacctagg cctgtcagac t
214723DNAartificial sequencePrimer 47caggttaatt tgaagtgcat ctg
234822DNAartificial sequencePrimer 48agcaccatgc tgcagtttat aa
224919DNAartificial sequencePrimer 49ggggaaacct tttgcttct
195022DNAartificial sequencePrimer 50ctgtgcctac tgtacttgaa ca
225123DNAartificial sequencePrimer 51tacatgaaca tgctgtttca gag
235224DNAartificial sequencePrimer 52catctaagtc aaatgactcc actg
245323DNAArtificial SequenceCD47 gRNA
sequencemisc_feature(21)..(21)n is a, c, g, or t 53tccatgcttt
gttactaata ngg 235423DNAArtificial SequenceCD47 gRNA
sequencemisc_feature(21)..(21)n is a, c, g, or t 54atcgagctaa
aatatcgtgt ngg 235523DNAArtificial SequenceCD47 gRNA
sequencemisc_feature(21)..(21)n is a, c, g, or t 55gatgatcgtt
tcaccttctc ngg 235623DNAArtificial SequenceCD47 gRNA
sequencemisc_feature(21)..(21)n is a, c, g, or t 56actcttatcc
atcttcaaag ngg 235723DNAArtificial SequenceCD47 gRNA
sequencemisc_feature(21)..(21)n is a, c, g, or t 57ttttgcacta
ctaaagtcag ngg 235823DNAArtificial SequenceCD47 gRNA
sequencemisc_feature(21)..(21)n is a, c, g, or t 58tttgcactac
taaagtcagt ngg 235923DNAArtificial SequenceCD47 gRNA
sequencemisc_feature(21)..(21)n is a, c, g, or t 59ttgcactact
aaagtcagtg ngg 236023DNAArtificial SequenceCD47 gRNA
sequencemisc_feature(21)..(21)n is a, c, g, or t 60actaaagtca
gtggggacag ngg 236123DNAArtificial SequenceCD47 gRNA
sequencemisc_feature(21)..(21)n is a, c, g, or t 61cttgtttaga
gctccatcaa ngg 236223DNAArtificial SequenceCD47 gRNA
sequencemisc_feature(21)..(21)n is a, c, g, or t 62tccatattag
taacaaagca ngg 236323DNAArtificial SequenceCD47 gRNA
sequencemisc_feature(21)..(21)n is a, c, g, or t 63atgctttgtt
actaatatgg ngg 236423DNAArtificial SequenceCD47 gRNA
sequencemisc_feature(21)..(21)n is a, c, g, or t 64tacgtaaagt
ggaaatttaa ngg 236523DNAArtificial SequenceCD47 gRNA
sequencemisc_feature(21)..(21)n is a, c, g, or t 65agagatattt
acacctttga ngg 236623DNAArtificial SequenceCD47 gRNA
sequencemisc_feature(21)..(21)n is a, c, g, or t 66gaagtctcac
aattactaaa ngg 236723DNAArtificial SequenceCD47 gRNA
sequencemisc_feature(21)..(21)n is a, c, g, or t 67aggagatgcc
tctttgaaga ngg 236823DNAArtificial SequenceCD47 gRNA
sequencemisc_feature(21)..(21)n is a, c, g, or t 68agtgatgctg
tctcacacac ngg 236923DNAArtificial SequenceCD47 gRNA
sequencemisc_feature(21)..(21)n is a, c, g, or t 69gtaacagaat
taaccagaga ngg 237025DNAArtificial SequenceCD47 morpholino sequence
70cgtcacaggc aggacccact gccca 25
* * * * *