U.S. patent application number 13/941287 was filed with the patent office on 2014-06-12 for methods of treating acute myeloid leukemia by blocking cd47.
The applicant listed for this patent is The Board of Trustees of the Leland Stanford Junior University. Invention is credited to Mark P. Chao, Siddhartha Jaiswal, Ravindra Majeti, Irving L. Weissman.
Application Number | 20140161825 13/941287 |
Document ID | / |
Family ID | 40885608 |
Filed Date | 2014-06-12 |
United States Patent
Application |
20140161825 |
Kind Code |
A1 |
Jaiswal; Siddhartha ; et
al. |
June 12, 2014 |
Methods of Treating Acute Myeloid Leukemia by Blocking CD47
Abstract
Methods are provided to manipulate phagocytosis of cancer cells,
including e.g. leukemias, solid tumors including carcinomas,
etc.
Inventors: |
Jaiswal; Siddhartha; (San
Francisco, CA) ; Weissman; Irving L.; (Stanford,
CA) ; Majeti; Ravindra; (Stanford, CA) ; Chao;
Mark P.; (Mountain View, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Board of Trustees of the Leland Stanford Junior
University |
Palo Alto |
CA |
US |
|
|
Family ID: |
40885608 |
Appl. No.: |
13/941287 |
Filed: |
July 12, 2013 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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12837409 |
Jul 15, 2010 |
8562997 |
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13941287 |
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PCT/US09/00319 |
Jan 15, 2009 |
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12837409 |
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61011324 |
Jan 15, 2008 |
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61189786 |
Aug 22, 2008 |
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Current U.S.
Class: |
424/174.1 |
Current CPC
Class: |
C07K 16/18 20130101;
A61P 15/00 20180101; A61K 47/6869 20170801; C07K 2317/31 20130101;
C07K 16/2896 20130101; C07K 16/3038 20130101; A61K 39/39558
20130101; A61K 2039/505 20130101; C07K 16/3069 20130101; C07K 16/30
20130101; C07K 2317/76 20130101; A61P 35/02 20180101; C07K 2317/73
20130101; A61K 47/6863 20170801; C07K 16/3061 20130101; C07K
16/3046 20130101; A61P 25/00 20180101; A61P 43/00 20180101; A61K
47/6867 20170801; A61K 47/6851 20170801; A61K 2039/507 20130101;
C07K 16/3053 20130101; A61K 47/6865 20170801; A61K 47/6861
20170801; A61P 35/00 20180101; C07K 16/2803 20130101; A61P 13/10
20180101 |
Class at
Publication: |
424/174.1 |
International
Class: |
C07K 16/18 20060101
C07K016/18 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
[0001] This invention was made with Government support under
contract CA086017 awarded by the National Institutes of Health. The
Government has certain rights in this invention.
Claims
1-15. (canceled)
16. A method of treating a human subject having a solid tumor
cancer, the method comprising: administering to the human subject
an antibody that prevents the binding of CD47 with SIRP.alpha., at
a dose that achieves a depletion of cancer cells of the solid
tumor.
17. The method of claim 16, wherein the cancer is a carcinoma.
18. The method of claim 17, wherein the carcinoma is ovarian
carcinoma, bladder carcinoma, pancreatic carcinoma, breast
carcinoma, colon carcinoma, squamous cell carcinoma, or
transitional cell carcinoma.
19. The method of claim 17, wherein the carcinoma is a squamous
cell carcinoma.
20. The method of claim 19, wherein the squamous cell carcinoma is
of the mouth, throat, larynx, cervix or lung.
21. The method of claim 16, wherein the cancer is a sarcoma.
22. The method of claim 16, wherein the cancer is a melanoma.
23. The method of claim 16, wherein the cancer is a brain
tumor.
24. The method of claim 23, wherein the brain tumor is selected
from astrocytoma, glioblastoma, medulloblastoma, and
meningioma.
25. A method of treating a human subject having a metastatic
cancer, the method comprising: administering to the human subject
an antibody that prevents the binding of CD47 with SIRP.alpha., at
a dose that inhibits metastases and/or formation of
micrometastases.
26. The method of claim 25, wherein the metastatic cancer is a
carcinoma.
27. The method of claim 26, wherein the carcinoma is ovarian
carcinoma, bladder carcinoma, pancreatic carcinoma, breast
carcinoma, colon carcinoma, squamous cell carcinoma, or
transitional cell carcinoma.
28. The method of claim 26, wherein the carcinoma is a squamous
cell carcinoma.
29. The method of claim 28, wherein the squamous cell carcinoma is
of the mouth, throat, larynx, cervix or lung.
30. The method of claim 25, wherein the metastatic cancer is a
sarcoma.
31. The method of claim 25, wherein the cancer is a melanoma.
32. The method of claim 25, wherein the cancer is a brain
tumor.
33. The method of claim 32, wherein the brain tumor is selected
from astrocytoma, glioblastoma, medulloblastoma, and meningioma.
Description
BACKGROUND
[0002] The reticuloendothelial system (RES) is a part of the immune
system. The RES consists of the phagocytic cells located in
reticular connective tissue, primarily monocytes and macrophages.
The RES consists of 1) circulating monocytes; 2) resident
macrophages in the liver, spleen, lymph nodes, thymus, submucosal
tissues of the respiratory and alimentary tracts, bone marrow, and
connective tissues; and 3) macrophage-like cells including
dendritic cells in lymph nodes, Langerhans cells in skin, and
microglial cells in the central nervous system. These cells
accumulate in lymph nodes and the spleen. The RES functions to
clear pathogens, particulate matter in circulation, and aged or
damaged hematopoietic cells.
[0003] To eliminate foreign cells or particles in the innate immune
response, macrophage-mediated phagocytosis is induced when the
phosphatidylserine receptor (PSR) reacts to phosphatidylserine
(PS), which can be externalized from the membranes of dead cells,
such as apoptotic and necrotic cells. In turn, the interaction
between PS and PSR plays a crucial role in the clearance of
apoptotic cells by macrophages. Once phagocytosis has been
performed by macrophages, the inflammatory response is
downregulated by an increase in factors such as IL-10, TGF-.beta.,
and prostaglandin E2 (PGE2). The strict balance between the
inflammatory and anti-inflammatory responses in both innate and
adaptive immunity plays a critical role in maintaining cellular
homeostasis and protecting a host from extrinsic invasion.
[0004] The causal relationship between inflammation and the
neoplastic progression is a concept widely accepted. Data now
support the concept of cancer immunosurveillance--that one of the
physiologic functions of the immune system is to recognize and
destroy transformed cells. However, some tumor cells are capable of
evading recognition and destruction by the immune system. Once
tumor cells have escaped, the immune system may participate in
their growth, for example by promoting the vascularization of
tumors.
[0005] Both adaptive and innate immune cells participate in the
surveillance and the elimination of tumor cells, but
monocytes/macrophages may be the first line of defense in tumors,
as they colonize rapidly and secrete cytokines that attract and
activate dendritic cells (DC) and NK cells, which in turn can
initiate the adaptive immune response against transformed
cells.
[0006] Tumors that escape from the immune machinery can be a
consequence of alterations occurring during the immunosurveillance
phase. As an example, some tumor cells develop deficiencies in
antigen processing and presentation pathways, which facilitate
evasion from an adaptive immune response, such as the absence or
abnormal functions of components of the IFN-.gamma. receptor
signaling pathway. Other tumors suppress the induction of
proinflammatory danger signals, leading, for example, to impaired
DC maturation. Finally, the inhibition of the protective functions
of the immune system may also facilitate tumor escape, such as the
overproduction of the anti-inflammatory cytokines IL-10 and
TGF-.beta., which can be produced by many tumor cells themselves
but also by macrophages or T regulatory cells.
[0007] A tumor can be viewed as an aberrant organ initiated by a
tumorigenic cancer cell that acquired the capacity for indefinite
proliferation through accumulated mutations. In this view of a
tumor as an abnormal organ, the principles of normal stem cell
biology can be applied to better understand how tumors develop.
Many observations suggest that analogies between normal stem cells
and tumorigenic cells are appropriate. Both normal stem cells and
tumorigenic cells have extensive proliferative potential and the
ability to give rise to new (normal or abnormal) tissues. Both
tumors and normal tissues are composed of heterogeneous
combinations of cells, with different phenotypic characteristics
and different proliferative potentials.
[0008] Stem cells are defined as cells that have the ability to
perpetuate themselves through self-renewal and to generate mature
cells of a particular tissue through differentiation. In most
tissues, stem cells are rare. As a result, stem cells must be
identified prospectively and purified carefully in order to study
their properties. Perhaps the most important and useful property of
stem cells is that of self-renewal. Through this property, striking
parallels can be found between stem cells and cancer cells: tumors
may often originate from the transformation of normal stem cells,
similar signaling pathways may regulate self-renewal in stem cells
and cancer cells, and cancers may comprise rare cells with
indefinite potential for self-renewal that drive tumorigenesis.
[0009] Study of cell surface markers specific to or specifically
upregulated in cancer cells is pivotal in providing targets for
reducing growth of or for depleting cancer cells. Provided herein
is a marker for myeloid leukemia, especially a marker for Acute
Myeloid Leukemia (AML). Our studies have revealed a role of this
marker in helping AML stem cells avoid clearance by phagocytosis.
Methods are provided for using this marker to increase phagocytosis
of AML stem cells (AML SCs), as well as to improve transplantation
of hematopoietic and progenitor stem cells.
[0010] Interestingly, certain markers are shown to be shared by
leukemia stem cells and hematopoietic stem cells (HSCs). During
normal development, HSCs migrate to ectopic niches in fetal and
adult life via the blood stream. Once in the blood stream, HSCs
must navigate the vascular beds of the spleen and liver before
settling in a niche. At these vascular beds, macrophages function
to remove damaged cells and foreign particles from the blood
stream. Furthermore, during inflammatory states, macrophages become
more phagocytically active. The newly arriving stem cells thus face
the possibility of being phagocytosed while en route, unless
additional protection can be generated. Exploration of mechanisms
by which the endogenous HSC avoid being cleared by phagocytosis can
provide insight into ways for improving transplantation success of
hematopoietic and progenitor stem cells. The present invention
satisfies these, and other, needs.
SUMMARY OF THE INVENTION
[0011] Methods are provided to manipulate phagocytosis of
hematopoietic cells, including circulating hematopoietic cells,
e.g. bone marrow cells. In some embodiments of the invention the
circulating cells are hematopoietic stem cells, or hematopoietic
progenitor cells, particularly in a transplantation context, where
protection from phagocytosis is desirable. In other embodiments the
circulating cells are leukemia cells, particularly acute leukemia
cells such as AML (acute myeloid leukemia) or ALL (acute
lymphocytic leukemia), where increased phagocytosis is desirable.
In certain embodiments of the invention, methods are provided to
manipulate macrophage phagocytosis of circulating hematopoietic
cells. In yet other embodiments of the invention, methods are
provided to manipulate phagocytosis of solid tumors.
[0012] In other embodiments, tumor cells, e.g. solid tumor cells,
leukemia cells, etc. are targeted for phagocytosis by blocking CD47
on the cell surface. It is shown that leukemia cells, particularly
AML, ALL, etc. cells, evade macrophage surveillance by upregulation
of CD47 expression. Administration of agents that mask the CD47
protein, e.g. antibodies that bind to CD47 and prevent interaction
between CD47 and SIRP.alpha. are administered to a patient, which
increases the clearance of acute leukemia cells via phagocytosis.
In other embodiments, cells of solid tumors, e.g. carcinoma cells,
are targeted for phagocytosis by blocking CD47 present on the cell
surface. In other aspects, an agent that masks CD47 is combined
with monoclonal antibodies directed against one or more additional
leukemia stem cell (LSC) markers, e.g. CD96, and the like, which
compositions can be synergistic in enhancing phagocytosis and
elimination of LSC as compared to the use of single agents.
[0013] In another embodiment, methods are provided for targeting or
depleting leukemia stem cells, the method comprising contacting a
population of cells, e.g. blood from a leukemia patient, with a
reagent that specifically binds CD47 in order to target or deplete
LSC. In certain aspects, the reagent is an antibody conjugated to a
cytotoxic agent, e.g. radioactive isotope, chemotherapeutic agent,
toxin, etc. In some embodiments, the depletion is performed on an
ex vivo population of cells, e.g. the purging of autologous stem
cell products (mobilized peripheral blood or bone marrow) for use
in autologous transplantation for patients with acute myeloid
leukemia. In another embodiment, methods are provided for targeting
cancer cells of a solid tumor in a human subject by administering
an antibody against CD47 to the subject.
[0014] In some embodiments of the invention, hematopoietic stem or
progenitor cells are protected from phagocytosis in circulation by
providing a host animal with a CD47 mimetic molecule, which
interacts with SIRP.alpha. on phagocytic cells, such as,
macrophages, and decreases phagocytosis. The CD47 mimetic may be
soluble CD47; CD47 coated on the surface of the cells to be
protected, a CD47 mimetic that binds to SIRP.alpha. at the CD47
binding site, and the like. In some embodiments of the invention,
CD47 is provided as a fusion protein, for example soluble CD47
fused to an Fc fragment, e.g., IgG1 Fc, IgG2 Fc, Ig A Fc etc.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1. FACS analysis of human HSC and progenitor CD47
expression from Myelodysplastic syndrome (MDS, blue), Chronic
Myelogenous Leukemia, Accelerated Phase (CML AP, green) and normal
bone marrow (red).
[0016] FIG. 2. ET vs. PV. FACS analysis of CD47 expression by human
myeloproliferative disorders such as essential thrombocythemia (ET,
blue) and polycythemia vera (PV, green) HSC, progenitor and lineage
positive cells compared with human normal bone marrow (red).
[0017] FIG. 3A. Progenitor Profiles of Normal Bone Marrow (left),
post-polycythemic myelofibrosis with myeloid metaplasia (PPMM) and
CML Blast Crisis. FIG. 3B. FACS analysis of human normal bone
marrow (red) versus UMPD (green) versus PV (blue=ML) versus
atypical CML (orange), HSC, progenitor and lineage positive cell
CD47 expression.
[0018] FIG. 4. Increased CD47 Expression by CMML Progenitors (blue)
compared with normal bone marrow (red) with disease
progression.
[0019] FIGS. 5A-5B. (A) Progenitor Profiles of Normal bone marrow
(left) versus AML (right). (B) FACS analysis of human normal bone
marrow (red) versus AML (blue) HSC, progenitor and lineage positive
cell (blast) CD47 expression.
[0020] FIG. 6. CD47 is More Highly Expressed on AML LSC Compared to
Their Normal Counterparts. A. Relative CD47 expression on normal
bone marrow HSC (Lin-CD34+CD38-CD90+) and MPP
(Lin-CD34+CD38-CD90-CD45RA-), as well as LSC (Lin-CD34+CD38-CD90-)
and bulk leukemia cells from human AML samples was determined by
flow cytometry. Mean fluorescence intensity was normalized for cell
size and against lineage positive cells to account for analysis on
different days. The same sample of normal bone marrow (red, n=3) or
AML (blue, n=13) is indicated by the same symbol in the different
populations. The differences between the mean expression of HSC
with LSC (p=0.003), HSC with bulk leukemia (p=0.001), MPP with LSC
(p=0.004), and MPP with bulk leukemia (p=0.002) were statistically
significant using a 2-sided Student's t-test. The difference
between the mean expression of AML LSC compared to bulk AML was not
statistically significant with p=0.50 using a paired 2-sided
Student's t-test. B. Clinical and molecular characteristics of
primary human AML samples manipulated in vitro and/or in vivo.
[0021] FIG. 7. Anti-CD47 Antibody Stimulates In Vitro Macrophage
Phagocytosis of Primary Human AML LSC. AML LSC were purified by
FACS from two primary human AML samples, labeled with the
fluorescent dye CFSE, and incubated with mouse bone marrow-derived
macrophages either in the presence of an isotype control (A) or
anti-CD47 antibody (B). These cells were assessed by
immunofluorescence microscopy for the presence of fluorescently
labeled LSC within the macrophages. (C) The phagocytic index was
determined for each condition by calculating the number of ingested
cells per 100 macrophages.
[0022] FIG. 8A-C. Monoclonal Antibodies Directed Against Human CD47
Preferentially Enable Phagocytosis of Human AML LSC by Human and
Mouse Macrophages. A,B. CFSE-labeled AML LSC were incubated with
human peripheral blood-derived macrophages (A) or mouse bone
marrow-derived macrophages (B) in the presence of IgG1 isotype
control, anti-CD45 IgG1, or anti-CD47 (B6H12.2) IgG1 antibody.
These cells were assessed by immunofluorescence microscopy for the
presence of fluorescently labeled LSC within the macrophages
(indicated by arrows). C. CFSE-labeled AML LSC or normal bone
marrow CD34+ cells were incubated with human (left) or mouse
(right) macrophages in the presence of the indicated antibodies and
then assessed for phagocytosis by immunofluorescence microscopy.
The phagocytic index was determined for each condition by
calculating the number of ingested cells per 100 macrophages. For
AML LSC, the differences between isotype or anti-CD45 antibody with
blocking anti-CD47 antibody treatment (B6H12.2 and BRIC126) were
statistically significant with p<0.001 for all pairwise
comparisons with human and mouse macrophages. For human
macrophages, the differences between AML LSC and normal CD34+ cells
were statistically significant for B6H12.2 (p<0.001) and BRIC126
(p=0.002).
[0023] FIG. 9. Anti-CD47 Antibody stimulates in vitro macrophage
phagocytosis of primary human AML LSC. AML LSC were purified by
FACS from four primary human AML samples, labeled with the
fluorescent dye CFSE, and incubated with human peripheral blood
macrophages either in the presence of an isotype control, isotype
matched anti-CD45, or anti-CD47 antibody. (A) These cells were
assessed by immunofluorescence microscopy for the presence of
fluorescently-labeled LSC within the macrophages. The phagocytic
index was determined for each condition by calculating the number
of ingested cells per 100 macrophages. (B) The macrophages were
harvested, stained with a fluorescently labeled anti-human
macrophage antibody, and analyzed by flow cytometry. hMac+CFSE+
double positive events identify macrophages that have phagocytosed
CFSE-labeled LSC. Each sample is represented by a different
color.
[0024] FIG. 10A-B: A Monoclonal Antibody Directed Against Human
CD47 Inhibits AML LSC Engraftment In Vivo. Three primary human AML
samples were incubated with IgG1 isotype control, anti-CD45 IgG1,
or anti-CD47 IgG1 antibody (B6H12.2) prior to transplantation into
newborn NOG mice. A portion of the cells was analyzed for coating
by staining with a secondary anti-mouse IgG antibody and analyzed
by flow cytometry (A). 13 weeks later, mice were sacrificed and the
bone marrow was analyzed for the percentage of human CD45+CD33+
myeloid leukemia cells by flow cytometry (B). The difference in
mean engraftment between anti-CD47-coated cells and both isotype
(p<0.001) and anti-CD45 (p=0.003) coated cells was statistically
significant.
[0025] FIG. 11. CD47 is upregulated in murine acute myeloid
leukemia. Typical stem and progenitor plots are shown for leukemic
hMRP8bcrabl.times.hMRP8bcl2 cells compared to control non-leukemic
animals. Lin- c-Kit+Sca-1+ gated cells from control bone marrow (a)
and leukemic spleen (b) and Lin- c-Kit+Sca-1- gated cells from
control bone marrow (c) and leukemic spleen (d) demonstrate
perturberances in normal hematopoiesis in leukemic mice. Frequency
is shown as a percentage of entire marrow or spleen mononuclear
fraction. (e) Quantitative RT-PCR shows that CD47 is upregulated in
leukemic BM cells. Data are shown from 3 sets of mice transplanted
with either leukemic or control hRMP8bcrabl.times.hMRP8bcl2 BM
cells and then sacrificed 2-6 weeks later. Results were normalized
to beta-actin and 18S rRNA expression. Fold change relative to
control transplanted whole Bcl-2+ BM cells was determined. Error
bars represent 1 s.d. (f) Histograms show expression of CD47 on
gated populations for leukemic (gray) and control (black) mice.
[0026] FIG. 12. GMP expansion and CD47 upregulation in human
myeloid leukemia. a) Representative FACS plots of myeloid
progenitors (CD34+CD38+Lin-) including common myeloid progenitors
(CMP), megakaryocyte-erythroid progenitors (MEP) and
granulocyte-macrophage progenitors (GMP) in normal bone marrow (BM)
versus aCML, BC CML and AML. b) Comparative FACS histograms of CD47
expression by normal (red; n=6) and acute myelogenous leukemic
(AML, blue; n=6) hematopoietic stem cells (HSC;
CD34+CD38-CD90+Lin-) and progenitors (CD34+CD38+Lin-). c)
Comparative FACS histograms of CD47 expression by normal (red) and
chronic myelogenous leukemia hematopoietic stem cells (HSC;
CD34+CD38-CD90+Lin) and committed progenitors (CD34+CD38+Lin-).
Upper panel: Normal (n=7) versus chronic phase CML (n=4) HSC,
progenitors and lineage positive cells. Middle panel: Normal (n=7)
versus accelerated phase CML (n=7) HSC, progenitors and lineage
positive cells. Lower panel: Normal (n=7) versus blast crisis CML
(n=4) HSC, progenitors and lineage positive cells.
[0027] FIG. 13. Over-expression of murine CD47 increases
tumorigenicity of MOLM-13 cells. a) MOLM-13 cells were transduced
with either control virus or virus expressing murine CD47 cDNA form
2. The resulting cell lines, termed Tet or Tet-CD47, were
transplanted competitively into RAG/common gamma chain deficient
mice with untransduced MOLM-13 cells (5.times.10.sup.5 Tet (n=6) or
Tet-47 (n=8) cells with 5.times.10.sup.5 MOLM-13). Mice were
analyzed for GFP and human CD45 chimerism when moribund. b) MOLM-13
chimerism in hematopoietic tissues was determined by human CD45
chimerism and measurement of tumor lesion size. c) Survival of mice
competitively transplanted with MOLM-13 plus Tet or Tet-CD47
MOLM-13 cells was plotted. Control mice died of large tumor burden
at the site of injection but had no engraftment in hematopoietic
tissues. d) Hematoxylin and eosin sections of Tet-CD47 MOLM-13
transplanted liver (200.times.). Periportal (arrow) and sinusoidal
(arrowhead) tumor infiltration is evident. e) 1.times.10.sup.6Tet
(n=5) or Tet-CD47 MOLM-13 (n=4) cells were injected into the right
femur of RAG2-/-, Gc-/- mice and the tissues were analyzed 50-75
days later and chimerism of MOLM-13 cells in bone marrow was
determined. f) Survival curve of mice transplanted intrafemorally
with Tet or Tet-CD47 MOLM-13 cells. g) Examples of liver tumor
formation and hepatomegaly in Tet-CD47 MOLM-13 transplanted mice
versus control transplanted mice. GFP fluorescence demonstrates
tumor nodule formation as well diffuse infiltration.
[0028] FIG. 14. CD47 over-expression prevents phagocytosis of live
unopsonized MOLM-13 cells. a) Tet or Tet-CD47 MOLM-13 cells were
incubated with bone marrow derived macrophages (BMDM) for 2, 4, or
6 hours and phagocytic index was determined. Error bars represent 1
s.d. (n=6 for each time point). b) FACS analysis of BMDMs incubated
with either Tet or Tet-CD47 cells. c) Photomicrographs of BMDMs
incubated with Tet or Tet-CD47 MOLM-13 cells at 2 and 24 hours
(400.times.). d) Tet or Tet-CD47 MOLM-13 cells were transplanted
into RAG2-/-, Gc-/- mice and marrow, spleen, and liver macrophages
were analyzed 2 hours later. GFP+ fraction of macrophages are
gated. Results are representative of 3 experiments.
[0029] FIG. 15. Higher expression of CD47 on MOLM-13 cells
correlates with tumorigenic potential and evasion of phagocytosis.
a) Tet-CD47 MOLM-13 cells were divided into high and low expressing
clones as described. Histograms show CD47 expression in MOLM-13
high (black), MOLM-13 low (gray), and mouse bone marrow (shaded)
cells. Value obtained for MFI/FSC.sup.2 (.times.10.sup.9) are
shown. b) Mice transplanted with CD47hi MOLM-13 cells were given
doxycycline for 2 weeks. The histograms show level of CD47
expression in untreated (shaded) and treated (shaded) mice, with
the values of MFI/FSC.sup.2 (.times.10.sup.9) indicated. c)
Survival of RAG2-/-, Gc-/- mice transplanted with 1.times.10.sup.6
CD47.sup.hi, CD47.sup.lo MOLM-13 cells, or CD47.sup.hi MOLM-13
cells with doxycycline administration after 2 weeks
post-transplant. d) Liver and spleen size of mice at necropsy or 75
days after transplant with 1.times.10.sup.6 CD47.sup.hi,
CD47.sup.lo MOLM-13 cells, or CD47.sup.hi MOLM-13 cells with
doxycycline administration after 2 weeks post-transplant. e) Bone
marrow and spleen chimerism of human cells in mice at necropsy or
75 days after transplant with 1.times.10.sup.6 CD47.sup.hi,
CD47.sup.lo MOLM-13 cells, or CD47.sup.loi MOLM-13 cells with
doxycycline administration after 2 weeks post-transplant. f) Murine
CD47 expression on CD47.sup.lo MOLM-13 cells engrafting in bone
marrow (open) compared with original cell line (shaded). The values
of MFI/FSC.sup.2 (.times.10.sup.9) are indicated. g)
2.5.times.10.sup.5 CD47.sup.hi or CD47.sup.lo MOLM-13 cells were
incubated with 5.times.10.sup.4 BMDMs for 2 hours. Phagocytic index
is shown. h) 2.5.times.10.sup.5 CD47.sup.hi RFP and CD47.sup.lo
MOLM-13 GFP cells were incubated with 5.times.10.sup.4 BMDMs for 2
hours. Phagocytic index is shown for three separate samples for
CD47.sup.hi RFP (red) and CD47.sup.lo MOLM-13 GFP (green) cells. i)
2.5.times.10.sup.5 CD47.sup.hi RFP and CD47.sup.lo MOLM-13 GFP
cells were incubated with 5.times.10.sup.4 BMDMs for 24 hours.
Photomicrographs show brightfield (top left), RFP (top right), GFP
(bottom left), and merged (bottom right) images.
[0030] FIG. 16. a) FACS analysis of CD47 expression of non-leukemic
Fas lpr/lpr hMRP8bcl-2 (blue) and leukemic Fas lpr/lpr hMRP8bcl-2
(green) bone marrow hematopoietic stem cells (c-kit+Sca+Lin-),
myeloid progenitors (c-kit+Sca-Lin-) or blasts (c-kit lo Sca-Lin-).
b) Mouse bone marrow was transduced with retrovirus containing p210
bcr/abl as previously described.sup.24. Mice were sacrificed when
moribund and the spleens were analyzed. Expression of CD47 in
c-Kit+Mac-1+ cells in the spleens of two leukemic mice (unshaded
histograms) and bone marrow from a wild-type mouse (shaded
histogram) are shown. c) Histograms show expression of CD47 on
gated populations for leukemic hMRP8bcrabl.times.hMRP8bcl2 mice
(red), hMRP8bcl2 non-leukemic (blue) and wild-type (green) mice.
CD47 was stained using FITC conjugated anti-mouse CD47
(Pharmingen).
[0031] FIG. 17. a) Expression of human CD47 (black histograms) on
human leukemia cell lines and cord blood HSCs is shown. Isotype
control staining is shown in gray. b) CD47 MFI over background was
normalized to cell size by dividing by FSC.sup.2. The value
obtained for each cell type is shown above the bar. c) HL-60 cells
engraft mouse bone marrow. 5.times.10.sup.5 cells were injected
intravenously into RAG2-/-, Gc-/- animals and mice were analyzed 4
weeks later. d) Cells were stained with CFSE and co-cultured with
BMDM. Phagocytic events were counted after 2 h. For irradiation,
Jurkat cells were given a dose of 2 Gray and incubated for 16 h
prior to the phagocytosis assay.
[0032] FIG. 18. (a) Analysis of stem and progenitor cells from bone
marrow of IAP+/+, IAP+/-, and IAP-/- mice. Stem cells (left) are
gated on lineage--c-Kit+Sca-1+ cells. Myeloid progenitors (right)
are gated on lineage--c-Kit+Sca-1+ cells. Frequency in whole bone
marrow is shown adjacent to each gated population. (b) Colony
output on day 7 of individually sorted LT-HSC. G-granulocyte,
M-macrophage, GM-granulocyte and macrophage, GEMM-granulocyte,
macrophage, erythroid, and megakaryocyte, Meg-megakaryocyte. (c)
Survival curve of recipient mice given a radiation dose of 9.5 Gray
and transplanted with the cells shown. Radiation control mice all
died within 12-15 days. n=5 for each group. (d) Examples of
CD45.1/CD45.2 chimerism plots at 4 weeks post-transplant. CD45.1
mice were transplanted with 50 LT-HSC (CD45.2) and 2.times.10.sup.5
CD45.1 helper marrow. Cells are gated on B220- CD3- Mac-1+ side
scatter mid/hi cells. IAP-/- cells fail to engraft. (e) Summary of
chimerism analysis of mice transplanted with either 50 or 500
IAP+/+ or IAP-/- cells. (f) IAP+/+ or IAP-/- c-Kit enriched cells
were incubated with wild-type BMDM. Results indicate mean
phagocytic index calculated from three separate samples. Error bars
represent 1 s.d. (g) Photomicrographs of phagocytosis assays taken
after 2 hours. Genotype of the -Kit enriched cells is shown.
[0033] FIG. 19. (a) Mice were mobilized with Cy/G and bone marrow
was analyzed on day 2. Expression level of CD47 on c-Kit+ cells is
shown. (b) Myeloid progenitor and stem cell gates are shown for day
2 mobilized bone marrow. Histograms on left show level of CD47
expression in marrow LT-HSC and GMP for steady-state (shaded
histogram), day 2 mobilized (black line), and day 5 mobilized (gray
line). (c) Relative MFI of CD47 for GMP on days 0-5 of Cy/G
mobilization. Results were normalized so that steady state GMP were
equal to 100. (d) Myeloid progenitor and stem cell gates are shown
for day 2 bone marrow post-LPS treatment. Histograms show level of
CD47 expression on day 2 post-LPS (black line), day 5 post-LPS
(dark gray shaded histogram), steady state (light gray shaded
histogram), and IAP-/- (black shaded histogram) LT-HSC and GMP. (e)
Evaluation of KLS cells in the hematopoietic organs of IAP+/+ and
IAP-/- mice mobilized on days 2 through 5. Two mice are analyzed
per genotype per day.
[0034] FIG. 20. (a) CD47 expression level of IAP+/+, IAP+/-, and
IAP-/- LT-HSC. The numbers shown are the MFI for each group. (b)
Donor chimerism analysis for transplants of IAP+/+ (top) or IAP+/-
(bottom) mice. Mice were bled at 2, 8, and 40 weeks post
transplant. 2.times.10.sup.6 donor cells were transplanted into
sub-lethally irradiated congenic recipients.
[0035] FIG. 21A-D: Identification and Separation of Normal and
Leukemic Progenitors From the Same Patient Based On Differential
CD47 Expression. A. CD47 expression on the Lin-CD34+CD38-
LSC-enriched fraction of specimen SU008 was determined by flow
cytometry. CD47hi- and CD47lo-expressing cells were identified and
purified using FACS. The left panels are gated on lineage negative
cells, while the right panels are gated on Lin-CD34+CD38- cells. B.
Lin-CD34+CD38-CD47lo and Lin-CD34+CD38-CD47hi cells were plated
into complete methylcellulose, capable of supporting the growth of
all myeloid colonies. 14 days later, myeloid colony formation was
determined by morphologic assessment. Representative CFU-G/M (left)
and BFU-E (right) are presented. C. Lin-CD34+CD38-CD47lo cells were
transplanted into 2 newborn NOG mice. 12 weeks later, the mice were
sacrificed and the bone marrow was analyzed for the presence of
human CD45+CD33+ myeloid cells and human CD45+CD19+ lymphoid cells
by flow cytometry. D. Normal bone marrow HSC, bulk 5U008 leukemia
cells, Lin-CD34+CD38-CD47hi cells, Lin-CD34+CD38-CD47lo cells, or
human CD45+ cells purified from the bone marrow of mice engrafted
with Lin-CD34+CD38-CD47lo cells were assessed for the presence of
the FLT3-ITD mutation by PCR. The wild type FLT3 and the FLT3-ITD
products are indicated.
[0036] FIG. 22: Increased CD47 Expression in Human AML is
Associated with Poor Clinical Outcomes. Event-free (A,C) and
overall (B,D) survival of 132 AML patients with normal cytogenetics
(A,B) and the subset of 74 patients without the FLT3-ITD mutation
(C,D). Patients were stratified into low CD47 and high CD47
expression groups based on an optimal threshold (28% high, 72% low)
determined by microarray analysis from an independent training data
set. The significance measures are based on log-likelihood
estimates of the p-value, when treating the model with CD47
expression as a binary classification.
[0037] FIG. 23A-E: A Monoclonal Antibody Directed Against Human
CD47 Eliminates AML In Vivo. Newborn NOG mice were transplanted
with AML LSC, and 8-12 weeks later, peripheral blood (A,B) and bone
marrow (C-E) were analyzed for baseline engraftment prior to
treatment with anti-CD47 (B6H12.2) or control IgG antibody (Day 0).
Mice were treated with daily 100 microgram intraperitoneal
injections for 14 days, at the end of which, they were sacrificed
and peripheral blood and bone marrow were analyzed for the
percentage of human CD45+CD33+ leukemia. A. Pre- and post-treatment
human leukemic chimerism in the peripheral blood from
representative anti-CD47 antibody and control IgG-treated mice as
determined by flow cytometry. B. Summary of human leukemic
chimerism in the peripheral blood assessed on multiple days during
the course of treatment demonstrated elimination of leukemia in
anti-CD47 antibody treated mice compared to control IgG treatment
(p=0.007). C. Pre- and post-treatment human leukemic chimerism in
the bone marrow from representative anti-CD47 antibody or control
IgG-treated mice as determined by flow cytometry. D. Summary of
human leukemic chimerism in the bone marrow on day 14 relative to
day 0 demonstrated a dramatic reduction in leukemic burden in
anti-CD47 antibody treated mice compared to control IgG treatment
(p<0.001). E. H&E sections of representative mouse bone
marrow cavities from mice engrafted with 5U004 post-treatment with
either control IgG (panels 1,2) or anti-CD47 antibody (panels 4,5).
IgG-treated marrows were packed with monomorphic leukemic blasts,
while anti-CD47-treated marrows were hypocellular, demonstrating
elimination of the human leukemia. In some anti-CD47
antibody-treated mice that contained residual leukemia, macrophages
were detected containing phagocytosed pyknotic cells, capturing the
elimination of human leukemia (panels 3,6 arrows).
[0038] FIG. 24. Increased CD47 expression predicts worse overall
survival in DLBCL and ovarian cancer. (A) A cohort of 230 patients
with diffuse large B-cell lymphoma (p=0.01). (B) A cohort of 133
patients with advanced stage (III/IV) ovarian carcinoma
(p=0.04).
[0039] FIG. 25: Anti-CD47 antibody enables the phagocytosis of
solid tumor stem cells in vitro. The indicated cells were incubated
with human macrophages in the presence of IgG1 isotype, anti-HLA,
or anti-CD47 antibodies and the phagocytic index was determined by
immunofluorescence microscopy. Statistics: Bladder cancer cells
IgG1 isotype compared to anti-HLA (p=0.93) and anti-CD47 (p=0.01);
normal bladder urothelium IgG1 isotype compared to anti-HLA
(p=0.50) and anti-CD47 (p=0.13); ovarian cancer cells IgG1 isotype
compared to anti-HLA (p=0.11) and anti-CD47 (p<0.001). Each
individual data point represents a distinct tumor or normal tissue
sample.
[0040] FIG. 26: CD47 expression is an independent prognostic
predictor in mixed and high-risk ALL. (A) Pediatric ALL patients
(n=360) with mixed risk and treatment were stratified into CD47
high- and low-expressing groups based on an optimal cut point.
Disease-free survival (DFS) was determined by Kaplan-Meier
analysis. CD47 high-expressing patients had a worse DFS compared to
CD47 low-expressing patients when CD47 expression was considered as
a continuous variable. (B) Pediatric ALL patients (n=207) with
high-risk (as defined by age>10 years, presenting WBC
count>50,000/.mu.l, hypodiploidy, and BCR-ABL positive disease)
and uniform treatment were stratified into CD47 high- and
low-expressing groups using a similar approach as in A. CD47
high-expressing patients had a worse overall survival compared to
CD47 low-expressing patients (p=0.0009). (C) Multivariate analysis
of prognostic covariates was performed from patients analyzed for
CD47 expression in (B). When incorporated into this multivariate
analysis, CD47 expression still remained prognostic (p=0.035). (D)
ALL patients from were stratified into groups either achieving a
complete remission (CR) or not achieving a CR (no CR). CD47
expression was higher in patients failing to receive a CR compared
to those who did (p=0.0056).
[0041] FIG. 27: Ex vivo coating of ALL cells with an anti-CD47
antibody inhibits leukemic engraftment. (A) ALL cells were
incubated with the indicated antibodies in vitro, and positive cell
coating was detected by staining a portion of the cells with a
fluorescently-labeled secondary antibody. A flow cytometry plot of
a representative ALL sample is shown. (B-C) Pre-coated ALL cells
were then transplanted into NSG mice, and human ALL chimerism was
assessed 6-10 weeks later in the peripheral blood (B) or bone
marrow (C). Ex vivo coating of ALL cells (ALL4 and ALL8) with
anti-CD47 antibody inhibited engraftment in the peripheral blood
compared to IgG1 isotype control (p=0.02). Ex vivo coating of ALL
cells with anti-CD47 antibody inhibited bone marrow engraftment
compared to IgG1 isotype control (p=0.02), while no difference in
engraftment levels were detected between anti-CD45 antibody and
IgG1 isotype control (p=0.67, considering both B and T-ALL
samples). Each symbol represents a different primary ALL sample,
with each point representing a different mouse. p-values were
calculated using the Fisher's exact test. Red diamond=ALL4, blue
diamond=ALL4.
[0042] FIG. 28: Anti-CD47 antibody eliminates ALL engraftment in
the peripheral blood and bone marrow. (A) NSG mice engrafted with
primary B and T-ALL patient samples were treated for 14 days with
daily intraperitoneal injections of 100 .mu.g IgG control or
anti-CD47 antibody. Peripheral blood human ALL chimerism
(huCD45+CD19/CD3+) pre- and post-treatment were measured by flow
cytometry. Peripheral blood chimerism is shown from representative
treatment mice. (B) Anti-CD47 antibody treatment reduced the level
of circulating leukemia compared to IgG control (p=0.0002). (C)
Anti-CD47 antibody treatment also reduced ALL engraftment in the
bone marrow compared to IgG control (p=0.0004). Each symbol
represents a different patient sample, with each data point
representing a different mouse. (D) (Top) Hematoxylin and eosin
bone marrow sections from representative mice engrafted with B-ALL
post-treatment. IgG-treated marrows were primarily packed with
monomorphic leukemic blasts, while anti-CD47 antibody-treated
marrows demonstrated areas of normal mouse hematopoiesis. (Bottom)
Leukemic infiltration was confirmed by immunohistochemical analysis
of human CD45 demonstrating robust human leukemia infiltration in
IgG-treated bone marrow compared to anti-CD47 antibody-treated
marrow.
[0043] FIG. 29: Anti-CD47 antibody eliminates ALL engraftment in
the spleen and liver. (A) NSG mice engrafted with primary B-ALL
cells from sample ALL8, ALL21, or ALL22 were treated for 14 days
with daily injections of IgG control or anti-CD47 antibody. Spleens
were then harvested, with representative spleens from IgG control
or anti-CD47 antibody treatment shown. (B) Spleen weights were
determined from mice treated with anti-CD47 antibody demonstrating
a reduction in spleen size compared to control IgG-treated mice
(p=0.04) to sizes similar to that of normal spleens (p=0.09).
Control IgG-treated mice demonstrate splenomegaly compared to
normal mice (p=0.0002, student t-test). (C-D) Levels of ALL
engraftment were determined at the end of antibody treatment in the
spleen (C) and liver (D). Compared to IgG control, treatment with
anti-CD47 antibody eliminated ALL disease in the spleen
(p<0.0001) and liver (p<0.0001, student t-test).
[0044] FIGS. 30A-30D: Antibodies targeted to human CD47 enable the
phagocytosis of human cancer cells. A: CFSE-labeled human patient
bladder cancer cells were incubated with human macrophages in the
presence of the indicated antibodies and assessed for the presence
of tumor cells within macrophages. B-D: Phagocytosis of patient
bladder cancer cells (B), ovarian cancer cells (C), or colon cancer
stem cells (D) resulting from indicated antibody treatment was
quantified. Each dot color represents a different primary tumor
sample. Open (non-colored) symbols represent normal tissue
controls. The phagocytic index was determined as the number of CFSE
labeled tumor cells present within 100 macrophages.
[0045] FIGS. 31A-31F: Antibodies targeted to CD47 inhibit the
growth of patient tumors. A-D: Tumor cells from ovarian (A),
pancreatic (B), breast (C), or colon (D) tumors were engrafted into
immunodeficient mice. These mice were then treated with control IgG
or anti-CD47 antibodies and tumor growth was assessed directly or
by bioluminescence. In all cases, anti-CD47 antibody treatment
substantially inhibited tumor growth. E-F: Tumor cells from patient
bladder were injected subcutaneously into immunodeficient mice and
treated with the indicated antibodies. Anti-CD47 antibody treatment
significantly inhibited metastasis to the lymph nodes (E) and
formation of micrometastases in the lungs (F). The total number of
metastases detected in each treatment group is indicated.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0046] Methods are provided to manipulate the phagocytosis of
cells, including circulating hematopoietic cells. In some
embodiments of the invention, leukemia cells, e.g. AML, B-ALL,
T-ALL, etc. are targeted for phagocytosis by blocking CD47 on the
cell surface. In other embodiments, cells of solid tumors are
targeted for phagocytosis by blocking CD47 on the cell surface. In
another embodiment, methods are provided for targeting or depleting
leukemia stem cells, e.g. AML stem cells, ALL stem cells, etc., the
method comprising contacting reagent blood cells with an antibody
that specifically binds CD47 in order to target or deplete LSC. In
another embodiment, methods are provided for targeting cancer cells
of a tumor in a human subject by administering an antibody specific
for CD47 to the subject.
[0047] In other embodiments, hematopoietic stem or progenitor cells
are protected from phagocytosis in circulation by providing a host
animal with a CD47 mimetic molecule, which interacts with
SIRP.alpha. on phagocytic cells, such as, macrophages, and
decreases phagocytosis.
[0048] Before the present invention is further described, it is to
be understood that this invention is not limited to particular
embodiments described, as such may, of course, vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
be limiting, since the scope of the present invention will be
limited only by the appended claims.
[0049] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0050] Methods recited herein may be carried out in any order of
the recited events which is logically possible, as well as the
recited order of events.
[0051] Unless defined otherwise, 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. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now
described.
[0052] All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
[0053] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. It is
further noted that the claims may be drafted to exclude any
optional element. As such, this statement is intended to serve as
antecedent basis for use of such exclusive terminology as "solely,"
"only" and the like in connection with the recitation of claim
elements, or use of a "negative" limitation.
[0054] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
DEFINITIONS
[0055] CD47 polypeptides. The three transcript variants of human CD
47 (variant 1, NM 001777; variant 2, NM 198793; and variant 3, NM
001025079) encode three isoforms of CD47 polypeptide. CD47 isoform
1 (NP 001768), the longest of the three isoforms, is 323 amino
acids long. CD47 isoform 2 (NP 942088) is 305 amino acid long. CD47
isoform 3 is 312 amino acids long. The three isoforms are identical
in sequence in the first 303 amino acids. Amino acids 1-8 comprise
the signal sequence, amino acids 9-142 comprise the CD47
immunoglobulin like domain, which is the soluble fragment, and
amino acids 143-300 is the transmembrane domain.
[0056] "CD47 mimetics" include molecules that function similarly to
CD47 by binding and activating SIRP.alpha. receptor. Molecules
useful as CD47 mimetics include derivatives, variants, and
biologically active fragments of naturally occurring CD47. A
"variant" polypeptide means a biologically active polypeptide as
defined below having less than 100% sequence identity with a native
sequence polypeptide. Such variants include polypeptides wherein
one or more amino acid residues are added at the N- or C-terminus
of, or within, the native sequence; from about one to forty amino
acid residues are deleted, and optionally substituted by one or
more amino acid residues; and derivatives of the above
polypeptides, wherein an amino acid residue has been covalently
modified so that the resulting product has a non-naturally
occurring amino acid. Ordinarily, a biologically active variant
will have an amino acid sequence having at least about 90% amino
acid sequence identity with a native sequence polypeptide,
preferably at least about 95%, more preferably at least about 99%.
The variant polypeptides can be naturally or non-naturally
glycosylated, i.e., the polypeptide has a glycosylation pattern
that differs from the glycosylation pattern found in the
corresponding naturally occurring protein. The variant polypeptides
can have post-translational modifications not found on the natural
CD47 protein.
[0057] Fragments of the soluble CD47, particularly biologically
active fragments and/or fragments corresponding to functional
domains, are of interest. Fragments of interest will typically be
at least about 10 aa to at least about 15 aa in length, usually at
least about 50 aa in length, but will usually not exceed about 142
aa in length, where the fragment will have a stretch of amino acids
that is identical to CD47. A fragment "at least 20 aa in length,"
for example, is intended to include 20 or more contiguous amino
acids from, for example, the polypeptide encoded by a cDNA for
CD47. In this context "about" includes the particularly recited
value or a value larger or smaller by several (5, 4, 3, 2, or 1)
amino acids. The protein variants described herein are encoded by
polynucleotides that are within the scope of the invention. The
genetic code can be used to select the appropriate codons to
construct the corresponding variants. The polynucleotides may be
used to produce polypeptides, and these polypeptides may be used to
produce antibodies by known methods.
[0058] A "fusion" polypeptide is a polypeptide comprising a
polypeptide or portion (e.g., one or more domains) thereof fused or
bonded to heterologous polypeptide. A fusion soluble CD47 protein,
for example, will share at least one biological property in common
with a native sequence soluble CD47 polypeptide. Examples of fusion
polypeptides include immunoadhesins, as described above, which
combine a portion of the CD47 polypeptide with an immunoglobulin
sequence, and epitope tagged polypeptides, which comprise a soluble
CD47 polypeptide or portion thereof fused to a "tag polypeptide".
The tag polypeptide has enough residues to provide an epitope
against which an antibody can be made, yet is short enough such
that it does not interfere with biological activity of the CD47
polypeptide. Suitable tag polypeptides generally have at least six
amino acid residues and usually between about 6-60 amino acid
residues.
[0059] A "functional derivative" of a native sequence polypeptide
is a compound having a qualitative biological property in common
with a native sequence polypeptide. "Functional derivatives"
include, but are not limited to, fragments of a native sequence and
derivatives of a native sequence polypeptide and its fragments,
provided that they have a biological activity in common with a
corresponding native sequence polypeptide. The term "derivative"
encompasses both amino acid sequence variants of polypeptide and
covalent modifications thereof. Derivatives and fusion of soluble
CD47 find use as CD47 mimetic molecules.
[0060] The first 142 amino acids of CD47 polypeptide comprise the
extracellular region of CD47 (SEQ ID NO: 1). The three isoforms
have identical amino acid sequence in the extracellular region, and
thus any of the isoforms are can be used to generate soluble CD47.
"Soluble CD47" is a CD47 protein that lacks the transmembrane
domain. Soluble CD47 is secreted out of the cell expressing it
instead of being localized at the cell surface. Soluble CD47 may be
fused to another polypeptide to provide for added functionality,
e.g. to increase the in vivo stability. Generally such fusion
partners are a stable plasma protein that is capable of extending
the in vivo plasma half-life of soluble CD47 protein when present
as a fusion, in particular wherein such a stable plasma protein is
an immunoglobulin constant domain. In most cases where the stable
plasma protein is normally found in a multimeric form, e.g.,
immunoglobulins or lipoproteins, in which the same or different
polypeptide chains are normally disulfide and/or noncovalently
bound to form an assembled multichain polypeptide. Soluble CD47
fused to human Ig G1 has been described (Motegi S. et al. EMBO J.
22(11): 2634-2644).
[0061] Stable plasma proteins are proteins typically having about
from 30 to 2,000 residues, which exhibit in their native
environment an extended half-life in the circulation, i.e. greater
than about 20 hours. Examples of suitable stable plasma proteins
are immunoglobulins, albumin, lipoproteins, apolipoproteins and
transferrin. The extracellular region of CD47 is typically fused to
the plasma protein at the N-terminus of the plasma protein or
fragment thereof which is capable of conferring an extended
half-life upon the soluble CD47. Increases of greater than about
100% on the plasma half-life of the soluble CD47 are
satisfactory.
[0062] Ordinarily, the soluble CD47 is fused C-terminally to the
N-terminus of the constant region of immunoglobulins in place of
the variable region(s) thereof, however N-terminal fusions may also
find use. Typically, such fusions retain at least functionally
active hinge, CH2 and CH3 domains of the constant region of an
immunoglobulin heavy chain, which heavy chains may include IgG1,
IgG2a, IgG2b, IgG3, IgG4, IgA, IgM, IgE, and IgD, usually one or a
combination of proteins in the IgG class. Fusions are also made to
the C-terminus of the Fc portion of a constant domain, or
immediately N-terminal to the CH1 of the heavy chain or the
corresponding region of the light chain. This ordinarily is
accomplished by constructing the appropriate DNA sequence and
expressing it in recombinant cell culture. Alternatively, the
polypeptides may be synthesized according to known methods.
[0063] The precise site at which the fusion is made is not
critical; particular sites may be selected in order to optimize the
biological activity, secretion or binding characteristics of CD47.
The optimal site will be determined by routine experimentation.
[0064] In some embodiments the hybrid immunoglobulins are assembled
as monomers, or hetero- or homo-multimers, and particularly as
dimers or tetramers. Generally, these assembled immunoglobulins
will have known unit structures. A basic four chain structural unit
is the form in which IgG, IgD, and IgE exist. A four chain unit is
repeated in the higher molecular weight immunoglobulins; IgM
generally exists as a pentamer of basic four-chain units held
together by disulfide bonds. IgA immunoglobulin, and occasionally
IgG immunoglobulin, may also exist in a multimeric form in serum.
In the case of multimers, each four chain unit may be the same or
different.
[0065] Suitable CD47 mimetics and/or fusion proteins may be
identified by compound screening by detecting the ability of an
agent to mimic the biological activity of CD47. One biological
activity of CD47 is the activation of SIRP.alpha. receptor on
macrophages. In vitro assays may be conducted as a first screen for
efficacy of a candidate agent, and usually an in vivo assay will be
performed to confirm the biological assay. Desirable agents are
effective in temporarily blocking SIRP a receptor activation.
Desirable agents are temporary in nature, e.g. due to biological
degradation.
[0066] In vitro assays for CD47 biological activity include, e.g.
inhibition of phagocytosis of porcine cells by human macrophages,
binding to SIRP .alpha. receptor, SIRP .alpha. tyrosine
phosphorylation, etc. An exemplary assay for CD47 biological
activity contacts a human macrophage composition in the presence of
a candidate agent. The cells are incubated with the candidate agent
for about 30 minutes and lysed. The cell lysate is mixed with
anti-human SIRP .alpha. antibodies to immunoprecipitate SIRP
.alpha.. Precipitated proteins are resolved by SDS PAGE, then
transferred to nitrocellulose and probed with antibodies specific
for phosphotyrosine. A candidate agent useful as CD47 mimetic
increases SIRP .alpha. tyrosine phosphorylation by at least 10%, or
up to 20%, or 50%, or 70% or 80% or up to about 90% compared to the
level of phosphorylation observed in the absence of candidate
agent. Another exemplary assay for CD47 biological activity
measures phagocytosis of hematopoietic cells by human macrophages.
A candidate agent useful as a CD47 mimetic results in the down
regulation of phagocytosis by at least about 10%, at least about
20%, at least about 50%, at least about 70%, at least about 80%, or
up to about 90% compared to level of phagocytosis observed in
absence of candidate agent.
[0067] Polynucleotide encoding soluble CD47 or soluble CD47-Fc can
be introduced into a suitable expression vector. The expression
vector is introduced into a suitable cell. Expression vectors
generally have convenient restriction sites located near the
promoter sequence to provide for the insertion of polynucleotide
sequences. Transcription cassettes may be prepared comprising a
transcription initiation region, CD47 gene or fragment thereof, and
a transcriptional termination region. The transcription cassettes
may be introduced into a variety of vectors, e.g. plasmid;
retrovirus, e.g. lentivirus; adenovirus; and the like, where the
vectors are able to transiently or stably be maintained in the
cells, usually for a period of at least about one day, more usually
for a period of at least about several days to several weeks.
[0068] The various manipulations may be carried out in vitro or may
be performed in an appropriate host, e.g. E. coli. After each
manipulation, the resulting construct may be cloned, the vector
isolated, and the DNA screened or sequenced to ensure the
correctness of the construct. The sequence may be screened by
restriction analysis, sequencing, or the like.
[0069] Soluble CD47 can be recovered and purified from recombinant
cell cultures by well-known methods including ammonium sulfate or
ethanol precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, affinity chromatography, protein G
affinity chromatography, for example, hydroxylapatite
chromatography and lectin chromatography. Most preferably, high
performance liquid chromatography ("HPLC") is employed for
purification.
[0070] Soluble CD47 can also be recovered from: products of
purified cells, whether directly isolated or cultured; products of
chemical synthetic procedures; and products produced by recombinant
techniques from a prokaryotic or eukaryotic host, including, for
example, bacterial, yeast higher plant, insect, and mammalian
cells.
[0071] A plurality of assays may be run in parallel with different
concentrations to obtain a differential response to the various
concentrations. As known in the art, determining the effective
concentration of an agent typically uses a range of concentrations
resulting from 1:10, or other log scale, dilutions. The
concentrations may be further refined with a second series of
dilutions, if necessary. Typically, one of these concentrations
serves as a negative control, i.e. at zero concentration or below
the level of detection of the agent or at or below the
concentration of agent that does not give a detectable change in
binding.
[0072] Compounds of interest for screening include biologically
active agents of numerous chemical classes, primarily organic
molecules, although including in some instances inorganic
molecules, organometallic molecules, immunoglobulins, chimeric CD47
proteins, CD47 related proteins, genetic sequences, etc. Also of
interest are small organic molecules, which comprise functional
groups necessary for structural interaction with proteins,
particularly hydrogen bonding, and typically include at least an
amine, carbonyl, hydroxyl or carboxyl group, frequently at least
two of the functional chemical groups. The candidate agents often
comprise cyclical carbon or heterocyclic structures and/or aromatic
or polyaromatic structures substituted with one or more of the
above functional groups. Candidate agents are also found among
biomolecules, including peptides, polynucleotides, saccharides,
fatty acids, steroids, purines, pyrimidines, derivatives,
structural analogs or combinations thereof.
[0073] Compounds are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds, including biomolecules,
including expression of randomized oligonucleotides and
oligopeptides. Alternatively, libraries of natural compounds in the
form of bacterial, fungal, plant and animal extracts are available
or readily produced. Additionally, natural or synthetically
produced libraries and compounds are readily modified through
conventional chemical, physical and biochemical means, and may be
used to produce combinatorial libraries. Known pharmacological
agents may be subjected to directed or random chemical
modifications, such as acylation, alkylation, esterification,
amidification, etc. to produce structural analogs.
[0074] By "manipulating phagocytosis" is meant an up-regulation or
a down-regulation in phagocytosis by at least about 10%, or up to
20%, or 50%, or 70% or 80% or up to about 90% compared to level of
phagocytosis observed in absence of intervention. Thus in the
context of decreasing phagocytosis of circulating hematopoietic
cells, particularly in a transplantation context, manipulating
phagocytosis means a down-regulation in phagocytosis by at least
about 10%, or up to 20%, or 50%, or 70% or 80% or up to about 90%
compared to level of phagocytosis observed in absence of
intervention.
[0075] CD47 inhibitors. Agents of interest as CD47 inhibitors
include specific binding members that prevent the binding of CD47
with SIRP a receptor. The term "specific binding member" or
"binding member" as used herein refers to a member of a specific
binding pair, i.e. two molecules, usually two different molecules,
where one of the molecules (i.e., first specific binding member)
through chemical or physical means specifically binds to the other
molecule (i.e., second specific binding member). CD47 inhibitors
useful in the methods of the invention include analogs, derivatives
and fragments of the original specific binding member.
[0076] In a preferred embodiment, the specific binding member is an
antibody. The term "antibody" or "antibody moiety" is intended to
include any polypeptide chain-containing molecular structure with a
specific shape that fits to and recognizes an epitope, where one or
more non-covalent binding interactions stabilize the complex
between the molecular structure and the epitope. Antibodies
utilized in the present invention may be polyclonal antibodies,
although monoclonal antibodies are preferred because they may be
reproduced by cell culture or recombinantly, and can be modified to
reduce their antigenicity.
[0077] Polyclonal antibodies can be raised by a standard protocol
by injecting a production animal with an antigenic composition.
See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, 1988. When utilizing an entire protein,
or a larger section of the protein, antibodies may be raised by
immunizing the production animal with the protein and a suitable
adjuvant (e.g., Freund's, Freund's complete, oil-in-water
emulsions, etc.) When a smaller peptide is utilized, it is
advantageous to conjugate the peptide with a larger molecule to
make an immunostimulatory conjugate. Commonly utilized conjugate
proteins that are commercially available for such use include
bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). In
order to raise antibodies to particular epitopes, peptides derived
from the full sequence may be utilized. Alternatively, in order to
generate antibodies to relatively short peptide portions of the
protein target, a superior immune response may be elicited if the
polypeptide is joined to a carrier protein, such as ovalbumin, BSA
or KLH. Alternatively, for monoclonal antibodies, hybridomas may be
formed by isolating the stimulated immune cells, such as those from
the spleen of the inoculated animal. These cells are then fused to
immortalized cells, such as myeloma cells or transformed cells,
which are capable of replicating indefinitely in cell culture,
thereby producing an immortal, immunoglobulin-secreting cell line.
In addition, the antibodies or antigen binding fragments may be
produced by genetic engineering. Humanized, chimeric, or xenogeneic
human antibodies, which produce less of an immune response when
administered to humans, are preferred for use in the present
invention.
[0078] In addition to entire immunoglobulins (or their recombinant
counterparts), immunoglobulin fragments comprising the epitope
binding site (e.g., Fab', F(ab').sub.2, or other fragments) are
useful as antibody moieties in the present invention. Such antibody
fragments may be generated from whole immunoglobulins by ricin,
pepsin, papain, or other protease cleavage. "Fragment," or minimal
immunoglobulins may be designed utilizing recombinant
immunoglobulin techniques. For instance "Fv" immunoglobulins for
use in the present invention may be produced by linking a variable
light chain region to a variable heavy chain region via a peptide
linker (e.g., poly-glycine or another sequence which does not form
an alpha helix or beta sheet motif).
[0079] The efficacy of a CD47 inhibitor is assessed by assaying
CD47 activity. The above-mentioned assays or modified versions
thereof are used. In an exemplary assay, AML SCs are incubated with
bone marrow derived macrophages, in the presence or absence of the
candidate agent. An inhibitor of the cell surface CD47 will
up-regulate phagocytosis by at least about 10%, or up to 20%, or
50%, or 70% or 80% or up to about 90% compared to the phagocytosis
in absence of the candidate agent. Similarly, an in vitro assay for
levels of tyrosine phosphorylation of SIRP.alpha. will show a
decrease in phosphorylation by at least about 10%, or up to 20%, or
50%, or 70% or 80% or up to about 90% compared to phosphorylation
observed in absence of the candidate agent.
[0080] In one embodiment of the invention, the agent, or a
pharmaceutical composition comprising the agent, is provided in an
amount effective to detectably inhibit the binding of CD47 to
SIRP.alpha. receptor present on the surface of phagocytic cells.
The effective amount is determined via empirical testing routine in
the art. The effective amount may vary depending on the number of
cells being transplanted, site of transplantation and factors
specific to the transplant recipient.
[0081] The terms "phagocytic cells" and "phagocytes" are used
interchangeably herein to refer to a cell that is capable of
phagocytosis. There are three main categories of phagocytes:
macrophages, mononuclear cells (histiocytes and monocytes);
polymorphonuclear leukocytes (neutrophils) and dendritic cells.
[0082] The term "biological sample" encompasses a variety of sample
types obtained from an organism and can be used in a diagnostic or
monitoring assay. The term encompasses blood and other liquid
samples of biological origin, solid tissue samples, such as a
biopsy specimen or tissue cultures or cells derived therefrom and
the progeny thereof. The term encompasses samples that have been
manipulated in any way after their procurement, such as by
treatment with reagents, solubilization, or enrichment for certain
components. The term encompasses a clinical sample, and also
includes cells in cell culture, cell supernatants, cell lysates,
serum, plasma, biological fluids, and tissue samples.
[0083] Hematopoietic stem cells (HSC), as used herein, refers to a
population of cells having the ability to self-renew, and to give
rise to all hematopoietic lineages. Such cell populations have been
described in detail in the art. Hematopoietic progenitor cells
include the myeloid committed progenitors (CMP), the lymphoid
committed progenitors (CLP), megakaryocyte progenitors, and
multipotent progenitors. The earliest known lymphoid-restricted
cell in adult mouse bone marrow is the common lymphocyte progenitor
(CLP), and the earliest known myeloid-restricted cell is the common
myeloid progenitor (CMP). Importantly, these cell populations
possess an extremely high level of lineage fidelity in in vitro and
in vivo developmental assays. A complete description of these cell
subsets may be found in Akashi et al. (2000) Nature 404(6774):193,
U.S. Pat. No. 6,465,247; and published application U.S. Ser. No.
09/956,279 (common myeloid progenitor); Kondo et al. (1997) Cell
91(5):661-7, and International application WO99/10478 (common
lymphoid progenitor); and is reviewed by Kondo et al. (2003) Annu
Rev Immunol. 21:759-806, each of which is herein specifically
incorporated by reference. The composition may be frozen at liquid
nitrogen temperatures and stored for long periods of time, being
capable of use on thawing. For such a composition, the cells will
usually be stored in a 10% DMSO, 50% FCS, 40% RPMI 1640 medium.
[0084] Populations of interest for use in the methods of the
invention include substantially pure compositions, e.g. at least
about 50% HSC, at least about 75% HSC, at least about 85% HSC, at
least about 95% HSC or more; or may be combinations of one or more
stem and progenitor cells populations, e.g. white cells obtained
from apheresis, etc. Where purified cell populations are desired,
the target population may be purified in accordance with known
techniques. For example, a population containing white blood cells,
particularly including blood or bone marrow samples, are stained
with reagents specific for markers present of hematopoietic stem
and progenitor cells, which markers are sufficient to distinguish
the major stem and progenitor groups. The reagents, e.g.
antibodies, may be detectably labeled, or may be indirectly labeled
in the staining procedure.
[0085] Any combination of markers may be used that are sufficient
to select for the stem/progenitor cells of interest. A marker
combination of interest may include CD34 and CD38, which
distinguishes hematopoietic stem cells, (CD34.sup.+, CD38.sup.-)
from progenitor cells, which are CD34.sup.+, CD38.sup.+). HSC are
lineage marker negative, and positive for expression of CD90.
[0086] In the myeloid lineage are three cell populations, termed
CMPs, GMPs, and MEPs. These cells are CD34.sup.+ CD38.sup.+, they
are negative for multiple mature lineage markers including early
lymphoid markers such as CD7, CD10, and IL-7R, and they are further
distinguished by the markers CD45RA, an isoform of CD45 that can
negatively regulate at least some classes of cytokine receptor
signaling, and IL-3R. These characteristics are CD45RA.sup.-
IL-3R.alpha..sup.lo (CMPs), CD45RA.sup.+IL-3R.alpha..sup.lo (GMPs),
and CD45RA.sup.- IL-3R.alpha..sup.- (MEPs). CD45RA.sup.-
IL-3R.alpha..sup.lo cells give rise to GMPs and MEPs and at least
one third generate both GM and MegE colonies on a single-cell
level. All three of the myeloid lineage progenitors stain
negatively for the markers Thy-1 (CD90), IL-7R.alpha. (CD127); and
with a panel of lineage markers, which lineage markers may include
CD2; CD3; CD4; CD7; CD8; CD10; CD11b; CD14; CD19; CD20; CD56; and
glycophorin A (GPA) in humans and CD2; CD3; CD4; CD8; CD19; IgM;
Ter110; Gr-1 in mice. With the exception of the mouse MEP subset,
all of the progenitor cells are CD34 positive. In the mouse all of
the progenitor subsets may be further characterized as Sca-1
negative, (Ly-6E and Ly-6A), and c-kit high. In the human, all
three of the subsets are CD38.sup.+.
[0087] Common lymphoid progenitors, CLP, express low levels of
c-kit (CD117) on their cell surface. Antibodies that specifically
bind c-kit in humans, mice, rats, etc. are known in the art.
Alternatively, the c-kit ligand, steel factor (Slf) may be used to
identify cells expressing c-kit. The CLP cells express high levels
of the IL-7 receptor alpha chain (CDw127). Antibodies that bind to
human or to mouse CDw127 are known in the art. Alternatively, the
cells are identified by binding of the ligand to the receptor,
IL-7. Human CLPs express low levels of CD34. Antibodies specific
for human CD34 are commercially available and well known in the
art. See, for example, Chen et al. (1997) Immunol Rev 157:41-51.
Human CLP cells are also characterized as CD38 positive and CD10
positive. The CLP subset also has the phenotype of lacking
expression of lineage specific markers, exemplified by B220, CD4,
CD8, CD3, Gr-1 and Mac-1. The CLP cells are characterized as
lacking expression of Thy-1, a marker that is characteristic of
hematopoietic stem cells. The phenotype of the CLP may be further
characterized as MeI-14.sup.-, CD43.sup.lo, HSA.sup.lo, CD45.sup.+
and common cytokine receptor .gamma. chain positive.
[0088] Megakaryocyte progenitor cells (MKP) cells are positive for
CD34 expression, and tetraspanin CD9 antigen. The CD9 antigen is a
227-amino acid molecule with 4 hydrophobic domains and 1
N-glycosylation site. The antigen is widely expressed, but is not
present on certain progenitor cells in the hematopoietic lineages.
The MKP cells express CD41, also referred to as the glycoprotein
IIb/IIIa integrin, which is the platelet receptor for fibrinogen
and several other extracellular matrix molecules, for which
antibodies are commercially available, for example from BD
Biosciences, Pharmingen, San Diego, Calif., catalog number 340929,
555466. The MKP cells are positive for expression of CD117, which
recognizes the receptor tyrosine kinase c-Kit. Antibodies are
commercially available, for example from BD Biosciences,
Pharmingen, San Diego, Calif., Cat. No. 340529. MKP cells are also
lineage negative, and negative for expression of Thy-1 (CD90).
[0089] The phrase "solid tumor" as used herein refers to an
abnormal mass of tissue that usually does not contain cysts or
liquid areas. Solid tumors may be benign or malignant. Different
types of solid tumors are named for the type of cells that form
them. Examples of solid tumors are sarcomas, carcinomas, lymphomas
etc.
[0090] Anti-CD47 antibodies. Certain antibodies that bind CD47
prevent its interaction with SIRP.alpha. receptor. Antibodies
include free antibodies and antigen binding fragments derived
therefrom, and conjugates, e.g. pegylated antibodies, drug,
radioisotope, or toxin conjugates, and the like.
[0091] Monoclonal antibodies directed against a specific epitope,
or combination of epitopes, will allow for the targeting and/or
depletion of cellular populations expressing the marker. Various
techniques can be utilized using monoclonal antibodies to screen
for cellular populations expressing the marker(s), and include
magnetic separation using antibody-coated magnetic beads, "panning"
with antibody attached to a solid matrix (i.e., plate), and flow
cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al.
Cell, 96:737-49 (1999)). These techniques allow for the screening
of particular populations of cells; in immunohistochemistry of
biopsy samples; in detecting the presence of markers shed by cancer
cells into the blood and other biologic fluids, and the like.
[0092] Humanized versions of such antibodies are also within the
scope of this invention. Humanized antibodies are especially useful
for in vivo applications in humans due to their low
antigenicity.
[0093] The phrase "bispecific antibody" refers to a synthetic or
recombinant antibody that recognizes more than one protein.
Examples include bispecific antibodies 2B1, 520C9xH22, mDX-H210,
and MDX447. Bispecific antibodies directed against a combination of
epitopes, will allow for the targeting and/or depletion of cellular
populations expressing the combination of epitopes. Exemplary
bi-specific antibodies include those targeting a combination of
CD47 and a cancer cell marker, such as, CD96, CD97, CD99, PTHR2,
HAVCR2 etc. Generation of bi-specific antibody is described in the
literature, for example, in U.S. Pat. No. 5,989,830, U.S. Pat. No.
5,798,229, which are incorporated herein by reference.
[0094] The terms "treatment", "treating", "treat" and the like are
used herein to generally refer to obtaining a desired pharmacologic
and/or physiologic effect. The effect may be prophylactic in terms
of completely or partially preventing a disease or symptom thereof
and/or may be therapeutic in terms of a partial or complete
stabilization or cure for a disease and/or adverse effect
attributable to the disease. "Treatment" as used herein covers any
treatment of a disease in a mammal, particularly a human, and
includes: (a) preventing the disease or symptom from occurring in a
subject which may be predisposed to the disease or symptom but has
not yet been diagnosed as having it; (b) inhibiting the disease
symptom, i.e., arresting its development; or (c) relieving the
disease symptom, i.e., causing regression of the disease or
symptom.
[0095] The terms "recipient", "individual", "subject", "host", and
"patient", used interchangeably herein and refer to any mammalian
subject for whom diagnosis, treatment, or therapy is desired,
particularly humans.
[0096] A "host cell", as used herein, refers to a microorganism or
a eukaryotic cell or cell line cultured as a unicellular entity
which can be, or has been, used as a recipient for a recombinant
vector or other transfer polynucleotides, and include the progeny
of the original cell which has been transfected. It is understood
that the progeny of a single cell may not necessarily be completely
identical in morphology or in genomic or total DNA complement as
the original parent, due to natural, accidental, or deliberate
mutation.
[0097] The terms "cancer", "neoplasm", "tumor", and "carcinoma",
are used interchangeably herein to refer to cells which exhibit
relatively autonomous growth, so that they exhibit an aberrant
growth phenotype characterized by a significant loss of control of
cell proliferation. In general, cells of interest for detection or
treatment in the present application include precancerous (e.g.,
benign), malignant, pre-metastatic, metastatic, and non-metastatic
cells. Detection of cancerous cells is of particular interest. The
term "normal" as used in the context of "normal cell," is meant to
refer to a cell of an untransformed phenotype or exhibiting a
morphology of a non-transformed cell of the tissue type being
examined. "Cancerous phenotype" generally refers to any of a
variety of biological phenomena that are characteristic of a
cancerous cell, which phenomena can vary with the type of cancer.
The cancerous phenotype is generally identified by abnormalities
in, for example, cell growth or proliferation (e.g., uncontrolled
growth or proliferation), regulation of the cell cycle, cell
mobility, cell-cell interaction, or metastasis, etc.
[0098] Cancers of interest for treatment by the methods of the
invention, e.g. treatment with an agent that binds to cell surface
CD47 to increase phagocytosis of the cancer cells; include
leukemias, particularly acute leukemias such as T-ALL, B-ALL, AML,
etc.; lymphomas (Hodgkins and non-Hodgkins); sarcomas; melanomas;
adenomas; carcinomas of solid tissue including ovarian carcinoma,
breast carcinoma, pancreatic carcinoma, colon carcinoma, squamous
cell carcinoma, transitional cell carcinoma, etc., hypoxic tumors,
squamous cell carcinomas of the mouth, throat, larynx, and lung,
genitourinary cancers such as cervical and bladder cancer,
hematopoietic cancers, head and neck cancers, and nervous system
cancers, such as gliomas, astrocytomas, meningiomas, etc., benign
lesions such as papillomas, and the like.
[0099] As used herein, a "target cell" is a cell expressing CD47 on
the surface, where masking or otherwise altering the CD47 positive
phenotype results in altered phagocytosis. Usually a target cell is
a mammalian cell, preferably a human cell.
[0100] A "biological sample" encompasses a variety of sample types
obtained from an individual and can be used in a diagnostic or
monitoring assay. The definition encompasses blood and other liquid
samples of biological origin, solid tissue samples such as a biopsy
specimen or tissue cultures or cells derived therefrom and the
progeny thereof. The definition also includes samples that have
been manipulated in any way after their procurement, such as by
treatment with reagents, solubilization, or enrichment for certain
components, such as proteins or polynucleotides. The term
"biological sample" encompasses a clinical sample, and also
includes cells in culture, cell supernatants, cell lysates, serum,
plasma, biological fluid, and tissue samples.
[0101] An "individual" is a vertebrate, preferably a mammal, more
preferably a human. Mammals include, but are not limited to,
rodents, primates, farm animals, sport animals, and pets.
[0102] An "effective amount" is an amount sufficient to effect
beneficial or desired clinical results. An effective amount can be
administered in one or more administrations. For purposes of this
invention, an effective amount of a CD47 binding agent is an amount
that is sufficient to palliate, ameliorate, stabilize, reverse,
slow or delay the progression of the disease state by modulating
phagocytosis of a target cell.
[0103] As used herein, "treatment" is an approach for obtaining
beneficial or desired clinical results. For purposes of this
invention, beneficial or desired clinical results include, but are
not limited to, alleviation of symptoms, diminishment of extent of
disease, stabilized (i.e., not worsening) state of disease,
preventing spread (i.e., metastasis) of disease, delay or slowing
of disease progression, amelioration or palliation of the disease
state, and remission (whether partial or total), whether detectable
or undetectable. "Treatment" can also mean prolonging survival as
compared to expected survival if not receiving treatment.
"Palliating" a disease means that the extent and/or undesirable
clinical manifestations of a disease state are lessened and/or time
course of the progression is slowed or lengthened, as compared to
not administering the methods of the present invention.
[0104] "Therapeutic target" refers to a gene or gene product that,
upon modulation of its activity (e.g., by modulation of expression,
biological activity, and the like), can provide for modulation of
the cancerous phenotype. As used throughout, "modulation" is meant
to refer to an increase or a decrease in the indicated phenomenon
(e.g., modulation of a biological activity refers to an increase in
a biological activity or a decrease in a biological activity).
Methods for Transplantation
[0105] Methods are provided to manipulate phagocytosis of
circulating hematopoietic cells. In some embodiments of the
invention the circulating cells are hematopoietic stem cells, or
hematopoietic progenitor cells, particularly in a transplantation
context, where protection from phagocytosis is desirable. In other
embodiments the circulating cells are leukemia cells, particularly
acute myeloid leukemia (AML), where increased phagocytosis is
desirable.
[0106] In some embodiments of the invention, hematopoietic stem or
progenitor cells are protected from phagocytosis in circulation by
providing a host animal with a CD47 mimetic molecule, which
interacts with SIRP.alpha. on macrophages and decreases macrophage
phagocytosis. The CD47 mimetic may be soluble CD47; CD47 coated on
the surface of the cells to be protected, a CD47 mimetic that binds
to SIRP.alpha. at the CD47 binding site, and the like. In some
embodiments of the invention, CD47 is provided as a fusion protein,
for example soluble CD47 fused to an Fc fragment, e.g., IgG1 Fc,
IgG2 Fc, Ig A Fc etc.
[0107] Methods for generating proteins lacking the transmembrane
region are well known in the art. For example, a soluble CD47 can
be generated by introducing a stop codon immediately before the
polynucleotide sequence encoding the transmembrane region.
Alternatively, the polynucleotide sequence encoding the
transmembrane region can be replaced by a polynucleotide sequence
encoding a fusion protein such as IgG1 Fc. Sequence for Fc
fragments from different sources are available via publicly
accessible database including Entrez, Embl, etc. For example, mRNA
encoding human IgG1 Fc fragment is provided by accession number
X70421.
[0108] The subject invention provide for methods for transplanting
hematopoietic stem or progenitor cells into a mammalian recipient.
A need for transplantation may be caused by genetic or
environmental conditions, e.g. chemotherapy, exposure to radiation,
etc. The cells for transplantation may be mixtures of cells, e.g.
buffy coat lymphocytes from a donor, or may be partially or
substantially pure. The cells may be autologous cells, particularly
if removed prior to cytoreductive or other therapy, or allogeneic
cells, and may be used for hematopoietic stem or progenitor cell
isolation and subsequent transplantation.
[0109] The cells may be combined with the soluble CD47 mimetic
prior to administration. For example, the cells may be combined
with the mimetic at a concentration of from about 10 .mu.g/ml,
about 100 .mu.g/ml, about 1 mg/ml, about 10 mg/ml, etc., at a
temperature of from about 4.degree., about 10.degree., about
25.degree. about 37.degree., for a period of time sufficient to
coat the cells, where in some embodiments the cells are maintained
on ice. In other embodiments the cells are contacted with the CD47
mimetic immediately prior to introduction into the recipient, where
the concentrations of mimetic are as described above.
[0110] The composition comprising hematopoietic stem or progenitor
cells and a CD47 mimetic is administered in any physiologically
acceptable medium, normally intravascularly, although they may also
be introduced into bone or other convenient site, where the cells
may find an appropriate site for regeneration and differentiation.
Usually, at least 1.times.10.sup.5 cells will be administered,
preferably 1.times.10.sup.6 or more. The composition may be
introduced by injection, catheter, or the like.
Myeloproliferative Disorders, Leukemias, and Myelodysplastic
Syndrome
[0111] Acute leukemias are rapidly progressing leukemia
characterized by replacement of normal bone marrow by blast cells
of a clone arising from malignant transformation of a hematopoietic
cell. The acute leukemias include acute lymphoblastic leukemia
(ALL) and acute myelogenous leukemia (AML). ALL often involves the
CNS, whereas acute monoblastic leukemia involves the gums, and AML
involves localized collections in any site (granulocytic sarcomas
or chloromas). In addition to CD47, we have discovered a number of
markers specific to AML SC. These include CD96, CD97, CD99, PTHR2,
HAVCR2 etc. These markers have been disclosed in U.S. Patent
Application No. 61/011,324, filed on Jan. 15, 2008 and are hereby
incorporated by reference.
[0112] The presenting symptoms are usually nonspecific (e.g.,
fatigue, fever, malaise, weight loss) and reflect the failure of
normal hematopoiesis. Anemia and thrombocytopenia are very common
(75 to 90%). The WBC count may be decreased, normal, or increased.
Blast cells are usually found in the blood smear unless the WBC
count is markedly decreased. The blasts of ALL can be distinguished
from those of AML by histochemical studies, cytogenetics,
immunophenotyping, and molecular biology studies. In addition to
smears with the usual stains, terminal transferase,
myeloperoxidase, Sudan black B, and specific and nonspecific
esterase.
[0113] ALL is the most common malignancy in children, with a peak
incidence from ages 3 to 5 yr. It also occurs in adolescents and
has a second, lower peak in adults. Typical treatment emphasizes
early introduction of an intensive multidrug regimen, which may
include prednisone, vincristine, anthracycline or asparaginase.
Other drugs and combinations are cytarabine and etoposide, and
cyclophosphamide. Relapse usually occurs in the bone marrow but may
also occur in the CNS or testes, alone or concurrent with bone
marrow. Although second remissions can be induced in many children,
subsequent remissions tend to be brief.
[0114] The incidence of AML increases with age; it is the more
common acute leukemia in adults. AML may be associated with
chemotherapy or irradiation (secondary AML). Remission induction
rates are lower than with ALL, and long-term disease-free survival
reportedly occurs in only 20 to 40% of patients. Treatment differs
most from ALL in that AML responds to fewer drugs. The basic
induction regimen includes cytarabine; along with daunorubicin or
idarubicin. Some regimens include 6-thioguanine, etoposide,
vincristine, and prednisone.
[0115] Polycythemia vera (PV) is an idiopathic chronic
myeloproliferative disorder characterized by an increase in Hb
concentration and RBC mass (erythrocytosis). PV occurs in about
2.3/100,000 people per year; more often in males (about 1.4:1). The
mean age at diagnosis is 60 yr (range, 15 to 90 yr; rarely in
childhood); 5% of patients are <40 yr at onset. The bone marrow
sometimes appears normal but usually is hypercellular; hyperplasia
involves all marrow elements and replaces marrow fat. There is
increased production and turnover of RBCs, neutrophils, and
platelets. Increased megakaryocytes may be present in clumps.
Marrow iron is absent in >90% of patients, even when phlebotomy
has not been performed.
[0116] Studies of women with PV who are heterozygous at the
X-chromosome-linked locus for G6PD have shown that RBCs,
neutrophils, and platelets have the same G6PD isoenzyme, supporting
a clonal origin of this disorder at a pluripotent stem cell
level.
[0117] Eventually, about 25% of patients have reduced RBC survival
and fail to adequately increase erythropoiesis; anemia and
myelofibrosis develop. Extramedullary hemopoiesis occurs in the
spleen, liver, and other sites with the potential for blood cell
formation.
[0118] Without treatment, 50% of symptomatic patients die within 18
mo of diagnosis. With treatment, median survival is 7 to 15 yr.
Thrombosis is the most common cause of death, followed by
complications of myeloid metaplasia, hemorrhage, and development of
leukemia.
[0119] The incidence of transformation into an acute leukemia is
greater in patients treated with radioactive phosphate (.sup.32P)
or alkylating agents than in those treated with phlebotomy alone.
PV that transforms into acute leukemia is more resistant to
induction chemotherapy than de novo leukemia.
[0120] Because PV is the only form of erythrocytosis for which
myelosuppressive therapy may be indicated, accurate diagnosis is
critical. Therapy must be individualized according to age, sex,
medical status, clinical manifestations, and hematologic
findings.
[0121] Myelodysplastic syndrome (MDS) is a group of syndromes
(preleukemia, refractory anemias, Ph-negative chronic myelocytic
leukemia, chronic myelomonocytic leukemia, myeloid metaplasia)
commonly seen in older patients. Exposure to carcinogens may by be
implicated. MDS is characterized by clonal proliferation of
hematopoietic cells, including erythroid, myeloid, and
megakaryocytic forms. The bone marrow is normal or hypercellular,
and ineffective hematopoiesis causes variable cytopenias, the most
frequent being anemia. The disordered cell production is also
associated with morphologic cellular abnormalities in marrow and
blood. Extramedullary hematopoiesis may occur, leading to
hepatomegaly and splenomegaly. Myelofibrosis is occasionally
present at diagnosis or may develop during the course of MDS. The
MDS clone is unstable and tends to progress to AML.
[0122] Anemia is the most common clinical feature, associated
usually with macrocytosis and anisocytosis. Some degree of
thrombocytopenia is usual; on blood smear, the platelets vary in
size, and some appear hypogranular. The WBC count may be normal,
increased, or decreased. Neutrophil cytoplasmic granularity is
abnormal, with anisocytosis and variable numbers of granules.
Eosinophils also may have abnormal granularity. A monocytosis is
characteristic of the chronic myelomonocytic leukemia subgroup, and
immature myeloid cells may occur in the less well differentiated
subgroups. The prognosis is highly dependent on classification and
on any associated disease. Response of MDS to AML chemotherapy is
similar to that of AML, after age and karyotype are considered.
Treatment of Cancer
[0123] The invention provides methods for reducing growth of cancer
cells by increasing their clearance by phagocytosis, through the
introduction of a CD47 blocking agent, e.g. an anti-CD47 antibody.
In certain embodiments the cancer cells may be AML stem cells. In
other embodiments, the cancer cells may be those of a solid tumor,
such as carcinomas, glioblastoma, melanoma, etc. By blocking the
activity of CD47, the downregulation of phagocytosis that is found
with certain tumor cells, e.g. AML cells, is prevented.
[0124] "Reducing growth of cancer cells" includes, but is not
limited to, reducing proliferation of cancer cells, and reducing
the incidence of a non-cancerous cell becoming a cancerous cell.
Whether a reduction in cancer cell growth has been achieved can be
readily determined using any known assay, including, but not
limited to, [.sup.3H]-thymidine incorporation; counting cell number
over a period of time; detecting and/or measuring a marker
associated with AML, etc.
[0125] Whether a substance, or a specific amount of the substance,
is effective in treating cancer can be assessed using any of a
variety of known diagnostic assays for cancer, including, but not
limited to biopsy, contrast radiographic studies, CAT scan, and
detection of a tumor marker associated with cancer in the blood of
the individual. The substance can be administered systemically or
locally, usually systemically.
[0126] As an alternative embodiment, an agent, e.g. a
chemotherapeutic drug that reduces cancer cell growth, can be
targeted to a cancer cell by conjugation to a CD47 specific
antibody. Thus, in some embodiments, the invention provides a
method of delivering a drug to a cancer cell, comprising
administering a drug-antibody complex to a subject, wherein the
antibody is specific for a cancer-associated polypeptide, and the
drug is one that reduces cancer cell growth, a variety of which are
known in the art. Targeting can be accomplished by coupling (e.g.,
linking, directly or via a linker molecule, either covalently or
non-covalently, so as to form a drug-antibody complex) a drug to an
antibody specific for a cancer-associated polypeptide. Methods of
coupling a drug to an antibody are well known in the art and need
not be elaborated upon herein.
[0127] In certain embodiments, a bi-specific antibody may be used.
For example a bi-specific antibody in which one antigen binding
domain is directed against CD47 and the other antigen binding
domain is directed against a cancer cell marker, such as, CD96
CD97, CD99, PTHR2, HAVCR2 etc. may be used.
[0128] Depletion of AMLSC is useful in the treatment of AML.
Depletion can be achieved by several methods. Depletion is defined
as a reduction in the target population by up to about 30%, or up
to about 40%, or up to about 50%, or up to about 75% or more. An
effective depletion is usually determined by the sensitivity of the
particular disease condition to the levels of the target
population. Thus in the treatment of certain conditions a depletion
of even about 20% could be beneficial.
[0129] A CD47 specific agent that specifically depletes the
targeted AMLSC is used to contact the patient blood in vitro or in
vivo, wherein after the contacting step, there is a reduction in
the number of viable AMLSC in the targeted population. An effective
dose of antibodies for such a purpose is sufficient to decrease the
targeted population to the desired level, for example as described
above. Antibodies for such purposes may have low antigenicity in
humans or may be humanized antibodies.
[0130] In one embodiment of the invention, antibodies for depleting
target population are added to patient blood in vivo. In another
embodiment, the antibodies are added to the patient blood ex vivo.
Beads coated with the antibody of interest can be added to the
blood, target cells bound to these beads can then be removed from
the blood using procedures common in the art. In one embodiment the
beads are magnetic and are removed using a magnet. Alternatively,
when the antibody is biotinylated, it is also possible to
indirectly immobilize the antibody onto a solid phase which has
adsorbed avidin, streptavidin, or the like. The solid phase,
usually agarose or sepharose beads are separated from the blood by
brief centrifugation. Multiple methods for tagging antibodies and
removing such antibodies and any cells bound to the antibodies are
routine in the art. Once the desired degree of depletion has been
achieved, the blood is returned to the patient. Depletion of target
cells ex vivo decreases the side effects such as infusion reactions
associated with the intravenous administration. An additional
advantage is that the repertoire of available antibodies is
expanded significantly as this procedure does not have to be
limited to antibodies with low antigenicity in humans or humanized
antibodies.
Example 1
[0131] Cd47 is a Marker of Myeloid Leukemias
[0132] Materials and Methods
[0133] Immunohistochemistry.
[0134] Cytospins of double sorted myeloid progenitor populations
(CMP, GMP), IL-3R.alpha. high CD45 RA+ cells and CD14+c-kit+lin-
cells were performed using a Shandon cytospin apparatus. Cytospins
were stained with Giemsa diluted 1/5 with H20 for 10 min followed
by staining with May-Grunwald for 20 minutes. Cytospins were
analyzed with the aid of a Zeiss microscope.
[0135] Human Bone Marrow and Peripheral Blood Samples.
[0136] Normal bone marrow samples were obtained with informed
consent from 20-25 year old paid donors who were hepatitis A, B, C
and HIV negative by serology (All Cells). CMML bone marrow samples
were obtained with informed consent, from previously untreated
patients, at Stanford University Medical Center.
[0137] Human Bone Marrow HSC and Myeloid Progenitor Flow-Cytometric
Analysis and Cell Sorting.
[0138] Mononuclear fractions were extracted following Ficoll
density centrifugation according to standard methods and analyzed
fresh or subsequent to rapid thawing of samples previously frozen
in 90% FCS and 10% DMSO in liquid nitrogen. In some cases, CD34+
cells were enriched from mononuclear fractions with the aid of
immunomagnetic beads (CD34+ Progenitor Isolation Kit, Miltenyi
Biotec, Bergisch-Gladbach, Germany). Prior to FACS analysis and
sorting, myeloid progenitors were stained with lineage marker
specific phycoerythrin (PE)-Cy5-conjugated antibodies including CD2
RPA-2.10; CD11b, ICRF44; CD20, 2H7; CD56, B159; GPA, GA-R2 (Becton
Dickinson--PharMingen, San Diego), CD3,S4.1;CD4, S3.5; CD7,
CD7-6B7; CD8, 3B5; CD10, 5-1B4, CD14, TUK4; CD19, SJ25-C1 (Caltag,
South San Francisco, Calif.) and APC-conjugated anti-CD34, HPCA-2
(Becton Dickinson-PharMingen), biotinylated anti-CD38, HIT2
(Caltag) in addition to PE-conjugated anti-IL-3R.alpha., 9F5
(Becton Dickinson-ParMingen) and FITC-conjugated anti-CD45RA, MEM56
(Caltag) followed by staining with Streptavidin--Texas Red to
visualize CD38-BIO stained cells and resuspension in propidium
iodide to exclude dead cells. Unstained samples and isotype
controls were included to assess background fluorescence.
[0139] Following staining, cells were analyzed and sorted using a
modified FACS Vantage (Becton Dickinson Immunocytometry Systems,
Mountain View, Calif.) equipped with a 599 nm dye laser and a 488
nm argon laser. Double sorted progenitor cells (HSC) were
identified as CD34+ CD38+ and lineage negative. Common myeloid
progenitors (CMP) were identified based on CD34+ CD38+
IL-3R.alpha.+ CD45RA- lin- staining and their progeny including
granulocyte/macrophage progenitors (GMP) were
CD34+CD38+IL-3R.alpha.+CD45RA+ while megakaryocyte/erythrocyte
progenitors (MEP) were identified based on CD34+ CD38+
IL-3R.alpha.- CD45RA- lin- staining (Manz, PNAS 11872).
[0140] CD47 Expression by Normal Versus Myeloproliferative and AML
Progenitors
[0141] Peripheral blood and bone marrow samples were obtained with
informed consent from patients with myeloproliferative disorders
and acute myelogenous leukemia at Stanford University Medical
Center according to Stanford IRB and HIPAA regulations. Peripheral
blood or bone marrow mononuclear cells (1-5.times.10.sup.6 cells)
were stained with lineage cocktail as above but excluding CD7,
CD11b and CD14. Subsequently, samples were stained with CD14 PE
(1/25), CD47 FITC (1/25), CD38 Bio (Bio) and c-kit APC (1/25) or
CD34 APC or FITC (1/50) for 45 min followed by washing and staining
with Streptavidin Texas Red (1/25) for 45 min and finally
resuspension in propidium iodide.
[0142] Discussion
[0143] Here we show that CD47 overexpression is characteristic of
progression of human myeloproliferative disorders to AML (see FIGS.
1-5B). CD47 controls integrin function but also the ability of
macrophages to phagocytose cells, depending on the level of CD47
expression. Thus, aberrant CD47 expression may allow LSC to evade
both innate and adaptive host immunity.
[0144] Human CD47 expression analysis was performed via FACS on
human normal, pre-leukemic myeloproliferative disorder (MPD) or AML
HSC, progenitors and lineage positive cells derived from marrow or
peripheral blood. MPD samples (n=63) included polycythemia vera
(PV; n=15), post-polycythemic myeloid metaplasia/myelofibrosis
(PPMM/MF; n=5), essential thrombocythemia (ET; n=8), atypical
chronic myelogenous leukemia (aCML; n=2), CML (n=7), chronic
eosinophilic leukemia (CEL; n=1), chronic myelomonocytic leukemia
(CMML; n=13) and acute myelogenous leukemia (AML; n=12). As we have
observed with the transgenic leukemic mouse models, progression of
human myeloproliferative disorders to AML (n=12) was associated
with an expansion of the GMP pool (70.6%+/-S.D. 2.15) compared with
normal bone marrow (14.7%+/-S.D. 2.3). Furthermore, FACS analysis
revealed that CD47 expression first increased 1.7 fold in AML
compared with normal HSC and then increased to 2.2 fold greater
than normal with commitment of AML progenitors to the myeloid
lineage. CD47 was over-expressed by AML primitive progenitors and
their progeny but not by the majority of MPD (MFI 2.3+/-S.D. 0.43)
compared with normal bone marrow (MFI 1.9+/-S.D. 0.07). Thus,
increased CD47 expression is a useful diagnostic marker for
progression to AML and in addition represents a novel therapeutic
target.
Example 2
[0145] Human and Mouse Leukemias Upregulate CD47 to Evade
Macrophage Killing CD47 Facilitates Engraftment, Inhibits
Phagocytosis, and is More Highly Expressed on AML LSC.
[0146] We determined expression of CD47 on human AML LSC and normal
HSC by flow cytometry. HSC (Lin-CD34+CD38-CD90+) from three samples
of normal human mobilized peripheral blood and AML LSC
(Lin-CD34+CD38-CD90-) from seven samples of human AML were analyzed
for surface expression of CD47 (FIG. 6). CD47 was expressed at low
levels on the surface of normal HSC; however, on average, it was
approximately 5-fold more highly expressed on AML LSC, as well as
bulk leukemic blasts.
[0147] Anti-Human CD47 Monoclonal Antibody Stimulates Phagocytosis
and Inhibits Engraftment of AML LSC.
[0148] In order to test the model that CD47 overexpression on AML
LSC prevents phagocytosis of these cells through its interaction
with SIRP.alpha. on effector cells, we have utilized a monoclonal
antibody directed against CD47 known to disrupt the
CD47-SIRP.alpha. interaction. The hybridoma producing a
mouse-anti-human CD47 monoclonal antibody, termed B6H12, was
obtained from ATCC and used to produce purified antibody. First, we
conducted in vitro phagocytosis assays. Primary human AML LSC were
purified by FACS from two samples of human AML, and then loaded
with the fluorescent dye CFSE. These cells were incubated with
mouse bone marrow-derived macrophages and monitored using
immunofluorescence microscopy (FIG. 7) and flow cytometry (FIG. 9)
to identify phagocytosed cells. In both cases, no phagocytosis was
observed in the presence of an isotype control antibody; however,
significant phagocytosis was detected with the addition of the
anti-CD47 antibody (FIG. 9). Thus, blockage of human CD47 with a
monoclonal antibody is capable of stimulating the phagocytosis of
these cells by mouse macrophages.
[0149] We next investigated the ability of the anti-CD47 antibody
to inhibit AML LSC engraftment in vivo. Two primary human AML
samples were either untreated or coated with the anti-CD47 antibody
prior to transplantation into NOG newborn mice. 13 weeks later, the
mice were sacrificed and analyzed for human leukemia bone marrow
engraftment by flow cytometry (FIG. 10). The control mice
demonstrated leukemic engraftment while mice transplanted with the
anti-CD47-coated cells showed little to no engraftment. These data
indicate that blockade of human CD47 with a monoclonal antibody is
able to inhibit AML LSC engraftment.
[0150] CD96 is a Human Acute Myeloid Leukemia Stem Cell-Specific
Cell Surface Molecule.
[0151] CD96, originally termed Tactile, was first identified as a T
cell surface molecule that is highly upregulated upon T cell
activation. CD96 is expressed at low levels on resting T and NK
cells and is strongly upregulated upon stimulation in both cell
types. It is not expressed on other hematopoietic cells, and
examination of its expression pattern showed that it is only
otherwise present on some intestinal epithelia. The cytoplasmic
domain of CD96 contains a putative ITIM motif, but it is not know
if this functions in signal transduction. CD96 promotes adhesion of
NK cells to target cells expressing CD155, resulting in stimulation
of cytotoxicity of activated NK cells.
[0152] Preferential Cell Surface Expression of Molecules Identified
from Gene Expression Analysis.
[0153] Beyond CD47 and CD96, several molecules described in U.S.
Patent Application No. 61/011,324 are known to be expressed on AML
LSC, including: CD123, CD44, CD99 and CD33.
[0154] Tumor progression is characterized by several hallmarks,
including growth signal independence, inhibition of apoptosis, and
evasion of the immune system, among others. We show here that
expression of CD47, a ligand for the macrophage inhibitory signal
regulatory protein alpha (SIRP.alpha.) receptor, is increased in
human and mouse myeloid leukaemia and allows cells to evade
phagocytosis and increase their tumorigenic potential. CD47, also
known as integrin associated protein (IAP), is an
immunoglobulin-like transmembrane pentaspanin that is broadly
expressed in mammalian tissues. We provide evidence that CD47 is
upregulated in mouse and human myeloid leukaemia stem and
progenitor cells, as well as leukemic blasts. Consistent with a
biological role for CD47 in myeloid leukaemia development and
maintenance, we demonstrate that ectopic over-expression of CD47
allows a myeloid leukaemia cell line to grow in mice that are T, B,
and NK-cell deficient, whereas it is otherwise cleared rapidly when
transplanted into these recipients. The leukemogenic potential of
CD47 is also shown to be dose-dependent, as higher expressing
clones have greater tumor forming potential than lower expressing
clones. We also show that CD47 functions in promoting
leukemogenesis by inhibiting phagocytosis of the leukemic cells by
macrophages.
[0155] CD47 is significantly upregulated in leukemic
Fas.sup.lpr/lpr.times.hMRP8bcl2 transgenic bone marrow, and in
leukemic hMRP8bcr/abl.times.hMRP8bcl2 mice. Transcripts for CD47
are increased in leukemic hMRP8bcr/abl.times.hMRP8bcl2 bone marrow
3-4 fold by quantitative RT-PCR and 6-7 fold in c-Kit enriched
leukemic marrow relative to healthy hMRP8bcl2+ bone marrow (FIG.
11e). Leukemic spleen had an expansion of the granulocyte
macrophage progenitor (GMP) population as well as c-Kit+ Sca-1+
Lin-stem and progenitor subsets relative to control mice, which
were of the same genotype as leukemic mice but failed to develop
disease (FIG. 11a-d). Expression levels for CD47 protein were found
to begin increasing in leukemic mice relative to control mice at
the stage of the Flk2- CD34- c-Kit+ Sca-1+ Lin- long-term
hematopoietic stem cell (LT-HSC) (FIG. 11f). This increased level
of expression was maintained in GMP and Mac-1+ blasts, but not
megakaryocyte/erythroid restricted progenitors (MEP) (FIG. 11f).
The increase in CD47 between leukemic and normal cells was between
3 to 20 fold. All mice that developed leukaemia that we have
examined from hMRP8bcr/abl.times.hMRP8bcl2 primary (n=3) and
secondary transplanted mice (n=3), Fas.sup.lpr/lpr.times.hMRP8bcl2
primary (n=14) and secondary (n=19) mice, and
hMRP8bcl2.times.hMRP8bcl2 primary (n=3) and secondary (n=12) mice
had increased CD47 expression. We have also found increased CD47
expression in mice that received p210bcr/abl
retrovirally-transduced mouse bone marrow cells that developed
leukemia.
[0156] FACS-mediated analysis of human hematopoietic progenitor
populations was performed on blood and marrow derived from normal
cord blood and mobilized peripheral blood (n=16) and
myeloproliferative disorders (MPDs) including polycythemia vera
(PV; n=16), myelofibrosis (MF; n=5), essential thrombocythemia (ET;
n=7), chronic myelomonocytic leukaemia (CMML; n=11) and atypical
chronic myeloid leukaemia (aCML; n=1) as well as blast crisis phase
chronic myeloid leukaemia (CML; n=19), chronic phase CML (n=7) and
acute myelogenous leukaemia (AML; n=13). This analysis demonstrated
that granulocyte-macrophage progenitors (GMP) expanded in MPDs with
myeloid skewed differentiation potential including atypical CML,
proliferative phase CMML and acute leukaemia including blast crisis
CML and AML (FIG. 12a). AML HSC and progenitors uniformly exhibited
higher levels of CD47 expression compared with normal controls
(FIG. 12b); every sample from BC-CML and AML had elevated levels of
CD47. Moreover, progression from chronic phase CML to blast crisis
was associated with a significant increase in CD47 expression (FIG.
12c). Using the methods described in this study, we have found that
human CD47 protein expression in CML-BC increased 2.2 fold in CD90+
CD34+ CD38- Lin- cells relative to normal (p=6.3.times.10.sup.-5),
2.3 fold in CD90- CD34+ CD38- Lin- cells relative to normal
(p=4.3.times.10.sup.-5), and 2.4 fold in CD 34+ CD38+ Lin- cells
(p=7.6.times.10.sup.-6) (FIGS. 12b-12c); however, using a newer
optimized staining protocol we have observed that CD47 is increased
approximately 10 fold in AML and BC-CML compared to normal human
HSCs and progenitors.
[0157] It was then asked whether forced expression of mouse CD47 on
human leukemic cells would confer a competitive advantage in
forming tumors in mice. MOLM-13 cells, which are derived from a
patient with AML 5a, were transduced with Tet-MCS-IRES-GFP (Tet) or
Tet-CD47-MCS-IRES-GFP (Tet-CD47) (FIG. 13a), and stable integrants
were propagated on the basis of GFP expression. The cells were then
transplanted intravenously in a competitive setting with
untransduced MOLM-13 cells into T, B, and NK deficient
recombination activating gene 2, common gamma chain deficient
(RAG2-/-, Gc-/-) mice. Only cells transduced with Tet-CD47 were
able to give rise to tumors in these mice, efficiently engrafting
bone marrow, spleen and peripheral blood (FIGS. 13a-b). The tumors
were also characterized by large tumor burden in the liver (FIGS.
13b, 13g), which is particularly significant because the liver is
thought to have the highest number of macrophages of any organ,
with estimates that Kupffer cells may comprise 80% of the total
tissue macrophage population. These cells also make up 30% of the
sinusoidal lining, thereby strategically placing them at sites of
entry into the liver. Hence, significant engraftment there would
have to disable a macrophage cytotoxic response. In addition to
developing tumor nodules, the Tet-CD47 MOLM-13 cells exhibited
patterns of hepatic involvement typically seen with human AML, with
leukemic cells infiltrating the liver with a sinusoidal and
perivenous pattern. (FIG. 13d). Overall, Tet-CD47 MOLM-13
transplanted mice died more quickly than Tet MOLM-13 transplanted
mice, which had virtually no engraftment of leukemic cells in
hematopoietic tissues (FIG. 13c). Tet-MOLM-13 mice still had
significant mortality, most likely due to localized growth at the
site of injection (retro-orbital sinus) with extension into the
brain.
[0158] Since CD47 has been shown to be important for the migration
of hematopoietic cells, and is known to modulate binding to
extracellular matrix proteins, either by direct interaction or via
its effect on integrins, one possibility for the lack of growth of
Tet MOLM-13 cells in mice was their inability to migrate to niches.
To test this possibility, Tet MOLM-13 or Tet-CD47 MOLM-13 cells
were directly injected into the femoral cavity of immunodeficient
mice. Tet-CD47 MOLM-13 cells were able to engraft all bones and
other hematopoietic tissues of recipient mice, whereas Tet MOLM-13
cells had minimal, if any, engraftment only at the site of
injection (FIG. 13e). Mice transplanted in this manner with
Tet-CD47 MOLM-13 cells died at approximately 50-60 days
post-transplant (n=4), whereas mice that received Tet MOLM-13 (n=5)
cells remained alive for at least 75 days without signs of disease
at which point they were euthanized for analysis. These results
suggest a function other than or in addition to migration or homing
for CD47 in MOLM-13 engraftment.
[0159] Complete lack of CD47 has been shown to result in
phagocytosis of transplanted murine erythrocytes and leukocytes,
via lack of interaction with SIRP.alpha. on macrophages. Thus, we
tested whether over-expression of CD47 could prevent phagocytosis
of live, unopsonized MOLM-13 cells. We incubated Tet or Tet-CD47
MOLM-13 cells with bone marrow derived macrophages (BMDM) for 2-24
hours and assessed phagocytosis by counting the number of ingested
GFP+ cells under a microscope or by evaluating the frequency of
GFP+ macrophages using a flow cytometer. Expression of CD47
dramatically lowered macrophage clearance of these cells at all
time points tested, whereas Tet-MOLM-13 were quickly phagocytosed
in a manner that increased over time (FIGS. 14a-c). We also
injected MOLM-13 cells into mice and analyzed hematopoietic organs
2 hours later for evidence of macrophage phagocytosis. Macrophages
in bone marrow, spleen, and liver all had higher GFP+ fraction when
injected with Tet MOLM-13 cells as compared to CD47 expressing
cells. This indicates that CD47 over-expression can compensate for
pro-phagocytic signals already present on leukemic cells, allowing
them to survive when they would otherwise be cleared by
macrophages.
[0160] Recent report indicates that lack of CD47 reactivity across
species might mediate xenorejections of transplanted cells.
Furthermore, a recent study has demonstrated that human CD47 is
unable to interact with SIRP.alpha. from C57Bl/6 mice, but is able
to react with receptor from non-obese diabetic (NOD) mice, which
are more permissive for human cell engraftment than C57Bl/6 mice.
Furthermore, we have also observed that HL-60 cells, a human
promyelocytic cell line with higher levels of human CD47 expression
than MOLM-13, are able to engraft mice and cause leukaemia. Jurkat
cells, a human T-lymphocyte cell line, are very high for human CD47
and are phagocytosed by murine macrophages in vitro at a much lower
rate than MOLM-13. Thus, our data indicate that the ability of
cells to engraft mice in vivo or evade phagocytosis in vitro by
mouse macrophages correlates with the level of human CD47
expression.
[0161] To model the tumorigenic effect of having high versus low
CD47 expression, we sorted clones of murine CD47 expressing MOLM-13
cells into high and low expressers. When adjusted for cell size,
CD47 density on the CD47.sup.lo MOLM-13 cells was approximately
equal to mouse bone marrow cells, whereas CD47.sup.hi MOLM-13 cells
had approximately 9 fold higher expression, an increase
commensurate with the change seen in CD47 expression on primary
leukemic cells compared to their normal counterparts (FIG. 15a).
When high or low expressing cells were transplanted into
recipients, only mice transplanted with high expressing cells
succumbed to disease by 75 days of age (FIG. 15c). Furthermore,
organomegaly was more pronounced in mice transplanted with high
expressing cells (FIG. 15d). Mice receiving CD47.sup.lo MOLM-13
cells still had notable liver masses. However, the masses were
invariably 1-2 large nodes that were well-encapsulated and
physically segregated from the liver parenchyma, in marked contrast
to tumor masses from CD47hi MOLM-13 cells which consisted of
hundreds of small masses scattered throughout the parenchyma. Thus,
these large tumor masses consist of cells which have found
macrophage free-niches to grow in separate from the main organ
body. As expected, the infiltration of MOLM-13 cells in bone marrow
and spleen of recipient mice was much higher for mice transplanted
with CD47.sup.hi MOLM-13 cells as well (FIG. 15e). We also examined
the level of CD47 expression in two mice that received CD47.sup.lo
MOLM-13 cells but had significant marrow engraftment. In both
cases, the persisting cells after 75 days had much higher levels of
CD47 than the original line (FIG. 15f), indicating that a strong
selection pressure exists in vivo for high levels of CD47
expression on leukemic cells. In total, these data indicate that
CD47 expression level is a significant factor in tumorigenic
potential, and that in a heterogeneous population of leukemic
cells, strong selection exists for those clones with high CD47
expression.
[0162] We then asked if higher CD47 expression level would provide
added protection against macrophage phagocytosis. We performed an
in vitro phagocytosis assay with CD47.sup.hi and CD47.sup.lo
MOLM-13 red fluorescent protein (RFP) expressing cells. After
incubation with macrophages, far greater numbers of CD47.sup.lo
cells were phagocytosed as compared to CD47.sup.hi cells (FIG.
15g). If phagocytic indices are compared for control MOLM-13 cells,
bulk (un-sorted) CD47 MOLM-13 cells, CD47.sup.lo, and CD47.sup.hi
MOLM-13 cells, then a direct correlation between CD47 expression
level and ability to evade phagocytosis can be seen (FIG. 14a, FIG.
15f). Furthermore, when CD47.sup.lo RFP MOLM-13 cells and
CD47.sup.hi GFP MOLM-13 cells were co-incubated with macrophages in
the same wells, the low expressing cells were far more likely to be
phagocytosed (FIG. 15h, 15i). Thus, in a mixed population of cells
with varying levels of CD47 expression, the low expressing cells
are more likely to be cleared by phagocytic clearance over
time.
[0163] We also titrated CD47 expression using another method. Since
CD47 is expressed in MOLM-13 cells using a Tet-OFF system, we
utilized the Tet-inducible promoter element to control expression
of CD47 in MOLM-13 cells. Beginning two weeks after transplantation
with CD47.sup.hi MOLM-13 cells, a cohort of mice was given
doxycycline and followed for up to 75 days post-transplant. During
this time course, none of the mice given doxycycline succumbed to
disease or had large infiltration of MOLM-13 cells in hematopoietic
organs (FIGS. 15b-d). At the doses of doxycycline used in this
experiment, muCD47 expression in MOLM-13 cells was reduced to
levels below that of normal mouse bone marrow, but notably not
completely absent (FIG. 15b). Thus, a sustained high level of CD47
expression is required for robust MOLM-13 survival in hematopoietic
organs.
[0164] Many examples of tumor clearance by T, B, and NK cells have
been described in the literature, indicating that a healthy immune
system is essential for regulating nascent tumor growth. However,
to date, few examples have been produced indicating that
macrophage-mediated phagocytosis can check tumor development.
Collectively, our studies reveal that ectopic expression of CD47
can enable otherwise immunogenic tumor cells to grow rapidly in a
T, B, and NK-cell deficient host. Furthermore, this is likely to
reflect a mechanism used by human myeloid leukemias to evade the
host immune system since CD47 is consistently upregulated in murine
and human myeloid leukemias, including all forms of chronic and
acute myeloid leukaemia tested thus far. Thus, it appears likely
that tumor cells are capable of being recognized as a target by
activated macrophages and cleared through phagocytosis. By
upregulating CD47, cancers are able to escape this form of innate
immune tumor surveillance.
[0165] This form of immune evasion is particularly important since
these cancers often occupy sites of high macrophage infiltration.
CD47 was first cloned as an ovarian tumor cell marker, indicating
that it may play a role in preventing phagocytosis of other tissue
cancers as well. Furthermore, solid tumors often metastasize to
macrophage rich tissues such as liver, lung, bone marrow, and lymph
nodes, indicating that they must be able to escape
macrophage-mediated killing in those tissues. Finding methods to
disrupt CD47-SIRP.alpha. interaction may thus prove broadly useful
in developing novel therapies for cancer. Preventing
CD47-SIRP.alpha. interaction is doubly effective since antigens
from phagocytosed tumor cells may be presented by macrophages to
activate an adaptive immune response, leading to further tumor
destruction.
Methods
[0166] Mice.
[0167] hMRP8bcrabl, hMRP8bcl2, and Fas.sup.lpr/lpr transgenic mice
were created as previously described and crossed to obtain double
transgenics. hMRP8bcl2 homozygotes were obtained by crossing
heterozygote mice to each other. C57Bl/6 Ka mice from our colony
were used as a source of wild-type cells. For transplant
experiments, cells were transplanted into C57Bl/6 RAG2.sup.-/-
common gamma chain (Gc).sup.-/- mice given a radiation dose of 4 Gy
using gamma rays from a cesium irradiator (Phillips). Primary mouse
leukemias were transplanted into CD45.2 C57Bl6/Ka mice given a
radiation dose of 9.5 Gy. Mice were euthanized when moribund.
[0168] Mouse Tissues.
[0169] Long bones were flushed with PBS supplemented with 2% fetal
calf serum staining media (SM) Spleens and livers were dissociated
using frosted glass slides in SM, then passed through a nylon mesh.
All samples were treated with ACK lysis buffer to lyse erythrocytes
prior to further analysis.
[0170] Quantitative RT-PCR Analysis.
[0171] Bone marrow was obtained from leukemic
hMRP8bcr/abl.times.hMRP8bcl2 mice or hMRP8bcl2 control mice. Cells
were c-Kit enriched using c-Kit microbeads and an autoMACS column
(Miltenyi). RNA was extracted using Trizol reagent (Invitrogen) and
reverse transcription performed using SuperScriptII reverse
polymerase (Invitrogen). cDNA corresponding to approximately 1000
cells was used per PCR reaction. Quantitative PCR was performed
with a SYBR green kit on an ABI Prism 7000 PCR (Applied Biosystems)
machine at 50.degree. C. for 2 minutes, followed by 95.degree. C.
for 10 minutes and then 40 cycles of 95.degree. C. for 15 minutes
followed by 60.degree. C. for 1 minute. Beta-actin and 18S RNA were
used as controls for cDNA quantity and results of CD47 expression
were normalized. Sequences for 18S RNA forward and reverse primers
were TTGACGGAAGGGCACCACCAG (SEQ ID NO: 8) and GCACCACCACCCACGGAATCG
(SEQ ID NO: 9), respectively, for beta-actin were
TTCCTTCTTGGGTATGGAAT (SEQ ID NO: 10) and GAGCAATGATCTTGATCCTC (SEQ
ID NO: 11), and for CD47 were AGGCCAAGTCCAGAAGCATTC (SEQ ID NO: 12)
and AATCATTCTGCTGCTCGTTGC (SEQ ID NO: 13).
[0172] Human Bone Marrow and Peripheral Blood Samples.
[0173] Normal bone marrow samples were obtained with informed
consent from 20-25 year old paid donors who were hepatitis A, B, C
and HIV negative by serology (All Cells). Blood and marrow cells
were donated by patients with chronic myelomonocytic leukemia
(CMML), chronic myeloid leukemia (CML), and acute myelogenous
leukemia (AML) and were obtained with informed consent, from
previously untreated patients.
[0174] Cell Lines.
[0175] MOLM-13 cells were obtained from DSMZ. HL-60 and Jurkat
cells were obtained from ATCC. Cells were maintained in Iscove's
modified Dulbecco's media (IMDM) plus 10% fetal bovine serum (FBS)
(Hyclone). To fractionate MOLM-13 cells into those with high and
low CD47 expression, Tet-CD47 MOLM-13 cells were stained with
anti-mouse CD47 Alexa-680 antibody (mIAP301). The highest and
lowest 5% of mouse CD47 expressing cells was sorted on a BD
FACSAria and re-grown in IMDM+10% FCS for 2 weeks. The cells were
sorted for three more rounds of selection following the same
protocol to obtain the high and low expressing cells used in this
study. To obtain red fluorescent protein (RFP) constructs, the
mCherry RFP DNA was cloned into Lentilox 3.7 (pLL3.7) empty vector.
Lentivirus obtained from this construct was then used to infect
cell lines.
[0176] Cell Staining and Flow Cytometry.
[0177] Staining for mouse stem and progenitor cells was performed
using the following monoclonal antibodies: Mac-1, Gr-1, CD3, CD4,
CD8, B220, and Ter119 conjugated to Cy5-PE (eBioscience) were used
in the lineage cocktail, c-Kit PE-Cy7 (eBioscience), Sca-1 Alexa680
(e13-161-7, produced in our lab), CD34 FITC(eBioscience),
CD16/32(FcGRII/III) APC (Pharmingen), and CD135(Flk-2) PE
(eBioscience) were used as previously described to stain mouse stem
and progenitor subsets. Mouse CD47 antibody (clone mIAP301) was
assessed using biotinylated antibody produced in our lab. Cells
were then stained with streptavidin conjugated Quantum Dot 605
(Chemicon). Samples were analyzed using a FACSAria (Beckton
Dickinson).
[0178] For human samples, mononuclear fractions were extracted
following Ficoll density centrifugation according to standard
methods and analyzed fresh or subsequent to rapid thawing of
samples previously frozen in 90% FCS and 10% DMSO in liquid
nitrogen. In some cases, CD34+ cells were enriched from mononuclear
fractions with the aid of immunomagnetic beads (CD34+ Progenitor
Isolation Kit, Miltenyi Biotec, Bergisch-Gladbach, Germany). Prior
to FACS analysis and sorting, myeloid progenitors were stained with
lineage marker specific phycoerythrin (PE)-Cy5-conjugated
antibodies including CD2 RPA-2.10; CD11b, ICRF44; CD20, 2H7; CD56,
B159; GPA, GA-R2 (Becton Dickinson--PharMingen, San Diego),
CD3,S4.1;CD4, S3.5; CD7, CD7-6B7; CD8, 3B5; CD10, 5-1B4, CD14,
TUK4; CD19, SJ25-C1 (Caltag, South San Francisco, Calif.) and
APC-conjugated anti-CD34, HPCA-2 (Becton Dickinson-PharMingen),
biotinylated anti-CD38, HIT2 (Caltag) in addition to PE-conjugated
anti-IL-3R.alpha., 9F5 (Becton Dickinson--ParMingen) and
FITC-conjugated anti-CD45RA, MEM56 (Caltag) followed by staining
with Streptavidin--Texas Red to visualize CD38-BIO stained
cells.
[0179] Following staining, cells were analyzed using a modified
FACS Vantage (Becton Dickinson Immunocytometry Systems, Mountain
View, Calif.) equipped with a 599 nm dye laser and a 488 nm argon
laser or a FACSAria. Hematopoietic stem cells (HSC) were identified
as CD34+ CD38+ CD90+ and lineage negative. Anti-human CD47 FITC
(clone B6H12, Pharmingen) was used to assess CD47 expression in all
human samples. Fold change for CD47 expression was determined by
dividing the average mean fluorescence intensity of CD47 for all
the samples of CML-BC, CML-CP, or AML by the average mean
fluorescence intensity of normal cells for a given cell population.
Common myeloid progenitors (CMP) were identified based on CD34+
CD38+IL-3R.alpha.+ CD45RA- lin- staining and their progeny
including granulocyte/macrophage progenitors (GMP) were
CD34+CD38+IL-3R.alpha.+ CD45RA+ Lin- while
megakaryocyte/erythrocyte progenitors (MEP) were identified based
on CD34+CD38+IL-3R.alpha.- CD45RA- Lin- staining.
[0180] To determine the density of mouse or human CD47, cells were
stained with saturating amounts of anti-CD47 antibody and analyzed
on a FACSAria. Since forward scatter is directly proportional to
cell diameter, and density is equal to expression level per unit of
surface area we used FloJo software to calculate geometric mean
fluorescent intensity of the CD47 channel and divided by the
geometric mean of the forward scatter value squared (FSC.sup.2) to
obtain an approximation for density of CD47 expression on the
membrane.
[0181] Engraftment of MOLM-13 cells was assessed by using
anti-human CD45 PE-Cy7 (Pharmingen), anti-mouse CD45.2 APC (clone
AL1-4A2), and anti-mouse CD47 Alexa-680 (mIAP301). All samples were
resuspended in propidium iodide containing buffer before analysis
to exclude dead cells. FACS data was analyzed using FloJo software
(Treestar).
[0182] Lentiviral Preparation and Transduction.
[0183] pRRL.sin-18.PPT.Tet07.IRES.GFP.pre, CMV, VSV, and tet
trans-activator (tTA) plasmids were obtained from Luigi Naldini.
The full length murine cDNA for CD47 form 2 was provided by Eric
Brown (UCSF). The CD47 cDNA construct was ligated into the
BamHI/NheI site of Tet-MCS-IRES-GFP. Plasmid DNA was transfected
into 293T cells using standard protocols. The supernatant was
harvested and concentrated using a Beckman LM-8 centrifuge
(Beckman). Cells were transduced with Tet or Tet-CD47-MCS-IRES-GFP
and tTA lentivirus for 48 hours. GFP+ cells were sorted to purity
and grown for several generations to ensure stability of the
transgenes.
[0184] Injections.
[0185] Cells were injected intravenously into the retro-orbital
sinuses of recipient mice or via the tail vein as noted. For
intra-femoral injections, cells were injected into the femoral
cavity of anesthetized mice in a volume of 20 .mu.l using a
27-gauge needle. An isofluorane gas chamber was used to anesthetize
mice when necessary.
[0186] MOLM-13 Cell Engraftment.
[0187] Animals were euthanized when moribund and bone marrow,
spleen, and liver harvested. Peripheral blood was obtained by tail
bleed of the animals 1 hour prior to euthanization. Engraftment of
MOLM-13 cells in marrow, spleen, and peripheral blood was
determined as described above. Tumor burden in the liver was
determined by calculating the area of each visible tumor nodule
using the formula ((length in mm+width in mm)/2)*.pi.. Area of each
nodule was then added together per liver.
[0188] Doxycycline administration. Doxycycline hydrochloride
(Sigma) was added to drinking water at a final concentration of 1
mg/mL. Drinking water was replaced every 4 days and protected from
light. In addition, mice received a 10 .mu.g bolus of doxycycline
by i.p. injection once a week.
[0189] Bone Marrow Derived Macrophages (BMDM).
[0190] Femurs and tibias were harvested from C57Bl/6 Ka mice and
the marrow was flushed and placed into a sterile suspension of PBS.
The bone marrow suspension was grown in IMDM plus 10% FBS with 10
ng/mL of recombinant murine macrophage colony stimulating factor
(MCSF, Peprotech) for 7-10 days.
[0191] In Vitro Phagocytosis Assays.
[0192] BMDM were harvested by incubation in trypsin/EDTA (Gibco)
for 5 minutes and gentle scraping. Macrophages were plated at
5.times.10.sup.4 cells per well in a 24-well tissue culture plate
(Falcon). After 24 hours, media was replaced with serum-free IMDM.
After an additional 2 hours, 2.5.times.10.sup.5 Tet or Tet-CD47
MOLM-13 cells were added to the macrophage containing wells and
incubated at 37 C..degree. for the indicated times. After
co-incubation, wells were washed thoroughly with IMDM 3 times and
examined under an Eclipse T5100 (Nikon) using an enhanced green
fluorescent protein (GFP) or Texas Red filter set (Nikon). The
number of GFP+ or RFP+ cells within macrophages was counted and
phagocytic index was calculated using the formula: phagocytic
index=number of ingested cells/(number of macrophages/100). At
least 200 macrophages were counted per well. For flow cytometry
analysis of phagocytosis macrophages were harvested after
incubation with MOLM-13 cells using trypsin/EDTA and gentle
scraping. Cells were stained with anti-Mac-1 PE antibody and
analyzed on a BD FACSAria. Fluorescent and brightfield images were
taken separately using an Eclipse T5100 (Nikon), a super high
pressure mercury lamp (Nikon), an endow green fluorescent protein
(eGFP) bandpass filter (Nikon) a Texas Red bandpass filter (Nikon),
and a RT Slider (Spot Diagnostics) camera. Images were merged with
Photoshop software (Adobe).
[0193] For in vivo assays, marrow from leg long bones, spleen, and
liver were harvested 2 hours after injecting target cells into
RAG2.sup.-/-, Gc.sup.-/- mice. They were prepared into single cell
suspensions in PBS plus 2% FCS. Cells were labeled with anti-human
CD45 Cy7-PE and anti-mouse F4/80 biotin (eBiosciences). Secondary
stain was performed with Streptavidin-APC (eBiosciences). Cells
that were human CD45-, F4/80+ were considered to be macrophages,
and GFP+ cells in this fraction was assessed.
Example 3
[0194] Hematopoietic Stem and Progenitor Cells Upregulate CD47 to
Facilitate Mobilization and Homing to Hematopoietic Tissues
[0195] We show here that hematopoietic stem cells (HSCs) from CD47
deficient (IAP.sup.-/-) mice fail to engraft wild-type recipients.
As expected, these cells are rapidly cleared by host macrophages,
whereas IAP.sup.+/+ HSCs are not. When stem and progenitor cells
are forced to divide and enter circulation using
cyclophosphamide/G-CSF or lipopolysaccharide, CD47 is rapidly
up-regulated on these cells. We propose that higher levels of CD47
in stem cells during stress hematopoiesis and mobilization provides
added protection against phagocytosis by activated macrophages of
the reticuloendothelial system. In support of this hypothesis, we
show that IAP.sup.+/- cells transplanted into wild-type recipients
lose engraftment over time, whereas wild-type donor cells do not.
We conclude that phagocytosis is a significant physiological
mechanism that clears hematopoietic progenitors over time, and that
CD47 over-expression is required to prevent phagocytic
clearance.
[0196] HSCs have the ability to migrate to ectopic niches in fetal
and adult life via the blood stream. Furthermore, HSCs can be
prodded into the circulation using a combination of cytotoxic
agents and cytokines that first expand HSC numbers in situ. Once in
the blood stream, HSCs must navigate the vascular beds of the
spleen and liver. Macrophages at these sites function to remove
damaged cells and foreign particles from the blood stream.
Furthermore, during inflammatory states, macrophages become more
phagocytically active. Hence, additional protection against
phagocytosis might be required for newly arriving stem cells at
these sites.
[0197] We determined if CD47 expression on bone marrow stem and
progenitor cells had a role in regulation of normal hematopoiesis.
CD47 expression has been shown to be essential for preventing
phagocytosis of red blood cells, T-cells, and whole bone marrow
cells in a transplant setting. Thus, we asked if lack of CD47 would
prevent HSCs from engrafting after being delivered intravenously.
To test this, we employed the CD47 knockout mouse (IAP.sup.-/-).
These mice develop normally and do not display any gross
abnormalities. They do, however, die very quickly after
intraperitoneal bacterial challenge because neutrophils fail to
migrate to the gut quickly. In addition, cells from these mice fail
to transplant into wild-type recipients, but they will engraft in
IAP-/- recipients.
[0198] We first examined stem and progenitor frequencies in
IAP.sup.+/- and IAP.sup.-/- mice. When examining for cells in the
stem and myeloid progenitor compartment, there was no difference
between these mice and wild-type mice (FIG. 18a). We then tested
stem cells from these mice for their ability to form colonies in an
in vitro assay. We sorted highly purified Flk2- CD34- KLS stem
cells from these mice and plated them onto methylcellulose in the
presence of a standard cytokine cocktail. We examined colony
formation at day 7 and found that there was no major difference
between wild-type and IAP-/- stem cells in the number and type of
colonies formed (FIG. 18b).
[0199] We then asked if bone marrow cells from IAP.sup.-/- mice
could rescue recipient mice from the effects of lethal irradiation.
Typically, a dose of 2.times.10.sup.5 bone marrow cells will rescue
100% of wild-type recipient mice in this assay. We found that
IAP.sup.-/- bone marrow could not rescue these recipients (FIG.
18c). However, administration of these cells did prolong lifespan;
normally, mice die between day 12 and 15 after irradiation, but
mice that received IAP.sup.-/- bone marrow lived about 7 to 10 days
longer (FIG. 18c). We do not yet know the reason for the
prolongation of lifespan in this case, although we have observed
that multipotent progenitors and megakaryocyte erythrocyte
progenitors can prolong survival after lethal irradiation, and that
contribution from these cells following transplant of whole bone
marrow may have contributed to the elongation of survival time.
[0200] Next, we sorted Flk-2.sup.- CD34.sup.- KLS stem cells from
wild-type and IAP.sup.-/- cells and transplanted them into
wild-type recipients along with 2.times.10.sup.5 competitor cells.
None of the mice which received IAP.sup.-/- HSCs, at either a dose
of 50 or 500 had any engraftment of donor cells, indicating that
CD47 was indeed required for the ability of these cells to
transplant (FIG. 18d-e). We speculated that this was due to
phagocytosis of the cells which lacked CD47, as has been shown for
erythrocytes and T-cells. To test this, we enriched c-Kit.sup.+
cells from the bone marrow of wild-type and IAP.sup.-/- mice and
co-incubated them with bone marrow derived macrophages. IAP.sup.-/-
stem and progenitor cells were readily phagocytosed in this assay,
whereas wild-type cells were only minimally phagocytosed (FIG.
18f-g). Interestingly, when incubated with IAP.sup.-/- macrophages,
there was significantly less phagocytosis of IAP.sup.-/- cells,
confirming that macrophages from these mice are indeed abnormal in
their phagocytic capacity.
[0201] Since mobilization of stem and progenitor cells involves
several steps in which they come into contact with macrophages
(egress from the marrow sinusoids, entry into the marrow and liver
sinusoids, and in the splenic marginal zone), we asked if CD47 is
up-regulated in the marrow of mice which have been induced to
undergo mobilization. The most commonly used protocol involves
administering the drug cyclophosphamide (Cy), which kills dividing
(mainly myeloid progenitor) cells, followed by treatment with
granulocyte colony stimulating factor (G-CSF). This involves
administering cyclophosphamide on the first day, and then giving
G-CSF every day thereafter. By convention, the first day after
cyclophosphamide administration is called day 0. The peak numbers
of stem cells in the bone marrow is achieved on day 2; from days
3-4 they egress from the bone marrow into the periphery, and their
numbers in the spleen and liver reach a peak at day 5;
myeloerythroid progenitors are also mobilized. There is a
characteristic rise in the frequency of stem cells and myeloid
progenitors during the mobilization response.
[0202] Thus, we administered this mobilization protocol to
wild-type mice and sacrificed mice on days 2 through 5. We found
that there was a notable increase of CD47 on c-Kit.sup.+ bone
marrow cells on day 2 (FIG. 19a). We found that there was
approximately a four-fold increase in the level of CD47 on stem and
progenitor cells on day 2 of mobilization (FIG. 19b). The increase
was seen at all levels of the myeloid progenitor hierarchy, as
LT-HSCs as well as GMPs displayed this increase in CD47 expression
(FIG. 19b). By day 5, when egress from the marrow has largely
halted, the levels of CD47 had returned to nearly normal levels. In
FIG. 19c, the mean fluorescence intensity of CD47 expression on
GMPs is shown on days 0 to 5 of mobilization. CD47 levels are
actually subnormal following myeloablation on day 0, but they
quickly rise to a peak on day 2. The expression quickly lowers
thereafter and the levels by day 5 are equivalent to steady
state.
[0203] Endotoxins are also thought to contribute to bone marrow
mobilization. This may represent a physiological response to
infection, where normal marrow output of immune cells needs to be
increased to clear the offending pathogens. Lipopolysaccharide
(LPS) is a cell wall component of gram-negative bacteria. It binds
to the lipid binding protein (LBP) in the serum, which can then
form a complex with CD1411 and toll-like receptor 4 (TLR-4) 12 on
monocytes, macrophages, and dendritic cells. This results in
activation of macrophages and results in a pro-inflammatory
response. LPS administration has also been shown to increase the
phagocytic capacity of macrophages. This may be due to the fact
that LBP-LPS complexes act as opsonins.
[0204] We tested if LPS administration in mice would affect CD47
expression in stem and progenitor cells. Mirroring the pattern seen
in Cy/G induced mobilization, LPS caused expansion of stem and
progenitor cells by 2 days post treatment, followed by migration to
the spleen and liver (FIG. 19d). On day 2 after LPS administration,
stem and progenitor cells in the marrow had up-regulated CD47 to a
similar degree as in Cy/G mobilization. By day 5, when the
inflammatory response has resolved, the levels of the protein had
dropped to steady-state levels (FIG. 19d).
[0205] Since CD47 was consistently up-regulated in the mobilization
response, we decided to test the ability of stem and progenitor
cells to mobilize following Cy/G. The CD47 knockout mouse has
defects in migration of neutrophils to sites of inflammation8 and
of dendritic cells to secondary lymphoid organs. The exact role of
CD47 in migration of these cells is unknown, but it may relate to
poor integrin association in the circulation (CD47 binds to several
integrins) or lack of interaction with SIRP.alpha. on endothelial
cells. Hence we reasoned that if CD47 was involved in the migration
capacity of these cells in the mobilization response, then IAP-/-
mice would display reduced numbers of cells in the peripheral
organs after Cy/G.
[0206] To test this hypothesis we administered Cy/G to both
wild-type and knockout mice and sacrificed mice on days 2-5. For
each mouse, we analyzed the number of stem and progenitor cells in
marrow, spleen, and liver. We decided to use the crude KLS
population as a surrogate for HSCs because numbers of CD34- cells
drops considerably in proliferative states, making accurate
calculation of LT-HSC numbers difficult. Since GMP are the most
expanded of all the populations in mobilization, we decided to
analyze their numbers as well. To calculate absolute progenitor
count, the total cellularity of marrow, spleen, and liver was
estimated by counting the mononuclear cell number in the whole
organ by hemocytometer. For bone marrow, leg long bones were
assumed to represent 15% of the total marrow. This number was then
multiplied by the frequency of the cell population to determine an
absolute count.
[0207] We found that there was little difference in mobilization of
KLS or GMP between wild-type and IAP.sup.-/- mice (FIG. 19e). There
was a modest decrease in the ability of IAP.sup.-/- mice to move
progenitors to the spleen by day 3, but by days 4 and 5 they had
restored normal numbers of cells to the periphery. The marrow and
liver compartments looked similar to wild-type mice. Hence,
IAP.sup.-/- mice do not have a significant mobilization defect.
[0208] Heterozygote IAP.sup.+/- erythrocytes have roughly the half
the amount of CD47 as wild-type erythrocytes and platelets. There
is also a dose dependent increase in the amount of phagocytosis
that occurs in immunoglobulin opsonized IAP.sup.+/- erythrocytes
and platelets relative to wild-type. Our observation that CD47
levels increase in states of stress and mobilization led us to
hypothesize that cells that were genetically hemizygous for CD47
might be more prone to phagocytosis and clearance by macrophages
over time. Hence, we asked if IAP.sup.+/- stem cells would be
disadvantaged relative to wild-type stem cells in long-term
contribution to hematopoiesis.
[0209] We first analyzed the levels of CD47 expressed on
IAP.sup.+/+, IAP.sup.+/-, and IAP.sup.-/- stem cells. FACS analysis
of CD34.sup.- Flk-2.sup.- KLS stem cells revealed that the MFI of
CD47 on heterozygote HSCs was indeed at roughly half the level of
wild-type stem cells (FIG. 20a). We then transplanted these cells
and examined their ability to engraft and produce hematopoietic
cells in a recipient. We gave congenic wild-type recipient mice 475
Gy, a sublethal dose of irradiation. We then transplanted one
cohort of recipients with 2.times.10.sup.6 wild-type whole bone
marrow cells, and another with the same dose of IAP.sup.+/- bone
marrow cells. Such a dose would be expected to contain roughly
50-100 HSCs. Since granulocyte chimerism in the peripheral blood is
a good surrogate marker of stem cell fitness, we analyzed cells
from the blood of these recipients at periodic intervals. When
wild-type marrow was transplanted into wild-type recipients,
granulocyte chimerism was maintained for up to 40 weeks. However,
when IAP.sup.+/- cells were transplanted, 3 out of 5 mice lost
donor chimerism over time, despite having a successful engraftment
initially (FIG. 20b).
[0210] We have observed that CD47 is up-regulated on the surface of
hematopoietic cells in the progression of leukemia. We have also
found an analogous increase in the level of CD47 expression when
mice were stimulated to mobilize stem and progenitor cells to the
periphery using Cy/G, or when they were challenged with LPS. But
why is CD47 upregulated in these states? Various studies have
described a dose-dependent effect for CD47 in the prevention of
phagocytosis. IAP.sup.+/- erythrocytes and platelets, which have
half the level of CD47 as wild-type cells, are phagocytosed more
readily than their normal counterparts. Evidence also indicates
that the level of CD47 expression on cells correlates well with the
ability of the cell to engage the SIRP.alpha. inhibitory receptor
on macrophages. Recently Danska et al reported that the ability of
NOD-SCID mice to support transplantation of human hematopoietic
cells correlated with a mutation in the SIRP alpha receptor in
these mice. Here we show that stem and progenitor cells that
express higher levels of CD47 are less likely to be cleared by
phagocytosis.
[0211] These studies point to a role for CD47 up-regulation in
protecting hematopoietic stem cells during states when they are
more prone to being phagocytosed by macrophages, such as
post-myeloablation and during mobilization. Macrophages have the
function of removing aged or damaged cells that they encounter; it
seems that they can eliminate damaged stem cells as well. Thus,
healthy recovering stem cells might up-regulate CD47 during a
mobilization response to prevent clearance, whereas damaged stem
cells fail to do so and are cleared. We speculate that this is a
mechanism by which the hematopoietic system self-regulates itself
to ensure that only healthy, undamaged cells are permitted to
survive and proliferate and utilize resources during high stress
states. The mobilization of HSC and progenitors into the
bloodstream and thence to hematopoietic sites following LPS induced
inflammation is very interesting; HSC migrate from blood to marrow
using integrin .alpha.4.beta.1 (Wagers and Weissman, Stem Cells
24(4):1087-94, 2006) and the chemokine receptor CXCR4 (Wright D E
et al., J Exp Med 195(9): 1145-54, 2002). We have shown previously
that integrin .alpha.4.beta.1 binds to VCAM1 on hematopoietic
stroma (Mikaye K et al. J Exp Med 173(3):599-607, 1991); VCAM1 is
also the vascular addressin on vessels that inflammatory T cells
use to recognize and enter local sites of cell death and
inflammation. In addition to expressing the integrin associated
protein CD47, itinerant HSC express functional integrin
.alpha.4.beta.1, leading to the speculation that migrating
hematopoietic stem and progenitors in states of inflammation may
not only re-seed marrow hematopoiesis, but also participate in
local inflammation as well.
Materials and Methods
[0212] Mice. C57Bl/6 CD45.1 and C57Bl/6 CD45.2 (wild-type) mice
were maintained in our colony. IAP-/- mice were obtained from Eric
Brown (University of California, San Francisco). These were bred on
C57B16/J background and crossed with our wild-type colony.
[0213] Screening. IAP+/- were crossed to each other to generate
IAP-/- and IAP+/- offspring. Mice were screened by PCR of tail DNA.
The following primers were used: 3'
Neo-GCATCGCATTGTCTGAGTAGGTGTCATTCTATTC (SEQ ID NO: 14); 5'
IAP-TCACCTTGTTGTTCCTGTACTAC AAGCA (SEQ ID NO: 15); 3'
IAP-TGTCACTTCGCAAGTGTAGTTCC (SEQ ID NO: 16).
[0214] Cell staining and sorting. Staining for mouse stem and
progenitor cells was performed using the following monoclonal
antibodies: Mac-1, Gr-1, CD3, CD4, CD8, B220, and Ter119 conjugated
to Cy5-PE (eBioscience) were used in the lineage cocktail, c-Kit
PE-Cy7 (eBioscience), Sca-1 Alexa680 (e13-161-7, produced in our
lab), CD34 FITC (eBioscience), CD16/32(FcGRII/III) APC
(Pharmingen), and CD135(Flk-2) PE (eBioscience) were used as
previously described to stain mouse stem and progenitor subsets 21
22. Mouse CD47 antibody (clone mIAP301) was assessed using
biotinylated antibody produced in our lab. Cells were then stained
with streptavidin conjugated Quantum Dot 605 (Chemicon). Samples
were analyzed using a FACSAria (Beckton Dickinson).
[0215] CD34- Flk2- KLS stem cells were double-sorted using a BD
FACSAria. Peripheral blood cells were obtained from tail vein bleed
and red cells were eliminated by Dextran T500 (Sigma) precipitation
and ACK lysis. Cells were stained with anti-CD45.1 APC, anti-CD45.2
FITC, anti-Ter119 PE (Pharmingen), anti-B220 Cy5-PE (eBiosciences),
anti-CD3 Cascade Blue, and anti-Mac-1 Cy7-PE (eBiosciences).
Granulocytes were Ter119- B220- CD3- Mac-1+ SSC hi. Cells were
analyzed using a BD FACSAria.
[0216] All samples were resuspended in propidium iodide containing
buffer before analysis to exclude dead cells. FACS data was
analyzed using FloJo software (Treestar).
[0217] In vitro colony forming assay. LT-HSC were directly clone
sorted into a 96-well plate containing methycellulose media
(Methocult 3100) that was prepared as described. The media was also
supplemented with recombinant mouse stem cell factor (SCF),
interleukin (IL)-3, IL-11, granulocyte-macrophage colony
stimulating factor (GM-CSF), thrombopoietin (Tpo) and
erythropoietin (Epo). Colonies were scored for CFU-G, CFU-M,
CFU-GM, CFU-GEMM, and Meg.
[0218] Cell transfers. For whole bone marrow transfers, IAP+/+,
IAP+/-, or IAP-/- cells were freshly isolated from leg long bones.
Cells were counted using a hemacytometer and resuspended in PBS+2%
FCS at 100 uL. For some experiments, CD45.1 cells from C57Bl/6 Ka
CD45.1 mice were used as donors into CD45.2 wild-type mice.
[0219] For sorted cells, cells were sorted into PBS buffer at the
correct dose (i.e. 50 or 500 cells per tube) and resuspended in 100
uL of PBS+2% FCS. For competition experiments, 2.times.10.sup.5
freshly isolated whole bone marrow cells from C57Bl/6 CD45.1 were
added to the 100 uL stem cell suspension.
[0220] C57Bl/6 Ka CD45.1 or C57Bl/6 J CD45.2 recipient mice were
irradiated using a cesium source at the doses indicated. Sub-lethal
dose was 4.75 Gray and lethal dose was a split dose of 9.5 Gray.
Cells were transferred using a 27-gauge syringe into the
retro-orbital sinuses of mice anesthetized with isofluorane.
[0221] Mobilization assay. Mice were mobilized with
cyclophosphamide (Sigma) (200 mg/kg) and G-CSF (Neupogen) (250
.mu.g/kg) as previously described. Bacterial LPS from E. coli
055:B5 (Sigma) was administered at a dose of 40 mg/kg into the
peritoneal cavity.
[0222] For analysis of mobilized organs, whole spleen, whole liver,
and leg long bones were prepared in a single cell suspension. Cell
density was determined using a hemacytometer to determine overall
cellularity of hematopoietic cells in these organs.
[0223] Enrichment of c-Kit.sup.+ cells. Whole mouse marrow was
stained with CD117 microbeads (Miltenyi). c-Kit.sup.+ cells were
selected on an AutoMACS Midi column (Miltenyi) using a magnetic
separator.
[0224] In vitro phagocytosis assay. BMDM were prepared as
previously described. c-Kit enriched bone marrow cells were stained
with CFSE (Invitrogen) prior to the assay. 2.5.times.10.sup.5 c-Kit
enriched cells were plated with 5.times.10.sup.4 macrophages.
Macrophages and c-Kit cells were obtained from either IAP.sup.+/+
or IAP.sup.-/- mice. Cells were incubated for 2 hours and
phagocytic index was determined. Photographs were taken as
described previously.
Example 4
[0225] CD47 is an Independent Prognostic Factor and Therapeutic
Antibody Target on Human Acute Myeloid Leukemia Cells
[0226] Acute myelogenous leukemia (AML) is organized as a cellular
hierarchy initiated and maintained by a subset of self-renewing
leukemia stem cells (LSC). We hypothesized that increased CD47
expression on AML LSC contributes to pathogenesis by inhibiting
their phagocytosis through the interaction of CD47 with an
inhibitory receptor on phagocytes. We found that CD47 was more
highly expressed on AML LSC than their normal counterparts, and
that increased CD47 expression predicted worse overall survival in
3 independent cohorts of adult AML patients. Furthermore, blocking
monoclonal antibodies against CD47 preferentially enabled
phagocytosis of AML LSC by macrophages in vitro, and inhibited
their engraftment in vivo. Finally, treatment of human
AML-engrafted mice with anti-CD47 antibody eliminated AML in vivo.
In summary, increased CD47 expression is an independent poor
prognostic factor that can be targeted on human AML stem cells with
monoclonal antibodies capable of stimulating phagocytosis of
LSC.
Results
[0227] CD47 is More Highly Expressed on AML LSC than their Normal
Counterparts and is Associated with the FLT3-ITD Mutation.
[0228] In our investigation of several mouse models of myeloid
leukemia, we identified increased expression of CD47 on mouse
leukemia cells compared to normal bone marrow. This prompted
investigation of CD47 expression on human AML LSC and their normal
counterparts. Using flow cytometry, CD47 was more highly expressed
on multiple specimens of AML LSC than normal bone marrow HSC and
MPP (FIG. 6). This increased expression extended to the bulk
leukemia cells, which expressed CD47 similarly to the LSC-enriched
fraction.
[0229] Examination of a subset of these samples indicated that CD47
surface expression correlated with CD47 mRNA expression. To
investigate CD47 expression across morphologic, cytogenetic, and
molecular subgroups of AML, gene expression data from a previously
described cohort of 285 adult patients were analyzed (Valk et al.,
2004 N Engl J Med 350, 1617-1628). No significant difference in
CD47 expression among FAB (French-American-British) subtypes was
found. In most cytogenetic subgroups, CD47 was expressed at similar
levels, except for cases harboring t(8;21)(q22;q22), a favorable
risk group which had a statistically significant lower CD47
expression. In molecularly characterized AML subgroups, no
significant association was found between CD47 expression and
mutations in the tyrosine kinase domain of FLT3 (FLT3-TKD),
over-expression of EVI1, or mutations in CEBPA, NRAS, or KRAS.
However, higher CD47 expression was strongly correlated with the
presence of FLT3-ITD (p<0.001), which is observed in nearly one
third of AML with normal karyotypes and is associated with worse
overall survival. This finding was separately confirmed in two
independent datasets of 214 and 137 AML patients (Table 1).
TABLE-US-00001 TABLE 1 Clinical and Molecular Characteristics of
AML Samples from the Validation Cohort and Comparison Between Low
CD47 and High CD47 Expression Groups Overall Low CD47 High CD47
Clinical Feature* n = 137 n = 95 n = 37 P.dagger. Age, yrs. 0.26
Median 46 47 46 Range 10-60 24-60 16-60 WBC, .times.10.sup.9/L
<0.01 Median 24 17 35 Range 1-238 1-178 1-238 Centrally reviewed
0.29 FAB Classification, no. (%) M0 11 (5) 9 (9) 2 (5) M1 20 (20)
16 (17) 2 (32) M2 36 (26) 22 (23) 11 (30) M4 33 (24) 26 (26) 8 (22)
M5 19 (14) 10 (17) 3 (8) M6 2 (1) 2 (2) 0 (0) Unclassified 6 (4) 4
(4) 0 (0) FLT3-ITD, no. (%) <0.05 Negative 84 (61) 63 (66) 17
(46) Positive 53 (39) 32 (34) 20 (54) FLT3-TKD, no. (%) 0.24
Negative 109 (57) 75 (89) 27 (79) Positive 17 (13) 10 (11) 7 (11)
NPM1, no. (%) 0.10 Wild-Type 55 (45) 41 (49) 10 (30) Mutated 66
(55) 43 (51) 23 (70) CEBPA, no. (%) 1 Wild-Type 100 (86) 70 (86) 27
(07) Mutated 16 (14) 11 (14) 4 (13) MLL-PTD, no. (%) 1 Negative 121
(93) 53 (92) 34 (94) Positive 9 (7) 7 (8) 2 (6) Event-free survival
0.004 Median, mos. 14 17.1 6.5 Disease-free at 36 (27-44) 41
(31-52) 22 (8-36) 3 yrs, % (95% CI) Overall survival 0.002 Median,
mos. 18.5 22.1 9.1 Alive at 3 yrs, 39 (31-48) 44 (33-55) 26 (12-41)
% (95% CI) Complete remission rate. no. (%) CR after 1st 60 (46%)
46 (48%) 14 (28%) 0.23 Induction, no. (%) CR after 2nd 84 (74%) 64
(75%) 20 (69%) 0.63 Induction, no. (%) Randomisstion to 2ndary
consolidative therapy Alloganeic-HSCT, 29 (22%) 25 (26%) 4 (11%)
0.09 no. (%) Autologous-HSCT, 23 (17%) 17 (18%) 6 (16%) 0.98 no.
(%) *Tabulated clinical and molecular characteristics at diagnosis.
WBC indicates white blood cell conar; FAB, French-American-British;
FLT3-ITD, internal tandem duplication of the FLT3 gene (for 10
cases with missing FLT3-ITD status, the predicted FLT3-ITD status
based on gene expression was substituted using method of Ballinger
et al, 2008); FLT3-TKD, tyrosine kinase domain stutation of the
FLT3 gene; NPMI, nutration of the NPMI gene; MLL-PTD, partial
tandem duplication of the MLL gene; and CEBPA, mutation of the
CEBPA gene. CR, complete remission. CR was assessed both after
first and second instruction regimens, which comprised ICE
(idarubicin, etoposide, cytarabine) or A-HAM (all-trans retinoic
acid and high-dose cytarabine plus mitoccautrone).
Aullologene-HSCT: autologene transplantation; Allogeneic-HSCT,
allogenic transplantation. .dagger.P value compares differences in
molecular and clinical characteristics at diagnosis between
patients with low and high CD47 mRNA expression values. CD47
expression was dichommised based on an optimal cut point for
overall survival stratification that we identified on an
independent microarry datases published (Valls et al, 2004) as
described to supplement methods.
[0230] Identification and Separation of Normal and Leukemic
Progenitors from the Same Patient Based On Differential CD47
Expression.
[0231] In the LSC-enriched Lin-CD34+CD38- fraction of specimen
SU008, a rare population of CD4710-expressing cells was detected,
in addition to the majority CD47.sup.hi-expressing cells (FIG.
21A). These populations were isolated by fluorescence-activated
cell sorting (FACS) to >98% purity and either transplanted into
newborn NOG mice or plated into complete methylcellulose. The
CD47.sup.hi cells failed to engraft in vivo or form any colonies in
vitro, as can be observed with some AML specimens.
[0232] However, the CD47.sup.lo cells engrafted with normal
myelo-lymphoid hematopoiesis in vivo and formed numerous
morphologically normal myeloid colonies in vitro (FIG. 21B,C). This
specimen harbored the FLT3-ITD mutation, which was detected in the
bulk leukemia cells (FIG. 21D). The purified CD47.sup.hi cells
contained the FLT3-ITD mutation, and therefore, were part of the
leukemic clone, while the CD47.sup.lo cells did not. Human cells
isolated from mice engrafted with the CD47.sup.lo cells contained
only wild type FLT3, indicating that the CD47.sup.lo cells
contained normal hematopoietic progenitors.
[0233] Increased CD47 Expression in Human AML is Associated with
Poor Clinical Outcomes.
[0234] We hypothesized that increased CD47 expression on human AML
contributes to pathogenesis. From this hypothesis, we predicted
that AML with higher expression of CD47 would be associated with
worse clinical outcomes. Consistent with this hypothesis, analysis
of a previously described group of 285 adult AML patients with
diverse cytogenetic and molecular abnormalities (Valk et al., 2004)
revealed that a dichotomous stratification of patients into low
CD47 and high CD47 expression groups was associated with a
significantly increased risk of death in the high expressing group
(p=0.03). The association of overall survival with this dichotomous
stratification of CD47 expression was validated in a second test
cohort of 242 adult patients (Metzeler et al., 2008 Blood) with
normal karyotypes (NK-AML) (p=0.01).
[0235] Applying this stratification to a distinct validation cohort
of 137 adult patients with normal karyotypes (Bullinger et al.,
2008 Blood 111, 4490-4495), we confirmed the prognostic value of
CD47 expression for both overall and event-free survival (FIG. 22).
Analysis of clinical characteristics of the low and high CD47
expression groups in this cross-validation cohort also identified
statistically significant differences in white blood cell (WBC)
count and FLT3-ITD status, and no differences in rates of complete
remission and type of consolidative therapy including allogeneic
transplantation (Table 1). Kaplan-Meier analysis demonstrated that
high CD47 expression at diagnosis was significantly associated with
worse event-free and overall survival (FIG. 22 A,B). Patients in
the low CD47 expression group had a median event-free survival of
17.1 months compared to 6.8 months in the high CD47 expression
group, corresponding to a hazard ratio of 1.94 (95% confidence
interval 1.30 to 3.77, p=0.004). For overall survival, patients in
the low CD47 expression group had a median of 22.1 months compared
to 9.1 months in the high CD47 expression group, corresponding to a
hazard ratio of 2.02 (95% confidence interval 1.37 to 4.03,
p=0.002). When CD47 expression was considered as a continuous
variable, increased expression was also associated with a worse
event-free (p=0.02) and overall survival (p=0.02).
[0236] Despite the association with FLT3-ITD (Table 1), increased
CD47 expression at diagnosis was significantly associated with
worse event-free and overall survival in the subgroup of 74
patients without FLT3-ITD, when considered either as a binary
classification (FIG. 22C,D) or as a continuous variable (p=0.02 for
both event-free and overall survival). In multivariable analysis
considering age, FLT3-ITD status, and CD47 expression as a
continuous variable, increased CD47 expression remained associated
with worse event-free survival with a hazard ratio of 1.33 (95%
confidence interval 1.03 to 1.73, p=0.03) and overall survival with
a hazard ratio of 1.31 (95% confidence interval 1.00 to 1.71,
p=0.05) (Table 2).
TABLE-US-00002 TABLE 2 Outcome Measure/Variables Considered HR 95%
CI P Event-free survival CD47 expression, continuous, 1.33
1.03-1.73 0.03 per 2-fold increase FLT3-ITD, positive vs. negative
2.21 1.39-3.53 <0.001 Age, per year 1.03 1.00-1.06 0.03 Overall
survival CD47 expression, continuous, 1.31 1.00-1.71 0.05 per
2-fold increase FLT3-ITD, positive vs. negative 2.29 1.42-3.68
<0.001 Age, per year 1.03 1.01-1.06 0.01
[0237] Monoclonal Antibodies Directed Against Human CD47
Preferentially Enable Phagocytosis of AML LSC by Human
Macrophages.
[0238] We hypothesized that increased CD47 expression on human AML
contributes to pathogenesis by inhibiting phagocytosis of leukemia
cells, leading us to predict that disruption of the
CD47-SIRP.alpha. interaction with a monoclonal antibody directed
against CD47 will preferentially enable the phagocytosis of AML
LSC. Several anti-human CD47 monoclonal antibodies have been
generated including some capable of blocking the CD47-SIRP.alpha.
interaction (B6H12.2 and BRIC126) and others unable to do so (2D3)
(Subramanian et al., 2006 Blood 107, 2548-2556). The ability of
these antibodies to enable phagocytosis of AML LSC, or normal human
bone marrow CD34+ cells, by human macrophages in vitro was tested.
Incubation of AML LSC with human macrophages in the presence of
IgG1 isotype control antibody or mouse anti-human CD45 IgG1
monoclonal antibody did not result in significant phagocytosis as
determined by either immunofluorescence microscopy (FIG. 8A) or
flow cytometry. However, addition of the blocking anti-CD47
antibodies B6H12.2 and BRIC126, but not the non-blocking 2D3,
enabled phagocytosis of AML LSC (FIG. 8A,C). No phagocytosis of
normal CD34+ cells was observed with any of the antibodies (FIG.
8C).
[0239] Monoclonal Antibodies Directed Against Human CD47 Enable
Phagocytosis of AML LSC by Mouse Macrophages.
[0240] The CD47-SIRP.alpha. interaction has been implicated as a
critical regulator of xenotransplantation rejection in several
cross species transplants; however, there are conflicting reports
of the ability of CD47 from one species to bind and stimulate
SIRP.alpha. of a different species. In order to directly assess the
effect of inhibiting the interaction of human CD47 with mouse
SIRP.alpha., the in vitro phagocytosis assays described above were
conducted with mouse macrophages. Incubation of AML LSC with mouse
macrophages in the presence of IgG1 isotype control antibody or
mouse anti-human CD45 IgG1 monoclonal antibody did not result in
significant phagocytosis as determined by either immunofluorescence
microscopy (FIG. 8B) or flow cytometry. However, addition of the
blocking anti-CD47 antibodies B6H12.2 and BRIC126, but not the
non-blocking 2D3, enabled phagocytosis of AML LSC (FIG. 8B,C).
[0241] A Monoclonal Antibody Directed Against Human CD47 Inhibits
AML LSC Engraftment and Eliminates AML in Vivo.
[0242] The ability of the blocking anti-CD47 antibody B6H12.2 to
target AML LSC in vivo was tested. First, a pre-coating strategy
was utilized in which AML LSC were purified by FACS and incubated
with IgG1 isotype control, anti-human CD45, or anti-human CD47
antibody. An aliquot of the cells was analyzed for coating by
staining with a secondary antibody demonstrating that both
anti-CD45 and anti-CD47 antibody bound the cells (FIG. 10A). The
remaining cells were transplanted into newborn NOG mice that were
analyzed for leukemic engraftment 13 weeks later (FIG. 10B). In all
but one mouse, the isotype control and anti-CD45 antibody coated
cells exhibited long-term leukemic engraftment. However, most mice
transplanted with cells coated with anti-CD47 antibody had no
detectable leukemia engraftment.
[0243] Next, a treatment strategy was utilized in which mice were
first engrafted with human AML LSC and then administered daily
intraperitoneal injections of 100 micrograms of either mouse IgG or
anti-CD47 antibody for 14 days, with leukemic engraftment
determined pre- and post-treatment. Analysis of the peripheral
blood showed near complete elimination of circulating leukemia in
mice treated with anti-CD47 antibody, often after a single dose,
with no response in control mice (FIG. 23A,B). Similarly, there was
a significant reduction in leukemic engraftment in the bone marrow
of mice treated with anti-CD47 antibody, while leukemic involvement
increased in control IgG-treated mice (FIG. 23 C,D). Histologic
analysis of the bone marrow identified monomorphic leukemic blasts
in control IgG-treated mice (FIG. 23E, panels 1,2) and cleared
hypocellular areas in anti-CD47 antibody-treated mice (FIG. 23E,
panels 4,5). In the bone marrow of some anti-CD47 antibody-treated
mice that contained residual leukemia, macrophages were detected
containing phagocytosed pyknotic cells, capturing the elimination
of human leukemia (FIG. 23E, panels 3,6).
[0244] We report here the identification of higher expression of
CD47 on AML LSC compared to their normal counterparts and
hypothesize that increased expression of CD47 on human AML
contributes to pathogenesis by inhibiting phagocytosis of these
cells through the interaction of CD47 with SIRP.alpha.. Consistent
with this hypothesis, we demonstrate that increased expression of
CD47 in human AML is associated with decreased overall survival. We
also demonstrate that disruption of the CD47-SIRP.alpha.
interaction with monoclonal antibodies directed against CD47
preferentially enables phagocytosis of AML LSC by macrophages in
vitro, inhibits the engraftment of AML LSC, and eliminates AML in
vivo. Together, these results establish the rationale for
considering the use of an anti-CD47 monoclonal antibody as a novel
therapy for human AML.
[0245] The pathogenic influence of CD47 appears mechanistically
distinct from the two main complementing classes of mutations in a
model proposed for AML pathogenesis. According to this model, class
I mutations, which primarily impact proliferation and apoptosis
(for example, FLT3 and NRAS), and class II mutations, which
primarily impair hematopoietic cell differentiation (for example,
CEBPA, MLL, and NPM1), cooperate in leukemogenesis. As demonstrated
here, CD47 contributes to pathogenesis via a distinct mechanism,
conferring a survival advantage to LSC and progeny blasts through
evasion of phagocytosis by the innate immune system. While
strategies for the evasion of immune responses have been described
for many human tumors, we believe that increased CD47 expression
represents the first such immune evasion mechanism with prognostic
and therapeutic implications for human AML.
[0246] Higher CD47 Expression is a Marker of Leukemia Stem Cells
and Prognostic for Overall Survival in AML. AML LSC are enriched in
the Lin-CD34+CD38- fraction, which in normal bone marrow contains
HSC and MPP. The identification of cell surface molecules that can
distinguish between leukemic and normal stem cells is essential for
flow cytometry-based assessment of minimal residual disease (MRD)
and for the development of prospective separation strategies for
use in cellular therapies. Several candidate molecules have
recently been identified, including CD123, CD96, CLL-1, and now
CD47. CD123 was the first molecule demonstrated to be more highly
expressed on AML LSC compared to normal HSC-enriched populations.
We previously identified AML LSC-specific expression of CD96
compared to normal HSC, and demonstrated that only CD96+, and not
CD96-, leukemia cells were able to engraft in vivo.
[0247] CLL-1 was identified as an AML LSC-specific surface molecule
expressed on most AML samples and not normal HSC; importantly, the
presence of Lin-CD34+CD38-CLL-1+ cells in the marrow of several
patients in hematologic remission was predictive of relapse. Here
we demonstrate that not only is CD47 more highly expressed on AML
LSC compared to normal HSC and MPP, but that this differential
expression can be used to separate normal HSC/MPP from LSC. This is
the first demonstration of the prospective separation of normal
from leukemic stem cells in the same patient sample, and offers the
possibility of LSC-depleted autologous HSC transplantation
therapies.
[0248] We initially identified higher expression of CD47 on AML
LSC, but noted that expression in bulk blasts was the same. Because
of this, we decided to utilize published gene expression data on
bulk AML to investigate the relationship between CD47 expression
and clinical outcomes. Consistent with our hypothesis, we found
that increased CD47 expression was independently predictive of a
worse clinical outcome in AML patients with a normal karyotype,
including the subset without the FLT3-ITD mutation, which is the
largest subgroup of AML patients. As this analysis was dependent on
the relative expression of CD47 mRNA, a quantitative PCR assay for
AML prognosis may be based on the level of CD47 expression. Such an
assay could be utilized in risk adapted therapeutic decision
making, particularly in the large subgroup of AML patients with
normal karyotypes who lack the FLT3-ITD mutation.
[0249] Targeting of CD47 on AML LSC with Therapeutic Monoclonal
Antibodies Cell surface molecules preferentially expressed on AML
LSC compared to their normal counterparts are candidates for
targeting with therapeutic monoclonal antibodies. Thus far, several
molecules have been targeted on AML including CD33, CD44, CD123,
and now CD47. CD33 is the target of the monoclonal antibody
conjugate gemtuzumab ozogamicin (Mylotarg), which is approved for
the treatment of relapsed AML in older patients. Targeting of CD44
with a monoclonal antibody was shown to markedly reduce AML
engraftment in mice, with evidence that it acts specifically on LSC
to induce differentiation. A monoclonal antibody directed against
CD123 was recently reported to have efficacy in reducing AML LSC
function in vivo. Here we report that a monoclonal antibody
directed against CD47 is able to stimulate phagocytosis of AML LSC
in vitro and inhibit engraftment in vivo.
[0250] Several lines of evidence suggest that targeting of CD47
with a monoclonal antibody likely acts by disrupting the
CD47-SIRP.alpha. interaction, thereby preventing a phagocytic
inhibitory signal. First, two blocking anti-CD47 antibodies enabled
AML LSC phagocytosis, while one non-blocking antibody did not, even
though all three bind the cells similarly. Second, in the case of
the B6H12.2 antibody used for most of our experiments, the
isotype-matched anti-CD45 antibody, which also binds LSC, failed to
produce the same effects. In fact, the B6H12.2 antibody is mouse
isotype IgG1, which is less effective at engaging mouse Fc
receptors than antibodies of isotype IgG2a or IgG2b.
[0251] For human clinical therapies, blocking CD47 on AML LSC with
humanized monoclonal antibodies promotes LSC phagocytosis through a
similar mechanism, as indicated by the human macrophage-mediated in
vitro phagocytosis (FIG. 8A,C). Higher CD47 expression is detected
on AML LSC; however, CD47 is expressed on normal tissues, including
bone marrow HSC. We identified a preferential effect of anti-CD47
antibodies in enabling the phagocytosis of AML LSC compared to
normal bone marrow CD34+ cells by human macrophages in vitro. In
fact, no increased phagocytosis of normal CD34+ cells compared to
isotype control was detected, demonstrating that blocking CD47 with
monoclonal antibodies is a viable therapeutic strategy for human
AML.
[0252] The experimental evidence presented here provides the
rationale for anti-CD47 monoclonal antibodies as monotherapy for
AML. However, such antibodies may be equally, if not more effective
as part of a combination strategy. The combination of an anti-CD47
antibody, able to block a strong inhibitory signal for
phagocytosis, with a second antibody able to bind a LSC-specific
molecule (for example CD96) and engage Fc receptors on phagocytes,
thereby delivering a strong positive signal for phagocytosis, may
result in a synergistic stimulus for phagocytosis and specific
elimination of AML LSC. Furthermore, combinations of monoclonal
antibodies to AML LSC that include blocking anti-CD47 and human
IgG1 antibodies directed against two other cell surface antigens
will be more likely to eliminate leukemia cells with pre-existing
epitope variants or antigen loss that are likely to recur in
patients treated with a single antibody.
Experimental Procedures
[0253] Human Samples.
[0254] Normal human bone marrow mononuclear cells were purchased
from AlICells Inc. (Emeryville, Calif.). Human acute myeloid
leukemia samples (FIG. 1A) were obtained from patients at the
Stanford University Medical Center with informed consent, according
to an IRB-approved protocol (Stanford IRB#76935 and 6453). Human
CD34- positive cells were enriched with magnetic beads (Miltenyi
Biotech).
[0255] Flow Cytometry Analysis and Cell Sorting.
[0256] A panel of antibodies was used for analysis and sorting of
AML LSC (Lin-CD34+CD38-CD90-, where lineage included CD3, CD19, and
CD20), HSC (Lin-CD34+CD38-CD90+), and MPP
(Lin-CD34+CD38-CD90-CD45RA-) as previously described (Majeti et
al., 2007). Analysis of CD47 expression was performed with an
anti-human CD47 PE antibody (clone B6H12, BD Biosciences, San Jose
Calif.).
[0257] Genomic DNA Preparation and Analysis of FLT3-ITD by PCR.
[0258] Genomic DNA was isolated from cell pellets using the Gentra
Puregene Kit according to the manufacturer's protocol (Gentra
Systems, Minneapolis, Minn.). FLT3-ITD status was screened by PCR
using primers that generated a wild-type product of 329 bp and ITD
products of variable larger sizes.
[0259] Anti-Human CD47 Antibodies.
[0260] Monoclonal mouse anti-human CD47 antibodies included:
BRIC126, IgG2b (Abcam, Cambridge, Mass.), 2D3, IgG1 (Ebiosciences.
San Diego, Calif.), and B6H12.2, IgG1. The B6H12.2 hybridoma was
obtained from the American Type Culture Collection (Rockville,
Md.). Antibody was either purified from hybridoma supernatant using
protein G affinity chromatography according to standard procedures
or obtained from BioXCell (Lebanon, N.H.).
[0261] Methylcellulose Colony Assay.
[0262] Methylcellulose colony formation was assayed by plating
sorted cells into a 6-well plate, each well containing 1 ml of
complete methylcellulose (Methocult GF+H4435, Stem Cell
Technologies). Plates were incubated for 14 days at 37.degree. C.,
then scored based on morphology.
[0263] In Vitro Phagocytosis Assays.
[0264] Human AML LSC or normal bone marrow CD34+ cells were
CFSE-labeled and incubated with either mouse or human macrophages
in the presence of 7 .mu.g/ml IgG1 isotype control, anti-CD45 IgG1,
or anti-CD47 (clones B6H12.2, BRIC126, or 2D3) antibody for 2
hours. Cells were then analyzed by fluorescence microscopy to
determine the phagocytic index (number of cells ingested per 100
macrophages). In some cases, cells were then harvested and stained
with either a mouse or human macrophage marker and phagocytosed
cells were identified by flow cytometry as macrophage+CFSE+.
Statistical analysis using Student's t-test was performed with
GraphPad Prism (San Diego, Calif.).
[0265] In Vivo Pre-Coating Engraftment Assay.
[0266] LSC isolated from AML specimens were incubated with 28 ug/mL
of IgG1 isotype control, anti-CD45 IgG1, or anti-CD47 IgG1
(B6H12.2) antibody at 4.degree. C. for 30 minutes. A small aliquot
of cells was then stained with donkey anti-mouse PE secondary
antibody (Ebioscience) and analyzed by flow cytometry to assess
coating. Approximately 10.sup.5 coated LSC were then transplanted
into each irradiated newborn NOD.Cg-Prkdcscidll2rgtm1Wjl/SzJ (NOG)
mouse. Mice were sacrificed 13 weeks post-transplantation and bone
marrow was analyzed for human leukemia engraftment (hCD45+hCD33+)
by flow cytometry (Majeti et al., 2007 Cell Stem Cell 1, 635-645).
The presence of human leukemia was confirmed by Wright-Giemsa
staining of hCD45+ cells and FLT3-ITD PCR. Statistical analysis
using Student's t-test was performed with GraphPad Prism (San
Diego, Calif.).
[0267] In Vivo Antibody Treatment of AML Engrafted Mice.
[0268] 1-25.times.10.sup.5 FACS-purified LSC were transplanted into
NOG pups. Eight to twelve weeks later, human AML engraftment
(hCD45+CD33+ cells) was assessed in the peripheral blood and bone
marrow by tail bleed and aspiration of the femur, respectively.
Engrafted mice were then treated with daily intraperitoneal
injections of 100 micrograms of anti-CD47 antibody or IgG control
for 14 days. On day 15 mice were sacrificed and the peripheral
blood and bone marrow were analyzed for AML.
[0269] AML Patients, Microarray Gene Expression Data, and
Statistical Analysis.
[0270] Gene expression and clinical data were analyzed for three
previously described cohorts of adult AML patients: (1) a training
dataset of 285 patients with diverse cytogenetic and molecular
abnormalities described by Valk et al., (2) a test dataset of 242
patients with normal karyotypes described by Metzeler et al., and
(3) a validation dataset of 137 patients with normal karyotypes
described by Bullinger et al. The clinical end points analyzed
included overall and event-free survival, with events defined as
the interval between study enrollment and removal from the study
owing to a lack of complete remission, relapse, or death from any
cause, with data censored for patients who did not have an event at
the last follow-up visit.
[0271] FLT3-ITD PCR.
[0272] All reactions were performed in a volume of 50 .mu.l
containing 5 .mu.l of 10.times.PCR buffer (50 mM KCL/10 nM Tris/2
mM MgCl2/0.01% gelatin), 1 .mu.l of 10 mM dNTPs, 2 units of Taq
polymerase (Invitrogen), 1 ul of 10 .mu.M forward primer 11F
(5'-GCAATTTAGGTATGAAAGCCAGC-3') (SEQ ID NO: 17) and reverse primer
12R (5'-CTTTCAGCATTTTGACGGCAACC-3') (SEQ ID NO: 18), and 10-50 ng
of genomic DNA. PCR conditions for amplification of the FLT3 gene
were 40 cycles of denaturation (30 sec at 95.degree. C.) annealing
(30 sec at 62.degree. C.), and extension (30 sec at 72.degree.
C.).
[0273] Preparation of Mouse and Human Macrophages.
[0274] BALB/c mouse bone marrow mononuclear cells were harvested
and grown in IMDM containing 10% FBS supplemented with 10 ng/mL
recombinant murine macrophage colony stimulating factor (M-CSF,
Peprotech, Rocky Hill, N.J.) for 7-10 days to allow terminal
differentiation of monocytes to macrophages. Human peripheral blood
mononuclear cells were prepared from discarded normal blood from
the Stanford University Medical Center. Monocytes were isolated by
adhering mononuclear cells to culture plates for one hour at
37.degree. C., after which non-adherent cells were removed by
washing. The remaining cells were >95% CD14 and CD11b positive.
Adherent cells were then incubated in IMDM plus 10% human serum
(Valley Biomedical, Winchester, Va.) for 7-10 days to allow
terminal differentiation of monocytes to macrophages.
[0275] In Vitro Phagocytosis Assay.
[0276] BMDM or peripheral blood macrophages were harvested by
incubation in trypsin/EDTA (Gibco/Invitrogen) for 5 minutes
followed by gentle scraping. 5.times.10.sup.4 macrophages were
plated in each well of a 24-well tissue culture plate in 10% IMDM
containing 10% FBS. After 24 hours, media was replaced with
serum-free IMDM and cells were cultured an additional 2 hours. LSC
were fluorescently labeled with CFSE according to the
manufacturer's protocol (Invitrogen). 2.times.10.sup.4 CFSE-labeled
LSC were added to the macrophage-containing wells along with 7
.mu.g/mL of IgG1 isotype (Ebiosciences), anti-CD45 (clone HI30,
Ebiosciences), or anti-CD47 antibody, and incubated for 2 hours.
Wells were then washed 3 times with IMDM and examined under an
Eclipse T5100 immunofluorescent microscope (Nikon) using an
enhanced green fluorescent protein filter able to detected CFSE
fluorescence. The number of CFSE positive cells within macrophages
was counted and the phagocytic index was determined as the number
of ingested cells per 100 macrophages. At least 200 macrophages
were counted per well. Fluorescent and brightfield images were
taken separately and merged with Image Pro Plus (Media Cybernetics,
Bethesda, Md.). In FIG. 22A,B, the three left images are presented
at 200.times. magnification, with the anti-CD47 right image at
400.times. magnification. For flow cytometry analysis of
phagocytosis, the cells were then harvested from each well using
trypsin/EDTA. Cell suspensions were then stained with a mouse
macrophage antibody anti-mouse F4/80-PECy7 (Ebiosciences) or
anti-human CD14-PECy7 (Ebiosciences) and analyzed on a FACSAria.
Phagocytosed LSC were defined as either CFSE+F4/80+ or CFSE+CD14+
cells when incubated with murine or human macrophages,
respectively.
[0277] Microarray Gene Expression Data.
[0278] Panel A of Supplemental FIG. 22 describes the main
microarray datasets analyzed herein, including the training, test,
and validation cohorts. Training Set: Gene expression data,
cytogenetics data, and molecular data for the 285 and 465 patients
with AML profiled with Affymetrix HG-U133A and HG-U133 Plus 2.0
microarrays by Valk et al. and Jongen-Lavrencic et al.
respectively, were obtained from the Gene Expression Omnibus using
the corresponding accession numbers (GSE1159 and GSE6891). Outcome
data were only available for the former dataset, and the
corresponding clinical information were kindly provided by the
authors. This cohort is presented as the "training" dataset. The
latter dataset was used to confirm univariate associations with
karyotype and molecular mutations described in the former. However,
these two datasets overlapped in that 247 of the 285 patients in
the first study were included in the second, and were accordingly
excluded in validation of the association of FLT3-ITD with CD47
expression in the 2nd dataset. Using NetAffx4, RefSeq5, and the
UCSC Genome Browser6, we identified 211075_s_at and 213857_s_at as
Affymetrix probe sets on the U133 Plus 2.0 microarray mapping
exclusively to constitutively transcribed exons of CD47. The
geometric mean of the base-2 logarithms of these two probe sets was
employed in estimating the mRNA expression level for CD47, and
corresponding statistical measures for associations with FAB
classification, karyotype, and molecular mutations. Because the
data provided by Valk et al. as GSE1159 were Affymetrix intensity
measurements, we converted these intensities to normalized base-2
logarithms of ratios to allow comparison to the corresponding
measurements from cDNA microarrays using a conventional scheme.
Specifically, we first (1) normalized raw data using CEL files from
all 291 microarrays within this dataset using gcRMA8, then (2)
generated ratios by dividing the intensity measurement for each
gene on a given array by the average intensity of the gene across
all arrays, (3) log-transformed (base 2) the resulting ratios, and
(4) median centered the expression data across arrays then across
genes. For the assessment of the prognostic value of CD47, we
employed the probe set 213857_s_at from the Affymetrix HG-U133A and
HG-U133 Plus 2.0 microarrays, given its similar expression
distribution (Supplemental FIG. 3B), and considering its position
within the mRNA transcript as compared with cDNA clones on the
Stanford cDNA microarrays as annotated within the NetAffx
resource.
[0279] Test Set: Gene expression and clinical data for the 242
adult patients with NKAML profiled with Affymetrix HG-U133A and
HG-U133 Plus 2.0 microarrays by Metzeler et al. were obtained from
the Gene Expression Omnibus using the corresponding accession
numbers (GSE12417). Since raw data were not available for this
dataset, for purposes of assessing the prognostic value of CD47, we
employed the normalized datasets provided by the authors (base 2
logarithms) and assessed expression of CD47 using the probe set
213857_s_at on the corresponding microarrays.
[0280] Validation Set: Gene expression data for the 137 patients
with normal karyotype AML profiled with cDNA microarrays by
Bullinger et al. were obtained from the Stanford Microarray
Database10. The corresponding clinical information including
outcome data and FLT3 mutation status were kindly provided by the
authors. Using the original annotations of microarray features as
well as SOURCE11, RefSeq5, and the UCSC Genome Browser6, we
identified IMAGE:811819 as a sequence verified cDNA clone mapping
to the constitutively transcribed 3' terminal exon of CD47 on the
corresponding cDNA microarrays.
[0281] Details of Treatment: AML patients described by Valk et al.
(training set), were treated according to several protocols of the
Dutch-Belgian Hematology-Oncology Cooperative group. The majority
(90%) of the NK-AML patients described by Metzeler et al. (test
set) were treated per protocol AMLCG-1999 of the German AML
Cooperative Group, with all patients receiving intensive
double-induction and consolidation chemotherapy. All 137 NK-AML
patients described by Bullinger et al. (validation set) received
standard-of-care intensified treatment regimens (protocol AML
HD98A), which included 2 courses of induction therapy with
idarubicin, cytarabine, and etoposide, one consolidation cycle of
high-dose cytarabine and mitoxantrone (HAM), followed by random
assignment to a late consolidation cycle of HAM versus autologous
hematopoietic cell transplantation in case no HLA identical family
donor was available for allogeneic hematopoietic cell
transplantation.
[0282] Statistical Analysis. We used two tailed t-tests and
analysis of variance for the estimation of significant differences
in CD47 expression level across subgroups of AML based on
morphologic, cytogenetic, and molecular categorizations.
Associations between the high and low CD47 groups and baseline
clinical, demographic, and molecular features were analyzed using
Fisher's exact and Mann-Whitney rank sum tests for categorical and
continuous variables, respectively. Two-sided p-values of less than
0.05 were considered to indicate statistical significance.
[0283] The prognostic value of CD47 expression was measured through
comparison of the event-free and overall survival of patients with
estimation of survival curves by the Kaplan-Meier product limit
method and the log-rank test. Within this analysis, we first
derived a binary classification of AML patients into High CD47 and
Low CD47 expression groups by comparing the expression of CD47 (as
measured by 213857_s_at within GSE1159) relative to an optimal
threshold. This threshold was determined using X-Tile16, a method
which we employed to maximize the chi-square statistic between the
two groups for the expected versus observed number of deaths. This
stratification segregates the 261 AML patients with available
outcome data into two unequally sized groups, with 72% of patients
with lowest expression considered CD47 low, and 28% with highest
expression considered CD47 high. These two groups have different
overall survival with a hazard ratio of 1.42 for the CD47 high
group, and a corresponding uncorrected p-value of 0.033, which
requires cross-validation to avoid the risk of overfitting.
[0284] Accordingly, we assessed the validity and robustness of risk
stratification using CD47 expression by applying this optimal
threshold to an independent test cohort of 242 NK-AML patients
described by Metzeler et al. Notably, despite the presence of other
variables potentially confounding associations with survival
(including more advanced age, and differing therapies), derivation
of an optimal cutpoint using the 242 NK-AML patients within the
test dataset yielded a similar stratification, with 74% of patients
with lowest expression considered CD47 low, and 26% with highest
expression considered CD47 high.
[0285] Next, we assessed the validity of this stratification in a
cross-validation cohort of 137 uniformly treated NK-AML patients
described by Bullinger et al. Within this validation dataset, we
could similarly define two groups of similar size (i.e., 72% and
28% with lowest and highest CD47 levels, respectively), and these
two groups had significantly different outcomes when assessed for
overall survival (FIG. 22B, p=0.002, hazard ratio 2.02, 95% CI 1.37
to 4.03), and event-free survival (FIG. 223A, p=0.004, hazard ratio
1.94, 95% CI 1.30 to 3.77). Of the 137 patients, 5 did not have
reliable measurements for CD47 when using the data selection and
normalization criteria described by the authors.
[0286] To determine the robustness of this association, we also
examined the predictive value of CD47 expression when the
validation cohort was divided into low and high CD47 expression
groups based on expression relative to the median, or as a
continuous variable. As above, higher CD47 expression was
associated with worse event-free and overall survival. Of the 137
patients studied, a subset of 123 patients had available survival
data, CD47 expression data, and FLT3-ITD status reported. Within
this cohort, we assessed the relationship of CD47 expression level
as a continuous variable with outcome using univariate Cox
proportional-hazards analysis, with event-free survival or overall
survival as the dependent variable. We used multivariate
Cox-proportional hazards analysis with event-free survival or
overall survival as the dependent variable and FLT3-ITD status,
age, and continuous expression level of CD47 as directly assessed
independent variables.
[0287] Associations of CD47 with other covariates (eg, NPM1, CEBPA)
were limited by sample size and missing data for covariates. The
Wald test was used to assess the significance of each covariate in
multivariate analyses. Univariate and multivariate
proportional-hazards analyses were done using the coxph function in
the R statistical package.
Example 5
CD47 is a Prognostic Factor and Therapeutic Antibody Target on
Solid Tumor Cancer Stem Cells
[0288] We have found that increased CD47 expression is associated
with worse clinical outcomes in diffuse large B-cell lymphoma
(DLBCL) and ovarian carcinoma (FIG. 24). Additionally, we have now
found that anti-CD47 antibodies enable the phagocytosis of cancer
stem cells from bladder cancer, ovarian carcinoma, and
medulloblastoma in vitro with human macrophages (FIG. 25).
Example 6
Therapeutic Antibody Targeting of CD47 Eliminates Human Acute
Lymphoblastic Leukemia
[0289] Although standard multi-agent chemotherapy cures a
significant number of patients with standard-risk pediatric ALL,
these same therapies are significantly less effective in both the
high-risk pediatric population and in all adults with ALL. Thus,
additional therapies are necessary to more effectively treat these
patient subsets. As an alternative to chemotherapy, monoclonal
antibodies have recently emerged as an attractive therapeutic
modality due to the ability to selectively target leukemia cells,
thereby minimizing systemic toxicity. Indeed, several monoclonal
antibodies are currently in clinical trials for the treatment of
ALL.
[0290] CD47 is identified herein as a therapeutic antibody target
in acute myeloid leukemia (AML). It is investigated whether a
blocking monoclonal antibody against CD47 could eliminate primary
human ALL in vitro and in vivo, in order to determine the use of an
anti-CD47 antibody as a therapy in standard and high-risk ALL.
Materials and Methods
[0291] Human Samples.
[0292] Normal human bone marrow cells were purchased from AlICells
Inc. (Emeryville, Calif., USA). Human ALL samples were obtained
from patients at the Stanford University Medical Center, with
informed consent, according to an IRB-approved protocol.
[0293] Flow Cytometry Analysis.
[0294] The following antibodies were used for analysis of ALL and
NBM cells: CD3 APC-Cy7 and CD19 APC (BD Biosciences, San Jose,
Calif., USA). Analysis of CD47 expression was performed with an
anti-human CD47 FITC antibody (clone B6H12.2, BD Biosciences). For
human engraftment analysis in mice, the following antibodies were
used: mouse Ter119 PeCy5, mouse CD45.1 PeCy7, human CD45 PB, human
CD19 APC, and human CD3 APC-Cy7 (Ebiosciences, San Diego, Calif.,
USA).
[0295] ALL Microarray Gene Expression Data and Statistical
Analysis.
[0296] We used previously described methods for the univariate and
multivariate statistical analyses of CD47 gene expression data and
its relationship to clinical and pathological variables. Briefly,
gene expression and clinical data were analyzed for three
previously described cohorts of ALL patients: 1) a dataset of 360
pediatric ALL patients with B- and T-ALL subtypes, diverse risk
profiles and corresponding therapies including a subset (n=205)
with available data on disease free survival. Microarray and
clinical data were obtained from St. Jude Children's Research
Hospital; 2) a dataset of 207 pediatric B-precursor ALL patients
with high-risk features uniformly treated through the Children's
Oncoloy Group Clinical Trial P9906 obtained from NCBI through the
Gene Expression Omnibus (GSE11877); and 3) 254 pediatric ALL
patients registered to Pediatric Oncology Group trials stratified
for the presence of recurrent cytogenetic abnormalities and
remission versus failure within each cytogenetic group with data
obtained from the National Cancer Institute caArray. Affymetrix
probeset summaries were derived from the corresponding microarray
raw CEL data files using a Custom Chip Definition File derived from
NCBI Reference Sequences (version 12), and then normalized using
MAS 5.0 using BioConductor. For survival analyses, NM 198793 at was
selected as the probeset to represent CD47 mRNA based on it
demonstrating highest expression among the 3 probesets for CD47 on
the microarrays, and based on its exonic structure capturing the
CD47 splice variant expressed in hematopoietic tissues.
[0297] We assessed the relationship of CD47 mRNA expression and
outcomes as continuous variables using univariate Cox
proportional-hazards analysis, with disease free or overall
survival as the dependent variable. Using the coxph function in the
R statistical package, the Wald test was used to assess the
significance of each covariate, represented by the base-2
logarithms of CD47 mRNA expression. For dichotomous stratification
of CD47 expression, we used maximally selected chi-square
statistics as implemented within X-tile to define an optimal
threshold. To guard against erroneous overestimation of p-values
through multiple hypothesis testing, we corrected the log-rank
Kaplan-Meier p-values using the Miller-Siegmund method well as
sub-sampling (n=1000) based internal cross-validation.
[0298] Therapeutic Antibodies.
[0299] Anti-human CD47 antibodies (B6H12.2, BRIC126, 2D3),
anti-SIRP.alpha. antibody, IgG control, and anti-CD45 antibodies
were used as described in the previous examples. The anti-CD47
antibody clone BRIC126 was obtained from AbD Serotec (Raleigh,
N.C., USA).
[0300] Generation of Mouse and Human Macrophages.
[0301] Isolation of mouse and human macrophages were performed as
previously described. Briefly, femurs and tibias from wild-type
Balb/C mice were harvested into a single cell suspension and
incubated for 7-10 days in IMDM 10% fetal calf serum with 10 ng/ml
murine M-CSF (Peprotech, Rocky Hill, N.J., USA) at 37.degree. C.
Cells were then washed and adherent cells trypsinized and plated
for in vitro phagocytosis assays. For human macrophages,
mononuclear cells were isolated from human peripheral blood by
ficoll density gradient centrifugation and plated onto 10 cm petri
dishes at 37.degree. C. for one hour. Non-adherent cells were then
washed off and the remaining adherent cells were incubated for 7-10
days in IMDM with 10% human AB serum. Cells were then trypsinized
and plated for in vitro phagocytosis assays.
[0302] In Vitro Phagocytosis Assays.
[0303] Phagocytosis assays were performed as described in the
previous examples. Briefly, bulk ALL cells were CFSE-labeled and
incubated with either mouse or human macrophages in the presence of
10 .mu.g/ml of the indicated antibodies at a target:effector cell
ratio of 4:1 (2.times.10.sup.5:5.times.10.sup.4). Incubation
occurred at 37.degree. C. for 2 hours and then analyzed by
fluorescent microscopy for phagocytosis using the phagocytic index:
number of cells ingested per 100 macrophages.
[0304] Ex Vivo Antibody Coating of ALL Cells.
[0305] Human ALL cells were incubated with 30 .mu.g/ml of either
IgG1 isotype control, anti-CD45, or anti-CD47 antibody for 30
minutes at 4.degree. C. Cells were washed and then
1-4.times.10.sup.6 cells were transplanted into
sublethally-irradiated NOD.Cg-Prkdc.sub.scidll2rg.sub.tm1Wjl/SzJ
(NSG) adults or pups and analyzed for ALL engraftment in the
peripheral blood and bone marrow 6-10 weeks later. Antibody coating
of ALL cells was confirmed by flow cytometry with a secondary
antibody prior to transplantation into mice. Sublethal irradiation
was 230 rads and 100 rads for NSG adults and pups,
respectively.
[0306] In Vivo Treatment of Human ALL Engrafted Mice.
[0307] 1-4.times.10.sup.6 bulk human ALL cells were transplanted
intravenously via the retro-orbital sinus into
sublethally-irradiated (230 rads) adult NSG mice. Alternatively,
ALL cells were transplanted into the facial vein of 2-4 day old
sublethally-irradiated (100 rads) NSG pups. Six to ten weeks later
peripheral blood and bone marrow ALL engraftment (B-ALL:
hCD45+CD19+; T-ALL: hCD45+CD3+) was assessed by tail bleed and
aspiration of the femur, respectively. Engrafted mice were treated
for 14 days with daily 100 .mu.g intraperitoneal injections of
either IgG control or anti-CD47 antibody (clone B6H12.2). On day
15, mice were sacrificed and analyzed for ALL engraftment in the
peripheral blood, bone marrow, spleen, and liver.
[0308] Bone Marrow Tissue Section Preparation and Staining.
[0309] Mouse tibias from antibody-treated NSG mice were harvested
and preserved in formalin. Hematoxylin and eosin staining and
immunohistochemistry of human CD45+ cells were performed by
Comparative Biosciences Inc. (Sunnyvale, Calif., USA).
Results
[0310] CD47 Expression is Increased on a Subset of Human ALL Cells
Compared to Normal Bone Marrow.
[0311] To determine whether CD47 may be involved in the
pathogenesis of ALL, we first investigated CD47 cell surface
expression on primary human ALL and normal bone marrow cells by
flow cytometry. We surveyed 17 diverse patients with ALL that
included both precursor B and T lineage subtypes. Compared to
normal mononuclear bone marrow cells, CD47 was more highly
expressed on human ALL samples, approximately 2-fold when
considering all samples, with similar expression between B and T
subtypes. However, assessing CD47mRNA expression in a large cohort
of ALL patients, we found that T-ALL patients expressed
significantly higher levels compared to B-ALL patients.
[0312] CD47 Expression is an Independent Prognostic Predictor in
Mixed and High-Risk ALL.
[0313] Since CD47 expression was increased on ALL samples, and
given the observed heterogeneity in CD47 expression across ALL
subtypes, we investigated whether the level of CD47 expression
correlated with clinical prognosis. First, CD47 expression was
investigated as a prognostic predictor in pediatric ALL patients
with mixed risk and treatment utilizing gene expression data from a
previously described patient cohort. 360 patients were stratified
into high and low CD47-expressing groups based on an optimal
cutpoint and clinical outcomes were determined. Among the subset of
this cohort with available outcome data (n=205), patients
expressing higher levels of CD47 had worse outcomes, whether CD47
expression was tested as a continuous variable (p=0.03; HR 1.78 per
2-fold change in CD47 expression; 95% CI 1.05-3.03), or as a
dichotomous variable relative to an internally validated optimal
threshold (uncorrected p=0.0005, corrected p=0.01; HR 3.05; 95% CI
1.49-6.26) (FIG. 26A).
[0314] Second, to investigate the prognostic power of CD47
expression in high-risk ALL patients, clinical outcome in a
uniformly treated previously described cohort of 207 high-risk
pediatric ALL patients was investigated. For this cohort, high-risk
was defined by age>10 years, presenting WBC
count>50,000/.mu.l, hypodiploidy, BCR-ABL positive disease, and
central nervous system or testicular involvement. In these
high-risk ALL patients, higher CD47 expression correlated with a
worse overall survival when CD47 expression was again considered as
either a continuous variable (p=0.0009, HR 3.59 per 2-fold change
in CD47 expression; 95% CI 1.70 to 7.61), or as a dichotomous one
relative to an internally validated optimal threshold (uncorrected
p=0.001, corrected p=0.01; HR 2.80; 95% CI 1.21 to 6.50) (FIG.
26B). In multivariate analysis, CD47 expression remained a
significant prognostic factor when age at diagnosis, gender, WBC
count, CNS involvement, and minimal residual disease were
considered as covariates (FIG. 26C).
[0315] Lastly, we utilized a third independent gene expression
dataset to investigate whether CD47 expression could predict
disease relapse. Indeed, CD47 expression was higher in patients
failing to achieve a complete remission (CR) compared to those that
did achieve a CR (FIG. 26D). Taken together, these separate
observations among distinct and diverse cohorts establish that
higher expression of CD47 is an independent predictor of adverse
outcomes in pediatric patients with standard- and high-risk ALL,
including induction failure, relapse, and death.
[0316] Blocking Monoclonal Antibodies Against CD47 Enable
Phagocytosis of ALL Cells.
[0317] Next, we investigated whether ALL cells could be eliminated
by macrophage phagocytosis enabled through blockade of the
CD47-SIRP.alpha. interaction with a blocking anti-CD47 antibody.
First, we incubated human macrophages with fluorescently-labeled
ALL cells in the presence of an IgG1 isotype control, anti-CD45
isotype-matched, or anti-CD47 antibody and measured phagocytosis by
immunofluorescence microscopy. Two different blocking anti-CD47
antibodies (B6H12.2 and BRIC126) enabled phagocytosis of ALL cells
compared to IgG1 isotype and anti-CD45 control antibodies as
measured by significant increases in the phagocytic index. In
addition, anti-CD47 antibodies enabled phagocytosis of all ALL
subtypes profiled, including those with cytogenetically high-risk
(Ph+ALL and MLL+ALL). Since several studies report that CD47-
SIRP.alpha. signaling may be species-specific, the ability of
anti-CD47 antibody-coated human cells to be phagocytosed by mouse
macrophages was determined before proceeding with in vivo antibody
treatment experiments in mouse xenotransplants. Similar to human
macrophages, two blocking anti-CD47 antibodies (B6H12.2 and
BRIC126) enabled phagocytosis of ALL cells by mouse macrophage
effectors compared to IgG1 isotype and anti-CD45 antibody controls.
Furthermore, no phagocytosis was observed with a non-blocking
anti-CD47 antibody (2D3). Lastly, blockade of SIRP.alpha. with an
anti-mouse SIRP.alpha. antibody also resulted in increased
phagocytosis, thus supporting the mechanism of increased
phagocytosis resulting from disruption of the CD47-SIRP.alpha.
interaction.
[0318] Ex Vivo Coating of ALL Cells with an Anti-CD47 Antibody
Inhibits Leukemic Engraftment.
[0319] The ability of a blocking anti-CD47 antibody to eliminate
ALL in vivo was investigated by two independent methods. First, the
ability of anti-CD47 antibody to inhibit ALL engraftment was
determined using an antibody pre-coating assay. ALL cells were
coated ex vivo with either IgG1 isotype control, anti-CD45, or
anti-CD47 antibody (B6H12.2), transplanted into
sublethally-irradiated immunodeficient NOD/SCID/II2.gamma.r null
(NSG) mice, and measured for ALL engraftment in the peripheral
blood and bone marrow 6-10 weeks later. Prior to transplantation,
coating of ALL cells with antibody was verified by flow cytometry
(FIG. 27A). Antibody pre-coating experiments were performed with
both primary B- and T-ALL samples to include the two major disease
subtypes. Anti-CD47 antibody significantly inhibited leukemic
engraftment of both B- and T-ALL cells in the peripheral blood
(FIG. 27B) and bone marrow (FIG. 27C) compared to IgG1 isotype or
anti-CD45 antibody controls. Pre-coating with the anti-CD47
antibody nearly completely eliminated ALL engraftment in vivo.
[0320] Anti-CD47 Antibody Eliminates ALL Engraftment in the
Peripheral Blood and Bone Marrow.
[0321] In the second method of investigating the in vivo efficacy
of an anti-CD47 antibody against human ALL, mice were first stably
engrafted with ALL cells and then treated with antibody.
1-4.times.10.sup.6 B- or T-ALL cells were transplanted into
sublethally-irradiated NSG newborn pups or adults. Six to ten weeks
later, ALL engraftment was measured in the peripheral blood and
bone marrow by flow cytometry. Those mice that had significant
levels of ALL engraftment were then selected for in vivo antibody
therapy (FIG. 28A), as determined by greater than 10% human
chimerism in the peripheral blood and/or bone marrow with
engraftment ranging from 10-97%. ALL engrafted mice were treated
with daily intraperitoneal injections of 100 .mu.g IgG control or
anti-CD47 antibody (B6H12.2) for 14 days. This dosing regimen was
selected based on our prior study demonstrating elimination of AML
in mouse xenotransplants. Tumor burden was then measured
post-treatment in the peripheral blood and bone marrow by flow
cytometry. Compared to IgG control, anti-CD47 antibody therapy
reduced the level of circulating leukemia, and in most cases
eliminated ALL from the peripheral blood (FIG. 28 A,B). This effect
was observed for mice transplanted with both B- and T-ALL cells.
Similarly, anti-CD47 antibody reduced or eliminated ALL engraftment
in the bone marrow, while ALL disease burden increased with IgG
control treatment (FIG. 28C). Bone marrow histology of
antibody-treated mice revealed infiltration of monomorphic leukemic
blasts in control IgG-treated mice (FIG. 28D). Anti-CD47
antibody-treated bone marrow demonstrated normal mouse
hematopoietic cells with cleared hypocellular areas.
Immunohistochemistry of mouse marrows confirmed near complete
invasion of human CD45-positive leukemic blasts in IgG-treated
marrow compared to few human CD45-positive leukemia cells observed
in anti-CD47 antibody-treated marrow (FIG. 28D).
[0322] Anti-CD47 Antibody Eliminates ALL Engraftment in the Spleen
and Liver.
[0323] Hepatomegaly and splenomegaly can cause clinical
complications and are a common finding in ALL, being observed in up
to 69% of patients at diagnosis. To determine whether anti-CD47
antibody could potentially treat hepatosplenomegaly in ALL, we
investigated the ability of anti-CD47 antibody to eliminate ALL
engrafted in the spleen and liver. Of the ALL samples utilized for
in vivo treatment studies, we identified three B-ALL patient
samples (ALL8, ALL21, and ALL22) that gave rise to disease in the
spleen and/or liver, with associated splenomegaly, when
transplanted into NSG mice. These mice were treated for 14 days
with the identical regimen of either IgG or anti-CD47 antibody as
in FIG. 28 with spleen weights and ALL chimerism in the spleen and
liver measured post-treatment. Control IgG-treated B-ALL-engrafted
mice exhibited significant splenomegaly compared to untransplanted
NSG mice (FIG. 29A,B). In contrast, anti-CD47 antibody treatment
reduced ALL-induced splenomegaly to spleen sizes similar to
untransplanted NSG mice (FIGS. 29A,B). To determine whether this
effect was due to direct elimination of ALL cells in the spleen,
the spleens of B-ALL-engrafted mice treated with IgG control or
anti-CD47 antibody were analyzed for ALL disease burden. Compared
to IgG-treated mice, anti-CD47 antibody significantly eliminated
B-ALL engraftment in the spleen (FIG. 29C). Similarly, anti-CD47
antibody significantly eliminated ALL in the liver compared to the
extensive leukemic infiltration observed with control IgG treatment
(FIG. 29D). These results indicate that anti-CD47 antibody is
highly effective in eliminating ALL in the spleen and liver, in
addition to the peripheral blood and bone marrow.
[0324] We report here that CD47 is expressed at high levels on a
large subset of human ALL subtypes, that cell surface CD47 is a
monoclonal antibody target for eliminating ALL blasts by enhancing
innate immune system recognition of leukemic blasts by
macrophage-mediated phagocytosis, and that CD47 itself is an
independent prognostic variable in ALL that can predict disease
free survival, overall survival, and relapse in both mixed and
high-risk ALL patients. Together, these data show that ALL
pathogenesis relies on mechanisms to evade innate immune
recognition and that modulation of the innate immune recognition of
tumor cells is a viable treatment modality.
[0325] Within the last few years, several cell surface proteins
have been identified as candidate targets, and some monoclonal
antibodies have proceeded into early and late phase clinical
trials. Most therapeutic antibodies in clinical development have
been focused on B-ALL. One such candidate is CD20, as its
expression is observed in approximately 40 to 50% of B-ALL cases.
Rituximab, an anti-CD20 antibody, initially approved for treatment
of B cell lymphoma, has demonstrated a significant survival
advantage when added to standard chemotherapy in some ALL clinical
trials, particularly the Burkitt's subtype. Although effective in
adult CD20+ B-ALL, there is a paucity of clinical data on the
efficacy of rituximab in pediatric ALL. In contrast to CD20, CD22
is expressed in a larger percentage of B-ALL cases and is present
on greater than 90% of B-ALL patients. Epratuzumab, a humanized
monoclonal anti-CD22 antibody, is currently being investigated in
clinical trials. Although early clinical studies with epratuzumab
as a single agent in relapsed ALL showed limited effect, anti-CD22
antibody-immunotoxin conjugates may improve the efficacy of
epratuzumab, since CD22 is reported to be rapidly internalized upon
antibody binding. Several immunoconjugates directed against CD22
are currently being explored in Phase I trials. In addition,
antibodies and immunotoxins to other antigens including CD19 are
currently being explored.
[0326] Although several therapeutic antibodies are in clinical
development for B-ALL, there are relatively few antibody therapies
for treatment of T-ALL. The most prominent antibody for T-ALL,
alemtuzumab, is targeted at CD52, as it is expressed on greater
than 95% of normal lymphocytes and at higher levels in T compared
to B lymphoblasts. Although pre-clinical data suggest potential
efficacy of alemtuzumab, early phase clinical trials do not report
a significant benefit as a single agent or in combination with
chemotherapy for the treatment of relapsed T-ALL.
[0327] In contrast to the targeted therapies developed for B-ALL
and T-ALL, our data strongly suggest that an anti-CD47 antibody can
be effective in eliminating both B- and T-ALL and thus could
increase the number of therapeutic options for both. Because
anti-CD47 antibody treatment may eliminate ALL blasts with limited
toxicity and is equally effective in targeting low, standard, and
high-risk ALL, these results provide a strong rationale for
development of an anti-CD47 antibody for the treatment of ALL
patients.
Example 7
Expression of CD47 on Solid Tumor Cells and Manipulation of
Phagocytosis of the Same
[0328] Several anti-human CD47 monoclonal antibodies have been
generated, including some capable of blocking the CD47-SIRP.alpha.
interaction (B6H12 and Bric126) and others unable to do so (2D3).
We tested the ability of these antibodies to enable phagocytosis of
ovarian, bladder, and colon cancer cells by macrophages in vitro,
and to alter the survival of animals engrafted with these cancer
cells in vivo. In contrast to cells treated with an IgG1 isotype
control or non-blocking anti-CD47 antibody (2D3), tumor cells
treated with blocking anti-CD47 antibodies B6H12 or Bric126 were
efficiently phagocytosed by mouse and human macrophages. Colon
cancer stem cells (Linneg, EpCAM+, CD44+, CD166+) were isolated by
Fluorescence Activated Cell Sorting (FACS) from patient tumor
samples.
[0329] The cell samples tested were as follows:
[0330] Ovarian. Patient ovarian cancer (OC) cells were engrafted
into the peritoneal cavity of NSG mice. Prior transduction of these
cells with a lentivirus designed to express GFP and luciferase
enabled the use of bioluminescent imaging to monitor tumor growth.
After confirming engraftment of the OC cells, mice were treated
daily with an intraperitoneal injection of 400 .mu.g anti-CD47
(clone Bric126) or control mouse IgG. Tumor growth was then
evaluated biweekly with bioluminescent imaging.
[0331] The fold change in total flux (photons/sec) is shown for
each mouse after each individual mouse was normalized to its
respective pretreatment value. IgG treated mice (n=9) are
represented by red circles. Anti-CD47 treated mice (n=10) are
represented by blue triangles. The horizontal line represents the
mean bioluminescent signal (plus standard error) of each treatment
group. The fold difference between the two treatment groups is
shown at each time point.
[0332] Pancreatic. Pancreatic cancer (PANC1) cells were transduced
with a lentivirus designed to express GFP and Luciferase.
Successfully transduced (GFP+) cells were isolated by FACS. 500,000
transduced PANC1 cells were directly injected into the pancreas of
NSG mice. After seven days, engraftment of PANC1 cells was
quantified by bioluminescent imaging. Mice were then treated daily
with 500 .mu.g control IgG (n=5) or anti-CD47, clone B6H12 (n=5).
Tumor growth was monitored and quantified weekly by bioluminescent
imaging. Each symbol represents an individual mouse.
[0333] Breast. 10.sup.6 cells from a patient breast cancer
xenograft were engrafted into the mammary fat pad of NSG mice.
Daily intraperitoneal injections of either 400 .mu.g control IgG or
anti-CD47 (B6H12) were initiated 2 weeks after cells were injected.
Antibody treatment was stopped after 8 weeks. Tumor formation
occurred in all control IgG treated mice. Importantly, the
anti-CD47 treated mice were evaluated 3 months after stopping
antibody treatment, and still no tumor formation was detected in
any of the mice, indicating that the anti-CD47 antibody
successfully targeted and eliminated the breast CSCs.
Representative images of the mammary fat pads of 3 mice from each
treatment group
[0334] Colon. Patient colon cancer cells were engrafted
subcutaneously on the back of NSG mice. Prior transduction of all
injected cells with a lentivirus designed to express GFP and
luciferase enabled the use of bioluminescent imaging to monitor
tumor growth. After confirming engraftment of the colon cancer
cells, mice were treated daily with an intraperitoneal injection of
500 .mu.g anti-CD47 (clone B6H12), anti CD44 (Hermes-3), control
mouse IgG, or a combination of anti-CD44 and anti-CD47 antibodies.
Tumor growth was then evaluated weekly with bioluminescent
imaging.
[0335] Bladder Metastasis. Tumor cells from a patient bladder
cancer sample which reliably forms metastases to they lymph nodes
and lungs were injected subcutaneously onto the back of NSG mice.
Treatment with anti-CD47 (400 .mu.g/day), anti-CD44 (150 .mu.g,
MWF), or Herceptin (200 .mu.g/week) antibodies was initiated upon
detection of a palpable tumor mass. The number of metastases was
determined by gross examination of excised lymph nodes at the
conclusion of the experiment. The number of micrometastases was
determined by a trained pathologist on sections cut from lungs
preserved in 10% buffered formalin phosphate.
[0336] Expression of CD47 on various cancer cells is shown in Table
3:
TABLE-US-00003 Flow Cytometry Of Dissociated Immunoflouresence
Cancer Tissue Staining On Frozen Percent CD47 Positive Tumor Tissue
CD47 Expression Tumor Type Cells On Cancer Cells Ovarian 58-95
Positive Breast 89 Not Determined Colon 73-97 Not Determined
Bladder 84-98 Positive Head & Neck 19-86 Not Determined Lung
Not Determined Positive Melanoma 98 Positive Glioblastoma 20-97 Not
Determined CD47 is expressed on human tumor cells. CD47 was
evaluated by flow cytometry (middle column) on dissociated patient
primary or xenograft tumors from various tissues. "Tumor cells" are
defined as live, lineage negative cells, where lineage represents
human CD45 negative CD31 negative (primary samples) or mouse CD45
negative, H-2K.sup.d/b negative (xenograft samples) cells.
Immunoflouresence staining (right column) was performed on sections
cut from a subset of primary and xenograft tumor samples preserved
in OCT immediately upon collection. Where indicated, CD47
expression was observed on bulk cancer cells.
[0337] As shown in FIG. 30, antibodies targeted to human CD47
enable the phagocytosis of OC cells. A: CFSE-labeled primary human
bladder cancer cells were incubated with human macrophages in the
presence of the indicated antibodies and assessed for the presence
of tumor cells within macrophages. B-C: Phagocytosis of patient
ovarian cancer cells (B) or colon cancer stem cells (C) resulting
from indicated antibody treatment was quantified. Each dot color
represents a different primary tumor sample. The phagocytic index
was determined as the number of OC cells present within 100
macrophages.
[0338] As shown in FIG. 31, antibodies targeted to CD47 inhibit the
growth of patient tumors. A-D: Tumor cells from ovarian (A),
pancreatic (B), breast (C), or colon (D) tumors were engrafted into
immunodeficient mice. These mice were then treated with control IgG
or anti-CD47 antibodies and tumor growth was assessed directly or
by bioluminescence. In all cases, anti-CD47 antibody treatment
substantially inhibited tumor growth. E-F: Tumor cells from patient
bladder were injected subcutaneously into immunodeficient mice and
treated with the indicated antibodies. Anti-CD47 antibody treatment
significantly inhibited metastasis to the lymph nodes (E) and
formation of micrometastases in the lungs (F). The total number of
metastases detected in each treatment group is indicated.
Example 8
Expression of CD47 in Brain Tumor
[0339] Acquisition of Brain Tumor Samples: Freshly resected brain
tumor samples were obtained from the department of Neurosurgery
under IRB approved protocols. Samples are minced using a sterile
scalpel and washed in HBSS to remove debris. Minced tissue is then
incubated in collagenase IV (img/ml) for 60-90 minutes at
37.degree. C. with constant agitation. Dissociated cells are then
passed sequentially through a 100, 70 and 40 .mu.m cell strainer
and washed in HBSS. Dead cells are removed by density
centrifugation in 0.9M sucrose and then treated with ACK-RBC lysis
buffer (invitrogen) to remove red blood cells. Cells are then
collected by centrifugation and resuspended in FACS buffer.
[0340] FACS Staining: Single cell suspension were stained with
CD133/1-APC and CD133/2-APC (Miltenyi) and CD47-PE (BD biosciences)
and analyzed on ARIA-II (BD Biosciences).
Results:
[0341] In 10 gliomas analyzed, 10/10 tumors were CD47.sup.+ with
varying degree of CD47 expression. An average of 66% of the cells
were CD47.sup.+, where as only 4/10 tumors were CD133.sup.+. In the
CD133.sup.+ tumors all CD133.sup.+ cells also expressed CD47.
[0342] This data suggests that anti-CD47 therapy can be a viable
avenue of investigation in human glioblastoma. This also suggests
that at least in CD133.sup.+ tumors targeting CD47 would also
target the cancer stem cell population.
TABLE-US-00004 Tumor % of the viable, CD45-cells: Date CD133+ CD47+
CD47+133+ (%) Nov. 15, 2006 45% 95% 53% Jun. 29, 2007 0% 97.1% 0
Oct. 1, 2007 0% 46% 0 Feb. 12, 2008 0% 20% 0 Mar. 27, 2008 0% 31% 0
Apr. 10, 2008 7.6% 68% 8.5% Apr. 28, 2008 9.1% 74% 8.8% Jun. 2,
2008 4.5% 92% 8.5% Jul. 15, 2008 0.0% 37% 0.0% Oct. 9, 2008 0.0%
95% 0.0%
[0343] An analysis of survival vs. gene expression data for cd47
and CD133/Prom1 shows that patients with glioblastoma whose tumors
express low CD47 have better survival (p=0.0239).
Sequence CWU 1
1
181142PRTHomo sapiens 1Met Trp Pro Leu Val Ala Ala Leu Leu Leu Gly
Ser Ala Cys Cys Gly1 5 10 15 Ser Ala Gln Leu Leu Phe Asn Lys Thr
Lys Ser Val Glu Phe Thr Phe 20 25 30 Cys Asn Asp Thr Val Val Ile
Pro Cys Phe Val Thr Asn Met Glu Ala 35 40 45 Gln Asn Thr Thr Glu
Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp 50 55 60 Ile Tyr Thr
Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp65 70 75 80 Phe
Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala 85 90
95 Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr
100 105 110 Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile
Ile Glu 115 120 125 Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Asn
Glu Asn 130 135 140 25346DNAHomo sapiens 2ggggagcagg cgggggagcg
ggcgggaagc agtgggagcg cgcgtgcgcg cggccgtgca 60gcctgggcag tgggtcctgc
ctgtgacgcg cggcggcggt cggtcctgcc tgtaacggcg 120gcggcggctg
ctgctccaga cacctgcggc ggcggcggcg accccgcggc gggcgcggag
180atgtggcccc tggtagcggc gctgttgctg ggctcggcgt gctgcggatc
agctcagcta 240ctatttaata aaacaaaatc tgtagaattc acgttttgta
atgacactgt cgtcattcca 300tgctttgtta ctaatatgga ggcacaaaac
actactgaag tatacgtaaa gtggaaattt 360aaaggaagag atatttacac
ctttgatgga gctctaaaca agtccactgt ccccactgac 420tttagtagtg
caaaaattga agtctcacaa ttactaaaag gagatgcctc tttgaagatg
480gataagagtg atgctgtctc acacacagga aactacactt gtgaagtaac
agaattaacc 540agagaaggtg aaacgatcat cgagctaaaa tatcgtgttg
tttcatggtt ttctccaaat 600gaaaatattc ttattgttat tttcccaatt
tttgctatac tcctgttctg gggacagttt 660ggtattaaaa cacttaaata
tagatccggt ggtatggatg agaaaacaat tgctttactt 720gttgctggac
tagtgatcac tgtcattgtc attgttggag ccattctttt cgtcccaggt
780gaatattcat taaagaatgc tactggcctt ggtttaattg tgacttctac
agggatatta 840atattacttc actactatgt gtttagtaca gcgattggat
taacctcctt cgtcattgcc 900atattggtta ttcaggtgat agcctatatc
ctcgctgtgg ttggactgag tctctgtatt 960gcggcgtgta taccaatgca
tggccctctt ctgatttcag gtttgagtat cttagctcta 1020gcacaattac
ttggactagt ttatatgaaa tttgtggctt ccaatcagaa gactatacaa
1080cctcctagga aagctgtaga ggaacccctt aatgcattca aagaatcaaa
aggaatgatg 1140aatgatgaat aactgaagtg aagtgatgga ctccgatttg
gagagtagta agacgtgaaa 1200ggaatacact tgtgtttaag caccatggcc
ttgatgattc actgttgggg agaagaaaca 1260agaaaagtaa ctggttgtca
cctatgagac ccttacgtga ttgttagtta agtttttatt 1320caaagcagct
gtaatttagt taataaaata attatgatct atgttgtttg cccaattgag
1380atccagtttt ttgttgttat ttttaatcaa ttaggggcaa tagtagaatg
gacaatttcc 1440aagaatgatg cctttcaggt cctagggcct ctggcctcta
ggtaaccagt ttaaattggt 1500tcagggtgat aactacttag cactgccctg
gtgattaccc agagatatct atgaaaacca 1560gtggcttcca tcaaaccttt
gccaactcag gttcacagca gctttgggca gttatggcag 1620tatggcatta
gctgagaggt gtctgccact tctgggtcaa tggaataata aattaagtac
1680aggcaggaat ttggttggga gcatcttgta tgatctccgt atgatgtgat
attgatggag 1740atagtggtcc tcattcttgg gggttgccat tcccacattc
ccccttcaac aaacagtgta 1800acaggtcctt cccagattta gggtactttt
attgatggat atgttttcct tttattcaca 1860taaccccttg aaaccctgtc
ttgtcctcct gttacttgct tctgctgtac aagatgtagc 1920accttttctc
ctctttgaac atggtctagt gacacggtag caccagttgc aggaaggagc
1980cagacttgtt ctcagagcac tgtgttcaca cttttcagca aaaatagcta
tggttgtaac 2040atatgtattc ccttcctctg atttgaaggc aaaaatctac
agtgtttctt cacttctttt 2100ctgatctggg gcatgaaaaa agcaagattg
aaatttgaac tatgagtctc ctgcatggca 2160acaaaatgtg tgtcaccatc
aggccaacag gccagccctt gaatggggat ttattactgt 2220tgtatctatg
ttgcatgata aacattcatc accttcctcc tgtagtcctg cctcgtactc
2280cccttcccct atgattgaaa agtaaacaaa acccacattt cctatcctgg
ttagaagaaa 2340attaatgttc tgacagttgt gatcgcctgg agtactttta
gacttttagc attcgttttt 2400tacctgtttg tggatgtgtg tttgtatgtg
catacgtatg agataggcac atgcatcttc 2460tgtatggaca aaggtggggt
acctacagga gagcaaaggt taattttgtg cttttagtaa 2520aaacatttaa
atacaaagtt ctttattggg tggaattata tttgatgcaa atatttgatc
2580acttaaaact tttaaaactt ctaggtaatt tgccacgctt tttgactgct
caccaatacc 2640ctgtaaaaat acgtaattct tcctgtttgt gtaataagat
attcatattt gtagttgcat 2700taataatagt tatttcttag tccatcagat
gttcccgtgt gcctctttta tgccaaattg 2760attgtcatat ttcatgttgg
gaccaagtag tttgcccatg gcaaacctaa atttatgacc 2820tgctgaggcc
tctcagaaaa ctgagcatac tagcaagaca gctcttcttg aaaaaaaaaa
2880tatgtataca caaatatata cgtatatcta tatatacgta tgtatataca
cacatgtata 2940ttcttccttg attgtgtagc tgtccaaaat aataacatat
atagagggag ctgtattcct 3000ttatacaaat ctgatggctc ctgcagcact
ttttccttct gaaaatattt acattttgct 3060aacctagttt gttactttaa
aaatcagttt tgatgaaagg agggaaaagc agatggactt 3120gaaaaagatc
caagctccta ttagaaaagg tatgaaaatc tttatagtaa aattttttat
3180aaactaaagt tgtacctttt aatatgtagt aaactctcat ttatttgggg
ttcgctcttg 3240gatctcatcc atccattgtg ttctctttaa tgctgcctgc
cttttgaggc attcactgcc 3300ctagacaatg ccaccagaga tagtggggga
aatgccagat gaaaccaact cttgctctca 3360ctagttgtca gcttctctgg
ataagtgacc acagaagcag gagtcctcct gcttgggcat 3420cattgggcca
gttccttctc tttaaatcag atttgtaatg gctcccaaat tccatcacat
3480cacatttaaa ttgcagacag tgttttgcac atcatgtatc tgttttgtcc
cataatatgc 3540tttttactcc ctgatcccag tttctgctgt tgactcttcc
attcagtttt atttattgtg 3600tgttctcaca gtgacaccat ttgtcctttt
ctgcaacaac ctttccagct acttttgcca 3660aattctattt gtcttctcct
tcaaaacatt ctcctttgca gttcctcttc atctgtgtag 3720ctgctctttt
gtctcttaac ttaccattcc tatagtactt tatgcatctc tgcttagttc
3780tattagtttt ttggccttgc tcttctcctt gattttaaaa ttccttctat
agctagagct 3840tttctttctt tcattctctc ttcctgcagt gttttgcata
catcagaagc taggtacata 3900agttaaatga ttgagagttg gctgtattta
gatttatcac tttttaatag ggtgagcttg 3960agagttttct ttctttctgt
tttttttttt tgtttttttt tttttttttt tttttttttt 4020ttttgactaa
tttcacatgc tctaaaaacc ttcaaaggtg attatttttc tcctggaaac
4080tccaggtcca ttctgtttaa atccctaaga atgtcagaat taaaataaca
gggctatccc 4140gtaattggaa atatttcttt tttcaggatg ctatagtcaa
tttagtaagt gaccaccaaa 4200ttgttatttg cactaacaaa gctcaaaaca
cgataagttt actcctccat ctcagtaata 4260aaaattaagc tgtaatcaac
cttctaggtt tctcttgtct taaaatgggt attcaaaaat 4320ggggatctgt
ggtgtatgta tggaaacaca tactccttaa tttacctgtt gttggaaact
4380ggagaaatga ttgtcgggca accgtttatt ttttattgta ttttatttgg
ttgagggatt 4440tttttataaa cagttttact tgtgtcatat tttaaaatta
ctaactgcca tcacctgctg 4500gggtcctttg ttaggtcatt ttcagtgact
aatagggata atccaggtaa ctttgaagag 4560atgagcagtg agtgaccagg
cagtttttct gcctttagct ttgacagttc ttaattaaga 4620tcattgaaga
ccagctttct cataaatttc tctttttgaa aaaaagaaag catttgtact
4680aagctcctct gtaagacaac atcttaaatc ttaaaagtgt tgttatcatg
actggtgaga 4740gaagaaaaca ttttgttttt attaaatgga gcattattta
caaaaagcca ttgttgagaa 4800ttagatccca catcgtataa atatctatta
accattctaa ataaagagaa ctccagtgtt 4860gctatgtgca agatcctctc
ttggagcttt tttgcatagc aattaaaggt gtgctatttg 4920tcagtagcca
tttttttgca gtgatttgaa gaccaaagtt gttttacagc tgtgttaccg
4980ttaaaggttt ttttttttat atgtattaaa tcaatttatc actgtttaaa
gctttgaata 5040tctgcaatct ttgccaaggt acttttttat ttaaaaaaaa
acataacttt gtaaatatta 5100ccctgtaata ttatatatac ttaataaaac
attttaagct attttgttgg gctatttcta 5160ttgctgctac agcagaccac
aagcacattt ctgaaaaatt taatttatta atgtattttt 5220aagttgctta
tattctaggt aacaatgtaa agaatgattt aaaatattaa ttatgaattt
5280tttgagtata atacccaata agcttttaat tagagcagag ttttaattaa
aagttttaaa 5340tcagtc 534635288DNAHomo sapiens 3ggggagcagg
cgggggagcg ggcgggaagc agtgggagcg cgcgtgcgcg cggccgtgca 60gcctgggcag
tgggtcctgc ctgtgacgcg cggcggcggt cggtcctgcc tgtaacggcg
120gcggcggctg ctgctccaga cacctgcggc ggcggcggcg accccgcggc
gggcgcggag 180atgtggcccc tggtagcggc gctgttgctg ggctcggcgt
gctgcggatc agctcagcta 240ctatttaata aaacaaaatc tgtagaattc
acgttttgta atgacactgt cgtcattcca 300tgctttgtta ctaatatgga
ggcacaaaac actactgaag tatacgtaaa gtggaaattt 360aaaggaagag
atatttacac ctttgatgga gctctaaaca agtccactgt ccccactgac
420tttagtagtg caaaaattga agtctcacaa ttactaaaag gagatgcctc
tttgaagatg 480gataagagtg atgctgtctc acacacagga aactacactt
gtgaagtaac agaattaacc 540agagaaggtg aaacgatcat cgagctaaaa
tatcgtgttg tttcatggtt ttctccaaat 600gaaaatattc ttattgttat
tttcccaatt tttgctatac tcctgttctg gggacagttt 660ggtattaaaa
cacttaaata tagatccggt ggtatggatg agaaaacaat tgctttactt
720gttgctggac tagtgatcac tgtcattgtc attgttggag ccattctttt
cgtcccaggt 780gaatattcat taaagaatgc tactggcctt ggtttaattg
tgacttctac agggatatta 840atattacttc actactatgt gtttagtaca
gcgattggat taacctcctt cgtcattgcc 900atattggtta ttcaggtgat
agcctatatc ctcgctgtgg ttggactgag tctctgtatt 960gcggcgtgta
taccaatgca tggccctctt ctgatttcag gtttgagtat cttagctcta
1020gcacaattac ttggactagt ttatatgaaa tttgtggctt ccaatcagaa
gactatacaa 1080cctcctagga ataactgaag tgaagtgatg gactccgatt
tggagagtag taagacgtga 1140aaggaataca cttgtgttta agcaccatgg
ccttgatgat tcactgttgg ggagaagaaa 1200caagaaaagt aactggttgt
cacctatgag acccttacgt gattgttagt taagttttta 1260ttcaaagcag
ctgtaattta gttaataaaa taattatgat ctatgttgtt tgcccaattg
1320agatccagtt ttttgttgtt atttttaatc aattaggggc aatagtagaa
tggacaattt 1380ccaagaatga tgcctttcag gtcctagggc ctctggcctc
taggtaacca gtttaaattg 1440gttcagggtg ataactactt agcactgccc
tggtgattac ccagagatat ctatgaaaac 1500cagtggcttc catcaaacct
ttgccaactc aggttcacag cagctttggg cagttatggc 1560agtatggcat
tagctgagag gtgtctgcca cttctgggtc aatggaataa taaattaagt
1620acaggcagga atttggttgg gagcatcttg tatgatctcc gtatgatgtg
atattgatgg 1680agatagtggt cctcattctt gggggttgcc attcccacat
tcccccttca acaaacagtg 1740taacaggtcc ttcccagatt tagggtactt
ttattgatgg atatgttttc cttttattca 1800cataacccct tgaaaccctg
tcttgtcctc ctgttacttg cttctgctgt acaagatgta 1860gcaccttttc
tcctctttga acatggtcta gtgacacggt agcaccagtt gcaggaagga
1920gccagacttg ttctcagagc actgtgttca cacttttcag caaaaatagc
tatggttgta 1980acatatgtat tcccttcctc tgatttgaag gcaaaaatct
acagtgtttc ttcacttctt 2040ttctgatctg gggcatgaaa aaagcaagat
tgaaatttga actatgagtc tcctgcatgg 2100caacaaaatg tgtgtcacca
tcaggccaac aggccagccc ttgaatgggg atttattact 2160gttgtatcta
tgttgcatga taaacattca tcaccttcct cctgtagtcc tgcctcgtac
2220tccccttccc ctatgattga aaagtaaaca aaacccacat ttcctatcct
ggttagaaga 2280aaattaatgt tctgacagtt gtgatcgcct ggagtacttt
tagactttta gcattcgttt 2340tttacctgtt tgtggatgtg tgtttgtatg
tgcatacgta tgagataggc acatgcatct 2400tctgtatgga caaaggtggg
gtacctacag gagagcaaag gttaattttg tgcttttagt 2460aaaaacattt
aaatacaaag ttctttattg ggtggaatta tatttgatgc aaatatttga
2520tcacttaaaa cttttaaaac ttctaggtaa tttgccacgc tttttgactg
ctcaccaata 2580ccctgtaaaa atacgtaatt cttcctgttt gtgtaataag
atattcatat ttgtagttgc 2640attaataata gttatttctt agtccatcag
atgttcccgt gtgcctcttt tatgccaaat 2700tgattgtcat atttcatgtt
gggaccaagt agtttgccca tggcaaacct aaatttatga 2760cctgctgagg
cctctcagaa aactgagcat actagcaaga cagctcttct tgaaaaaaaa
2820aatatgtata cacaaatata tacgtatatc tatatatacg tatgtatata
cacacatgta 2880tattcttcct tgattgtgta gctgtccaaa ataataacat
atatagaggg agctgtattc 2940ctttatacaa atctgatggc tcctgcagca
ctttttcctt ctgaaaatat ttacattttg 3000ctaacctagt ttgttacttt
aaaaatcagt tttgatgaaa ggagggaaaa gcagatggac 3060ttgaaaaaga
tccaagctcc tattagaaaa ggtatgaaaa tctttatagt aaaatttttt
3120ataaactaaa gttgtacctt ttaatatgta gtaaactctc atttatttgg
ggttcgctct 3180tggatctcat ccatccattg tgttctcttt aatgctgcct
gccttttgag gcattcactg 3240ccctagacaa tgccaccaga gatagtgggg
gaaatgccag atgaaaccaa ctcttgctct 3300cactagttgt cagcttctct
ggataagtga ccacagaagc aggagtcctc ctgcttgggc 3360atcattgggc
cagttccttc tctttaaatc agatttgtaa tggctcccaa attccatcac
3420atcacattta aattgcagac agtgttttgc acatcatgta tctgttttgt
cccataatat 3480gctttttact ccctgatccc agtttctgct gttgactctt
ccattcagtt ttatttattg 3540tgtgttctca cagtgacacc atttgtcctt
ttctgcaaca acctttccag ctacttttgc 3600caaattctat ttgtcttctc
cttcaaaaca ttctcctttg cagttcctct tcatctgtgt 3660agctgctctt
ttgtctctta acttaccatt cctatagtac tttatgcatc tctgcttagt
3720tctattagtt ttttggcctt gctcttctcc ttgattttaa aattccttct
atagctagag 3780cttttctttc tttcattctc tcttcctgca gtgttttgca
tacatcagaa gctaggtaca 3840taagttaaat gattgagagt tggctgtatt
tagatttatc actttttaat agggtgagct 3900tgagagtttt ctttctttct
gttttttttt tttgtttttt tttttttttt tttttttttt 3960ttttttgact
aatttcacat gctctaaaaa ccttcaaagg tgattatttt tctcctggaa
4020actccaggtc cattctgttt aaatccctaa gaatgtcaga attaaaataa
cagggctatc 4080ccgtaattgg aaatatttct tttttcagga tgctatagtc
aatttagtaa gtgaccacca 4140aattgttatt tgcactaaca aagctcaaaa
cacgataagt ttactcctcc atctcagtaa 4200taaaaattaa gctgtaatca
accttctagg tttctcttgt cttaaaatgg gtattcaaaa 4260atggggatct
gtggtgtatg tatggaaaca catactcctt aatttacctg ttgttggaaa
4320ctggagaaat gattgtcggg caaccgttta ttttttattg tattttattt
ggttgaggga 4380tttttttata aacagtttta cttgtgtcat attttaaaat
tactaactgc catcacctgc 4440tggggtcctt tgttaggtca ttttcagtga
ctaataggga taatccaggt aactttgaag 4500agatgagcag tgagtgacca
ggcagttttt ctgcctttag ctttgacagt tcttaattaa 4560gatcattgaa
gaccagcttt ctcataaatt tctctttttg aaaaaaagaa agcatttgta
4620ctaagctcct ctgtaagaca acatcttaaa tcttaaaagt gttgttatca
tgactggtga 4680gagaagaaaa cattttgttt ttattaaatg gagcattatt
tacaaaaagc cattgttgag 4740aattagatcc cacatcgtat aaatatctat
taaccattct aaataaagag aactccagtg 4800ttgctatgtg caagatcctc
tcttggagct tttttgcata gcaattaaag gtgtgctatt 4860tgtcagtagc
catttttttg cagtgatttg aagaccaaag ttgttttaca gctgtgttac
4920cgttaaaggt tttttttttt atatgtatta aatcaattta tcactgttta
aagctttgaa 4980tatctgcaat ctttgccaag gtactttttt atttaaaaaa
aaacataact ttgtaaatat 5040taccctgtaa tattatatat acttaataaa
acattttaag ctattttgtt gggctatttc 5100tattgctgct acagcagacc
acaagcacat ttctgaaaaa tttaatttat taatgtattt 5160ttaagttgct
tatattctag gtaacaatgt aaagaatgat ttaaaatatt aattatgaat
5220tttttgagta taatacccaa taagctttta attagagcag agttttaatt
aaaagtttta 5280aatcagtc 528845313DNAHomo sapiens 4ggggagcagg
cgggggagcg ggcgggaagc agtgggagcg cgcgtgcgcg cggccgtgca 60gcctgggcag
tgggtcctgc ctgtgacgcg cggcggcggt cggtcctgcc tgtaacggcg
120gcggcggctg ctgctccaga cacctgcggc ggcggcggcg accccgcggc
gggcgcggag 180atgtggcccc tggtagcggc gctgttgctg ggctcggcgt
gctgcggatc agctcagcta 240ctatttaata aaacaaaatc tgtagaattc
acgttttgta atgacactgt cgtcattcca 300tgctttgtta ctaatatgga
ggcacaaaac actactgaag tatacgtaaa gtggaaattt 360aaaggaagag
atatttacac ctttgatgga gctctaaaca agtccactgt ccccactgac
420tttagtagtg caaaaattga agtctcacaa ttactaaaag gagatgcctc
tttgaagatg 480gataagagtg atgctgtctc acacacagga aactacactt
gtgaagtaac agaattaacc 540agagaaggtg aaacgatcat cgagctaaaa
tatcgtgttg tttcatggtt ttctccaaat 600gaaaatattc ttattgttat
tttcccaatt tttgctatac tcctgttctg gggacagttt 660ggtattaaaa
cacttaaata tagatccggt ggtatggatg agaaaacaat tgctttactt
720gttgctggac tagtgatcac tgtcattgtc attgttggag ccattctttt
cgtcccaggt 780gaatattcat taaagaatgc tactggcctt ggtttaattg
tgacttctac agggatatta 840atattacttc actactatgt gtttagtaca
gcgattggat taacctcctt cgtcattgcc 900atattggtta ttcaggtgat
agcctatatc ctcgctgtgg ttggactgag tctctgtatt 960gcggcgtgta
taccaatgca tggccctctt ctgatttcag gtttgagtat cttagctcta
1020gcacaattac ttggactagt ttatatgaaa tttgtggctt ccaatcagaa
gactatacaa 1080cctcctagga aagctgtaga ggaacccctt aatgaataac
tgaagtgaag tgatggactc 1140cgatttggag agtagtaaga cgtgaaagga
atacacttgt gtttaagcac catggccttg 1200atgattcact gttggggaga
agaaacaaga aaagtaactg gttgtcacct atgagaccct 1260tacgtgattg
ttagttaagt ttttattcaa agcagctgta atttagttaa taaaataatt
1320atgatctatg ttgtttgccc aattgagatc cagttttttg ttgttatttt
taatcaatta 1380ggggcaatag tagaatggac aatttccaag aatgatgcct
ttcaggtcct agggcctctg 1440gcctctaggt aaccagttta aattggttca
gggtgataac tacttagcac tgccctggtg 1500attacccaga gatatctatg
aaaaccagtg gcttccatca aacctttgcc aactcaggtt 1560cacagcagct
ttgggcagtt atggcagtat ggcattagct gagaggtgtc tgccacttct
1620gggtcaatgg aataataaat taagtacagg caggaatttg gttgggagca
tcttgtatga 1680tctccgtatg atgtgatatt gatggagata gtggtcctca
ttcttggggg ttgccattcc 1740cacattcccc cttcaacaaa cagtgtaaca
ggtccttccc agatttaggg tacttttatt 1800gatggatatg ttttcctttt
attcacataa ccccttgaaa ccctgtcttg tcctcctgtt 1860acttgcttct
gctgtacaag atgtagcacc ttttctcctc tttgaacatg gtctagtgac
1920acggtagcac cagttgcagg aaggagccag acttgttctc agagcactgt
gttcacactt 1980ttcagcaaaa atagctatgg ttgtaacata tgtattccct
tcctctgatt tgaaggcaaa 2040aatctacagt gtttcttcac ttcttttctg
atctggggca tgaaaaaagc aagattgaaa 2100tttgaactat gagtctcctg
catggcaaca aaatgtgtgt caccatcagg ccaacaggcc 2160agcccttgaa
tggggattta ttactgttgt atctatgttg catgataaac attcatcacc
2220ttcctcctgt agtcctgcct cgtactcccc ttcccctatg attgaaaagt
aaacaaaacc 2280cacatttcct atcctggtta gaagaaaatt aatgttctga
cagttgtgat cgcctggagt 2340acttttagac ttttagcatt cgttttttac
ctgtttgtgg atgtgtgttt gtatgtgcat 2400acgtatgaga taggcacatg
catcttctgt atggacaaag gtggggtacc tacaggagag 2460caaaggttaa
ttttgtgctt ttagtaaaaa catttaaata caaagttctt tattgggtgg
2520aattatattt gatgcaaata tttgatcact taaaactttt aaaacttcta
ggtaatttgc 2580cacgcttttt gactgctcac caataccctg taaaaatacg
taattcttcc tgtttgtgta 2640ataagatatt catatttgta gttgcattaa
taatagttat ttcttagtcc atcagatgtt 2700cccgtgtgcc tcttttatgc
caaattgatt gtcatatttc atgttgggac caagtagttt 2760gcccatggca
aacctaaatt tatgacctgc tgaggcctct cagaaaactg agcatactag
2820caagacagct cttcttgaaa aaaaaaatat gtatacacaa atatatacgt
atatctatat 2880atacgtatgt atatacacac atgtatattc ttccttgatt
gtgtagctgt ccaaaataat 2940aacatatata gagggagctg tattccttta
tacaaatctg atggctcctg cagcactttt 3000tccttctgaa aatatttaca
ttttgctaac ctagtttgtt actttaaaaa tcagttttga 3060tgaaaggagg
gaaaagcaga tggacttgaa aaagatccaa gctcctatta gaaaaggtat
3120gaaaatcttt atagtaaaat tttttataaa ctaaagttgt accttttaat
atgtagtaaa 3180ctctcattta tttggggttc gctcttggat ctcatccatc
cattgtgttc tctttaatgc 3240tgcctgcctt ttgaggcatt cactgcccta
gacaatgcca ccagagatag tgggggaaat 3300gccagatgaa accaactctt
gctctcacta gttgtcagct tctctggata agtgaccaca 3360gaagcaggag
tcctcctgct tgggcatcat tgggccagtt ccttctcttt aaatcagatt
3420tgtaatggct cccaaattcc atcacatcac atttaaattg cagacagtgt
tttgcacatc 3480atgtatctgt tttgtcccat aatatgcttt ttactccctg
atcccagttt ctgctgttga 3540ctcttccatt cagttttatt tattgtgtgt
tctcacagtg acaccatttg tccttttctg 3600caacaacctt tccagctact
tttgccaaat tctatttgtc ttctccttca aaacattctc 3660ctttgcagtt
cctcttcatc tgtgtagctg ctcttttgtc tcttaactta ccattcctat
3720agtactttat gcatctctgc ttagttctat tagttttttg gccttgctct
tctccttgat 3780tttaaaattc cttctatagc tagagctttt ctttctttca
ttctctcttc ctgcagtgtt 3840ttgcatacat cagaagctag gtacataagt
taaatgattg agagttggct gtatttagat 3900ttatcacttt ttaatagggt
gagcttgaga gttttctttc tttctgtttt ttttttttgt 3960tttttttttt
tttttttttt tttttttttt tgactaattt cacatgctct aaaaaccttc
4020aaaggtgatt atttttctcc tggaaactcc aggtccattc tgtttaaatc
cctaagaatg 4080tcagaattaa aataacaggg ctatcccgta attggaaata
tttctttttt caggatgcta 4140tagtcaattt agtaagtgac caccaaattg
ttatttgcac taacaaagct caaaacacga 4200taagtttact cctccatctc
agtaataaaa attaagctgt aatcaacctt ctaggtttct 4260cttgtcttaa
aatgggtatt caaaaatggg gatctgtggt gtatgtatgg aaacacatac
4320tccttaattt acctgttgtt ggaaactgga gaaatgattg tcgggcaacc
gtttattttt 4380tattgtattt tatttggttg agggattttt ttataaacag
ttttacttgt gtcatatttt 4440aaaattacta actgccatca cctgctgggg
tcctttgtta ggtcattttc agtgactaat 4500agggataatc caggtaactt
tgaagagatg agcagtgagt gaccaggcag tttttctgcc 4560tttagctttg
acagttctta attaagatca ttgaagacca gctttctcat aaatttctct
4620ttttgaaaaa aagaaagcat ttgtactaag ctcctctgta agacaacatc
ttaaatctta 4680aaagtgttgt tatcatgact ggtgagagaa gaaaacattt
tgtttttatt aaatggagca 4740ttatttacaa aaagccattg ttgagaatta
gatcccacat cgtataaata tctattaacc 4800attctaaata aagagaactc
cagtgttgct atgtgcaaga tcctctcttg gagctttttt 4860gcatagcaat
taaaggtgtg ctatttgtca gtagccattt ttttgcagtg atttgaagac
4920caaagttgtt ttacagctgt gttaccgtta aaggtttttt tttttatatg
tattaaatca 4980atttatcact gtttaaagct ttgaatatct gcaatctttg
ccaaggtact tttttattta 5040aaaaaaaaca taactttgta aatattaccc
tgtaatatta tatatactta ataaaacatt 5100ttaagctatt ttgttgggct
atttctattg ctgctacagc agaccacaag cacatttctg 5160aaaaatttaa
tttattaatg tatttttaag ttgcttatat tctaggtaac aatgtaaaga
5220atgatttaaa atattaatta tgaatttttt gagtataata cccaataagc
ttttaattag 5280agcagagttt taattaaaag ttttaaatca gtc 53135323PRTHomo
sapiens 5Met Trp Pro Leu Val Ala Ala Leu Leu Leu Gly Ser Ala Cys
Cys Gly1 5 10 15 Ser Ala Gln Leu Leu Phe Asn Lys Thr Lys Ser Val
Glu Phe Thr Phe 20 25 30 Cys Asn Asp Thr Val Val Ile Pro Cys Phe
Val Thr Asn Met Glu Ala 35 40 45 Gln Asn Thr Thr Glu Val Tyr Val
Lys Trp Lys Phe Lys Gly Arg Asp 50 55 60 Ile Tyr Thr Phe Asp Gly
Ala Leu Asn Lys Ser Thr Val Pro Thr Asp65 70 75 80 Phe Ser Ser Ala
Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala 85 90 95 Ser Leu
Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr 100 105 110
Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu 115
120 125 Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Asn Glu Asn Ile
Leu 130 135 140 Ile Val Ile Phe Pro Ile Phe Ala Ile Leu Leu Phe Trp
Gly Gln Phe145 150 155 160 Gly Ile Lys Thr Leu Lys Tyr Arg Ser Gly
Gly Met Asp Glu Lys Thr 165 170 175 Ile Ala Leu Leu Val Ala Gly Leu
Val Ile Thr Val Ile Val Ile Val 180 185 190 Gly Ala Ile Leu Phe Val
Pro Gly Glu Tyr Ser Leu Lys Asn Ala Thr 195 200 205 Gly Leu Gly Leu
Ile Val Thr Ser Thr Gly Ile Leu Ile Leu Leu His 210 215 220 Tyr Tyr
Val Phe Ser Thr Ala Ile Gly Leu Thr Ser Phe Val Ile Ala225 230 235
240 Ile Leu Val Ile Gln Val Ile Ala Tyr Ile Leu Ala Val Val Gly Leu
245 250 255 Ser Leu Cys Ile Ala Ala Cys Ile Pro Met His Gly Pro Leu
Leu Ile 260 265 270 Ser Gly Leu Ser Ile Leu Ala Leu Ala Gln Leu Leu
Gly Leu Val Tyr 275 280 285 Met Lys Phe Val Ala Ser Asn Gln Lys Thr
Ile Gln Pro Pro Arg Lys 290 295 300 Ala Val Glu Glu Pro Leu Asn Ala
Phe Lys Glu Ser Lys Gly Met Met305 310 315 320 Asn Asp
Glu6305PRTHomo sapiens 6Met Trp Pro Leu Val Ala Ala Leu Leu Leu Gly
Ser Ala Cys Cys Gly1 5 10 15 Ser Ala Gln Leu Leu Phe Asn Lys Thr
Lys Ser Val Glu Phe Thr Phe 20 25 30 Cys Asn Asp Thr Val Val Ile
Pro Cys Phe Val Thr Asn Met Glu Ala 35 40 45 Gln Asn Thr Thr Glu
Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp 50 55 60 Ile Tyr Thr
Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp65 70 75 80 Phe
Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala 85 90
95 Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr
100 105 110 Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile
Ile Glu 115 120 125 Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Asn
Glu Asn Ile Leu 130 135 140 Ile Val Ile Phe Pro Ile Phe Ala Ile Leu
Leu Phe Trp Gly Gln Phe145 150 155 160 Gly Ile Lys Thr Leu Lys Tyr
Arg Ser Gly Gly Met Asp Glu Lys Thr 165 170 175 Ile Ala Leu Leu Val
Ala Gly Leu Val Ile Thr Val Ile Val Ile Val 180 185 190 Gly Ala Ile
Leu Phe Val Pro Gly Glu Tyr Ser Leu Lys Asn Ala Thr 195 200 205 Gly
Leu Gly Leu Ile Val Thr Ser Thr Gly Ile Leu Ile Leu Leu His 210 215
220 Tyr Tyr Val Phe Ser Thr Ala Ile Gly Leu Thr Ser Phe Val Ile
Ala225 230 235 240 Ile Leu Val Ile Gln Val Ile Ala Tyr Ile Leu Ala
Val Val Gly Leu 245 250 255 Ser Leu Cys Ile Ala Ala Cys Ile Pro Met
His Gly Pro Leu Leu Ile 260 265 270 Ser Gly Leu Ser Ile Leu Ala Leu
Ala Gln Leu Leu Gly Leu Val Tyr 275 280 285 Met Lys Phe Val Ala Ser
Asn Gln Lys Thr Ile Gln Pro Pro Arg Asn 290 295 300 Asn305
7312PRTHomo sapiens 7Met Trp Pro Leu Val Ala Ala Leu Leu Leu Gly
Ser Ala Cys Cys Gly1 5 10 15 Ser Ala Gln Leu Leu Phe Asn Lys Thr
Lys Ser Val Glu Phe Thr Phe 20 25 30 Cys Asn Asp Thr Val Val Ile
Pro Cys Phe Val Thr Asn Met Glu Ala 35 40 45 Gln Asn Thr Thr Glu
Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp 50 55 60 Ile Tyr Thr
Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp65 70 75 80 Phe
Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala 85 90
95 Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr
100 105 110 Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile
Ile Glu 115 120 125 Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Asn
Glu Asn Ile Leu 130 135 140 Ile Val Ile Phe Pro Ile Phe Ala Ile Leu
Leu Phe Trp Gly Gln Phe145 150 155 160 Gly Ile Lys Thr Leu Lys Tyr
Arg Ser Gly Gly Met Asp Glu Lys Thr 165 170 175 Ile Ala Leu Leu Val
Ala Gly Leu Val Ile Thr Val Ile Val Ile Val 180 185 190 Gly Ala Ile
Leu Phe Val Pro Gly Glu Tyr Ser Leu Lys Asn Ala Thr 195 200 205 Gly
Leu Gly Leu Ile Val Thr Ser Thr Gly Ile Leu Ile Leu Leu His 210 215
220 Tyr Tyr Val Phe Ser Thr Ala Ile Gly Leu Thr Ser Phe Val Ile
Ala225 230 235 240 Ile Leu Val Ile Gln Val Ile Ala Tyr Ile Leu Ala
Val Val Gly Leu 245 250 255 Ser Leu Cys Ile Ala Ala Cys Ile Pro Met
His Gly Pro Leu Leu Ile 260 265 270 Ser Gly Leu Ser Ile Leu Ala Leu
Ala Gln Leu Leu Gly Leu Val Tyr 275 280 285 Met Lys Phe Val Ala Ser
Asn Gln Lys Thr Ile Gln Pro Pro Arg Lys 290 295 300 Ala Val Glu Glu
Pro Leu Asn Glu305 310 821DNAArtificial Sequence18S RNA forward
primer 8ttgacggaag ggcaccacca g 21921DNAArtificial Sequence18S RNA
reverse primer 9gcaccaccac ccacggaatc g 211020DNAArtificial
Sequencebeta-actin forward primer 10ttccttcttg ggtatggaat
201120DNAArtificial Sequencebeta-actin reverse primer 11gagcaatgat
cttgatcctc 201221DNAArtificial SequenceCD47 forward primer
12aggccaagtc cagaagcatt c 211321DNAArtificial SequenceCD47 reverse
primer 13aatcattctg ctgctcgttg c 211434DNAArtificial Sequence3' Neo
PCR primer 14gcatcgcatt gtctgagtag gtgtcattct attc
341528DNAArtificial Sequence5' IAP PCR primer 15tcaccttgtt
gttcctgtac tacaagca 281623DNAArtificial Sequence3' IAP PCR Primer
16tgtcacttcg caagtgtagt tcc 231723DNAArtificial SequenceFLT3-ITD
PCR Forward Primer 11F 17gcaatttagg tatgaaagcc agc
231823DNAArtificial SequenceFLT3-ITD PCR Reverse Primer 12R
18ctttcagcat tttgacggca acc 23
* * * * *