U.S. patent application number 17/473459 was filed with the patent office on 2022-08-04 for compositions and methods for regulating erythropoiesis.
The applicant listed for this patent is University of Virginia Patent Foundation. Invention is credited to Thomas J. Braciale, Taeg S. Kim.
Application Number | 20220242964 17/473459 |
Document ID | / |
Family ID | 1000006274822 |
Filed Date | 2022-08-04 |
United States Patent
Application |
20220242964 |
Kind Code |
A1 |
Braciale; Thomas J. ; et
al. |
August 4, 2022 |
COMPOSITIONS AND METHODS FOR REGULATING ERYTHROPOIESIS
Abstract
The present application discloses a previously unknown function
of CD24 expressed on a subset of dendritic cells. The invention
encompasses regulating CD24 on these cells to regulate
erythropoiesis, induce EPO production and levels, increase RBC
levels, and to treat, for example, stress-mediated erythropoiesis.
The compositions and methods of the invention are useful, for
example, in treating anemia.
Inventors: |
Braciale; Thomas J.;
(Charlottesville, VA) ; Kim; Taeg S.;
(Charlottesville, VA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Virginia Patent Foundation |
Charlottesville |
VA |
US |
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|
Family ID: |
1000006274822 |
Appl. No.: |
17/473459 |
Filed: |
September 13, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16139428 |
Sep 24, 2018 |
11117976 |
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17473459 |
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14651708 |
Jun 12, 2015 |
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PCT/US2013/074245 |
Dec 11, 2013 |
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16139428 |
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61736246 |
Dec 12, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2896 20130101;
A61K 45/06 20130101; A61K 2039/505 20130101; A61K 38/00 20130101;
C07K 2317/75 20130101; A61K 39/3955 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 39/395 20060101 A61K039/395; A61K 45/06 20060101
A61K045/06 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under Grant
Nos. AI015608, AI083024, and HL033391, awarded by The National
Institutes of Health. The government has certain rights in the
invention.
Claims
1-37. (canceled)
38. A method of treating a disease, disorder, or condition
associated with a decrease in erythrocyte production, said method
comprising administering to a subject in need thereof a
pharmaceutical composition comprising a pharmaceutically-acceptable
carrier, an effective amount of at least one agonist of CD24, and
optionally at least one additional therapeutic agent, thereby
treating a disease, disorder, or condition associated with a
decrease in erythrocyte production.
39-42. (canceled)
43. The method of claim 38, wherein said agonist is a monoclonal
antibody selected from the group consisting of M1/69, 91, 30-F1,
J11d, eBioSN3, and ML5, or biologically active fragments or
homologs thereof.
44-55. (canceled)
56. The method of claim 38, wherein said subject has a disease,
disorder, or condition associated with a decrease in erythrocyte
production.
57. The method of claim 56, wherein said disease, disorder, or
condition is selected from the group consisting of aplastic anemia,
hypoplastic anemia, chronic renal failure, end-stage renal disease,
transplantation, renal transplantation, cancer, acquired immune
deficiency syndrome, chemotherapy, radiotherapy, bone marrow
transplantation, sepsis, rheumatoid arthritis, chronic persistent
infection such as HIV, tuberculosis, hepatitis B, hepatitis C,
chronic hepatitis, and chronic anemia in the elderly.
58. The method of claim 57, wherein said disease, disorder, or
condition is anemia.
59. The method of claim 58, wherein said anemia is associated with
hypoxic stress.
60-74. (canceled)
75. The method of claim 38, wherein said additional therapeutic
agent is selected from the group consisting of G-CSF, IL-4, SCF,
Flt3L, EPO, BMP4, anti-microbial agents, and host-derived danger
associated-pattern molecules.
76. The method of claim 75, wherein said host-derived danger
associated-pattern molecule is HMGB1 or Hsp70.
77. A method of stimulating stem cell factor synthesis, said method
comprising contacting a CD24.sup.+ dendritic cell with an effective
amount of an agonist of CD24 and optionally an additional
agent.
78. The method of claim 77, wherein said dendritic cell is
BDCA3.sup.+.
79. The method of claim 77, wherein said additional agent is a
host-derived danger associated-pattern molecule.
80. The method of claim 77, wherein said agonist is an antibody, or
a biologically active fragment or homolog thereof, directed against
CD24.
81. The method of claim 80, wherein said antibody is selected from
the group consisting of a polyclonal antibody, a monoclonal
antibody, a chimeric antibody, a single-chain antibody, a synthetic
antibody, and a humanized antibody.
82. The method of claim 81, wherein said monoclonal antibody is
selected from the group consisting of M1/69, 91, 30-F1, J11d,
eBioSN3, and ML5, or biologically active fragments or homologs
thereof.
83. The method of claim 82, wherein said fragment is an F(ab)2
fragment.
84. The method of claim 77, where said method stimulates said
dendritic cell.
85. The method of claim 77, wherein said CD24 has the amino acid
sequence of SEQ ID NO:2 or SEQ ID NO:4, or homologs thereof.
86. The method of claim 81, wherein said antibody is directed
against a CD24 peptide having the sequence of SEQ ID NO:2 or SEQ ID
NO:4, or homologs or fragments thereof.
87-93. (canceled)
94. A method of identifying of an agonist of CD24 useful for
stimulating erythropoiesis, said method comprising contacting a
dendritic cell expressing CD24 with a test compound, measuring the
stem cell factor level in said cell following said contact, wherein
an increase in stem cell factor in said cell relative to a control
cell not contacted with said test compound, is an indication that
said test compound is an agonist of CD24, thereby identifying of an
agonist of CD24 useful for stimulating erythropoiesis.
95. The method of claim 94, wherein said test compound is a small
molecule, drug, prodrug, or an antibody directed against CD24.
96-101. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 16/139,428, filed Sep. 24, 2018, which
is a continuation application of U.S. patent application Ser. No.
14/651,708, filed Jun. 12, 2015, which is a national stage filing
of PCT International Application No. PCT/US2013/074245, filed Dec.
11, 2013, which claims priority under 35 U.S.C. .sctn. 119(e) to
U.S. Provisional Application Ser. No. 61/736,246 filed Dec. 12,
2012, the disclosures of each of which are incorporated by
reference in their entirety herein.
BACKGROUND
[0003] Anemia is defined as a diminished number of red blood cells
(RBC) or less than the normal quantity of hemoglobin in the blood,
and is the single-most common hematological disorder. Because
hemoglobin (found inside RBCs) normally carries oxygen from the
lungs to the tissues, anemia leads to hypoxia (lack of oxygen) in
organs. Since all human cells depend on oxygen for survival,
varying degrees of anemia can have a wide range of clinical
consequences. People with anemia don't get enough oxygen-rich blood
and they may feel tired or experience shortness of breath during
routine activities. With severe or long-lasting anemia, the lack of
oxygen in the blood can damage the heart, brain, and other organs
and may even cause death. The NIH's National Heart Lung and Blood
Institute has identified 3 major causes of anemia: acute or chronic
blood loss (e.g., hemorrhage or vascular leak), excessive blood
cell destruction (hemolysis) or deficient red blood cell production
(ineffective hematopoiesis). Anemia is a common complication of
cancer and frequently occurs following chemotherapy treatment for
cancer. Anemia is also common in patients undergoing dialysis,
acute severe inflammation such as sepsis, individuals with
rheumatologic disorders, other chronic infectious or inflammatory
diseases, and, importantly, among the elderly. The incidence of
anemia increases with age.
[0004] Erythropoiesis, the production of erythrocytes, occurs
continuously in vivo to offset the turnover (decrease) of
erythrocytes in circulation (.about.120 days of lifespan in
adults). This process is a tightly regulated physiological
mechanism to ensure an adequate supply of erythrocytes to deliver
oxygen to tissues. Erythropoietin (EPO), a glycoprotein hormone
with a potent stimulatory ability for RBC production, is produced
in the kidney. Under basal/homeostatic conditions, EPO stimulates
the division and differentiation of committed erythroid progenitors
into mature erythrocytes primarily in the bone marrow (and to a
lesser extent in the spleen). EPO production and release into the
circulation is augmented in response to hypoxia with concomitant
accelerated production of the erythrocytes. This stress-induced
generation of erythrocytes occurs both in the bone marrow and
importantly in "extramedullary hematopoiesis sites" notably the
spleen and to a lesser extent, the liver. A loss of kidney function
as is seen in chronic renal failure (CRF), for example, typically
results in decreased production of EPO and a concomitant reduction
in red blood cells.
[0005] Maintenance of an adequate supply of oxygen to the body
tissues is vital to survival. In the United States alone, several
million people suffer from anemia secondary to renal failure,
chronic inflammatory disease and malignancies (U.S. Pat. No.
4,987,121, hereby incorporated by reference in its entirety). Since
to a large degree the oxygen-carrying capacity of blood is governed
by the concentration of erythrocytes in the blood, the appropriate
regulation of erythropoiesis is also crucial.
[0006] Human urinary EPO was purified by Miyake et al. (J. Biol.
Chem. 252, 5558 (1977)) from patients with aplastic anemia.
However, the amount of purified EPO protein obtained from this
source was insufficient for therapeutic applications. The
identification and cloning of the gene encoding human EPO and
expression of recombinant protein was disclosed in U.S. Pat. No.
4,703,008 to Lin, the disclosure of which is incorporated herein by
reference.
[0007] Cell adhesion proteins are dynamic molecules involved in
several aspects of cellular function including migration,
inflammation, and tissue development. For example, the maturation
of hematopoietic cells is associated with the regulated expression
of numerous genes, some of which encode cell surface proteins that
mediate maturation-stage-specific signals into and out of the cell.
This is accomplished by binding of the cell surface protein to a
variety of ligands such as soluble interleukins and adhesion
receptors either on other cells or within the extracellular matrix.
One such cell adhesion molecule found in most cells of
hematopoietic lineages is CD24, a glycoprotein consisting of 31 to
35 amino acid residues anchored to the plasma membrane by glycosyl
phosphatidylinositol (Kay et al., J. Immunol., 1991, 147,
1412-1416).
[0008] CD24, also known as Heat Stable Antigen (HSA), is a
glycoprotein expressed at the surface of many cell types including
dendritic cells (DCs). DCs perform many critical roles of immune
system regulation. CD24 has been described, inter alia, in B-cell
development and B-cell neoplasia, in the developing pancreas and
brain, and in regenerating muscle, keratinocytes, renal tubules,
and a large variety of malignant tumors
[0009] There is a long felt need in the art for compositions and
methods useful for stimulating erythropoiesis. The present
application satisfies these needs.
SUMMARY OF THE INVENTION
[0010] Disclosed herein is the discovery of a previously unknown
function of CD24 expressed by a distinct subset of splenic and bone
marrow (BM) dendritic cells; that is, engagement of CD24 on this
spleen (or bone marrow) resident cell type results in the
stimulation of erythropoietin (EPO) production and concomitant
vigorous production of red blood cells (RBCs/erythrocytes) in the
spleen and bone marrow of treated mice. The current invention
provides a novel strategy to enhance stress-mediated erythropoiesis
or stimulate erythropoiesis.
[0011] Further disclosed herein is the unexpected resulted that
engagement of the CD24 on this subset of splenic (or bone marrow)
dendritic cells activate the cells to in turn enhance the activity
of erythrocytic stem cell precursors in the spleen/BM and to
promote endogenous EPO production. This invention has significant
therapeutic potential in the treatment of diseases and disorders of
RBC levels, particularly acute and chronic anemia, because of its
ability to target the stimulation, activation, and proliferation of
erythroid progenitors in the spleen/BM as its primary effect as
well as to enhance the production of endogenous EPO and to augment
the potency of EPO administered therapeutically to subjects
suffering from chronic anemia without endogenous EPO stores.
[0012] It is also disclosed herein that treatment of mice with an
anti-CD24 antibody stimulates an increase in circulating endogenous
EPO levels in the blood, with a prior/concomitant rapid expansion
of erythroid progenitors and a concomitant dramatic increase in
reticulocytes (immature RBCs).
[0013] One key advantage of the compositions and methods of the
present invention is to activate and to expand the numbers of early
erythroid progenitors, the induction of long-term production of
endogenous erythropoietin in the body compared to other therapies
requiring repetitive injections of exogenous recombinant
erythropoietin. Another important advantage is the use of the
strategy to augment the effect of recombinant EPO administered
therapeutically to treat anemia.
[0014] Because the invention is based, at least in part, on the
discovery disclosed herein that certain DC cells are a target for
stimulating erythropoiesis, the invention provides compositions and
methods for targeting those cells and for increasing the numbers of
the cells or of active cells. This includes stimulating
proliferation to increase the numbers as well as methods for
obtaining and administering cells to a subject in need thereof.
[0015] The present invention is related to a method of simulating
stress-induced erythropoiesis in mammals. In one embodiment, the
method mimics the body's response to stresses causing acute or
chronic hypoxia such as hemorrhage or anemia. Identified herein are
agents, for example, a unique monoclonal antibody directed to CD24,
which in part through their action on CD24 displayed by a specific
subset of splenic (or bone marrow) DC dramatically enhances both
EPO production and extramedullary hematopoiesis.
[0016] In one embodiment, the method of the invention provides for
administration of at least one agonist of CD24 activity. The method
comprises administering to a subject a pharmaceutical composition
comprising a pharmaceutically-acceptable carrier, an effective
amount of at least one agonist of CD24, and optionally at least one
additional therapeutic agent. In one aspect, the method stimulates
CD24 expressed on dendritic cells. In one aspect, the dendritic
cells in the mouse express the cell surface molecule CD8.alpha. and
in the human the cell surface molecule BDCA3/CD141. In one aspect,
the agonist for CD24 on this subset of dendritic cells is a small
molecule, drug, prodrug, or an antibody, or a biologically active
fragment or homolog thereof, directed against CD24. In one aspect,
the antibody is a monoclonal antibody. In another aspect, the
invention provides for stimulating the CD24 receptor on a distinct
subset of splenic (or bone marrow) dendritic cells with at least
one agonist directed to CD24 other than an agonistic antibody
directed against CD24. One of skill in the art will appreciate that
other agonists can be used as well, such as other antibodies, other
proteins or peptides that bind with CD24 or activate it, small
molecules, drugs, or prodrugs. Optionally, additional therapeutic
agents can be administered, including, but not limited to,
anti-inflammatory agents, G-CSF, IL-4, SCF, Flt3L, EPO, BMP4,
anti-microbial agents, and host-derived danger associated-pattern
molecules.
[0017] One of ordinary skill in the art will appreciate that any
agent stimulating CD24 or its signaling pathway on the same cells
is also encompassed by the present invention, including, but not
limited to drugs and other peptides or ligands of CD24.
[0018] In one aspect, an agonist of the invention, or other
compounds with similar agonist activity for CD24, may be
administrated to an individual to increase erythropoiesis through
its effect on endogenous EPO/G-CSF/SCF and splenic erythroid
precursors. An agonist of the invention also has the potential to
be used as an adjuvant to enhance the effect of recombinant EPO
used in the treatment of chronic anemia. An increase in
erythropoiesis can be determined, for example, by measuring the
concentration of EPO in the serum and by measuring the hemoglobin
or reticulocyte/erythrocyte levels in the blood of the recipients
before and after treatment with the agent which activates CD24.
[0019] In one embodiment, the present invention provides
compositions and methods useful for enhancing the activity of
erythrocytic stem cell precursors and promoting the production of
erythrocytes and erythroid progenitor cells. In one aspect, the
method is useful for promoting stress erythropoiesis and treating
conditions responding to hypoxic stress. In another aspect, the
method is useful for enhancing the activity of erythrocytic stem
cell precursors in the spleen and promoting the production of
erythrocytes and erythroid progenitor cells.
[0020] In one aspect, the method comprises administering to a
subject in need thereof a pharmaceutical composition comprising an
effective amount of an agonist of CD24 on a distinct subset of
splenic, kidney, or bone marrow dendritic cells, a
pharmaceutically-acceptable carrier, and optionally at least one
additional therapeutic agent. In one aspect, the erythroid
progenitor cells are basophilic erythroblasts in the spleen. In
another aspect, the erythroid progenitor cells are
polychromatophilic erythroblasts in the spleen. In another aspect,
the erythroid progenitor cells are orthochromatic erythroblasts in
the spleen. In one embodiment, the distinct subset of splenic
dendritic cells is the BDCA3/CD141 dendritic cell subset in
humans.
[0021] An agonist of CD24 can be any type of compound that
activates CD24 as disclosed herein or that stimulates CD24
activity. An agonist of CD24 activity includes compounds such as
drugs or proteins that act either upstream or downstream of CD24.
In one embodiment, the agonist is an antibody. In one aspect, the
antibody is a single chain antibody, a monoclonal antibody, F(ab)2
fragments of a monoclonal antibody, a bi-specific antibody, a
chimeric antibody, a synthetic antibody, a polyclonal antibody, or
a humanized antibody, or active fragments or homologs thereof. In
one aspect, the antibody is human. In one aspect, the monoclonal
antibody is M1/69 (IgG2b), 91 (IgG2a), 30-F1 (IgG2c), J11d (IgM),
eBioSN3, or ML5, or biologically active fragments or homologs
thereof. Recombinant monoclonal antibodies (mAb) are playing an
increasing role in the management of many diseases including
malignancies, inflammatory bowel disease, rheumatoid arthritis, and
asthma.
[0022] In one embodiment, an antibody of the invention is directed
against CD24 having the amino acid sequence of SEQ ID NO:2 (human
CD24) or SEQ ID NO:4, or homologs or fragments thereof.
[0023] In one aspect, a monoclonal antibody is directed against
CD24. In one aspect, the antibody is an F(ab)2 fragment of a
monoclonal antibody (mAb) to CD24. In one aspect, the CD24 is human
CD24.
[0024] Therefore, the present invention further encompasses
development of additional anti-human CD24 specific agonist
monoclonal antibodies and humanized monoclonal anti-human CD24
antibodies for use in the invention. The antibody(s), or
biologically active fragments or homologs thereof, can be subjected
to preclinical testing, for example in vitro with human or animal
cells or in animal models, as well as clinical testing. The
invention also encompasses the identification and development of
other compounds which mimic the agonist effect of anti-CD24
monoclonal antibodies on erythropoiesis induced by CD24 engagement
such as the natural ligand for CD24 or derivatives thereof and
small molecules with this CD24-dependent signaling stimulatory
capacity.
[0025] Methods for preparing antibodies are described herein (see
Embodiments and Examples) and are known in the art, including
isolation and identification of single chain molecules produced by
techniques such as phage or yeast display.
[0026] In one embodiment, the present invention provides
compositions and methods for stimulating erythropoiesis. In one
aspect, the stimulation is via a CD24 regulatory pathway. In one
aspect, the present invention provides administering a
pharmaceutical composition comprising a therapeutically effective
amount of a compound of the invention to a subject in need thereof.
In one aspect, the compound stimulates CD24 or an upstream or
downstream component of the CD24 signal transduction pathway. In
one aspect, a compound of the invention directly interacts with
CD24. In another aspect, a compound stimulates an event or molecule
upstream from CD24. In one aspect, a compound of the invention
stimulates a downstream event in the CD24 signal transduction
pathway. In one aspect, CD24 is stimulated using a drug, agent,
antibody, homolog, derivative, or fragment thereof, or other
compound or molecule that elicits the same effect on CD24 disclosed
herein and stimulates erythropoiesis as described herein. In one
aspect, the antibody is a monoclonal antibody, or biologically
active derivative, homolog, or fragment thereof. In one aspect, a
monoclonal agonistic antibody binds to CD24 and stimulates
CD24-dependent cellular signaling events, resulting in the
production of erythropoietin, G-CSF, SCF, expansion of erythroid
progenitors and generation of red blood cells. In one aspect, the
present invention provides a method of increasing erythroid
progenitors.
[0027] In one aspect, an advantage of the present invention over
previous methods of inducing erythropoiesis or stimulating
erythropoietin production and levels is that the present method
directly enhances the activity and stimulates the production of
early erythroid progenitors. Another advantage is that the present
method can be performed without multiple or repetitive injections
of exogenous EPO. A third advantage is that the present method can
be used as a combination therapy in conjunction with EPO
administration.
[0028] In one embodiment the present invention provides
compositions and methods for treating a disease, disorder, or
condition associated with a decrease in erythrocyte production. The
method comprises administering to a subject a pharmaceutical
composition comprising a pharmaceutically-acceptable carrier, an
effective amount of at least one agonist of CD24, and optionally at
least one additional therapeutic agent. In one aspect, the method
stimulates CD24 expressed on dendritic cells. In one aspect, the
method the dendritic cells express the cell surface molecule
BDCA3/CD141. In one aspect, the agonist is a small molecule, drug,
prodrug, or an antibody, or a biologically active fragment or
homolog thereof, directed against CD24.
[0029] The present application discloses the unexpected result that
CD24 and its signal transduction pathway can be regulated to
stimulate, inter alia, erythropoiesis. To that end, the present
invention encompasses the use of or the targeting of nucleic acids
and proteins and various homologues, derivatives, and fragments
thereof. In one aspect, the homologues, derivatives, and fragments
have the same activity as the complete or mature sequence. In one
aspect, the have the function or activity based on the context in
which they are used or described.
[0030] The present invention further encompasses compositions and
methods to induce the CD24 signal transduction pathway and to
increase CD24 expression and levels. One of ordinary skill in the
art will appreciate that any ligand or molecule which is an agonist
of CD24 can be used. In one aspect, an expression vector comprising
a nucleic acid sequence encoding the CD24 protein, or a
biologically active fragment or homolog thereof can be used and a
cell of interest can be targeted or a transfected cell can be
administered to a subject.
[0031] Various antibodies directed against CD24, or biologically
active derivatives, homologs, or fragments of the antibodies, are
encompassed by the methods of the invention. Human CD24 has the
amino acid sequence of SEQ ID NO:2 and mouse CD24 has the amino
acid sequence of SEQ ID NO:4. These antibodies directed against the
CD24 sequences include, but are not limited to, monoclonal
antibodies, polyclonal antibodies, and humanized antibodies. The
antibodies may be directed against or made against appropriate
fragments or homologs of CD24. The methods of the invention also
encompass the identification and development of other compounds
which mimic the effect of anti-CD24 monoclonal antibodies on
erythropoiesis induced by CD24 engagement, such as the natural
ligand(s) for CD24 or derivatives thereof and small molecules with
this CD24 receptor stimulatory capacity.
[0032] In one aspect, the increase in number of erythroid
progenitors, reticulocytes, or erythrocytes following treatment
with a CD24 agonist is from at least about 5, 10, 15, 20, 25, 30,
35, 40, 45, 50, 55, 60, 65, 75, or 100%. In one aspect, the
increase in cell numbers is at least 2 fold. In one aspect, the
increase is at least 5 fold. In one aspect, the increase is at
least 10 fold. Disclosed herein are the results of studies
elucidating the mechanism for the observed erythropoiesis and
demonstrating the relative safety of administering an anti-CD24
antibody in mice. Additional studies have verified the safety of
the method (data not shown).
[0033] In one aspect, the present invention provides for the use of
an agonist of CD24 to stimulate an increase in SCF, G-CSF, and EPO
levels. In one aspect, SCF levels are increased. In one aspect, EPO
levels are increased. In one aspect, G-CSF levels are increased. In
one aspect, two of the three increase and in another aspect, all
three increase. In one aspect, the agonist is an antibody directed
against CD24. The present invention provides compositions and
methods for stimulating an increase in EPO levels and in
reticulocyte levels through a CD24 pathway. In one aspect, the
invention also provides for increasing the numbers of the DC subset
described herein, including stimulation of the cells to proliferate
or administering exogenous cells. In one aspect, the compositions
and methods are useful for increasing c-Kit expressing erythroid
progenitors. In one aspect, SCF and G-CSF are increased by
stimulating CD24 on the target DCs of the invention. In one aspect,
the increase in SCF is cell surface SCF, and SCF and G-CSF are
secreted by the target DCs.
[0034] In one aspect, the increase in SCF levels following
treatment with a CD24 agonist is from at least about 5, 10, 15, 20,
25, 30, 35, 40, 45, 50, 55, 60, 65, 75, or 100%.
[0035] In one aspect, the increase in EPO levels following
treatment with a CD24 agonist is from at least about 5, 10, 15, 20,
25, 30, 35, 40, 45, 50, 55, 60, 65, 75, or 100%.
[0036] In one aspect, the increase in G-CSF levels following
treatment with a CD24 agonist is from at least about 5, 10, 15, 20,
25, 30, 35, 40, 45, 50, 55, 60, 65, 75, or 100%.
[0037] Other compounds can be administered in conjunction with the
agonist of CD24 to augment various aspects of erythropoiesis. These
compounds include, but are not limited to, GM-CSF, IL-4, SCF,
Flt3L, EPO, and BMP4. Other compounds that can be used in
combination with agonists of the invention include host-derived
danger associated-pattern molecules (HMGB1, Hsp70, etc.). Other
therapeutic agents (antibiotics, anti-inflammatories, etc.) and
drugs can also be administered in conjunction with the agonist
therapy of the invention.
[0038] In one embodiment, blocking agents of CD24 can be
administered prior to treatment with the agonist of CD24. For
example, recombinant human CD24 fused to an immunoglobulin molecule
could be administered.
[0039] In one embodiment, the present invention provides
compositions and methods useful for treating anemia. In one aspect,
the anemia comprises aplastic anemia. In another aspect, the anemia
comprises hypoplastic anemia. In one aspect, the present invention
provides contacting an erythroid progenitor cells with a compound
of the invention in an amount effective to augment erythropoiesis.
In one aspect, the anemia is associated with chronic renal failure.
In one aspect, the anemia is associated with end-stage renal
disease. In another aspect, the anemia is associated with
transplantation. In one aspect, the transplantation is renal
transplantation. In another aspect, the anemia is associated with
cancer. In one aspect, the anemia is associated with acquired
immune deficiency syndrome. In another aspect, the anemia is
associated with chemotherapy. In one aspect, the anemia is
associated with radiotherapy. In another aspect, the anemia is
associated with bone marrow transplantation. In another aspect, the
anemia is acute and associated with sepsis. In one aspect, the
anemia is sickle cell anemia. In another aspect the anemia is
associated with rheumatoid arthritis, chronic persistent infection
such as HIV, tuberculosis, hepatitis B and C, and chronic, and
chronic anemia in the elderly (not treated by iron replacement
and/or nutritional supplementation). In one aspect, the disease,
disorder, or condition is anemia.
[0040] A dosage regimen for augmenting erythropoiesis with the
active agents is based on a variety of factors, including the type
of injury, the age, weight, sex, medical condition of the
individual, the severity of the condition, the route of
administration, and the particular compound employed. Thus, the
dosage regimen may vary, but can be determined routinely by a
physician using standard methods.
[0041] In one aspect, an antibody of the invention can be
administered at a dose of about 0.1 mg/kg to about 100 mg/kg body
weight. In another aspect, an antibody of the invention can be
administered at a dose of about 1.0 mg/kg to about 50 mg/kg. In yet
another aspect, an antibody of the invention can be administered at
a dose of about 5.0 mg/kg to about 25 mg/kg body weight. In another
aspect, an antibody of the invention can be administered at a dose
of about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5,0, 5,5, 6.0,
6,5, 7.0, 7.5, 8,0, 8.5, 9.0, 9,5, 10.0, 10.5, 11.0, 11.5, 12.0,
12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5,
18.0, 18.5, 19.0, 19.5, and 20.0 mg/kg body weight. The invention
further encompasses similar increments within each range of doses
described herein. In one embodiment, the agonist or additional
therapeutic agent is administered at a dose of about 1 .mu.g/kg
body weight to about 1 g/kg body weight.
[0042] The present invention encompasses administering an agonist
of the invention more than once to a subject. In one aspect, a
composition comprising at least one agonist of the invention is
administered at least twice. In another aspect, a composition is
administered at least five times. In yet another aspect, a
composition is administered at least 10 times. In one aspect, a
composition of the invention is administered at least once per day.
In another aspect, a composition is administered at least once per
week. In yet another aspect, a composition is administered at least
twice per week. In another aspect, a composition is administered at
least once per month. In yet another aspect, a composition is
administered at least twice per month.
[0043] In one aspect, after administration of the agonist to the
subject, a second round of the agonist is administered to stimulate
a second wave of production of erythrocytes and erythroid
progenitor cells. In one aspect, a subject may receive three or
more rounds of treatment with the agonist, in another aspect five
or more rounds, in yet another aspect, 10 or more rounds, and in a
further aspect, 25 or more rounds. In one aspect, the total number
of doses can be from about 2 to about 100; about 5 to about 75;
about 10 to about 50 times; and about 20 to about 40.
[0044] The invention further encompasses the use of unit doses, for
example unit doses of about 0.01, 0.05, 0.1, 0.5, 0.75, 1.0, 1.25,
1.5, 1.75, 2.0, 5.0, 10, 15, 20, 25, 30, 50, or 100 grams.
[0045] The treatment regimen will vary depending on the disease
being treated, based on a variety of factors, including the type of
injury, the age, weight, sex, medical condition of the individual,
the severity of the condition, the route of administration, and the
particular compound employed. The treatment can include
administration of a pharmaceutical composition of the invention
once or more than once. Other therapeutic drugs and agents can be
administered as well. Agents which stimulate erythropoietin, SCF
and G-CSF can also be administered.
[0046] In another embodiment, erythropoiesis is augmented ex vivo
by obtaining a sample of bone marrow cells, as is known in the art,
potentiating erythropoietin-induced differentiation with at least
one active agent of the invention and infusing the treated cells
back into a subject in need thereof. The methods also encompass the
use of the subset of DC cells of the invention disclosed
herein.
[0047] The full-length peptide of (80 amino acid residues in
humans; SEQ ID NO:2) comprises a putative signal peptide (amino
acid residues 1-26) and a mature peptide of 54 amino acid residues
(amino acid residues 27-80 of the full-length peptide).
[0048] Useful full-length sequences of the invention include, but
are not limited to, human and mouse nucleic acid and amino acid
sequences for CD24.
[0049] Those four CD24 sequences (SEQ ID NOs: 1-4) are as
follows:
TABLE-US-00001 human nucleic acid; GenBank accession no.
NM_013230.2; (2194 bp; mRNA)- SEQ ID NO: 1
gggtctcgccggctcgccgcgctccccaccttgcctgcgcccgcccggagccagcggttctccaagcacccagc-
atcctgc
tagacgcgccgcgcaccgacggaggggacatgggcagagcaatggtggccaggctcgggctggggctgctgctg-
ctggc
actgctcctacccacgcagatttattccagtgaaacaacaactggaacttcaagtaactcctcccagagtactt-
ccaactctggg
ttggccccaaatccaactaatgccaccaccaaggcggctggtggtgccctgcagtcaacagccagtctcttcgt-
ggtctcactc
tctcttctgcatctctactcttaagagactcaggccaagaaacgtcttctaaatttccccatcttctaaaccca-
atccaaatggcgtc
tggaagtccaatgtggcaaggaaaaacaggtcttcatcgaatctactaattccacaccttttattgacacagaa-
aatgttgagaat
cccaaatttgattgatttgaagaacatgtgagaggtttgactagatgatggatgccaatattaaatctgctgga-
gtttcatgtacaa
gatgaaggagaggcaacatccaaaatagttaagacatgatttccttgaatgtggcttgagaaatatggacactt-
aatactacctt
gaaaataagaatagaaataaaggatgggattgtggaatggagattcagttttcatttggttcattaattctata-
aggccataaaaca
ggtaatataaaaagatccatgattctatttatatgtacatgagaaggaacttccaggtgttactgtaattcctc-
aacgtattgtttcg
acagcactaatttaatgccgatatactctagatgaagttttacattgttgagctattgctgttctcttgggaac-
tgaactcactttcctc
ctgaggctttggatttgacattgcatttgaccttttatgtagtaattgacatgtgccagggcaatgatgaatga-
gaatctaccccca
gatccaagcatcctgagcaactcttgattatccatattgagtcaaatggtaggcatttcctatcacctgtttcc-
attcaacaagagc
actacattcatttagctaaacggattccaaagagtagaattgcattgaccacgactaatttcaaaatgctttta-
ttattattatttttta
gacagtctcactttgtcgcccaggccggagtgcagtggtgcgatctcagatcagtgtaccatttgcctcccggg-
ctcaagcgat
tctcctgcctcagcctcccaagtagctgggattacaggcacctgccaccatgcccggctaatttttgtaatttt-
agtagagacag
ggtttcaccatgttgcccaggctggtttcgaactcctgacctcaggtgatccacccgcctcggcctcccaaagt-
gctgggatta
caggcttgagcccccgcgcccagccatcaaaatgctttttatttctgcatatgttgaatactttttacaattta-
aaaaaatgatctgttt
tgaaggcaaaattgcaaatcttgaaattaagaaggcaaaaatgtaaaggagtcaaaactataaatcaagtattt-
gggaagtgaa
gactggaagctaatttgcattaaattcacaaacttttatactctttctgtatatacattttttttctttaaaaa-
acaactatggatcagaat
agccacatttagaacactttttgttatcagtcaatatttttagatagttagaacctggtcctaagcctaaaagt-
gggcttgattctgca
gtaaatctfttacaactgcctcgacacacataaaccttfttaaaaatagacactccccgaagtctfttgttcgc-
atggtcacacactg
atgcttagatgttccagtaatctaatatggccacagtagtcttgatgaccaaagtcctttttttccatctttag-
aaaactacatgggaa
caaacagatcgaacagttttgaagctactgtgtgtgtgaatgaacactcttgctttattccagaatgctgtaca-
tctattttggattgt
atattgtgtttgtgtatttacgctttgattcatagtaacttcttatggaattgatttgcattgaacacaaactg-
taaataaaaagaaatg gctgaaagagcaaaaaaaaaaaaa human amino acid; GenBank
Accession No. ACI46150.1; (80 amino acids)- SEQ ID NO: 2
MetGlyArgAlaMetValAlaArgLeuGlyLeuGlyLeuLeuLeuLeuAlaLeuLeuLeuProThrGln
IleTyrSerSerGluThrThrThrGlyThrSerSerAsnSerSerGlnSerThrSerAsnSerGlyLeuAlaPr-
o
AsnProThrAsnAlaThrThrLysAlaAlaGlyGlyAlaLeuGlnSerThrAlaSerLeuPheValValSer
LeuSerLeuLeuHisLeuTyrSer (single letter sequence of:
mgramvarlglgllllalllptqiyssetttgtssnssqstsnsglapnptnattkaaggalqstaslfvvsls-
llhlys) mouse nucleic acid; GenBank Accession No. NM_009846; (1825
bp; mRNA)- SEQ ID NO: 3
ccaccttgcctgcgcccgcgcgagcttagcagatctccacttaccgaacatctagagagtcgcgccgcgcgccg-
acggagc
ggacatgggcagagcgatggtggccaggctagggctggggttgctgcttctggcactgctcctacccacgcaga-
tttactgc
aaccaaacatctgttgcaccgtttcccggtaaccagaatatttctgcttccccaaatccaagtaacgctaccac-
cagagggggt
ggcagctccctgcagtccacagctggtctcctggctctctctctctctcttctacatctctactgttagagact-
caggccaggaaa
cgtctctacttccccatcttctacacctaccccaaatggcaaccacaagtccaatgtgatcaggaagaaacagg-
tccacctcga
attggctgttaccatatctcaacagaaaacacggagaattcgaaattcgacgggattaaaggacgcgtgaaagg-
tttgagaga
agagagatgccgctattgaatctgctggagttttacatcccaagatgaagacagcattcagaattgatgtgatt-
tccttgaatgtg
gcttaggaaatgtggacacttaaaactctcacttgaaattgggcacaggtttgatgtagagataaggacggggt-
gcggaatgg
agacccattttgtcattgattcatctgaccgataaggccatagtgcagttaggtgatattcgaaagcttctttg-
atgctctttatgtat
atgttggaaggaactaccaggcgttgcttaaattcccaatgtgttgtttcgttactactaatttaataccgtaa-
gctctaggtaaagt
tccatgttgttgaactctgactgttctctttggaattgaacgttttgcatcctcctcctgtggctttaggtctg-
acattgtatttgaccttt
actagtaattaacatgtgccaggcaatggtggattggaacccatccccaagtccagccaccactgaataaatct-
gatttcaaaa
gtcaaacagtagacatttcccattgtcgtttctcactcaccacaagcaccaaattcactagagtacactggttc-
cagagagcaga
atcatgttggccttggctaatttcaaaatgctgtcttttactttggtatatgttgagggcttttcataatttaa-
agtgtgttctgttagcaa
ggcaaaaattatgagtcttaattctacaggcaaatatgcaaaggagccaaaactgtaaacccagcatttgggat-
gtgaagactg
gaagctaactctcattgaattcacaaagtcttttatacaatttctgtacatactttttttttttttaagagaaa-
aacaaacggtggatca
gaatagccacgtttggaatactttggttatccattcatatttttagatagttattggtcctgtgcctgaaaggg-
ggcttggttctaccg
taagtttttccaatttccttgatatacacataccttctaaaacctagacatttcctgaaaaaaatcttttgttc-
gcatggtcacacactg
atgcttacccgtacagtagtcttgataaccagagtcattttctccatctttagaaaccttcctgggaagaagga-
gagctcacagac
ccgaagctactgtgtgtgtgaatgaacactccccttgcctcacacctgaatgctgtacatctatttgattgtaa-
attgtgtttgtgtat
ttatgctttgattcatagtaacttctcatgttatggaattgatttgcattgaacacaaactgtaaataaaagaa-
agaaatggcggaga aaaaaaaaa mouse amino acid; GenBank Accession No.
NP_033976; (76 amino acids)- SEQ ID NO: 4
MetGlyArgAlaMetValAlaArgLeuGlyLeuGlyLeuLeuLeuLeuAlaLeuLeuLeuProThrGln
IleTyrCysAsnG1nThrSerValAlaProPheProGlyAsnGlnAsnIleSerAlaSerProAsnProSer
AsnAlaThrThrArgGlyGlyGlySerSerLeuGlnSerThrAlaGlyLeuLeuAlaLeuSerLeuSerLeuLe-
u HisLeuTyrCys (single letter sequence of:
mgramvarlglgllllalllptqiycnqtsvapfpgnqnisaspnpsnattrgggsslqstagllalslsllhl-
yc)
[0050] The invention further provides for the use of the proteins
or peptides where one or more conservative amino acid substitutions
are made in the sequence and that the substitution has no effect on
the desired biological activity, where such activity is desired. In
one aspect, one conservative amino acid substitution is made. In
one aspect, at least two conservative amino acid substitutions are
made. When two or more substitutions are made, they do not have to
be at adjacent amino acid residue positions.
[0051] In one aspect, an antibody or other agonist of CD24 activity
that stimulates erythropoiesis, SCF, or EPO can be identified using
methods and assays described herein and the identified agents can
be used to practice the methods of the invention. The invention
therefore provides methods for identifying agents useful to
practice the invention and provides for the use thereof.
[0052] The invention also encompasses the identification and
development of other compounds (drugs, peptides, ligands, etc.)
which mimic the agonist effect of anti-CD24 monoclonal antibody on
erythropoiesis induced by CD24 engagement such as the natural
ligand for CD24 or derivatives thereof and small molecules with
this CD24-dependent signaling stimulatory capacity. The examples
provide assays for screening for the activity of such agents that
activate CD24, particularly in the dendritic cell populations used
herein.
[0053] In one embodiment, the present invention provides a method
of identifying of an agonist of CD24 useful for stimulating
erythropoiesis wherein a dendritic cell expressing CD24 is
contacted with a test compound, the stem cell factor level is
measured in the cell following the contact, and an increase in stem
cell factor in the cell relative to a control cell not contacted
with the test compound, is an indication that the test compound is
an agonist of CD24. The stem cell factor level can also be compared
to a standard and to cells treated with a compound that does not
stimulate stem cell factor or erythropoiesis, even if the compound
interacts with CD24 or binds to CD24. In one aspect, the test
compound is an antibody directed against CD24. In one aspect, the
test compound interacts with CD24. In one aspect, CD24 protein
levels are measured. In another aspect, CD24 messenger RNA levels
are measured.
[0054] According to the present invention, potential therapeutic
agents may be screened for the ability to stimulate splenic
erythrocyte progenitor (CD45-/+ c-Kit+ Ter119- and/or Ter119+CD45-)
expansion in the spleen following administration of the agents to
mice.
[0055] Alternatively, potential therapeutic agents may be screened
for the ability to increase the expression level of the murine stem
cell factor (mSCF) on the splenic CD8.alpha.+ DC subset following
treating the isolated conventional dendritic cells with candidate
compounds. In one aspect, agents are screened for the ability to
increase the expression level of the mSCF on the splenic
CD8.alpha.-CD11b+ DC subset following treating the isolated
conventional dendritic cells.
[0056] In addition, potential therapeutic compounds/agonists may be
screened for the ability to increase the expression level of the
human stem cell factor (hSCF) on human dendritic cells expressing
the cell surface molecule BDCA3/CD141.
[0057] Controls can include antibodies that are directed against
CD24 but do not stimulate erythropoiesis or SCF (see examples).
[0058] In a further aspect, the present invention provides kits
with components for promoting erythropoiesis, wherein the kits
comprise an effective amount of at least one active agent to
stimulate CD24 or the CD24 pathway of the invention, and
instructions for using the active agent as a therapeutic. In one
embodiment, the kit further comprises a pharmaceutically acceptable
carrier, such as those adjuvants described above. In another
embodiment, the kit further comprises a means for delivery of the
active agent to a mammal. Such devices include, but are not limited
to matrical or micellar solutions, polyethylene glycol polymers,
carboxymethyl cellulose preparations, crystalloid preparations
(e.g., saline, Ringer's lactate solution, phosphate-buffered
saline, etc.), viscoelastics, polyethylene glycols, and
polypropylene glycols. Optionally, at least one additional
therapeutic agent can be administered and can be provided in the
kit. In a further embodiment, the kits also comprise an amount of
erythropoietin effective to accelerate erythropoiesis.
[0059] As disclosed herein, a kit comprising agonists of the
invention is useful for, inter alia, treating anemia, stimulating
erythropoiesis, stimulating CD24-mediated stress erythropoiesis,
stimulating extramedullary erythropoiesis, stimulating SCF,
stimulating EPO, stimulating G-CSF, stimulating erythroid
progenitor proliferation, and increasing the number of
erythrocytes, reticulocytes, and erythroid progenitor cells.
[0060] Various aspects and embodiments of the invention are
described in further detail below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0061] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0062] FIG. 1, comprising FIGS. 1A to 1E. Administration of the
anti-CD24 mAb induces stress erythropoiesis in mice, and expression
of CD 24 by cells of hematopoietic origin is required for induction
of extra medullary hematopoiesis. Mice were infused
intraperitoneally (i.p.) with 150 .mu.g control rat IgG or
.alpha.CD24 (clone M1/69) and necropsied on days 1, 3 or 5 after Ab
treatment. (A) Representative macroscopic appearance of the spleens
over time after .alpha.CD24 mAb infusion in wt B6 mice (n>20).
(B) M1/69 treatment promotes stress erythropoiesis. Single cell
suspensions prepared from spleens at d5 post treatment were
analyzed for cell types expressed by flow cytometry and enumerated
(n.gtoreq.5): leukocyte (CD45.sup.+Ter119.sup.-, group I);
erythrocytes (CD45.sup.+Ter119.sup.-, group II), and erythroid
progenitor cells (CD45.sup.-Ter119.sup.+, group III). (C) We
further examined for the erythroid compartment, based on the
differential expression of Ter119 and the transferrin receptor,
CD71. Erythroblast subsets, consisting of basophilic
(Ter119.sup.+CD71.sup.hi, group I--least mature),
polychromatophilic (Ter119.sup.+CD71.sup.med, group ii), and
orthochromatic (Ter119.sup.+CD71.sup.lo, group iii) erythroblasts
in spleen. (D) Erythropoiesis induced by M1/69 treatment requires
CD24 expression. To investigate if these effects are specific to
M1/69 mAb interaction with CD24, mice deficient in CD24 expression
were given Ig or .alpha.CD24 mAb and examined at d5 p.t. for gross
appearance of spleens shown in (D) and absolute number of
CD45.sup.-Ter119.sup.+ (data not shown, n=3-5). (E) CD24 expression
by the cells of hematopoietic origin, but not the cells in stromal
compartment, is required for M1/69-induced stress erythropoiesis.
To assess the contribution of CD24-expressing cell type from the
respective compartment, BM chimeric mice were established by
transferring 2.times.10.sup.6 donor BM cells derived from either WT
or CD24.sup.-/- mice into lethally irradiated either WT or
CD24.sup.-/- recipient mice. At 6 weeks after BM cell
reconstitution, these chimeric mice were infused with Ig or M1/69.
At d5, mice were necropsied and evaluated for gross appearance of
spleens (right panel) and for erythroid progenitor cells
frequency/numbers (left panel) (n=3/group). CD24 expression on
cells of bone marrow/hematopoietic origin was necessary and
sufficient for stress-induced hematopoiesis mediated by M1/69
treatment. It is also noteworthy that erythropoiesis induced by
.alpha.CD24 mAb depends on F(ab).sub.2, but not Fc, fragments (data
not shown). Data represent mean.+-.SD. ***P<0.001.
[0063] FIG. 2, comprising FIGS. 2A to 2B. Stress erythropoiesis in
the peripheral blood after .alpha.CD24 mAb treatment. The effect of
M1/69 treatment in erythropoiesis was also observed in peripheral
blood. WT mice were infused i.p. with 150 .mu.g control Ig or
.alpha.CD24 and examined at the indicated time points. Peripheral
blood smears were prepared at d5 p.t. and stained by methylene
blue. Typical staining of residual RNA in reticulocytes--immature
RBC for about a day in the blood stream before developing into
mature RBCs--is examined by microscope (data now shown). (A)
Percentages of reticulocytes in the peripheral blood were also
assessed by flow cytometry using thiazole orange staining (to
selectively stain nucleic acid, in this case RNA) for about an 1 hr
at room temperature. A representative flow cytometric analysis of
examining reticulocytes in blood at d5 is shown. Data are
representative of >20 independently repeated experiments.
Numbers denote the percentage of reticulocytes staining with
thiazole orange dye in peripheral blood. (B) Kinetic analyses of
percent reticulocytes circulating in the blood of mice treated with
Ig or .alpha.CD24 at the indicated dates (n=4-7/day).
[0064] FIG. 3, comprising FIGS. 3A to 3E. Stress erythropoiesis
induced by .alpha.CD24 mAb treatment depends on the spleen and, to
a lesser extent, bone marrow. To study the extent of CD24-induced
erythropoiesis in the spleen as well as BM, respectively, WT mice
were injected i.p. with 150 .mu.g control Ig or .alpha.CD24. (A)
Representative of the frequency of CD45.sup.-Ter119.sup.+ erythroid
progenitors in spleens (top panels in A) and BM (bottom panels in
A), as measured by flow cytometry at d5 after .alpha.CD24
treatment. (B) Total erythroid progenitors per spleen and BM,
respectively, at d5 are depicted (n=3-5). (C-E) To evaluate the
impact of spleen on stress erythropoiesis, mice were splenectomized
prior to antibody administration. WT B6 mice that had undergone
splenectomy 5 weeks earlier were treated as in A. At d5, percentage
of reticulocytes in the blood (C and D) and absolute cell number of
CD45.sup.-Ter119.sup.+ cells in BM (E) was assessed by flow
cytometric analyses (n=3-5). Mean.+-.SD is shown (***P<0.001,
**P<0.01 and **P<0.05).
[0065] FIG. 4, comprising FIGS. 4A to 4F. A novel role of
conventional dendritic cells, in particular CD8.alpha.+ DC, in
CD24-mediated stress erythropoiesis in vivo. To investigate the
specific cell type(s) governing stress erythropoiesis in vivo, we
employed mice deficient in or depleted of various cell types (T, B,
and inflammatory monocytes, neutrophils and NK cells). Our analyses
found that none of these hematopoietic cell lineages were important
regulators of stress erythropoiesis induced by M1/69 treatment
(data not shown). Next, we assessed the impact of dendritic cells
(DCs) on the stress erythropoiesis. DCs are well recognized as
professional antigen presenting cells, composed of heterogeneous
subpopulations. In lymphoid organs such as spleens, three major
subsets of DCs are classified; two conventional DC (cDC) subsets
including CD8.alpha..sup.+CD11b.sup.- (CD8.alpha..sup.+ cDC, group
I) and CD8.alpha..sup.-CD11b.sup.+ (CD11b.sup.+ cDC, group II) (top
panels in A) and plasmacytoid DC (pDC) (data not shown). Notably,
it is the CD8.alpha..sup.+ cDC among DC subsets in the spleen which
express cell surface CD24 at the highest level (lower panel in A).
To study the role of CD24.sup.hi CD8.alpha..sup.+ cDC in
.alpha.CD24 mAb-induced stress erythropoiesis, we obtained mice
lacking transcriptional factor Batf3 gene (Batf3 KO mice), which
are developmentally devoid of CD8.alpha..sup.+ cDC in the spleen
and of nodes and a related lineage of DC which populate certain
non-lymphoid tissues such as lung and kidney etc. (B). WT and Batf3
KO mice were injected i.p. with 150 .mu.g control Ig or
.alpha.CD24. After 5 days, gross appearance of spleens (data not
shown), a representative of the percentage of circulating
reticulocytes in the blood (C) and absolute number of
CD45.sup.-Ter119.sup.+ erythroid progenitors in the spleens (D)
were assessed by flow cytometry (n=4-6). The results of these
analyses indicate that CD8.alpha..sup.+ cDC in the spleen and
possibly a related tissue-specific DC subset play a critical role
in promoting stress erythropoiesis. To exclude a possibility of an
additional unanticipated role of the Batf3 gene on erythropoiesis,
i.e., other than its role in the generation of a specific DC
lineage, we employed CD11c-DTR mice, which are engineered to
express non-human primate diphtheria toxin receptor (DTR) driven
off of murine CD11c (a conventional marker for murine DC) promoter.
Upon diphtheria toxin (DTx) administration (100 ng/mouse via i.p.),
DTR expressing CD11c.sup.+ cells (that is cDC) are conditionally
selectively ablated within 24 hrs (E). These cDC-ablated mice were
infused with Ig or .alpha.CD24 mAb 1 day after first DTx
administration. With second dose of DTx at d1 post treatment, mice
were necropsied at d5 for gross appearance of the spleens (data not
shown). The percentage of circulating reticulocytes in the blood
(data not shown) and absolute number of CD45.sup.-Ter119.sup.+
erythroid progenitors in the spleens (F) were assessed by flow
cytometry (n=3-5). The analysis demonstrates that CD11c.sup.+
cells, i.e., cDC, play a critical role in regulating the
development of stress hematopoiesis mediated by CD 24 engagement.
Data represent mean.+-.SD.
[0066] FIG. 5, comprising FIGS. 5A to 5D. Conventional DCs are
required for the expansion of c-Kit expressing erythroid
progenitors in the spleen during extramedullary stress
erythropoiesis. Recent studies show that stress erythropoiesis
depends on a population of stress erythroid progenitor cells that
are distinct from the counterpart present in BM. The development,
expansion, and differentiation of these progenitors are regulated
in part by a complex of less understood signals. We initially
attempted to identify these progenitors based on c-Kit (CD117)
expression as engagement of this receptor has been demonstrated to
be essential for stress erythropoiesis. (A-C) WT mice were injected
i.p. with 150 .mu.g control Ig or .alpha.CD24. (A) Anti-CD24 mAb
treatment promotes the expansion of cKit.sup.+CD45.sup.+/- cells in
the spleen. At d3 and 5 post treatments, single cells suspensions
prepared from the necropsied spleens were stained with
fluorochrome-conjugated mAb recognizing c-Kit (left panel). In
contrast to Ig treated mice, mice undergoing M1/69 treatment have a
dramatic increase in the number of c-Kit.sup.+ cells, peaking at d3
(n>6), with little to no expression of the standard
hematopoietic lineage marker CD45 (i.e., CD45.sup.+/-c-Kit.sup.+
cells (right panel). (B) CD45.sup.+/-c-Kit.sup.+ progenitors
exhibited greater (compared to DC and B cells) forward (F SC) and
side scatter (SSC) plot by flow cytometry analyses and expressed
different levels of Ter119 as well as CD71, indicative of
progenitor cells with erythroid lineage commitment such as
proerythroblasts. (C) To determine if c-Kit.sup.+ proerythroblasts
undergo proliferative expansion, the M1/69-treated mice were fed
with nucleic acid analog, BrdU, at d5 to label proliferating cells.
After 24 hr BrdU injection, c-Kit.sup.+ erythroid progenitors were
examined for active uptake of BrdU in combination with staining for
a proliferation-associated nuclear antigen, Ki-67. In contrast to
c-Kit.sup.+ cells in the Ig-treated spleen, c-Kit.sup.+ erythroid
progenitors isolated from the mice treated with M1/69 underwent
active proliferation. (D) Furthermore, in support of the importance
of splenic cDC in orchestrating stress erythropoiesis, ablation of
cDC by treatment of CD11c-DTR mice with diphtheria toxin resulted
in minimal expansion of these progenitors when stimulated by
.alpha.CD24 mAb infusion (n=3-5). Collectively, our data strongly
support the view that cDCs, particularly lymph node-resident
CD8.alpha.+ cDC subset and/or a developmentally related
tissue-resident cDC subset, play an essential role in regulating
extramedullary stress erythropoiesis.
[0067] FIG. 6, comprising FIGS. 6A to 6F. Stem cell factor produced
by cDC in the spleen is required for extramedullary stress
erythropoiesis. We next investigated the molecular mechanism
underlying cDC-dependent proerythroblast proliferation. Soluble
mediators--i.e., Erythropoietin (Epo), stem cell factor (SCF) and
bone morphogenetic protein 4 (BMP 4), IL-3 and GM-CSF--have each
been implicated as necessary to expand erythroid progenitors during
erythropoiesis. We first examined the profile of gene expression by
cDC and non-DC subpopulations after magnetically sorting out the
cell types from total splenic cells prepared at d1 (data now shown)
or d2 (A) or after M1/69 infusion. The splenic cells were sorted
into DCs (based on CD11c.sup.+), T cells (CD90.sup.+), B cells
(B220.sup.+) and remaining cell types, i.e., both CD45.sup.+
hematopoietic origin cells and CD45.sup.- splenic stromal cells. We
measured expression levels of mRNA encoding for SCF, Epo, BMP4,
IL-3, and GM-CSF, respectively. Our data revealed that M1/69
treatment induced the expression of SCF exclusively by cDC (A). In
contrast, cDC in the spleen did not upregulate mRNA for Epo, BMP4,
IL-3 and GM-CSF (data now shown). (B-D) This suggests that SCF
produced by splenic cDC via CD24 signaling contributes to
M1/69-induced erythropoiesis. To investigate the role of SCF-c-Kit
signaling in stress erythropoiesis, c-Kit KO mice were infused with
.alpha.CD24 mAb. c-Kit deficiency resulted in an a markedly reduced
percentage of reticulocytes in peripheral blood of mAb treated KO
mice compared to treated wild type control mice when analyzed at d5
after mAb infusion (B). The importance of c-kit-mediated signaling
in this process was further validated by the analysis of the gross
appearance of spleens (C) and the accumulation of c-Kit.sup.-
erythroid progenitors (CD45.sup.-Ter119.sup.+, left panel in D) and
c-Kit.sup.+ proerythroblasts (right panel in D) in the spleens at
d5 after mAb infusion. As shown in C and D, the absence of
c-Kit-SCF signaling axis resulted in the almost complete abrogation
of M1/69-stimulated erythropoiesis. (E and F) We complemented the
findings in c-Kit KO mice on the extramedullary stress
erythropoiesis, by analyzing the impact of the pharmacological
inhibitor of c-Kit signaling, imatinib (Gleevec). The drug (1
mg/kg) was administrated i.p. daily for 4 days into M1/69-treated
WT mice. Consistent with the observations in c-Kit KO mice,
administration of Imatinib into mAb treated WT B6 mice
significantly reduced the reticulocytes in the blood (E) and
inhibited the expansion/accumulation of CD45.sup.-Ter119.sup.+
erythroid progenitors (left panel in F) and c-Kit.sup.+
proerythroblasts (right panel in F) in the spleen. In summary, our
data suggest that SCF produced by splenic cDC stimulated by
engagement of the CD24 molecule on the cells with the M1/69 mAb
promotes the expansion of c-Kit.sup.+ erythroid progenitors in the
spleen through a engagement of the c-Kit receptor on the early
splenic erythroid progenitors.
[0068] FIG. 7, comprising FIGS. 7A to 7E. Stress erythropoiesis
triggered by M1/69 treatment requires Epo production by the
kidneys, and Epo production is in turn dependent on the presence of
cDC. Epo is a glycoprotein hormone that serves as a primary
regulator of differentiation, proliferation, and survival of
erythroid progenitor cells, and is made mainly by stromal cells
within the kidneys. Epo and c-Kit signaling are both necessary for
efficient erythropoiesis and work synergistically in this process.
To fully account for the robust expansion of erythroid lineage
progenitor cells in the spleen and the subsequent reticulocytosis
following mAb treatment, we postulated that engagement of CD24
stimulates directly or indirectly Epo production by stromal cells
in the kidney, which acts in concert with SCF produced locally in
spleen to orchestrate extramedullary hematopoiesis. (A) When serum
Epo levels were measured after M1/69 injection, we indeed detected
a robust increase in Epo, peaking d3 after injection and then
gradually decreasing over the several days. This burst of Epo
production, as expected, proceeds the appearance of reticulocytes
in the peripheral blood (see FIG. 2). (B) In FIG. 6, we have
demonstrated that cDCs are critical in stress erythropoiesis
induced by M1/69 treatment at least via the provision of SCF to
stimulate the expansion of c-Kit receptor-expressing erythroid
progenitors in the spleen. We reasoned that in parallel to splenic
cDC, the counterpart of cDC subsets in the kidney may play a
crucial role in promoting the production of Epo by epo-producing
renal stromal cells. To test this hypothesis, we first measured Epo
levels in the circulation of mice deficient in cDC, either
genetically (i.e., Batf3 KO mice) or following cDC ablation (i.e.,
DTx-treated CD11c-DTR mice). We found a significant decrease in Epo
production in Batf3 KO mice (C) and nearly complete ablation of Epo
production in DTx-treated CD11c-DTR mice (D). These data
demonstrate that cDCs localized in respective tissues play a
crucial role in CD24-mediated stress erythropoiesis via at least
stimulation of Epo induction in the kidneys and SCF production in
the spleen. To dissect the contribution of Epo and SCF,
respectively, we measured Epo concentration in c-Kit KO mice, which
have an intact cDC compartment in the kidney (data not shown). In
stark contrast to the absence of the expansion of c-Kit.sup.+
erythroid progenitors in the spleen, M1/69 treated c-Kit KO mice
have demonstrated an elevated Epo level in the circulation (FIG.
7E). This is in keeping with the failure of expansion of
c-Kit.sup.+ erythroid progenitors in the spleens of c-Kit KO mice
which would be the primary consumers of Epo through binding of Epo
to its receptor on the cells. This result indicates that the
production of Epo alone, although necessary, is not sufficient to
induce extramedullary stress erythropoiesis after CD24
engagement.
[0069] FIG. 8. Model for CD24-mediated stress erythropoiesis in
vivo.
[0070] Without wishing to be bound by any particular theory, this
schematic depicts a novel model for extramedullary stress
erythropoiesis using an agonist of CD24 (the monoclonal antibody
M1/69 in this schematic).
[0071] FIG. 9, comprising FIGS. 9A to 9B (also referred to as
Supplemental FIG. 1A to 1B). Analysis of the stimulation of splenic
erythrocyte progenitor (Ter119.sup.+CD45.sup.- cells) expansion in
the spleen following administration of M1/69 or 1 of 3 other
monoclonal anti-CD24 antibodies, 91, 30-F 1, and J11 d,
respectively. These data are included in Supplemental FIGS. 1A and
1B.
[0072] FIG. 10 (also referred to as Supplemental FIG. 2).
Demonstrates the results of an analysis of the reticulocyte
response to repeated administration of monoclonal antibody.
[0073] FIG. 11 (also referred to as Supplemental FIG. 3). Isolated
conventional dendritic cells from the spleens of mice were treated
with anti-CD24 monoclonal antibody overnight in cultures. We
monitored the expression of the murine stem cell factor (mSCF) on
the splenic CD8.alpha..sup.+ DC subset and the more abundant
CD8.alpha..sup.-CD11b.sup.+ splenic DCs. As the figure indicates,
mSCF is abundantly expressed on the surface of CD8.alpha..sup.+ DC
subset.
[0074] FIG. 12, comprising FIGS. 12A to 12B (also referred to as
Supplemental FIG. 4A to 4B). A strategy was employed for generating
BDCA3.sup.+ DC from human peripheral blood mononuclear cells in
vitro using a cocktail of growth factors consisting of GM-CSF,
IL-4, SCF, and Flt3L. These growth factors are highly potent
stimulators of proliferation and differentiation of circulating
mononuclear stem cells into a variety of cell types. Nonetheless,
as Supplemental FIG. 4A demonstrates we can detect a small
population of BDCA3.sup.+ DC-like cells in in vitro culture with a
larger population of BDCA3.sup.- cells. As Supplemental FIG. 4B
demonstrates we can detect up-regulation of hSCF in response to
treatment with 2 different monoclonal antibodies to human CD24:
eBioSN3 and ML5. As Supplemental FIG. 4B indicates the eBioSN3
monoclonal antibody regulates hSCF expression selectively on
BDCA3.sup.+ DC. It will also note that the background expression of
cell surface hSCF is high in both DC subsets. This we believe is
due to the strong growth promoting activity of the growth factor
cocktails. We anticipate that when this analysis is repeated with
resting BDCA3.sup.+ DC isolated from human spleen, the background
will be considerably lower. Nevertheless, these findings
demonstrate that the corresponding up-regulation of cell surface
hSCF by engagement of the CD24 receptor occurs as well in the
corresponding population of human dendritic cells.
DETAILED DESCRIPTION
Abbreviations and Acronyms
[0075] Ab--antibody [0076] BM--bone marrow [0077] BMP--bone
morphogenic protein [0078] CD--cluster of differentiation [0079]
CRF--chronic renal failure [0080] cDC--conventional DC [0081]
DC--dendritic cell [0082] DTR--diphtheria toxin receptor [0083]
DTx--diphtheria toxin [0084] EPO--erythropoietin [0085]
Flt3--Fms-Like Tyrosine Kinase 3 [0086] Flt3L--Flt3 Ligand [0087]
FSC--forward scatter [0088] GM-CSF--granulocyte macrophage colony
stimulating factor, also referred to as G-CSF [0089] HMGB1--high
mobility group protein group B1, also referred to as high mobility
group protein [0090] hEPO--human erythropoietin [0091] hSCF--human
stem cell factor [0092] HSP--heat shock protein [0093] KO--knockout
[0094] HSA--heat stable antigen (also referred to as CD24) [0095]
IL-3--interleukin-3 [0096] IL-4--interleukin-4 [0097]
mAb--monoclonal antibody [0098] mSCF--murine stem cell factor
[0099] RBC--red blood cell, also referred to as erythrocyte [0100]
SCF--stem cell factor [0101] SSC--side scatter [0102] WT--wild
type
Definitions
[0103] In describing and claiming the invention, the following
terminology will be used in accordance with the definitions set
forth below.
[0104] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0105] The term "about," as used herein, means approximately, in
the region of, roughly, or around. When the term "about" is used in
conjunction with a numerical range, it modifies that range by
extending the boundaries above and below the numerical values set
forth. For example, in one aspect, the term "about" is used herein
to modify a numerical value above and below the stated value by a
variance of 20%.
[0106] The terms "additional therapeutically active compound" or
"additional therapeutic agent", as used in the context of the
present invention, refers to the use or administration of a
compound for an additional therapeutic use for a particular injury,
disease, or disorder being treated. Such a compound, for example,
could include one being used to treat an unrelated disease or
disorder, or a disease or disorder which may not be responsive to
the primary treatment for the injury, disease or disorder being
treated.
[0107] As used herein, the term "adjuvant" refers to a substance
that elicits an enhanced immune response when used in combination
with a specific antigen.
[0108] As use herein, the terms "administration of" and or
"administering" a compound should be understood to mean providing a
compound of the invention or a prodrug of a compound of the
invention to a subject in need of treatment.
[0109] As used herein, the term "aerosol" refers to suspension in
the air. In particular, aerosol refers to the particlization or
atomization of a formulation of the invention and its suspension in
the air.
[0110] As used herein, an "agonist" is a composition of matter
which, when administered to a mammal such as a human, enhances or
extends a biological activity attributable to the level or presence
of a target compound or molecule of interest in the subject.
[0111] As used herein, "alleviating a disease or disorder symptom,"
means reducing the severity of the symptom or the frequency with
which such a symptom is experienced by a subject, or both.
[0112] As used herein, amino acids are represented by the full name
thereof, by the three letter code corresponding thereto, or by the
one-letter code corresponding thereto, as indicated in the
following table:
TABLE-US-00002 Full Name Three-Letter Code One-Letter Code Aspartic
Acid Asp D Glutamic Acid Glu E Lysine Lys K Arginine Arg R
Histidine His H Tyrosine Tyr Y Cysteine Cys C Asparagine Asn N
Glutamine Gln Q Serine Ser S Threonine Thr T Glycine Gly G Alanine
Ala A Valine Val V Leucine Leu L Isoleucine Ile I Methionine Met M
Proline Pro P Phenylalanine Phe F Tryptophan Trp W
[0113] The term "amino acid" is used interchangeably with "amino
acid residue," and may refer to a free amino acid and to an amino
acid residue of a peptide. It will be apparent from the context in
which the term is used whether it refers to a free amino acid or a
residue of a peptide.
[0114] Amino acids have the following general structure:
##STR00001##
[0115] Amino acids may be classified into seven groups on the basis
of the side chain R: (1) aliphatic side chains, (2) side chains
containing a hydroxylic (OH) group, (3) side chains containing
sulfur atoms, (4) side chains containing an acidic or amide group,
(5) side chains containing a basic group, (6) side chains
containing an aromatic ring, and (7) proline, an imino acid in
which the side chain is fused to the amino group.
[0116] The nomenclature used to describe the peptide compounds of
the present invention follows the conventional practice wherein the
amino group is presented to the left and the carboxy group to the
right of each amino acid residue. In the formulae representing
selected specific embodiments of the present invention, the
amino-and carboxy-terminal groups, although not specifically shown,
will be understood to be in the form they would assume at
physiologic pH values, unless otherwise specified.
[0117] The term "basic" or "positively charged" amino acid as used
herein, refers to amino acids in which the R groups have a net
positive charge at pH 7.0, and include, but are not limited to, the
standard amino acids lysine, arginine, and histidine.
[0118] As used herein, an "analog", or "analogue" of a chemical
compound is a compound that, by way of example, resembles another
in structure but is not necessarily an isomer (e.g., 5-fluorouracil
is an analog of thymine).
[0119] An "antagonist" is a composition of matter which when
administered to a mammal such as a human, inhibits a biological
activity attributable to the level or presence of a compound or
molecule of interest in the subject.
[0120] The term "antibody," as used herein, refers to an
immunoglobulin molecule which is able to specifically bind to a
specific epitope on an antigen. Antibodies can be intact
immunoglobulins derived from natural sources or from recombinant
sources and can be immunoreactive portions of intact
immunoglobulins. Antibodies are typically tetramers of
immunoglobulin molecules. The antibodies in the present invention
may exist in a variety of forms including, for example, polyclonal
antibodies, monoclonal antibodies, Fv, Fab and F(ab).sub.2, as well
as single chain antibodies and humanized antibodies.
[0121] An "antibody heavy chain," as used herein, refers to the
larger of the two types of polypeptide chains present in all
antibody molecules.
[0122] An "antibody light chain," as used herein, refers to the
smaller of the two types of polypeptide chains present in all
antibody molecules.
[0123] By the term "synthetic antibody" as used herein, is meant an
antibody which is generated using recombinant DNA technology, such
as, for example, an antibody expressed by a bacteriophage as
described herein. The term should also be construed to mean an
antibody which has been generated by the synthesis of a DNA
molecule encoding the antibody and which DNA molecule expresses an
antibody protein, or an amino acid sequence specifying the
antibody, wherein the DNA or amino acid sequence has been obtained
using synthetic DNA or amino acid sequence technology which is
available and well known in the art.
[0124] The term "antigen" as used herein is defined as a molecule
that provokes an immune response. This immune response may involve
either antibody production, or the activation of specific
immunologically-competent cells, or both. An antigen can be derived
from organisms, subunits of proteins/antigens, killed or
inactivated whole cells or lysates.
[0125] The term "antigenic determinant" as used herein refers to
that portion of an antigen that makes contact with a particular
antibody (i.e., an epitope). When a protein or fragment of a
protein, or chemical moiety is used to immunize a host animal,
numerous regions of the antigen may induce the production of
antibodies that bind specifically to a given region or
three-dimensional structure on the protein; these regions or
structures are referred to as antigenic determinants. An antigenic
determinant may compete with the intact antigen (i.e., the
"immunogen" used to elicit the immune response) for binding to an
antibody.
[0126] The term "antimicrobial agents" as used herein refers to any
naturally-occurring, synthetic, or semi-synthetic compound or
composition or mixture thereof, which is safe for human or animal
use as practiced in the methods of this invention, and is effective
in killing or substantially inhibiting the growth of microbes.
"Antimicrobial" as used herein, includes antibacterial, antifungal,
and antiviral agents.
[0127] As used herein, the term "antisense oligonucleotide" or
antisense nucleic acid means a nucleic acid polymer, at least a
portion of which is complementary to a nucleic acid which is
present in a normal cell or in an affected cell. "Antisense" refers
particularly to the nucleic acid sequence of the non-coding strand
of a double stranded DNA molecule encoding a protein, or to a
sequence which is substantially homologous to the non-coding
strand. As defined herein, an antisense sequence is complementary
to the sequence of a double stranded DNA molecule encoding a
protein. It is not necessary that the antisense sequence be
complementary solely to the coding portion of the coding strand of
the DNA molecule. The antisense sequence may be complementary to
regulatory sequences specified on the coding strand of a DNA
molecule encoding a protein, which regulatory sequences control
expression of the coding sequences. The antisense oligonucleotides
of the invention include, but are not limited to, phosphorothioate
oligonucleotides and other modifications of oligonucleotides.
[0128] An "aptamer" is a compound that is selected in vitro to bind
preferentially to another compound (for example, the identified
proteins herein). Often, aptamers are nucleic acids or peptides
because random sequences can be readily generated from nucleotides
or amino acids (both naturally occurring or synthetically made) in
large numbers but of course they need not be limited to these.
[0129] The term "binding" refers to the adherence of molecules to
one another, such as, but not limited to, enzymes to substrates,
ligands to receptors, antibodies to antigens, DNA binding domains
of proteins to DNA, and DNA or RNA strands to complementary
strands.
[0130] "Binding partner," as used herein, refers to a molecule
capable of binding to another molecule.
[0131] The term "biocompatible", as used herein, refers to a
material that does not elicit a substantial detrimental response in
the host.
[0132] As used herein, the term "biologically active fragments" or
"bioactive fragment" of the polypeptides encompasses natural or
synthetic portions of the full-length protein that are capable of
specific binding to their natural ligand or of performing the
function of the protein.
[0133] The term "biological sample," as used herein, refers to
samples obtained from a subject, including, but not limited to,
sputum, CSF, blood, serum, plasma, gastric aspirates, throat swabs,
skin, hair, tissue, blood, plasma, serum, cells, sweat and
urine.
[0134] "Blood components" refers to main/important components such
as red cells, white cells, platelets, and plasma and to other
components that can be derived such as serum.
[0135] As used herein, the term "carrier molecule" refers to any
molecule that is chemically conjugated to the antigen of interest
that enables an immune response resulting in antibodies specific to
the native antigen.
[0136] The term "cell surface protein" means a protein found where
at least part of the protein is exposed at the outer aspect of the
cell membrane. Examples include growth factor receptors.
[0137] As used herein, the term "chemically conjugated," or
"conjugating chemically" refers to linking the antigen to the
carrier molecule. This linking can occur on the genetic level using
recombinant technology, wherein a hybrid protein may be produced
containing the amino acid sequences, or portions thereof, of both
the antigen and the carrier molecule. This hybrid protein is
produced by an oligonucleotide sequence encoding both the antigen
and the carrier molecule, or portions thereof. This linking also
includes covalent bonds created between the antigen and the carrier
protein using other chemical reactions, such as, but not limited to
glutaraldehyde reactions. Covalent bonds may also be created using
a third molecule bridging the antigen to the carrier molecule.
These cross-linkers are able to react with groups, such as but not
limited to, primary amines, sulfhydryls, carbonyls, carbohydrates,
or carboxylic acids, on the antigen and the carrier molecule.
Chemical conjugation also includes non-covalent linkage between the
antigen and the carrier molecule.
[0138] A "coding region" of a gene consists of the nucleotide
residues of the coding strand of the gene and the nucleotides of
the non-coding strand of the gene which are homologous with or
complementary to, respectively, the coding region of an mRNA
molecule which is produced by transcription of the gene.
[0139] The term "competitive sequence" refers to a peptide or a
modification, fragment, derivative, or homolog thereof that
competes with another peptide for its cognate binding site.
[0140] "Complementary" as used herein refers to the broad concept
of subunit sequence complementarity between two nucleic acids,
e.g., two DNA molecules. When a nucleotide position in both of the
molecules is occupied by nucleotides normally capable of base
pairing with each other, then the nucleic acids are considered to
be complementary to each other at this position. Thus, two nucleic
acids are complementary to each other when a substantial number (at
least 50%) of corresponding positions in each of the molecules are
occupied by nucleotides which normally base pair with each other
(e.g., A:T and G:C nucleotide pairs). Thus, it is known that an
adenine residue of a first nucleic acid region is capable of
forming specific hydrogen bonds ("base pairing") with a residue of
a second nucleic acid region which is antiparallel to the first
region if the residue is thymine or uracil. Similarly, it is known
that a cytosine residue of a first nucleic acid strand is capable
of base pairing with a residue of a second nucleic acid strand
which is antiparallel to the first strand if the residue is
guanine. A first region of a nucleic acid is complementary to a
second region of the same or a different nucleic acid if, when the
two regions are arranged in an antiparallel fashion, at least one
nucleotide residue of the first region is capable of base pairing
with a residue of the second region. Preferably, the first region
comprises a first portion and the second region comprises a second
portion, whereby, when the first and second portions are arranged
in an antiparallel fashion, at least about 50%, and preferably at
least about 75%, at least about 90%, or at least about 95% of the
nucleotide residues of the first portion are capable of base
pairing with nucleotide residues in the second portion. More
preferably, all nucleotide residues of the first portion are
capable of base pairing with nucleotide residues in the second
portion.
[0141] A "compound," as used herein, refers to any type of
substance or agent that is commonly considered a drug, or a
candidate for use as a drug, as well as combinations and mixtures
of the above, as well as to biologics. When referring to a compound
of the invention, and unless otherwise specified, the term
"compound" is intended to encompass not only the specified
molecular entity but also its pharmaceutically acceptable,
pharmacologically active analogs, including, but not limited to,
salts, polymorphs, esters, amides, prodrugs, adducts, conjugates,
active metabolites, and the like, where such modifications to the
molecular entity are appropriate.
[0142] As used herein, the term "conservative amino acid
substitution" is defined herein as an amino acid exchange within
one of the following five groups:
[0143] I. Small aliphatic, nonpolar or slightly polar residues:
[0144] Ala, Ser, Thr, Pro, Gly;
[0145] II. Polar, negatively charged residues and their amides:
[0146] Asp, Asn, Glu, Gln;
[0147] III. Polar, positively charged residues: [0148] His, Arg,
Lys;
[0149] IV. Large, aliphatic, nonpolar residues: [0150] Met Leu,
Ile, Val, Cys
[0151] V. Large, aromatic residues: [0152] Phe, Tyr, Trp
[0153] A "control" cell is a cell having the same cell type as a
test cell. The control cell may, for example, be examined at
precisely or nearly the same time the test cell is examined. The
control cell may also, for example, be examined at a time distant
from the time at which the test cell is examined, and the results
of the examination of the control cell may be recorded so that the
recorded results may be compared with results obtained by
examination of a test cell.
[0154] A "test" cell is a cell being examined.
[0155] The term "delivery vehicle" refers to any kind of device or
material which can be used to deliver compounds in vivo or can be
added to a composition comprising compounds administered to a plant
or animal. This includes, but is not limited to, implantable
devices, aggregates of cells, matrix materials, gels, etc.
[0156] As used herein, a "derivative" of a compound refers to a
chemical compound that may be produced from another compound of
similar structure in one or more steps, as in replacement of H by
an alkyl, acyl, or amino group.
[0157] As used herein, a "detectable marker" or a "reporter
molecule" is an atom or a molecule that permits the specific
detection of a compound comprising the marker in the presence of
similar compounds without a marker. Detectable markers or reporter
molecules include, e.g., radioactive isotopes, antigenic
determinants, enzymes, nucleic acids available for hybridization,
chromophores, fluorophores, chemiluminescent molecules,
electrochemically detectable molecules, and molecules that provide
for altered fluorescence-polarization or altered
light-scattering.
[0158] The term "directed against CD24" means that the compound
being recited, whether a small molecule, drug, prodrug, or an
antibody, or a biologically active fragment or homolog thereof,
binds to and/or activates CD24 or stimulates its activity, or all
of the above.
[0159] A "disease" is a state of health of an animal wherein the
animal cannot maintain homeostasis, and wherein if the disease is
not ameliorated then the animal's health continues to
deteriorate.
[0160] In contrast, a "disorder" in an animal is a state of health
in which the animal is able to maintain homeostasis, but in which
the animal's state of health is less favorable than it would be in
the absence of the disorder. Left untreated, a disorder does not
necessarily cause a further decrease in the animal's state of
health.
[0161] As used herein, the term "domain" refers to a part of a
molecule or structure that shares common physicochemical features,
such as, but not limited to, hydrophobic, polar, globular and
helical domains or properties such as ligand binding, signal
transduction, cell penetration and the like. Specific examples of
binding domains include, but are not limited to, DNA binding
domains and ATP binding domains.
[0162] As used herein, an "effective amount" or "therapeutically
effective amount" means an amount sufficient to produce a selected
effect, such as alleviating symptoms of a disease or disorder. In
the context of administering compounds in the form of a
combination, such as multiple compounds, the amount of each
compound, when administered in combination with another
compound(s), may be different from when that compound is
administered alone. Thus, an effective amount of a combination of
compounds refers collectively to the combination as a whole,
although the actual amounts of each compound may vary. The term
"more effective" means that the selected effect is alleviated to a
greater extent by one treatment relative to the second treatment to
which it is being compared.
[0163] As used herein, the term "effector domain" refers to a
domain capable of directly interacting with an effector molecule,
chemical, or structure in the cytoplasm which is capable of
regulating a biochemical pathway.
[0164] "Encoding" refers to the inherent property of specific
sequences of nucleotides in a polynucleotide, such as a gene, a
cDNA, or an mRNA, to serve as templates for synthesis of other
polymers and macromolecules in biological processes having either a
defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a
defined sequence of amino acids and the biological properties
resulting therefrom. Thus, a gene encodes a protein if
transcription and translation of mRNA corresponding to that gene
produces the protein in a cell or other biological system. Both the
coding strand, the nucleotide sequence of which is identical to the
mRNA sequence and is usually provided in sequence listings, and the
non-coding strand, used as the template for transcription of a gene
or cDNA, can be referred to as encoding the protein or other
product of that gene or cDNA.
[0165] An "enhancer" is a DNA regulatory element that can increase
the efficiency of transcription, regardless of the distance or
orientation of the enhancer relative to the start site of
transcription.
[0166] The term "epitope" as used herein is defined as small
chemical groups on the antigen molecule that can elicit and react
with an antibody. An antigen can have one or more epitopes. Most
antigens have many epitopes; i.e., they are multivalent. In
general, an epitope is roughly five amino acids or sugars in size.
One skilled in the art understands that generally the overall
three-dimensional structure, rather than the specific linear
sequence of the molecule, is the main criterion of antigenic
specificity.
[0167] The term "erythropoietin" as used herein includes EPO of
every origin, especially human or animal EPO. The term used herein
encompasses not only the naturally occurring, that is wild-type
forms of EPO, but also its homologues, fragments, derivatives,
analogs, modifications, muteins, mutants or others, as long as they
show the biological effects of wild-type erythropoietin or an
activity described herein. It also includes synthetic EPO.
[0168] As used herein, an "essentially pure" preparation of a
particular protein or peptide is a preparation wherein at least
about 95%, and preferably at least about 99%, by weight, of the
protein or peptide in the preparation is the particular protein or
peptide.
[0169] As used in the specification and the appended claims, the
terms "for example," "for instance," "such as," "including" and the
like are meant to introduce examples that further clarify more
general subject matter. Unless otherwise specified, these examples
are provided only as an aid for understanding the invention, and
are not meant to be limiting in any fashion.
[0170] The terms "formula" and "structure" are used interchangeably
herein.
[0171] A "fragment" or "segment" is a portion of an amino acid
sequence, comprising at least one amino acid, or a portion of a
nucleic acid sequence comprising at least one nucleotide. The terms
"fragment" and "segment" are used interchangeably herein.
[0172] As used herein, the term "fragment," as applied to a protein
or peptide, can ordinarily be at least about 3-15 amino acids in
length, at least about 15-25 amino acids, at least about 25-50
amino acids in length, at least about 50-75 amino acids in length,
at least about 75-100 amino acids in length, and greater than 100
amino acids in length.
[0173] As used herein, the term "fragment" as applied to a nucleic
acid, may ordinarily be at least about 20 nucleotides in length,
typically, at least about 50 nucleotides, more typically, from
about 50 to about 100 nucleotides, preferably, at least about 100
to about 200 nucleotides, even more preferably, at least about 200
nucleotides to about 300 nucleotides, yet even more preferably, at
least about 300 to about 350, even more preferably, at least about
350 nucleotides to about 500 nucleotides, yet even more preferably,
at least about 500 to about 600, even more preferably, at least
about 600 nucleotides to about 620 nucleotides, yet even more
preferably, at least about 620 to about 650, and most preferably,
the nucleic acid fragment will be greater than about 650
nucleotides in length.
[0174] As used herein, a "functional" molecule is a molecule in a
form in which it exhibits a property or activity by which it is
characterized. A functional enzyme, for example, is one that
exhibits the characteristic catalytic activity by which the enzyme
is characterized.
[0175] "Homologous" as used herein, refers to the subunit sequence
similarity between two polymeric molecules, e.g., between two
nucleic acid molecules, e.g., two DNA molecules or two RNA
molecules, or between two polypeptide molecules. When a subunit
position in both of the two molecules is occupied by the same
monomeric subunit, e.g., if a position in each of two DNA molecules
is occupied by adenine, then they are homologous at that position.
The homology between two sequences is a direct function of the
number of matching or homologous positions, e.g., if half (e.g.,
five positions in a polymer ten subunits in length) of the
positions in two compound sequences are homologous then the two
sequences are 50% homologous, if 90% of the positions, e.g., 9 of
10, are matched or homologous, the two sequences share 90%
homology. By way of example, the DNA sequences 3'ATTGCC5' and
3'TATGGC share 50% homology.
[0176] As used herein, "homology" is used synonymously with
"identity."
[0177] The determination of percent identity between two nucleotide
or amino acid sequences can be accomplished using a mathematical
algorithm. For example, a mathematical algorithm useful for
comparing two sequences is the algorithm of Karlin and Altschul
(1990, Proc. Natl. Acad. Sci. USA 87:2264-2268), modified as in
Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA
90:5873-5877). This algorithm is incorporated into the NBLAST and
XBLAST programs of Altschul, et al. (1990, J. Mol. Biol.
215:403-410), and can be accessed, for example at the National
Center for Biotechnology Information (NCBI) world wide web site.
BLAST nucleotide searches can be performed with the NBLAST program
(designated "blastn" at the NCBI web site), using the following
parameters: gap penalty=5; gap extension penalty=2; mismatch
penalty=3; match reward=1; expectation value 10.0; and word size=11
to obtain nucleotide sequences homologous to a nucleic acid
described herein. BLAST protein searches can be performed with the
XBLAST program (designated "blastn" at the NCBI web site) or the
NCBI "blastp" program, using the following parameters: expectation
value 10.0, BLOSUM62 scoring matrix to obtain amino acid sequences
homologous to a protein molecule described herein. To obtain gapped
alignments for comparison purposes, Gapped BLAST can be utilized as
described in Altschul et al. (1997, Nucleic Acids Res.
25:3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used to
perform an iterated search which detects distant relationships
between molecules (Id.) and relationships between molecules which
share a common pattern. When utilizing BLAST, Gapped BLAST,
PSI-Blast, and PHI-Blast programs, the default parameters of the
respective programs (e.g., XBLAST and NBLAST) can be used.
[0178] The percent identity between two sequences can be determined
using techniques similar to those described above, with or without
allowing gaps. In calculating percent identity, typically exact
matches are counted.
[0179] By the term "immunizing a subject against an antigen" is
meant administering to the subject a composition, a protein
complex, a DNA encoding a protein complex, an antibody or a DNA
encoding an antibody, which elicits an immune response in the
subject, and, for example, provides protection to the subject
against a disease caused by the antigen or which prevents the
function of the antigen.
[0180] The term "immunologically active fragments thereof" will
generally be understood in the art to refer to a fragment of a
polypeptide antigen comprising at least an epitope, which means
that the fragment at least comprises 4 contiguous amino acids from
the sequence of the polypeptide antigen.
[0181] As used herein, the term "induction of apoptosis" means a
process by which a cell is affected in such a way that it begins
the process of programmed cell death, which is characterized by the
fragmentation of the cell into membrane-bound particles that are
subsequently eliminated by the process of phagocytosis.
[0182] As used herein, the term "inhaler" refers both to devices
for nasal and pulmonary administration of a drug, e.g., in
solution, powder and the like. For example, the term "inhaler" is
intended to encompass a propellant driven inhaler, such as is used
to administer antihistamine for acute asthma attacks, and plastic
spray bottles, such as are used to administer decongestants.
[0183] The term "inhibit," as used herein, refers to the ability of
a compound of the invention to reduce or impede a described
function, such as having inhibitory sodium channel activity.
Preferably, inhibition is by at least 10%, more preferably by at
least 25%, even more preferably by at least 50%, and most
preferably, the function is inhibited by at least 75%. The terms
"inhibit", "reduce", and "block" are used interchangeably
herein.
[0184] The term "inhibit a complex," as used herein, refers to
inhibiting the formation of a complex or interaction of two or more
proteins, as well as inhibiting the function or activity of the
complex. The term also encompasses disrupting a formed complex.
However, the term does not imply that each and every one of these
functions must be inhibited at the same time.
[0185] The term "inhibit a protein," as used herein, refers to any
method or technique which inhibits protein synthesis, levels,
activity, or function, as well as methods of inhibiting the
induction or stimulation of synthesis, levels, activity, or
function of the protein of interest. The term also refers to any
metabolic or regulatory pathway which can regulate the synthesis,
levels, activity, or function of the protein of interest. The term
includes binding with other molecules and complex formation.
Therefore, the term "protein inhibitor" refers to any agent or
compound, the application of which results in the inhibition of
protein function or protein pathway function. However, the term
does not imply that each and every one of these functions must be
inhibited at the same time.
[0186] As used herein "injecting or applying" includes
administration of a compound of the invention by any number of
routes and means including, but not limited to, topical, oral,
buccal, intravenous, intramuscular, intra arterial, intramedullary,
intrathecal, intraventricular, transdermal, subcutaneous,
intraperitoneal, intranasal, enteral, topical, sublingual, vaginal,
ophthalmic, pulmonary, or rectal means.
[0187] As used herein, an "instructional material" includes a
publication, a recording, a diagram, or any other medium of
expression which can be used to communicate the usefulness of the
peptide of the invention in the kit for effecting alleviation of
the various diseases or disorders recited herein. Optionally, or
alternately, the instructional material may describe one or more
methods of alleviating the diseases or disorders in a cell or a
tissue of a mammal. The instructional material of the kit of the
invention may, for example, be affixed to a container which
contains the identified compound invention or be shipped together
with a container which contains the identified compound.
Alternatively, the instructional material may be shipped separately
from the container with the intention that the instructional
material and the compound be used cooperatively by the
recipient.
[0188] An "isolated nucleic acid" refers to a nucleic acid segment
or fragment which has been separated from sequences which flank it
in a naturally occurring state, e.g., a DNA fragment which has been
removed from the sequences which are normally adjacent to the
fragment, e.g., the sequences adjacent to the fragment in a genome
in which it naturally occurs. The term also applies to nucleic
acids which have been substantially purified from other components
which naturally accompany the nucleic acid, e.g., RNA or DNA or
proteins, which naturally accompany it in the cell. The term
therefore includes, for example, a recombinant DNA which is
incorporated into a vector, into an autonomously replicating
plasmid or virus, or into the genomic DNA of a prokaryote or
eukaryote, or which exists as a separate molecule (e.g., as a cDNA
or a genomic or cDNA fragment produced by PCR or restriction enzyme
digestion) independent of other sequences. It also includes a
recombinant DNA which is part of a hybrid gene encoding additional
polypeptide sequence.
[0189] A "ligand" is a compound that specifically binds to a target
receptor.
[0190] A "receptor" is a compound that specifically binds to a
ligand.
[0191] A ligand or a receptor (e.g., an antibody) "specifically
binds to" or "is specifically immunoreactive with" a compound when
the ligand or receptor functions in a binding reaction which is
determinative of the presence of the compound in a sample of
heterogeneous compounds. Thus, under designated assay (e.g.,
immunoassay) conditions, the ligand or receptor binds
preferentially to a particular compound and does not bind in a
significant amount to other compounds present in the sample. For
example, a polynucleotide specifically binds under hybridization
conditions to a compound polynucleotide comprising a complementary
sequence; an antibody specifically binds under immunoassay
conditions to an antigen bearing an epitope against which the
antibody was raised. A variety of immunoassay formats may be used
to select antibodies specifically immunoreactive with a particular
protein. For example, solid-phase ELISA immunoassays are routinely
used to select monoclonal antibodies specifically immunoreactive
with a protein. See Harlow and Lane (1988, Antibodies, A Laboratory
Manual, Cold Spring Harbor Publications, New York) for a
description of immunoassay formats and conditions that can be used
to determine specific immunoreactivity.
[0192] As used herein, the term "linkage" refers to a connection
between two groups. The connection can be either covalent or
non-covalent, including but not limited to ionic bonds, hydrogen
bonding, and hydrophobic/hydrophilic interactions.
[0193] As used herein, the term "linker" refers to a molecule that
joins two other molecules either covalently or noncovalently, e.g.,
through ionic or hydrogen bonds or van der Waals interactions,
e.g., a nucleic acid molecule that hybridizes to one complementary
sequence at the 5' end and to another complementary sequence at the
3' end, thus joining two non-complementary sequences.
[0194] The term "measuring the level of expression" or "determining
the level of expression" as used herein refers to any measure or
assay which can be used to correlate the results of the assay with
the level of expression of a gene or protein of interest. Such
assays include measuring the level of mRNA, protein levels, etc.
and can be performed by assays such as northern and western blot
analyses, binding assays, immunoblots, etc. The level of expression
can include rates of expression and can be measured in terms of the
actual amount of an mRNA or protein present. Such assays are
coupled with processes or systems to store and process information
and to help quantify levels, signals, etc. and to digitize the
information for use in comparing levels.
[0195] The term "modulate", as used herein, refers to changing the
level of an activity, function, or process. The term "modulate"
encompasses both inhibiting and stimulating an activity, function,
or process.
[0196] The term "nucleic acid" typically refers to large
polynucleotides. By "nucleic acid" is meant any nucleic acid,
whether composed of deoxyribonucleosides or ribonucleosides, and
whether composed of phosphodiester linkages or modified linkages
such as phosphotriester, phosphoramidate, siloxane, carbonate,
carboxymethylester, acetamidate, carbamate, thioether, bridged
phosphoramidate, bridged methylene phosphonate, bridged
phosphoramidate, bridged phosphoramidate, bridged methylene
phosphonate, phosphorothioate, methylphosphonate,
phosphorodithioate, bridged phosphorothioate or sulfone linkages,
and combinations of such linkages. The term nucleic acid also
specifically includes nucleic acids composed of bases other than
the five biologically occurring bases (adenine, guanine, thymine,
cytosine and uracil).
[0197] As used herein, the term "nucleic acid" encompasses RNA as
well as single and double-stranded DNA and cDNA. Furthermore, the
terms, "nucleic acid," "DNA," "RNA" and similar terms also include
nucleic acid analogs, i.e. analogs having other than a
phosphodiester backbone. For example, the so-called "peptide
nucleic acids," which are known in the art and have peptide bonds
instead of phosphodiester bonds in the backbone, are considered
within the scope of the present invention. By "nucleic acid" is
meant any nucleic acid, whether composed of deoxyribonucleosides or
ribonucleosides, and whether composed of phosphodiester linkages or
modified linkages such as phosphotriester, phosphoramidate,
siloxane, carbonate, carboxymethylester, acetamidate, carbamate,
thioether, bridged phosphoramidate, bridged methylene phosphonate,
bridged phosphoramidate, bridged phosphoramidate, bridged methylene
phosphonate, phosphorothioate, methylphosphonate,
phosphorodithioate, bridged phosphorothioate or sulfone linkages,
and combinations of such linkages. The term nucleic acid also
specifically includes nucleic acids composed of bases other than
the five biologically occurring bases (adenine, guanine, thymine,
cytosine and uracil). Conventional notation is used herein to
describe polynucleotide sequences: the left-hand end of a
single-stranded polynucleotide sequence is the 5'-end; the
left-hand direction of a double-stranded polynucleotide sequence is
referred to as the 5'-direction. The direction of 5' to 3' addition
of nucleotides to nascent RNA transcripts is referred to as the
transcription direction. The DNA strand having the same sequence as
an mRNA is referred to as the "coding strand"; sequences on the DNA
strand which are located 5' to a reference point on the DNA are
referred to as "upstream sequences"; sequences on the DNA strand
which are 3' to a reference point on the DNA are referred to as
"downstream sequences."
[0198] The term "nucleic acid construct," as used herein,
encompasses DNA and RNA sequences encoding the particular gene or
gene fragment desired, whether obtained by genomic or synthetic
methods.
[0199] Unless otherwise specified, a "nucleotide sequence encoding
an amino acid sequence" includes all nucleotide sequences that are
degenerate versions of each other and that encode the same amino
acid sequence. Nucleotide sequences that encode proteins and RNA
may include introns.
[0200] The term "oligonucleotide" typically refers to short
polynucleotides, generally, no greater than about 50 nucleotides.
It will be understood that when a nucleotide sequence is
represented by a DNA sequence (i.e., A, T, G, C), this also
includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces
"T."
[0201] By describing two polynucleotides as "operably linked" is
meant that a single-stranded or double-stranded nucleic acid moiety
comprises the two polynucleotides arranged within the nucleic acid
moiety in such a manner that at least one of the two
polynucleotides is able to exert a physiological effect by which it
is characterized upon the other. By way of example, a promoter
operably linked to the coding region of a gene is able to promote
transcription of the coding region.
[0202] As used herein, "parenteral administration" of a
pharmaceutical composition includes any route of administration
characterized by physical breaching of a tissue of a subject and
administration of the pharmaceutical composition through the breach
in the tissue. Parenteral administration thus includes, but is not
limited to, administration of a pharmaceutical composition by
injection of the composition, by application of the composition
through a surgical incision, by application of the composition
through a tissue-penetrating non-surgical wound, and the like. In
particular, parenteral administration is contemplated to include,
but is not limited to, subcutaneous, intraperitoneal,
intramuscular, intrasternal injection, and kidney dialytic infusion
techniques.
[0203] The term "per application" as used herein refers to
administration of a compositions, drug, or compound to a
subject.
[0204] The term "pharmaceutical composition" shall mean a
composition comprising at least one active ingredient, whereby the
composition is amenable to investigation for a specified,
efficacious outcome in a mammal (for example, without limitation, a
human). Those of ordinary skill in the art will understand and
appreciate the techniques appropriate for determining whether an
active ingredient has a desired efficacious outcome based upon the
needs of the artisan.
[0205] As used herein, the term "pharmaceutically acceptable
carrier" includes any of the standard pharmaceutical carriers, such
as a phosphate buffered saline solution, water, emulsions such as
an oil/water or water/oil emulsion, and various types of wetting
agents. The term also encompasses any of the agents approved by a
regulatory agency of the US Federal government or listed in the US
Pharmacopeia for use in animals, including humans.
[0206] As used herein, the term "physiologically acceptable" ester
or salt means an ester or salt form of the active ingredient which
is compatible with any other ingredients of the pharmaceutical
composition, which is not deleterious to the subject to which the
composition is to be administered.
[0207] "Plurality" means at least two.
[0208] A "polynucleotide" means a single strand or parallel and
anti-parallel strands of a nucleic acid. Thus, a polynucleotide may
be either a single-stranded or a double-stranded nucleic acid.
[0209] "Polypeptide" refers to a polymer composed of amino acid
residues, related naturally occurring structural variants, and
synthetic non-naturally occurring analogs thereof linked via
peptide bonds, related naturally occurring structural variants, and
synthetic non-naturally occurring analogs thereof.
[0210] "Synthetic peptides or polypeptides" means a non-naturally
occurring peptide or polypeptide. Synthetic peptides or
polypeptides can be synthesized, for example, using an automated
polypeptide synthesizer. Various solid phase peptide synthesis
methods are known to those of skill in the art.
[0211] By "presensitization" is meant pre-administration of at
least one innate immune system stimulator prior to challenge with
an agent. This is sometimes referred to as induction of
tolerance.
[0212] The term "prevent," as used herein, means to stop something
from happening, or taking advance measures against something
possible or probable from happening. In the context of medicine,
"prevention" generally refers to action taken to decrease the
chance of getting a disease or condition.
[0213] A "preventive" or "prophylactic" treatment is a treatment
administered to a subject who does not exhibit signs, or exhibits
only early signs, of a disease or disorder. A prophylactic or
preventative treatment is administered for the purpose of
decreasing the risk of developing pathology associated with
developing the disease or disorder.
[0214] "Primer" refers to a polynucleotide that is capable of
specifically hybridizing to a designated polynucleotide template
and providing a point of initiation for synthesis of a
complementary polynucleotide. Such synthesis occurs when the
polynucleotide primer is placed under conditions in which synthesis
is induced, i.e., in the presence of nucleotides, a complementary
polynucleotide template, and an agent for polymerization such as
DNA polymerase. A primer is typically single-stranded, but may be
double-stranded. Primers are typically deoxyribonucleic acids, but
a wide variety of synthetic and naturally occurring primers are
useful for many applications. A primer is complementary to the
template to which it is designed to hybridize to serve as a site
for the initiation of synthesis, but need not reflect the exact
sequence of the template. In such a case, specific hybridization of
the primer to the template depends on the stringency of the
hybridization conditions. Primers can be labeled with, e.g.,
chromogenic, radioactive, or fluorescent moieties and used as
detectable moieties.
[0215] A "prodrug" refers to an agent that is converted into the
parent drug in vivo. Prodrugs are often useful because, in some
situations, they may be easier to administer than the parent drug.
They may, for instance, be bioavailable by oral administration
whereas the parent is not. The prodrug may also have improved
solubility in pharmaceutical compositions over the parent drug, or
may demonstrate increased palatability or be easier to
formulate.
[0216] As used herein, the term "promoter/regulatory sequence"
means a nucleic acid sequence which is required for expression of a
gene product operably linked to the promoter/regulator sequence. In
some instances, this sequence may be the core promoter sequence and
in other instances, this sequence may also include an enhancer
sequence and other regulatory elements which are required for
expression of the gene product. The promoter/regulatory sequence
may, for example, be one which expresses the gene product in a
tissue specific manner.
[0217] A "constitutive" promoter is a promoter which drives
expression of a gene to which it is operably linked, in a constant
manner in a cell. By way of example, promoters which drive
expression of cellular housekeeping genes are considered to be
constitutive promoters.
[0218] An "inducible" promoter is a nucleotide sequence which, when
operably linked with a polynucleotide which encodes or specifies a
gene product, causes the gene product to be produced in a living
cell substantially only when an inducer which corresponds to the
promoter is present in the cell.
[0219] A "tissue-specific" promoter is a nucleotide sequence which,
when operably linked with a polynucleotide which encodes or
specifies a gene product, causes the gene product to be produced in
a living cell substantially only if the cell is a cell of the
tissue type corresponding to the promoter.
[0220] A "prophylactic" treatment is a treatment administered to a
subject who does not exhibit signs of a disease or exhibits only
early signs of the disease for the purpose of decreasing the risk
of developing pathology associated with the disease.
[0221] As used herein, "protecting group" with respect to a
terminal amino group refers to a terminal amino group of a peptide,
which terminal amino group is coupled with any of various
amino-terminal protecting groups traditionally employed in peptide
synthesis. Such protecting groups include, for example, acyl
protecting groups such as formyl, acetyl, benzoyl, trifluoroacetyl,
succinyl, and methoxysuccinyl; aromatic urethane protecting groups
such as benzyloxycarbonyl; and aliphatic urethane protecting
groups, for example, tert-butoxycarbonyl or adamantyloxycarbonyl.
See Gross and Mienhofer, eds., The Peptides, vol. 3, pp. 3-88
(Academic Press, New York, 1981) for suitable protecting
groups.
[0222] As used herein, "protecting group" with respect to a
terminal carboxy group refers to a terminal carboxyl group of a
peptide, which terminal carboxyl group is coupled with any of
various carboxyl-terminal protecting groups. Such protecting groups
include, for example, tert-butyl, benzyl or other acceptable groups
linked to the terminal carboxyl group through an ester or ether
bond.
[0223] The term "protein" typically refers to large polypeptides.
Conventional notation is used herein to portray polypeptide
sequences: the left-hand end of a polypeptide sequence is the
amino-terminus; the right-hand end of a polypeptide sequence is the
carboxyl-terminus.
[0224] The term "protein regulatory pathway", as used herein,
refers to both the upstream regulatory pathway which regulates a
protein, as well as the downstream events which that protein
regulates. Such regulation includes, but is not limited to,
transcription, translation, levels, activity, posttranslational
modification, and function of the protein of interest, as well as
the downstream events which the protein regulates.
[0225] The terms "protein pathway" and "protein regulatory pathway"
are used interchangeably herein.
[0226] As used herein, the term "purified" and like terms relate to
an enrichment of a molecule or compound relative to other
components normally associated with the molecule or compound in a
native environment. The term "purified" does not necessarily
indicate that complete purity of the particular molecule has been
achieved during the process. A "highly purified" compound as used
herein refers to a compound that is greater than 90% pure.
[0227] The term "regulate" refers to either stimulating or
inhibiting a function or activity of interest.
[0228] As used herein, the term "purified" and like terms relate to
an enrichment of a molecule or compound relative to other
components normally associated with the molecule or compound in a
native environment. The term "purified" does not necessarily
indicate that complete purity of the particular molecule has been
achieved during the process. A "highly purified" compound as used
herein refers to a compound that is greater than 90% pure. In
particular, purified sperm cell DNA refers to DNA that does not
produce significant detectable levels of non-sperm cell DNA upon
PCR amplification of the purified sperm cell DNA and subsequent
analysis of that amplified DNA. A "significant detectable level" is
an amount of contaminate that would be visible in the presented
data and would need to be addressed/explained during analysis of
the forensic evidence.
[0229] "Recombinant polynucleotide" refers to a polynucleotide
having sequences that are not naturally joined together. An
amplified or assembled recombinant polynucleotide may be included
in a suitable vector, and the vector can be used to transform a
suitable host cell.
[0230] A recombinant polynucleotide may serve a non-coding function
(e.g., promoter, origin of replication, ribosome-binding site,
etc.) as well.
[0231] A host cell that comprises a recombinant polynucleotide is
referred to as a "recombinant host cell." A gene which is expressed
in a recombinant host cell wherein the gene comprises a recombinant
polynucleotide, produces a "recombinant polypeptide."
[0232] A "recombinant polypeptide" is one which is produced upon
expression of a recombinant polynucleotide.
[0233] A "receptor" is a compound that specifically binds to a
ligand.
[0234] A "ligand" is a compound that specifically binds to a target
receptor.
[0235] A "recombinant cell" is a cell that comprises a transgene.
Such a cell may be a eukaryotic or a prokaryotic cell. Also, the
transgenic cell encompasses, but is not limited to, an embryonic
stem cell comprising the transgene, a cell obtained from a chimeric
mammal derived from a transgenic embryonic stem cell where the cell
comprises the transgene, a cell obtained from a transgenic mammal,
or fetal or placental tissue thereof, and a prokaryotic cell
comprising the transgene.
[0236] The term "regulate" refers to either stimulating or
inhibiting a function or activity of interest.
[0237] As used herein, the term "reporter gene" means a gene, the
expression of which can be detected using a known method. By way of
example, the Escherichia coli lacZ gene may be used as a reporter
gene in a medium because expression of the lacZ gene can be
detected using known methods by adding the chromogenic substrate
o-nitrophenyl-.beta.-galactoside to the medium (Gerhardt et al.,
eds., 1994, Methods for General and Molecular Bacteriology,
American Society for Microbiology, Washington, D.C., p. 574).
[0238] A "sample," as used herein, refers preferably to a
biological sample from a subject, including, but not limited to,
normal tissue samples, diseased tissue samples, biopsies, blood,
saliva, feces, semen, tears, and urine. A sample can also be any
other source of material obtained from a subject which contains
cells, tissues, or fluid of interest. A sample can also be obtained
from cell or tissue culture.
[0239] As used herein, the term "secondary antibody" refers to an
antibody that binds to the constant region of another antibody (the
primary antibody).
[0240] By the term "signal sequence" is meant a polynucleotide
sequence which encodes a peptide that directs the path a
polypeptide takes within a cell, i.e., it directs the cellular
processing of a polypeptide in a cell, including, but not limited
to, eventual secretion of a polypeptide from a cell. A signal
sequence is a sequence of amino acids which are typically, but not
exclusively, found at the amino terminus of a polypeptide which
targets the synthesis of the polypeptide to the endoplasmic
reticulum. In some instances, the signal peptide is proteolytically
removed from the polypeptide and is thus absent from the mature
protein.
[0241] By "small interfering RNAs (siRNAs)" is meant, inter alia,
an isolated dsRNA molecule comprised of both a sense and an
anti-sense strand. In one aspect, it is greater than 10 nucleotides
in length. siRNA also refers to a single transcript which has both
the sense and complementary antisense sequences from the target
gene, e.g., a hairpin. siRNA further includes any form of dsRNA
(proteolytically cleaved products of larger dsRNA, partially
purified RNA, essentially pure RNA, synthetic RNA, recombinantly
produced RNA) as well as altered RNA that differs from naturally
occurring RNA by the addition, deletion, substitution, and/or
alteration of one or more nucleotides.
[0242] As used herein, the term "solid support" relates to a
solvent insoluble substrate that is capable of forming linkages
(preferably covalent bonds) with various compounds. The support can
be either biological in nature, such as, without limitation, a cell
or bacteriophage particle, or synthetic, such as, without
limitation, an acrylamide derivative, agarose, cellulose, nylon,
silica, or magnetized particles.
[0243] By the term "specifically binds to", as used herein, is
meant when a compound or ligand functions in a binding reaction or
assay conditions which is determinative of the presence of the
compound in a sample of heterogeneous compounds.
[0244] The term "standard," as used herein, refers to something
used for comparison. For example, it can be a known standard agent
or compound which is administered and used for comparing results
when administering a test compound, or it can be a standard
parameter or function which is measured to obtain a control value
when measuring an effect of an agent or compound on a parameter or
function. Standard can also refer to an "internal standard", such
as an agent or compound which is added at known amounts to a sample
and is useful in determining such things as purification or
recovery rates when a sample is processed or subjected to
purification or extraction procedures before a marker of interest
is measured. Internal standards are often a purified marker of
interest which has been labeled, such as with a radioactive
isotope, allowing it to be distinguished from an endogenous
marker.
[0245] The term "stimulate CD24" refers to synthesis, levels,
activity, or function of the CD24; and for synthesis or levels can
refer to mRNA and protein. Additionally, the terms "stimulate" or
"stimulation" mean to cause an increase in synthesis, levels,
activity, or function of the molecule or cell of interest, based on
the context in which the term is used.
[0246] A "subject" of analysis, diagnosis, or treatment is an
animal. Such animals include mammals, preferably a human.
[0247] As used herein, a "subject in need thereof" is a patient,
animal, mammal, or human, who will benefit from the method of this
invention.
[0248] As used herein, a "substantially homologous amino acid
sequences" includes those amino acid sequences which have at least
about 95% homology, preferably at least about 96% homology, more
preferably at least about 97% homology, even more preferably at
least about 98% homology, and most preferably at least about 99% or
more homology to an amino acid sequence of a reference antibody
chain. Amino acid sequence similarity or identity can be computed
by using the BLASTP and TBLASTN programs which employ the BLAST
(basic local alignment search tool) 2.0.14 algorithm. The default
settings used for these programs are suitable for identifying
substantially similar amino acid sequences for purposes of the
present invention.
[0249] "Substantially homologous nucleic acid sequence" means a
nucleic acid sequence corresponding to a reference nucleic acid
sequence wherein the corresponding sequence encodes a peptide
having substantially the same structure and function as the peptide
encoded by the reference nucleic acid sequence; e.g., where only
changes in amino acids not significantly affecting the peptide
function occur. Preferably, the substantially identical nucleic
acid sequence encodes the peptide encoded by the reference nucleic
acid sequence. The percentage of identity between the substantially
similar nucleic acid sequence and the reference nucleic acid
sequence is at least about 50%, 65%, 75%, 85%, 95%, 99% or more.
Substantial identity of nucleic acid sequences can be determined by
comparing the sequence identity of two sequences, for example by
physical/chemical methods (i.e., hybridization) or by sequence
alignment via computer algorithm. Suitable nucleic acid
hybridization conditions to determine if a nucleotide sequence is
substantially similar to a reference nucleotide sequence are: 7%
sodium dodecyl sulfate SDS, 0.5 M NaPO.sub.4, 1 mM EDTA at
50.degree. C. with washing in 2.times.standard saline citrate
(SSC), 0.1% SDS at 50.degree. C.; preferably in 7% (SDS), 0.5 M
NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 1.times.SSC,
0.1% SDS at 50.degree. C.; preferably 7% SDS, 0.5 M NaPO.sub.4, 1
mM EDTA at 50.degree. C. with washing in 0.5.times.SSC, 0.1% SDS at
50.degree. C.; and more preferably in 7% SDS, 0.5 M NaPO.sub.4, 1
mM EDTA at 50.degree. C. with washing in 0.1.times.SSC, 0.1% SDS at
65.degree. C. Suitable computer algorithms to determine substantial
similarity between two nucleic acid sequences include, GCS program
package (Devereux et al., 1984 Nucl. Acids Res. 12:387), and the
BLASTN or FASTA programs (Altschul et al., 1990 Proc. Natl. Acad.
Sci. USA. 1990 87:14:5509-13; Altschul et al., J. Mol. Biol. 1990
215:3:403-10; Altschul et al., 1997 Nucleic Acids Res.
25:3389-3402). The default settings provided with these programs
are suitable for determining substantial similarity of nucleic acid
sequences for purposes of the present invention.
[0250] The term "substantially pure" describes a compound, e.g., a
protein or polypeptide that has been separated from components
which naturally accompany it. Typically, a compound is
substantially pure when at least 10%, more preferably at least 20%,
more preferably at least 50%, more preferably at least 60%, more
preferably at least 75%, more preferably at least 90%, and most
preferably at least 99% of the total material (by volume, by wet or
dry weight, or by mole percent or mole fraction) in a sample is the
compound of interest. Purity can be measured by any appropriate
method, e.g., in the case of polypeptides by column chromatography,
gel electrophoresis, or HPLC analysis. A compound, e.g., a protein,
is also substantially purified when it is essentially free of
naturally associated components or when it is separated from the
native contaminants which accompany it in its natural state.
[0251] The term "symptom," as used herein, refers to any morbid
phenomenon or departure from the normal in structure, function, or
sensation, experienced by the patient and indicative of disease. In
contrast, a "sign" is objective evidence of disease. For example, a
bloody nose is a sign. It is evident to the patient, doctor, nurse
and other observers.
[0252] A "therapeutic" treatment is a treatment administered to a
subject who exhibits signs of pathology for the purpose of
diminishing or eliminating those signs.
[0253] A "therapeutically effective amount" of a compound is that
amount of compound which is sufficient to provide a beneficial
effect to the subject to which the compound is administered.
[0254] As used herein, the term "transgene" means an exogenous
nucleic acid sequence comprising a nucleic acid which encodes a
promoter/regulatory sequence operably linked to nucleic acid which
encodes an amino acid sequence, which exogenous nucleic acid is
encoded by a transgenic mammal.
[0255] As used herein, the term "transgenic mammal" means a mammal,
the germ cells of which comprise an exogenous nucleic acid.
[0256] As used herein, a "transgenic cell" is any cell that
comprises a nucleic acid sequence that has been introduced into the
cell in a manner that allows expression of a gene encoded by the
introduced nucleic acid sequence.
[0257] The term to "treat," as used herein, means reducing the
frequency with which symptoms are experienced by a patient or
subject or administering an agent or compound to reduce the
frequency with which symptoms are experienced.
[0258] As used herein, the term "treating" can include prophylaxis
of the specific disorder or condition, or alleviation of the
symptoms associated with a specific disorder or condition and/or
preventing or eliminating said symptoms. A "prophylactic" treatment
is a treatment administered to a subject who does not exhibit signs
of a disease or exhibits only early signs of the disease for the
purpose of decreasing the risk of developing pathology associated
with the disease.
[0259] A "therapeutic" treatment is a treatment administered to a
subject who exhibits signs of pathology for the purpose of
diminishing or eliminating those signs.
[0260] A "therapeutically effective amount" of a compound is that
amount of compound which is sufficient to provide a beneficial
effect to the subject to which the compound is administered.
[0261] The term to "treat," as used herein, means reducing the
frequency with which symptoms are experienced by a patient or
subject or administering an agent or compound to reduce the
frequency with which symptoms are experienced. By the term
"vaccine," as used herein, is meant a composition which when
inoculated into a subject has the effect of stimulating an immune
response in the subject, which serves to fully or partially protect
the subject against a condition, disease or its symptoms. In one
aspect, the condition is conception. The term vaccine encompasses
prophylactic as well as therapeutic vaccines. A combination vaccine
is one which combines two or more vaccines, or two or more
compounds or agents.
[0262] A "vector" is a composition of matter which comprises an
isolated nucleic acid and which can be used to deliver the isolated
nucleic acid to the interior of a cell. Numerous vectors are known
in the art including, but not limited to, linear polynucleotides,
polynucleotides associated with ionic or amphiphilic compounds,
plasmids, and viruses. Thus, the term "vector" includes an
autonomously replicating plasmid or a virus. The term should also
be construed to include non-plasmid and non-viral compounds which
facilitate transfer or delivery of nucleic acid to cells, such as,
for example, polylysine compounds, liposomes, and the like.
Examples of viral vectors include, but are not limited to,
adenoviral vectors, adeno-associated virus vectors, retroviral
vectors, recombinant viral vectors, and the like. Examples of
non-viral vectors include, but are not limited to, liposomes,
polyamine derivatives of DNA and the like.
[0263] "Expression vector" refers to a vector comprising a
recombinant polynucleotide comprising expression control sequences
operatively linked to a nucleotide sequence to be expressed. An
expression vector comprises sufficient cis-acting elements for
expression; other elements for expression can be supplied by the
host cell or in an in vitro expression system. Expression vectors
include all those known in the art, such as cosmids, plasmids
(e.g., naked or contained in liposomes) and viruses that
incorporate the recombinant polynucleotide.
[0264] Embodiments
[0265] The present invention is directed to compositions and
methods for regulating erythropoiesis by stimulating CD24 or a CD24
pathway. One of ordinary skill in the art will appreciate that the
methods described herein can be modified in some instances and
practiced with the invention.
[0266] In some embodiments, the therapeutic methods described
herein may be used to promote stress erythropoiesis. In some
embodiments, the therapeutic methods described herein may be used
to treat conditions responding to hypoxic stress by administering
to a subject in need thereof a therapeutic composition. Conditions
responding to hypoxic stress include, but are not limited to,
anemia, blood loss, and aging. According to the present invention,
potential therapeutic agents may be screened for the ability to
stimulate splenic erythrocyte progenitor expansion in the
spleen.
[0267] In addition, potential therapeutic agents may be screened
for the ability to increase the expression level of the human stem
cell factor (hSCF) on the human splenic DC expressing the cell
surface molecule BDCA3 (CD141).
[0268] An agonist of CD24 includes any compound or group of
compounds that can stimulate stress erythropoiesis as disclosed
herein using antibodies directed against CD24. Agonists of CD24
activity can include, for example, peptides, antisense
oligonucleotides, nucleic acids encoding peptides described herein,
aptamers, antibodies, kinase inhibitors, and
drugs/agents/compounds. Many assays and methods are described
herein or are known in the art that allow one of ordinary skill in
the art to monitor whether a compound regulates the components of
the signal transduction and regulatory pathway.
[0269] In some embodiments, the present invention is a method of
enhancing the activity of erythrocytic stem cell precursors by
administering to a subject in need thereof a fragment of the
monoclonal antibody (mAb) against CD24. According to some aspects
of the present invention, compounds of interest include, but are
not limited to, the F(ab)2 fragment of the monoclonal antibody
(mAb) to CD24.
[0270] In some instances, the production of cells including, but
not limited to, erythrocytes and erythroid progenitor cells is
promoted. The production of any convenient erythroid progenitor
cells may be promoted by the method. In some instances, the
erythroid progenitor cells of interest include, but are not limited
to, basophilic erythroblasts of the spleen, in some instances
polychromatophilic erythroblasts, and in some instances, the
erythroid progenitor cells are orthochromatic erythroblasts.
[0271] According to some aspects of the invention, the CD24 being
engaged by the therapeutic compound is on a subset of splenic
DCs.
[0272] In some embodiments, the present invention is a method of
enhancing the activity of erythrocytic stem cell precursors and
promoting the production of erythrocytes and erythroid progenitor
cells by administering to a subject in need thereof an effective
amount of compound or peptide that is a downstream component of the
CD24 signal pathway.
[0273] The present invention further encompasses use of the yeast
two-hybrid system to identify regulators of the proteins and
pathways described herein. Such regulators can be drugs, compounds,
peptides, nucleic acids, etc. Such regulators can include
endogenous regulators. Generally, the yeast two-hybrid assay can
identify novel protein-protein interactions and compounds that
alter those interactions. By using a number of different proteins
as potential binding partners, it is possible to detect
interactions that were previously uncharacterized. Additionally,
the yeast two-hybrid assay can be used to characterize interactions
already known to occur. Characterization could include determining
which protein domains are responsible for the interaction, by using
truncated proteins, or under what conditions interactions take
place, by altering the intracellular environment. These assays can
also be used to screen modulators of the interactions.
[0274] This invention encompasses methods of screening compounds to
identify those compounds that act as agonists (stimulate) or
antagonists (inhibit) of the protein interactions and pathways
described herein. Screening assays for antagonist compound
candidates are designed to identify compounds that bind or complex
with the peptides described herein, or otherwise interfere with the
interaction of the peptides with other cellular proteins. Such
screening assays will include assays amenable to high-throughput
screening of chemical libraries, making them particularly suitable
for identifying small molecule drug candidates.
[0275] The assays can be performed in a variety of formats,
including protein-protein binding assays, biochemical screening
assays, immunoassays, and cell-based assays, which are well
characterized in the art.
[0276] In binding assays, the interaction is binding and the
complex formed can be isolated or detected in the reaction mixture.
In a particular embodiment, one of the peptides of the complexes
described herein, or the test compound or drug candidate is
immobilized on a solid phase, e.g., on a microtiter plate, by
covalent or non-covalent attachments. Non-covalent attachment
generally is accomplished by coating the solid surface with a
solution of the peptide and drying. Alternatively, an immobilized
antibody, e.g., a monoclonal antibody, specific for the peptide to
be immobilized can be used to anchor it to a solid surface. The
assay is performed by adding the non-immobilized component, which
may be labeled by a detectable label, to the immobilized component,
e.g., the coated surface containing the anchored component. When
the reaction is complete, the non-reacted components are removed,
e.g., by washing, and complexes anchored on the solid surface are
detected. When the originally non-immobilized component carries a
detectable label, the detection of label immobilized on the surface
indicates that complexing occurred. Where the originally
non-immobilized component does not carry a label, complexing can be
detected, for example, by using a labeled antibody specifically
binding the immobilized complex.
[0277] If the candidate compound interacts with, but does not bind
to a particular peptide identified herein, its interaction with
that peptide can be assayed by methods well known for detecting
protein-protein interactions. Such assays include traditional
approaches, such as, e.g., cross-linking, co-immunoprecipitation,
and co-purification through gradients or chromatographic columns.
In addition, protein-protein interactions can be monitored by using
a yeast-based genetic system described by Fields and co-workers
(Fields and Song, Nature (London), 340:245-246 (1989); Chien et
al., Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)) as disclosed
by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89: 5789-5793
(1991). Complete kits for identifying protein-protein interactions
between two specific proteins using the two-hybrid technique are
available. This system can also be extended to map protein domains
involved in specific protein interactions as well as to pinpoint
amino acid residues that are crucial for these interactions.
[0278] Compounds that interfere with the interaction of a peptide
identified herein and other intra- or extracellular components can
be tested as follows: usually a reaction mixture is prepared
containing the product of the gene and the intra- or extracellular
component under conditions and for a time allowing for the
interaction and binding of the two products. To test the ability of
a candidate compound to inhibit binding, the reaction is run in the
absence and in the presence of the test compound. In addition, a
placebo may be added to a third reaction mixture, to serve as
positive control. The binding (complex formation) between the test
compound and the intra- or extracellular component present in the
mixture is monitored as described hereinabove. The formation of a
complex in the control reaction(s) but not in the reaction mixture
containing the test compound indicates that the test compound
interferes with the interaction of the test compound and its
reaction partner.
[0279] To assay for antagonists, the peptide may be added to a cell
along with the compound to be screened for a particular activity
and the ability of the compound to inhibit the activity of interest
in the presence of the peptide indicates that the compound is an
antagonist to the peptide. The peptide can be labeled, such as by
radioactivity.
[0280] Anti-CD24 (ML5) (Santa Cruz Biotechnology) is a mouse
monoclonal antibody raised against CD24 of human origin. This
antibody can also be labeled or purchased with a label, such as
conjugated with Alexa Fluor.RTM. 488. Unlabeled anti-CD24 mAbs can
be obtained from the B cell panel of the V International Workshop
on Human Leucocyte Differentiation Antigens.
[0281] Any convenient monoclonal antibody (mAb) against CD24 may be
utilized. Alternatively, any convenient polyclonal antibody against
CD24 may be utilized. Methods of generating antibodies (i.e.,
monoclonal and polyclonal) are well known in the art. Antibodies
may be generated via any one of several methods known in the art,
which methods can employ induction of in-vivo production of
antibody molecules, screening of immunoglobulin libraries (Orlandi
D. R. et al., 1989. Proc. Natl. Acad. Sci. U.S.A. 86:3833-3837;
Winter G. et al., 1991. Nature 349:293-299) or generation of
monoclonal antibody molecules by continuous cell lines in culture.
These include, but are not limited to, the hybridoma technique, the
human B-cell hybridoma technique, and the Epstein-Barr virus
(EBV)-hybridoma technique (Kohler G. et al., 1975. Nature
256:495-497; Kozbor D. et al., 1985. J. Immunol. Methods 81:31-42;
Cote R J. et al., 1983. Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030;
Cole S P. et al., 1984. Mol. Cell. Biol. 62:109-120). Anti-CD24
antibodies, both polyclonal and monoclonal, suitable for use in the
methods and compositions of the present invention are commercially
available, for example, from Santa Cruz Biotechnology (Santa Cruz,
Calif.), AbDSerotec (Kidlington, UK) and Life Span BioSciences, Inc
(Seattle Wash.).
[0282] It will be appreciated that for human therapy or
diagnostics, humanized antibodies are preferably used. Humanized
forms of nonhuman (e.g., murine) antibodies are genetically
engineered chimeric antibodies or antibody fragments
having--preferably minimal--portions derived from nonhuman
antibodies. Humanized antibodies include antibodies in which
complementary determining regions of a human antibody (recipient
antibody) are replaced by residues from a complementarity
determining region of a nonhuman species (donor antibody) such as
mouse, rat or rabbit having the desired functionality. In some
instances, Fv framework residues of the human antibody are replaced
by corresponding nonhuman residues. Humanized antibodies may also
comprise residues which are found neither in the recipient antibody
nor in the imported complementarity determining region or framework
sequences. In general, the humanized antibody will comprise
substantially all of at least one, and typically two, variable
domains, in which all or substantially all of the complementarity
determining regions correspond to those of a nonhuman antibody and
all, or substantially all, of the framework regions correspond to
those of a relevant human consensus sequence. Humanized antibodies
optimally also include at least a portion of an antibody constant
region, such as an Fc region, typically derived from a human
antibody (see, for example, Jones et al., 1986. Nature 321:522-525;
Riechmann et al., 1988. Nature 332:323-329; and Presta, 1992. Curr.
Op. Struct. Biol. 2:593-596).
[0283] Methods for humanizing nonhuman antibodies are well known in
the art. Generally, a humanized antibody has one or more amino acid
residues introduced into it from a source which is nonhuman. These
nonhuman amino acid residues are often referred to as imported
residues which are typically taken from an imported variable
domain. Humanization can be essentially performed as described
(see, for example: Jones et al., 1986. Nature 321:522-525;
Riechmann et al., 1988. Nature 332:323-327; Verhoeyen et al., 1988.
Science 239:1534-1536; U.S. Pat. No. 4,816,567) by substituting
human complementarity determining regions with corresponding rodent
complementarity determining regions. Accordingly, such humanized
antibodies are chimeric antibodies, wherein substantially less than
an intact human variable domain has been substituted by the
corresponding sequence from a nonhuman species. In practice,
humanized antibodies may be typically human antibodies in which
some complementarity determining region residues and possibly some
framework residues are substituted by residues from analogous sites
in rodent antibodies.
[0284] Human antibodies can also be produced using various
techniques known in the art, including phage or yeast display
libraries [see, for example, Hoogenboom and Winter, 1991. J. Mol.
Biol. 227:381; Marks et al., 1991. J. Mol. Biol. 222:581; Cole et
al., "Monoclonal Antibodies and Cancer Therapy", Alan R. Liss, pp.
77 (1985); Boerner et al., 1991. J. Immunol. 147:86-95). Humanized
antibodies can also be made by introducing sequences encoding human
immunoglobulin loci into transgenic animals, e.g., into mice in
which the endogenous immunoglobulin genes have been partially or
completely inactivated. Upon antigenic challenge, human antibody
production is observed in such animals which closely resembles that
seen in humans in all respects, including gene rearrangement, chain
assembly, and antibody repertoire. Ample guidance for practicing
such an approach is provided in the literature of the art (for
example, refer to: U.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825,
5,625,126, 5,633,425, and 5,661,016; Marks et al., 1992.
Bio/Technology 10:779-783; Lonberg et al., 1994. Nature
368:856-859; Morrison, 1994. Nature 368:812-13; Fishwild et al.,
1996. Nature Biotechnology 14:845-51; Neuberger, 1996. Nature
Biotechnology 14:826; Lonberg and Huszar, 1995. Intern. Rev.
Immunol. 13:65-93).
[0285] Once antibodies are obtained, they may be tested for
activity, for example via ELISA.
[0286] According to some aspects of the present invention, the
method includes providing to the subject a therapeutic compound in
combination with a pharmaceutically acceptable carrier.
[0287] According to some aspects of the present invention, the
antibody or combination can be provided using any one of a variety
of delivery methods. Delivery methods and suitable formulations are
described herein below with respect to pharmaceutical
compositions.
[0288] Techniques for formulation and administration of drugs may
be found in "Remington's Pharmaceutical Sciences," Mack Publishing
Co., Easton, Pa., latest edition, which is incorporated herein by
reference.
[0289] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, especially transnasal, intestinal or
parenteral delivery, including intramuscular, subcutaneous and
intramedullary injections as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal, or
intraocular injections.
[0290] Alternately, one may administer a preparation in a local
rather than systemic manner, for example, via injection of the
preparation directly into a specific region of a patient's
body.
[0291] Pharmaceutical compositions of the present invention may be
manufactured by processes well known in the art, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
[0292] Pharmaceutical compositions for use in accordance with the
present invention may be formulated in conventional manner using
one or more physiologically acceptable carriers comprising
excipients and auxiliaries, which facilitate processing of the
active ingredients into preparations which, can be used
pharmaceutically. Proper formulation is dependent upon the route of
administration chosen.
[0293] It will be appreciated, of course, that the proteins or
peptides of the invention may incorporate amino acid residues which
are modified without affecting activity. For example, the termini
may be derivatized to include blocking groups, i.e. chemical
substituents suitable to protect and/or stabilize the N- and
C-termini from "undesirable degradation", a term meant to encompass
any type of enzymatic, chemical or biochemical breakdown of the
compound at its termini which is likely to affect the function of
the compound, i.e. sequential degradation of the compound at a
terminal end thereof.
[0294] Blocking groups include protecting groups conventionally
used in the art of peptide chemistry which will not adversely
affect the in vivo activities of the peptide. For example, suitable
N-terminal blocking groups can be introduced by alkylation or
acylation of the N-terminus. Examples of suitable N-terminal
blocking groups include C.sub.1-C.sub.5 branched or unbranched
alkyl groups, acyl groups such as formyl and acetyl groups, as well
as substituted forms thereof, such as the acetamidomethyl (Acm)
group. Desamino analogs of amino acids are also useful N-terminal
blocking groups, and can either be coupled to the N-terminus of the
peptide or used in place of the N-terminal reside. Suitable
C-terminal blocking groups, in which the carboxyl group of the
C-terminus is either incorporated or not, include esters, ketones
or amides. Ester or ketone-forming alkyl groups, particularly lower
alkyl groups such as methyl, ethyl and propyl, and amide-forming
amino groups such as primary amines (--NH.sub.2), and mono- and
dialkylamino groups such as methylamino, ethylamino, dimethylamino,
diethylamino, methylethylamino and the like are examples of
C-terminal blocking groups. Descarboxylated amino acid analogues
such as agmatine are also useful C-terminal blocking groups and can
be either coupled to the peptide's C-terminal residue or used in
place of it. Further, it will be appreciated that the free amino
and carboxyl groups at the termini can be removed altogether from
the peptide to yield desamino and descarboxylated forms thereof
without affect on peptide activity.
[0295] Other modifications can also be incorporated without
adversely affecting the activity and these include, but are not
limited to, substitution of one or more of the amino acids in the
natural L-isomeric form with amino acids in the D-isomeric form.
Thus, the peptide may include one or more D-amino acid resides, or
may comprise amino acids which are all in the D-form. Retro-inverso
forms of peptides in accordance with the present invention are also
contemplated, for example, inverted peptides in which all amino
acids are substituted with D-amino acid forms.
[0296] Acid addition salts of the present invention are also
contemplated as functional equivalents. Thus, a peptide in
accordance with the present invention treated with an inorganic
acid such as hydrochloric, hydrobromic, sulfuric, nitric,
phosphoric, and the like, or an organic acid such as an acetic,
propionic, glycolic, pyruvic, oxalic, malic, malonic, succinic,
maleic, fumaric, tartaric, citric, benzoic, cinnamic, mandelic,
methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicyclic and
the like, to provide a water soluble salt of the peptide is
suitable for use in the invention.
[0297] Modifications (which do not normally alter primary sequence)
include in vivo, or in vitro chemical derivatization of
polypeptides, e.g., acetylation, or carboxylation. Also included
are modifications of glycosylation, e.g., those made by modifying
the glycosylation patterns of a polypeptide during its synthesis
and processing or in further processing steps; e.g., by exposing
the polypeptide to enzymes which affect glycosylation, e.g.,
mammalian glycosylating or deglycosylating enzymes. Also embraced
are sequences which have phosphorylated amino acid residues, e.g.,
phosphotyrosine, phosphoserine, or phosphothreonine.
[0298] Also included are polypeptides which have been modified
using ordinary molecular biological techniques so as to improve
their resistance to proteolytic degradation or to optimize
solubility properties or to render them more suitable as a
therapeutic agent. Analogs of such polypeptides include those
containing residues other than naturally occurring L-amino acids,
e.g., D-amino acids or non-naturally occurring synthetic amino
acids. The peptides of the invention are not limited to products of
any of the specific exemplary processes listed herein.
[0299] Antibodies and their Preparation
[0300] Antibodies directed against proteins, polypeptides, or
peptide fragments thereof of the invention may be generated using
methods that are well known in the art. For instance, U.S. patent
application Ser. No. 07/481,491, which is incorporated by reference
herein in its entirety, discloses methods of raising antibodies to
peptides. For the production of antibodies, various host animals,
including but not limited to rabbits, mice, and rats, can be
immunized by injection with a polypeptide or peptide fragment
thereof. To increase the immunological response, various adjuvants
may be used depending on the host species, including but not
limited to Freund's (complete and incomplete), mineral gels such as
aluminum hydroxide, surface active substances such as lysolecithin,
pluronic polyols, polyanions, peptides, oil emulsions, keyhole
limpet hemocyanins, dinitrophenol, and potentially useful human
adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium
parvum.
[0301] The antigenic fragments of the proteins of the invention may
include, for example, peptide antigens that are at least about 5,
10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,
95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 or up to
about 200 amino acids in length. Of course, these are prepared
based on the length of the starting protein or peptide. Also
included are full-length unprocessed protein as well as mature
processed protein. These various length antigenic fragments may be
designed in tandem order of linear amino acid sequence of the
immunogen of choice, such as SAS1R, or staggered in linear sequence
of the protein. In addition, antibodies to three-dimensional
epitopes, i.e., non-linear epitopes, can also be prepared, based
on, e.g., crystallographic data of proteins. Hosts may also be
injected with peptides of different lengths encompassing a desired
target sequence.
[0302] For the preparation of monoclonal antibodies, any technique
which provides for the production of antibody molecules by
continuous cell lines in culture may be utilized. For example, the
hybridoma technique originally developed by Kohler and Milstein
(1975, Nature 256:495-497), the trioma technique, the human B-cell
hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72),
and the EBV-hybridoma technique (Cole et al., 1985, in Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) may
be employed to produce human monoclonal antibodies. In another
embodiment, monoclonal antibodies are produced in germ-free
animals.
[0303] In one embodiment, any new monoclonal antibody described
herein, or made using the methods described herein, and the
hybridomas making the antibodies, as well as those not described
herein, will be deposited with the American Type Culture Collection
(10801 University Boulevard, Manassas, Va. 20110-2209) and assigned
Accession Numbers. The deposits will be maintained under the terms
of the Budapest Treaty on the International Recognition of the
Deposit of Microorganisms for the Purposes of Patent Procedure and
made available for use under those terms. This assures maintenance
of a viable culture of the deposit for 30 years from the date of
deposit. The deposits will be made available by ATCC under the
terms of the Budapest Treaty, and subject to an agreement between
the University of Virginia and ATCC, which assures permanent and
unrestricted availability of the progeny of the culture of the
deposit to the public upon issuance of the pertinent U.S. patent or
upon laying open to the public of any U.S. or foreign patent
application, whichever comes first, and assures availability of the
progeny to one determined by the U.S. Commissioner of Patents and
Trademarks to be entitled thereto according to 35 USC section 122
and the Commissioner's rules pursuant thereto (including 37 CFR
section 1.14 with particular reference to 886 OG 638). The assignee
of the present application has agreed that if a culture of the
materials on deposit should die or be lost or destroyed when
cultivated under suitable conditions, the materials will be
promptly replaced on notification with another of the same.
Availability of the deposited material is not to be construed as a
license to practice the invention in contravention of the rights
granted under the authority of any government in accordance with
its patent laws. Nucleic acid and amino acid sequences will be
deposited with GenBank and made accessible to the public.
[0304] In accordance with the invention, human antibodies may be
used and obtained by utilizing human hybridomas (Cote et al., 1983,
Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or by transforming
human B cells with EBV virus in vitro (Cole et al., 1985, in
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.
77-96). Furthermore, techniques developed for the production of
"chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad.
Sci. U.S.A. 81:6851-6855; Neuberger et al., 1984, Nature
312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing
the genes from a mouse antibody molecule specific for epitopes of
SLLP polypeptides together with genes from a human antibody
molecule of appropriate biological activity can be employed; such
antibodies are within the scope of the present invention. Once
specific monoclonal antibodies have been developed, the preparation
of mutants and variants thereof by conventional techniques is also
available.
[0305] Humanized (chimeric) antibodies are immunoglobulin molecules
comprising a human and non-human portion. More specifically, the
antigen combining region (or variable region) of a humanized
chimeric antibody is derived from a non-human source (e.g., murine)
and the constant region of the chimeric antibody (which confers
biological effector function to the immunoglobulin) is derived from
a human source. The humanized chimeric antibody should have the
antigen binding specificity of the non-human antibody molecule and
the effector function conferred by the human antibody molecule. A
large number of methods of generating chimeric antibodies are well
known to those of skill in the art (see, e.g., U.S. Pat. Nos.
5,502,167, 5,500,362, 5,491,088, 5,482,856, 5,472,693, 5,354,847,
5,292,867, 5,231,026, 5,204,244, 5,202,238, 5,169,939, 5,081,235,
5,075,431, and 4,975,369). Detailed methods for preparation of
chimeric (humanized) antibodies can be found in U.S. Pat. No.
5,482,856.
[0306] In another embodiment, this invention provides for fully
human antibodies. Human antibodies consist entirely of
characteristically human polypeptide sequences. The human
antibodies of this invention can be produced in using a wide
variety of methods (see, e.g., U.S. Pat. No. 5,001,065, for
review).
[0307] In one embodiment, techniques described for the production
of single-chain antibodies (U.S. Pat. No. 4,946,778, incorporated
by reference herein in its entirety) are adapted to produce
protein-specific single-chain antibodies. In another embodiment,
the techniques described for the construction of Fab expression
libraries (Huse et al., 1989, Science 246:1275-1281) are utilized
to allow rapid and easy identification of monoclonal Fab fragments
possessing the desired specificity for specific antigens, proteins,
derivatives, or analogs of the invention.
[0308] Antibody fragments which contain the idiotype of the
antibody molecule can be generated by known techniques. For
example, such fragments include but are not limited to: the
F(ab').sub.2 fragment which can be produced by pepsin digestion of
the antibody molecule; the Fab' fragments which can be generated by
reducing the disulfide bridges of the F(ab').sub.2 fragment; the
Fab fragments which can be generated by treating the antibody
molecule with papain and a reducing agent; and Fv fragments.
[0309] The generation of polyclonal antibodies is accomplished by
inoculating the desired animal with the antigen and isolating
antibodies which specifically bind the antigen therefrom.
[0310] Monoclonal antibodies directed against full length or
peptide fragments of a protein or peptide may be prepared using any
well known monoclonal antibody preparation procedures, such as
those described, for example, in Harlow et al. (1988, In:
Antibodies, A Laboratory Manual, Cold Spring Harbor, N.Y.) and in
Tuszynski et al. (1988, Blood, 72:109-115). Quantities of the
desired peptide may also be synthesized using chemical synthesis
technology. Alternatively, DNA encoding the desired peptide may be
cloned and expressed from an appropriate promoter sequence in cells
suitable for the generation of large quantities of peptide.
Monoclonal antibodies directed against the peptide are generated
from mice immunized with the peptide using standard procedures as
referenced herein.
[0311] A nucleic acid encoding the monoclonal antibody obtained
using the procedures described herein may be cloned and sequenced
using technology which is available in the art, and is described,
for example, in Wright et al. (1992, Critical Rev. in Immunol.
12(3,4):125-168) and the references cited therein. Further, the
antibody of the invention may be "humanized" using the technology
described in Wright et al., (supra) and in the references cited
therein, and in Gu et al. (1997, Thrombosis and Hematocyst
77(4):755-759).
[0312] To generate a phage antibody library, a cDNA library is
first obtained from mRNA which is isolated from cells, e.g., the
hybridoma, which express the desired protein to be expressed on the
phage surface, e.g., the desired antibody. cDNA copies of the mRNA
are produced using reverse transcriptase. cDNA which specifies
immunoglobulin fragments are obtained by PCR and the resulting DNA
is cloned into a suitable bacteriophage vector to generate a
bacteriophage DNA library comprising DNA specifying immunoglobulin
genes. The procedures for making a bacteriophage library comprising
heterologous DNA are well known in the art and are described, for
example, in Sambrook et al. (1989, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor, N.Y.).
[0313] Bacteriophage which encode the desired antibody, may be
engineered such that the protein is displayed on the surface
thereof in such a manner that it is available for binding to its
corresponding binding protein, e.g., the antigen against which the
antibody is directed. Thus, when bacteriophage which express a
specific antibody are incubated in the presence of a cell which
expresses the corresponding antigen, the bacteriophage will bind to
the cell. Bacteriophage which do not express the antibody will not
bind to the cell. Such panning techniques are well known in the
art.
[0314] Processes such as those described above, have been developed
for the production of human antibodies using M13 bacteriophage
display (Burton et al., 1994, Adv. Immunol. 57:191-280).
Essentially, a cDNA library is generated from mRNA obtained from a
population of antibody-producing cells. The mRNA encodes rearranged
immunoglobulin genes and thus, the cDNA encodes the same. Amplified
cDNA is cloned into M13 expression vectors creating a library of
phage which express human Fab fragments on their surface. Phage
which display the antibody of interest are selected by antigen
binding and are propagated in bacteria to produce soluble human Fab
immunoglobulin. Thus, in contrast to conventional monoclonal
antibody synthesis, this procedure immortalizes DNA encoding human
immunoglobulin rather than cells which express human
immunoglobulin.
[0315] The procedures just presented describe the generation of
phage which encode the Fab portion of an antibody molecule.
However, the invention should not be construed to be limited solely
to the generation of phage encoding Fab antibodies. Rather, phage
which encode single chain antibodies (scFv/phage antibody
libraries) are also included in the invention. Fab molecules
comprise the entire Ig light chain, that is, they comprise both the
variable and constant region of the light chain, but include only
the variable region and first constant region domain (CH1) of the
heavy chain. Single chain antibody molecules comprise a single
chain of protein comprising the Ig Fv fragment. An Ig Fv fragment
includes only the variable regions of the heavy and light chains of
the antibody, having no constant region contained therein. Phage
libraries comprising scFv DNA may be generated following the
procedures described in Marks et al., 1991, J. Mol. Biol.
222:581-597. Panning of phage so generated for the isolation of a
desired antibody is conducted in a manner similar to that described
for phage libraries comprising Fab DNA.
[0316] The invention should also be construed to include synthetic
phage display libraries in which the heavy and light chain variable
regions may be synthesized such that they include nearly all
possible specificities (Barbas, 1995, Nature Medicine 1:837-839; de
Kruif et al. 1995, J. Mol. Biol. 248:97-105).
[0317] In the production of antibodies, screening for the desired
antibody can be accomplished by techniques known in the art, e.g.,
ELISA (enzyme-linked immunosorbent assay). Antibodies generated in
accordance with the present invention may include, but are not
limited to, polyclonal, monoclonal, chimeric (i.e., "humanized"),
and single chain (recombinant) antibodies, Fab fragments, and
fragments produced by a Fab expression library.
[0318] The peptides of the present invention may be readily
prepared by standard, well-established techniques, such as
solid-phase peptide synthesis (SPPS) as described by Stewart et al.
in Solid Phase Peptide Synthesis, 2nd Edition, 1984, Pierce
Chemical Company, Rockford, Ill.; and as described by Bodanszky and
Bodanszky in The Practice of Peptide Synthesis, 1984,
Springer-Verlag, New York. At the outset, a suitably protected
amino acid residue is attached through its carboxyl group to a
derivatized, insoluble polymeric support, such as cross-linked
polystyrene or polyamide resin. "Suitably protected" refers to the
presence of protecting groups on both the .alpha.-amino group of
the amino acid, and on any side chain functional groups. Side chain
protecting groups are generally stable to the solvents, reagents
and reaction conditions used throughout the synthesis, and are
removable under conditions that will not affect the final peptide
product. Stepwise synthesis of the oligopeptide is carried out by
the removal of the N-protecting group from the initial amino acid,
and couple thereto of the carboxyl end of the next amino acid in
the sequence of the desired peptide. This amino acid is also
suitably protected. The carboxyl of the incoming amino acid can be
activated to react with the N-terminus of the support-bound amino
acid by formation into a reactive group such as formation into a
carbodiimide, a symmetric acid anhydride or an "active ester" group
such as hydroxybenzotriazole or pentafluorophenyl esters.
[0319] Examples of solid phase peptide synthesis methods include
the BOC method that utilized tert-butyloxycarbonyl as the
.alpha.-amino protecting group, and the FMOC method which utilizes
9-fluorenylmethyloxycarbonyl to protect the .alpha.-amino of the
amino acid residues, both methods of which are well-known by those
of skill in the art.
[0320] To ensure that the proteins or peptides obtained from either
chemical or biological synthetic techniques is the desired peptide,
analysis of the peptide composition should be conducted. Such amino
acid composition analysis may be conducted using high resolution
mass spectrometry to determine the molecular weight of the peptide.
Alternatively, or additionally, the amino acid content of the
peptide can be confirmed by hydrolyzing the peptide in aqueous
acid, and separating, identifying and quantifying the components of
the mixture using HPLC, or an amino acid analyzer. Protein
sequenators, which sequentially degrade the peptide and identify
the amino acids in order, may also be used to determine definitely
the sequence of the peptide.
[0321] Prior to its use, the peptide can be purified to remove
contaminants. In this regard, it will be appreciated that the
peptide will be purified to meet the standards set out by the
appropriate regulatory agencies. Any one of a number of a
conventional purification procedures may be used to attain the
required level of purity including, for example, reversed-phase
high-pressure liquid chromatography (HPLC) using an alkylated
silica column such as C.sub.4-, C.sub.8- or C.sub.18-silica. A
gradient mobile phase of increasing organic content is generally
used to achieve purification, for example, acetonitrile in an
aqueous buffer, usually containing a small amount of
trifluoroacetic acid. Ion-exchange chromatography can be also used
to separate peptides based on their charge.
[0322] Substantially pure peptide obtained as described herein may
be purified by following known procedures for protein purification,
wherein an immunological, enzymatic or other assay is used to
monitor purification at each stage in the procedure. Protein
purification methods are well known in the art, and are described,
for example in Deutscher et al. (ed., 1990, Guide to Protein
Purification, Harcourt Brace Jovanovich, San Diego).
[0323] The invention further encompasses the use of aptamers. In
one embodiment, an aptamer is a compound that is selected in vitro
to bind preferentially to another compound (in this case the
identified proteins). In one aspect, aptamers are nucleic acids or
peptides, because random sequences can be readily generated from
nucleotides or amino acids (both naturally occurring or
synthetically made) in large numbers but of course they need not be
limited to these. In another aspect, the nucleic acid aptamers are
short strands of DNA that bind protein targets. In one aspect, the
aptamers are oligonucleotide aptamers. Oligonucleotide aptamers are
oligonucleotides which can bind to a specific protein sequence of
interest. A general method of identifying aptamers is to start with
partially degenerate oligonucleotides, and then simultaneously
screen the many thousands of oligonucleotides for the ability to
bind to a desired protein. The bound oligonucleotide can be eluted
from the protein and sequenced to identify the specific recognition
sequence. Transfer of large amounts of a chemically stabilized
aptamer into cells can result in specific binding to a polypeptide
of interest, thereby blocking its function. [For example, see the
following publications describing in vitro selection of aptamers:
Klug et al., Mol. Biol. Reports 20:97-107 (1994); Wallis et al.,
Chem. Biol. 2:543-552 (1995); Ellington, Curr. Biol. 4:427-429
(1994); Lato et al., Chem. Biol. 2:291-303 (1995); Conrad et al.,
Mol. Div. 1:69-78 (1995); and Uphoff et al., Curr. Opin. Struct.
Biol. 6:281-287 (1996)]. Aptamers offer advantages over other
oligonucleotide-based approaches that artificially interfere with
target gene function due to their ability to bind protein products
of these genes with high affinity and specificity. However, RNA
aptamers can be limited in their ability to target intracellular
proteins since even nuclease-resistant aptamers do not efficiently
enter the intracellular compartments. Moreover, attempts at
expressing RNA aptamers within mammalian cells through vector-based
approaches have been hampered by the presence of additional
flanking sequences in expressed RNA aptamers, which may alter their
functional conformation.
[0324] The idea of using single-stranded nucleic acids (DNA and RNA
aptamers) to target protein molecules is based on the ability of
short sequences (20 mers to 80 mers) to fold into unique 3D
conformations that enable them to bind targeted proteins with high
affinity and specificity. RNA aptamers have been expressed
successfully inside eukaryotic cells, such as yeast and
multicellular organisms, and have been shown to have inhibitory
effects on their targeted proteins in the cellular environment.
[0325] This invention encompasses methods of screening compounds to
identify those compounds that act as agonists (stimulate) or
antagonists (inhibit) of the protein interactions and pathways
described herein. Screening assays for antagonist compound
candidates are designed to identify compounds that bind or complex
with the peptides described herein, or otherwise interfere with the
interaction of the peptides with other cellular proteins. Such
screening assays will include assays amenable to high-throughput
screening of chemical libraries, making them particularly suitable
for identifying small molecule drug candidates.
[0326] The assays can be performed in a variety of formats,
including protein-protein binding assays, biochemical screening
assays, high-throughput assays, immunoassays, and cell-based
assays, which are well characterized in the art.
[0327] All assays for antagonists are common in that they call for
contacting the compound or drug candidate with a peptide identified
herein under conditions and for a time sufficient to allow these
two components to interact.
[0328] In binding assays, the interaction is binding and the
complex formed can be isolated or detected in the reaction mixture.
In a particular embodiment, one of the peptides of the complexes
described herein, or the test compound or drug candidate is
immobilized on a solid phase, e.g., on a microtiter plate, by
covalent or non-covalent attachments. Non-covalent attachment
generally is accomplished by coating the solid surface with a
solution of the peptide and drying. Alternatively, an immobilized
antibody, e.g., a monoclonal antibody, specific for the peptide to
be immobilized can be used to anchor it to a solid surface. The
assay is performed by adding the non-immobilized component, which
may be labeled by a detectable label, to the immobilized component,
e.g., the coated surface containing the anchored component. When
the reaction is complete, the non-reacted components are removed,
e.g., by washing, and complexes anchored on the solid surface are
detected. When the originally non-immobilized component carries a
detectable label, the detection of label immobilized on the surface
indicates that complexing occurred. Where the originally
non-immobilized component does not carry a label, complexing can be
detected, for example, by using a labeled antibody specifically
binding the immobilized complex.
[0329] If the candidate compound interacts with, but does not bind
to a particular peptide identified herein, its interaction with
that peptide can be assayed by methods well known for detecting
protein-protein interactions. Such assays include traditional
approaches, such as, e.g., cross-linking, co-immunoprecipitation,
and co-purification through gradients or chromatographic columns.
In addition, protein-protein interactions can be monitored by using
a yeast-based genetic system described by Fields and co-workers
(Fields and Song, Nature (London), 340:245-246 (1989); Chien et
al., Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)) as disclosed
by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89: 5789-5793
(1991). Complete kits for identifying protein-protein interactions
between two specific proteins using the two-hybrid technique are
available. This system can also be extended to map protein domains
involved in specific protein interactions as well as to pinpoint
amino acid residues that are crucial for these interactions.
[0330] Compounds that interfere with the interaction of a peptide
identified herein and other intra- or extracellular components can
be tested as follows: usually a reaction mixture is prepared
containing the product of the gene and the intra- or extracellular
component under conditions and for a time allowing for the
interaction and binding of the two products. To test the ability of
a candidate compound to inhibit binding, the reaction is run in the
absence and in the presence of the test compound. In addition, a
placebo may be added to a third reaction mixture, to serve as
positive control. The binding (complex formation) between the test
compound and the intra- or extracellular component present in the
mixture is monitored as described hereinabove. The formation of a
complex in the control reaction(s) but not in the reaction mixture
containing the test compound indicates that the test compound
interferes with the interaction of the test compound and its
reaction partner.
[0331] Other assays and libraries are encompassed within the
invention, such as the use of phylomers.RTM. and reverse yeast
two-hybrid assays (see Watt, 2006, Nature Biotechnology, 24:177;
Watt, U.S. Pat. No. 6,994,982; Watt, U.S. Pat. Pub. No.
2005/0287580; Watt, U.S. Pat. No. 6,510,495; Barr et al., 2004, J.
Biol. Chem., 279:41:43178-43189; the contents of each of these
publications is hereby incorporated by reference herein in their
entirety). Phylomers.RTM. are derived from sub domains of natural
proteins, which makes them potentially more stable than
conventional short random peptides. Phylomers.RTM. are sourced from
biological genomes that are not human in origin. This feature
significantly enhances the potency associated with Phylomers.RTM.
against human protein targets. Phylogica's current Phylomer.RTM.
library has a complexity of 50 million clones, which is comparable
with the numerical complexity of random peptide or antibody Fab
fragment libraries. An Interacting Peptide Library, consisting of
63 million peptides fused to the B42 activation domain, can be used
to isolate peptides capable of binding to a target protein in a
forward yeast two hybrid screen. The second is a Blocking Peptide
Library made up of over 2 million peptides that can be used to
screen for peptides capable of disrupting a specific protein
interaction using the reverse two-hybrid system.
[0332] The Phylomer.RTM. library consists of protein fragments,
which have been sourced from a diverse range of bacterial genomes.
The libraries are highly enriched for stable subdomains (15-50
amino acids long). This technology can be integrated with high
throughput screening techniques such as phage display and reverse
yeast two-hybrid traps.
[0333] The present application discloses compositions and methods
for regulating the proteins described herein, and those not
disclosed which are known in the art are encompassed within the
invention. For example, various modulators/effectors are known,
e.g. antibodies, biologically active nucleic acids, such as
antisense molecules, RNAi molecules, or ribozymes, aptamers,
peptides or low-molecular weight organic compounds recognizing said
polynucleotides or polypeptides.
[0334] The present invention also provides nucleic acids encoding
peptides, proteins, and antibodies of the invention. By "nucleic
acid" is meant any nucleic acid, whether composed of
deoxyribonucleosides or ribonucleosides, and whether composed of
phosphodiester linkages or modified linkages such as
phosphotriester, phosphoramidate, siloxane, carbonate,
carboxymethylester, acetamidate, carbamate, thioether, bridged
phosphoramidate, bridged methylene phosphonate, bridged
phosphoramidate, bridged phosphoramidate, bridged methylene
phosphonate, phosphorothioate, methylphosphonate,
phosphorodithioate, bridged phosphorothioate or sulfone linkages,
and combinations of such linkages. The term nucleic acid also
specifically includes nucleic acids composed of bases other than
the five biologically occurring bases (adenine, guanine, thymine,
cytosine and uracil).
[0335] It is not intended that the present invention be limited by
the nature of the nucleic acid employed. The target nucleic acid
may be native or synthesized nucleic acid. The nucleic acid may be
from a viral, bacterial, animal or plant source. The nucleic acid
may be DNA or RNA and may exist in a double-stranded,
single-stranded or partially double-stranded form. Furthermore, the
nucleic acid may be found as part of a virus or other
macromolecule. See, e.g., Fasbender et al., 1996, J. Biol. Chem.
272:6479-89 (polylysine condensation of DNA in the form of
adenovirus).
[0336] In some circumstances, as where increased nuclease stability
is desired, nucleic acids having modified internucleoside linkages
may be preferred. Nucleic acids containing modified internucleoside
linkages may also be synthesized using reagents and methods that
are well known in the art. For example, methods for synthesizing
nucleic acids containing phosphonate phosphorothioate,
phosphorodithioate, phosphoramidate methoxyethyl phosphoramidate,
formacetal, thioformacetal, diisopropylsilyl, acetamidate,
carbamate, dimethylene-sulfide (--CH2-S--CH2),
dimethylene-sulfoxide (--CH2-SO--CH2), dimethylene-sulfone
(--CH2-SO2-CH2), 2'-O-alkyl, and 2'-deoxy2'-fluoro phosphorothioate
internucleoside linkages are well known in the art (see Uhlmann et
al., 1990, Chem. Rev. 90:543-584; Schneider et al., 1990,
Tetrahedron Lett. 31:335 and references cited therein).
[0337] The nucleic acids may be purified by any suitable means, as
are well known in the art. For example, the nucleic: acids can be
purified by reverse phase or ion exchange HPLC, size exclusion
chromatography or gel electrophoresis. Of course, the skilled
artisan will recognize that the method of purification will depend
in part on the size of the DNA to be purified. The term nucleic
acid also specifically includes nucleic acids composed of bases
other than the five biologically occurring bases (adenine, guanine,
thymine, cytosine and uracil).
[0338] The present invention also encompasses pharmaceutical and
therapeutic compositions comprising the compounds of the present
invention.
[0339] The present invention is also directed to pharmaceutical
compositions comprising the compounds of the present invention.
More particularly, such compounds can be formulated as
pharmaceutical compositions using standard pharmaceutically
acceptable carriers, fillers, solubilizing agents and stabilizers
known to those skilled in the art.
[0340] When used in vivo for therapy, the antibodies of the
invention are administered to the subject in therapeutically
effective amounts (i.e., amounts that have a desired therapeutic
effect). In one aspect, they will be administered parenterally. The
dose and dosage regimen will depend, for example, upon the degree
of the anemia, the characteristics of the particular antibody or
other compound used, e.g., its therapeutic index, the subject, and
the subject's history. In one embodiment, at least one antibody or
other agonist compound is administered once, or more than once, or
even continuously over a period of 1-2 weeks. Optionally, the
administration is made during the course of adjunct therapy such as
antimicrobial treatment, or administration of, for example, a
cytokine(s), or other EPO or erythropoiesis regulatory agent.
[0341] For parenteral administration, an antibody can be
formulated, for example, in a unit dosage injectable form
(solution, suspension, emulsion) in association with a
pharmaceutically acceptable parenteral vehicle. Such vehicles are
inherently nontoxic, and non-therapeutic. Examples of such vehicle
are water, saline, Ringer's solution, dextrose solution, and 5%
human serum albumin. Nonaqueous vehicles such as fixed oils and
ethyl oleate can also be used. Liposomes can be used as carriers.
The vehicle can contain minor amounts of additives such as
substances that enhance isotonicity and chemical stability, e.g.,
buffers and preservatives. The antibodies will typically be
formulated in such vehicles at concentrations of about 1.0 mg/ml to
about 10 mg/ml.
[0342] In one aspect, the invention provides for the use of IgM
antibodies; however, IgG molecules by being smaller can be more
able than IgM molecules to localize to certain types of infected
cells. Therefore, in one aspect, IgG antibodies are useful in the
practice of the invention.
[0343] The antibody compositions used can be formulated and dosages
established in a fashion consistent with good medical practice
taking into account the condition or disorder to be treated, the
condition of the individual patient, the site of delivery of the
composition, the method of administration, and other factors known
to practitioners. The antibody compositions are prepared for
administration according to the description of preparation of
polypeptides for administration, infra.
[0344] As is well understood in the art, biospecific capture
reagents include antibodies, binding fragments of antibodies which
bind to activated integrin receptors on metastatic cells (e.g.,
single chain antibodies, Fab' fragments, F(ab)'2 fragments, and
scFv proteins and affibodies (Affibody, Teknikringen 30, floor 6,
Box 700 04, Stockholm SE-10044, Sweden; See U.S. Pat. No.
5,831,012, incorporated herein by reference in its entirety and for
all purposes)). Depending on intended use, they also can include
receptors and other proteins that specifically bind another
biomolecule.
[0345] The hybrid antibodies and hybrid antibody fragments include
complete antibody molecules having full length heavy and light
chains, or any fragment thereof, such as Fab, Fab', F(ab')2, Fd,
scFv, antibody light chains and antibody heavy chains. Chimeric
antibodies which have variable regions as described herein and
constant regions from various species are also suitable. See for
example, U.S. Application No. 20030022244.
[0346] Initially, a predetermined target object is chosen to which
an antibody can be raised. Techniques for generating monoclonal
antibodies directed to target objects are well known to those
skilled in the art. Examples of such techniques include, but are
not limited to, those involving display libraries, xeno or human
mice, hybridomas, and the like. Target objects include any
substance which is capable of exhibiting antigenicity and are
usually proteins or protein polysaccharides. Examples include
receptors, enzymes, hormones, growth factors, peptides and the
like. It should be understood that not only are naturally occurring
antibodies suitable for use in accordance with the present
disclosure, but engineered antibodies and antibody fragments which
are directed to a predetermined object are also suitable.
[0347] The present invention is also directed to pharmaceutical
compositions comprising the compounds of the present invention.
More particularly, such compounds can be formulated as
pharmaceutical compositions using standard pharmaceutically
acceptable carriers, fillers, solubilizing agents and stabilizers
known to those skilled in the art.
[0348] In accordance with one embodiment, a method of treating a
subject in need of treatment is provided. The method comprises
administering a pharmaceutical composition comprising at least one
compound of the present invention to a subject in need thereof.
Compounds identified by the methods of the invention can be
administered with known compounds or other medications as well.
[0349] The invention also encompasses the use of pharmaceutical
compositions of an appropriate compound, and homologs, fragments,
analogs, or derivatives thereof to practice the methods of the
invention, the composition comprising at least one appropriate
compound, and homolog, fragment, analog, or derivative thereof and
a pharmaceutically-acceptable carrier.
[0350] The pharmaceutical compositions useful for practicing the
invention may be administered to deliver a dose of between 1
ng/kg/day and 100 mg/kg/day.
[0351] The invention encompasses the preparation and use of
pharmaceutical compositions comprising a compound useful for
treatment of the diseases disclosed herein as an active ingredient.
Such a pharmaceutical composition may consist of the active
ingredient alone, in a form suitable for administration to a
subject, or the pharmaceutical composition may comprise the active
ingredient and one or more pharmaceutically acceptable carriers,
one or more additional ingredients, or some combination of these.
The active ingredient may be present in the pharmaceutical
composition in the form of a physiologically acceptable ester or
salt, such as in combination with a physiologically acceptable
cation or anion, as is well known in the art.
[0352] As used herein, the term "physiologically acceptable" ester
or salt means an ester or salt form of the active ingredient which
is compatible with any other ingredients of the pharmaceutical
composition, which is not deleterious to the subject to which the
composition is to be administered.
[0353] The formulations of the pharmaceutical compositions
described herein may be prepared by any method known or hereafter
developed in the art of pharmacology. In general, such preparatory
methods include the step of bringing the active ingredient into
association with a carrier or one or more other accessory
ingredients, and then, if necessary or desirable, shaping or
packaging the product into a desired single- or multi-dose
unit.
[0354] It will be understood by the skilled artisan that such
pharmaceutical compositions are generally suitable for
administration to animals of all sorts. Subjects to which
administration of the pharmaceutical compositions of the invention
is contemplated include, but are not limited to, humans and other
primates, mammals including commercially relevant mammals such as
cattle, pigs, horses, sheep, cats, and dogs, birds including
commercially relevant birds such as chickens, ducks, geese, and
turkeys. The invention is also contemplated for use in
contraception for nuisance animals such as rodents.
[0355] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in bulk, as a single unit dose, or as a
plurality of single unit doses. As used herein, a "unit dose" is
discrete amount of the pharmaceutical composition comprising a
predetermined amount of the active ingredient. The amount of the
active ingredient is generally equal to the dosage of the active
ingredient which would be administered to a subject or a convenient
fraction of such a dosage such as, for example, one-half or
one-third of such a dosage.
[0356] The relative amounts of the active ingredient, the
pharmaceutically acceptable carrier, and any additional ingredients
in a pharmaceutical composition of the invention will vary,
depending upon the identity, size, and condition of the subject
treated and further depending upon the route by which the
composition is to be administered. By way of example, the
composition may comprise between 0.1% and 100% (w/w) active
ingredient.
[0357] In addition to the active ingredient, a pharmaceutical
composition of the invention may further comprise one or more
additional pharmaceutically active agents. Particularly
contemplated additional agents include anti-emetics and scavengers
such as cyanide and cyanate scavengers.
[0358] Controlled- or sustained-release formulations of a
pharmaceutical composition of the invention may be made using
conventional technology.
[0359] As used herein, "additional ingredients" include, but are
not limited to, one or more of the following: excipients; surface
active agents; dispersing agents; inert diluents; granulating and
disintegrating agents; binding agents; lubricating agents;
sweetening agents; flavoring agents; coloring agents;
preservatives; physiologically degradable compositions such as
gelatin; aqueous vehicles and solvents; oily vehicles and solvents;
suspending agents; dispersing or wetting agents; emulsifying
agents, demulcents; buffers; salts; thickening agents; fillers;
emulsifying agents; antioxidants; antibiotics; antifungal agents;
stabilizing agents; and pharmaceutically acceptable polymeric or
hydrophobic materials. Other "additional ingredients" which may be
included in the pharmaceutical compositions of the invention are
known in the art and described, for example in Genaro, ed., 1985,
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa., which is incorporated herein by reference.
[0360] Typically, dosages of the compound of the invention which
may be administered to an animal, preferably a human, range in
amount from 1 .mu.g to about 100 g per kilogram of body weight of
the subject. While the precise dosage administered will vary
depending upon any number of factors, including, but not limited
to, the type of animal and type of disease state being treated, the
age of the subject and the route of administration. In one aspect,
the dosage of the compound will vary from about 1 mg to about 10 g
per kilogram of body weight of the subject. In another aspect, the
dosage will vary from about 10 mg to about 1 g per kilogram of body
weight of the subject.
[0361] The compound may be administered to a subject as frequently
as several times daily, or it may be administered less frequently,
such as once a day, once a week, once every two weeks, once a
month, or even lees frequently, such as once every several months
or even once a year or less. The frequency of the dose will be
readily apparent to the skilled artisan and will depend upon any
number of factors, such as, but not limited to, the type and
severity of the condition or disease being treated, the type and
age of the subject, etc.
[0362] The invention is also directed to methods of administering
the compounds of the invention to a subject. In one embodiment, the
invention provides a method of treating a subject by administering
compounds identified using the methods of the invention.
Pharmaceutical compositions comprising the present compounds are
administered to an individual in need thereof by any number of
routes including, but not limited to, topical, oral, intravenous,
intramuscular, intra arterial, intramedullary, intrathecal,
intraventricular, transdermal, subcutaneous, intraperitoneal,
intranasal, enteral, topical, sublingual, or rectal means.
[0363] For oral administration, the active ingredient can be
administered in solid dosage forms, such as capsules, tablets, and
powders, or in liquid dosage forms, such as elixirs, syrups, and
suspensions. Active component(s) can be encapsulated in gelatin
capsules together with inactive ingredients and powdered carriers,
such as glucose, lactose, sucrose, mannitol, starch, cellulose or
cellulose derivatives, magnesium stearate, stearic acid, sodium
saccharin, talcum, magnesium carbonate, and the like. Examples of
additional inactive ingredients that may be added to provide
desirable color, taste, stability, buffering capacity, dispersion
or other known desirable features are red iron oxide, silica gel,
sodium lauryl sulfate, titanium dioxide, edible white ink and the
like. Similar diluents can be used to make compressed tablets. Both
tablets and capsules can be manufactured as sustained release
products to provide for continuous release of medication over a
period of hours. Compressed tablets can be sugar coated or film
coated to mask any unpleasant taste and protect the tablet from the
atmosphere, or enteric-coated for selective disintegration in the
gastrointestinal tract. Liquid dosage forms for oral administration
can contain coloring and flavoring to increase patient
acceptance.
[0364] The invention also includes a kit comprising the composition
of the invention and an instructional material which describes
adventitially administering the composition to a cell or a tissue
of a mammal. In another embodiment, this kit comprises a
(preferably sterile) solvent suitable for dissolving or suspending
the composition of the invention prior to administering the
compound to the mammal.
[0365] As used herein, an "instructional material" includes a
publication, a recording, a diagram, or any other medium of
expression which can be used to communicate the usefulness of the
peptide of the invention in the kit for effecting alleviation of
the various diseases or disorders recited herein. Optionally, or
alternately, the instructional material may describe one or more
methods of alleviation the diseases or disorders in a cell or a
tissue of a mammal. The instructional material of the kit of the
invention may, for example, be affixed to a container which
contains the peptide of the invention or be shipped together with a
container which contains the peptide. Alternatively, the
instructional material may be shipped separately from the container
with the intention that the instructional material and the compound
be used cooperatively by the recipient.
[0366] Other techniques known in the art may be used in the
practice of the present invention, including those described in
international patent application WO 2006/091535
(PCT/US2006/005970), the entirety of which is incorporated by
reference herein.
[0367] It will be appreciated, of course, that the proteins or
peptides of the invention may incorporate amino acid residues which
are modified without affecting activity. For example, the termini
may be derivatized to include blocking groups, i.e. chemical
substituents suitable to protect and/or stabilize the N- and
C-termini from "undesirable degradation", a term meant to encompass
any type of enzymatic, chemical or biochemical breakdown of the
compound at its termini which is likely to affect the function of
the compound, i.e. sequential degradation of the compound at a
terminal end thereof.
[0368] Blocking groups include protecting groups conventionally
used in the art of peptide chemistry which will not adversely
affect the in vivo activities of the peptide. For example, suitable
N-terminal blocking groups can be introduced by alkylation or
acylation of the N-terminus. Examples of suitable N-terminal
blocking groups include C.sub.1-C.sub.5 branched or unbranched
alkyl groups, acyl groups such as formyl and acetyl groups, as well
as substituted forms thereof, such as the acetamidomethyl (Acm)
group. Desamino analogs of amino acids are also useful N-terminal
blocking groups, and can either be coupled to the N-terminus of the
peptide or used in place of the N-terminal reside. Suitable
C-terminal blocking groups, in which the carboxyl group of the
C-terminus is either incorporated or not, include esters, ketones
or amides. Ester or ketone-forming alkyl groups, particularly lower
alkyl groups such as methyl, ethyl and propyl, and amide-forming
amino groups such as primary amines (--NH.sub.2), and mono- and
dialkylamino groups such as methylamino, ethylamino, dimethylamino,
diethylamino, methylethylamino and the like are examples of
C-terminal blocking groups. Descarboxylated amino acid analogues
such as agmatine are also useful C-terminal blocking groups and can
be either coupled to the peptide's C-terminal residue or used in
place of it. Further, it will be appreciated that the free amino
and carboxyl groups at the termini can be removed altogether from
the peptide to yield desamino and descarboxylated forms thereof
without affect on peptide activity.
[0369] Acid addition salts of the present invention are also
contemplated as functional equivalents. Thus, a peptide in
accordance with the present invention treated with an inorganic
acid such as hydrochloric, hydrobromic, sulfuric, nitric,
phosphoric, and the like, or an organic acid such as an acetic,
propionic, glycolic, pyruvic, oxalic, malic, malonic, succinic,
maleic, fumaric, tartaric, citric, benzoic, cinnamic, mandelic,
methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicyclic and
the like, to provide a water soluble salt of the peptide is
suitable for use in the invention.
[0370] Modifications (which do not normally alter primary sequence)
include in vivo, or in vitro chemical derivatization of
polypeptides, e.g., acetylation, or carboxylation. Also included
are modifications of glycosylation, e.g., those made by modifying
the glycosylation patterns of a polypeptide during its synthesis
and processing or in further processing steps; e.g., by exposing
the polypeptide to enzymes which affect glycosylation, e.g.,
mammalian glycosylating or deglycosylating enzymes. Also embraced
are sequences which have phosphorylated amino acid residues, e.g.,
phosphotyrosine, phosphoserine, or phosphothreonine.
[0371] Also included are polypeptides which have been modified
using ordinary molecular biological techniques so as to improve
their resistance to proteolytic degradation or to optimize
solubility properties or to render them more suitable as a
therapeutic agent. Analogs of such polypeptides include those
containing residues other than naturally occurring L-amino acids,
e.g., D-amino acids or non-naturally occurring or non-standard
synthetic amino acids. The peptides of the invention are not
limited to products of any of the specific exemplary processes
listed herein.
[0372] The invention includes the use of beta-alanine (also
referred to as .beta.-alanine, .beta.-Ala, bA, and .beta.A, having
the structure:
##STR00002##
[0373] Sequences are provided herein which use the symbol
".beta.A", but in the Sequence Listing submitted herewith ".beta.A"
is provided as "Xaa" and reference in the text of the Sequence
Listing indicates that Xaa is beta alanine.
[0374] Peptides useful in the present invention, such as standards,
or modifications for analysis, may be readily prepared by standard,
well-established techniques, such as solid-phase peptide synthesis
(SPPS) as described by Stewart et al. in Solid Phase Peptide
Synthesis, 2nd Edition, 1984, Pierce Chemical Company, Rockford,
Ill.; and as described by Bodanszky and Bodanszky in The Practice
of Peptide Synthesis, 1984, Springer-Verlag, New York. At the
outset, a suitably protected amino acid residue is attached through
its carboxyl group to a derivatized, insoluble polymeric support,
such as cross-linked polystyrene or polyamide resin. "Suitably
protected" refers to the presence of protecting groups on both the
.alpha.-amino group of the amino acid, and on any side chain
functional groups. Side chain protecting groups are generally
stable to the solvents, reagents and reaction conditions used
throughout the synthesis, and are removable under conditions which
will not affect the final peptide product. Stepwise synthesis of
the oligopeptide is carried out by the removal of the N-protecting
group from the initial amino acid, and couple thereto of the
carboxyl end of the next amino acid in the sequence of the desired
peptide. This amino acid is also suitably protected. The carboxyl
of the incoming amino acid can be activated to react with the
N-terminus of the support-bound amino acid by formation into a
reactive group such as formation into a carbodiimide, a symmetric
acid anhydride or an "active ester" group such as
hydroxybenzotriazole or pentafluorophenyl esters.
[0375] Examples of solid phase peptide synthesis methods include
the BOC method which utilized tert-butyloxycarbonyl as the
.alpha.-amino protecting group, and the FMOC method which utilizes
9-fluorenylmethyloxycarbonyl to protect the .alpha.-amino of the
amino acid residues, both methods of which are well-known by those
of skill in the art.
[0376] Incorporation of N- and/or C-blocking groups can also be
achieved using protocols conventional to solid phase peptide
synthesis methods. For incorporation of C-terminal blocking groups,
for example, synthesis of the desired peptide is typically
performed using, as solid phase, a supporting resin that has been
chemically modified so that cleavage from the resin results in a
peptide having the desired C-terminal blocking group. To provide
peptides in which the C-terminus bears a primary amino blocking
group, for instance, synthesis is performed using a
p-methylbenzhydrylamine (MBHA) resin so that, when peptide
synthesis is completed, treatment with hydrofluoric acid releases
the desired C-terminally amidated peptide. Similarly, incorporation
of an N-methylamine blocking group at the C-terminus is achieved
using N-methylaminoethyl-derivatized DVB, resin, which upon HF
treatment releases a peptide bearing an N-methylamidated
C-terminus. Blockage of the C-terminus by esterification can also
be achieved using conventional procedures. This entails use of
resin/blocking group combination that permits release of side-chain
peptide from the resin, to allow for subsequent reaction with the
desired alcohol, to form the ester function. FMOC protecting group,
in combination with DVB resin derivatized with methoxyalkoxybenzyl
alcohol or equivalent linker, can be used for this purpose, with
cleavage from the support being effected by TFA in dichloromethane.
Esterification of the suitably activated carboxyl function e.g.
with DCC, can then proceed by addition of the desired alcohol,
followed by deprotection and isolation of the esterified peptide
product.
[0377] Incorporation of N-terminal blocking groups can be achieved
while the synthesized peptide is still attached to the resin, for
instance by treatment with a suitable anhydride and nitrile. To
incorporate an acetyl blocking group at the N-terminus, for
instance, the resin-coupled peptide can be treated with 20% acetic
anhydride in acetonitrile. The N-blocked peptide product can then
be cleaved from the resin, deprotected and subsequently
isolated.
[0378] To ensure that the peptide obtained from either chemical or
biological synthetic techniques is the desired peptide, analysis of
the peptide composition should be conducted. Such amino acid
composition analysis may be conducted using high resolution mass
spectrometry to determine the molecular weight of the peptide.
Alternatively, or additionally, the amino acid content of the
peptide can be confirmed by hydrolyzing the peptide in aqueous
acid, and separating, identifying and quantifying the components of
the mixture using HPLC, or an amino acid analyzer. Protein
sequenators, which sequentially degrade the peptide and identify
the amino acids in order, may also be used to determine definitely
the sequence of the peptide.
[0379] Prior to its use, the peptide may be purified to remove
contaminants. In this regard, it will be appreciated that the
peptide will be purified so as to meet the standards set out by the
appropriate regulatory agencies. Any one of a number of a
conventional purification procedures may be used to attain the
required level of purity including, for example, reversed-phase
high performance liquid chromatography (HPLC) using an alkylated
silica column such as C.sub.4-, C.sub.8- or C.sub.18-silica. A
gradient mobile phase of increasing organic content is generally
used to achieve purification, for example, acetonitrile in an
aqueous buffer, usually containing a small amount of
trifluoroacetic acid. Ion-exchange chromatography can be also used
to separate peptides based on their charge.
[0380] Substantially pure protein obtained as described herein may
be purified by following known procedures for protein purification,
wherein an immunological, enzymatic or other assay is used to
monitor purification at each stage in the procedure. Protein
purification methods are well known in the art, and are described,
for example in Deutscher et al. (ed., 1990, Guide to Protein
Purification, Harcourt Brace Jovanovich, San Diego).
[0381] As discussed, modifications or optimizations of peptide
ligands of the invention are within the scope of the application.
Modified or optimized peptides are included within the definition
of peptide binding ligand. Specifically, a peptide sequence
identified can be modified to optimize its potency, pharmacokinetic
behavior, stability and/or other biological, physical and chemical
properties.
[0382] Amino Acid Substitutions
[0383] In certain embodiments, the disclosed methods and
compositions may involve preparing peptides with one or more
substituted amino acid residues. In various embodiments, the
structural, physical and/or therapeutic characteristics of peptide
sequences may be optimized by replacing one or more amino acid
residues.
[0384] Other modifications can also be incorporated without
adversely affecting the activity and these include, but are not
limited to, substitution of one or more of the amino acids in the
natural L-isomeric form with amino acids in the D-isomeric form.
Thus, the peptide may include one or more D-amino acid resides, or
may comprise amino acids which are all in the D-form. Retro-inverso
forms of peptides in accordance with the present invention are also
contemplated, for example, inverted peptides in which all amino
acids are substituted with D-amino acid forms.
[0385] The skilled artisan will be aware that, in general, amino
acid substitutions in a peptide typically involve the replacement
of an amino acid with another amino acid of relatively similar
properties (i.e., conservative amino acid substitutions). The
properties of the various amino acids and effect of amino acid
substitution on protein structure and function have been the
subject of extensive study and knowledge in the art. For example,
one can make the following isosteric and/or conservative amino acid
changes in the parent polypeptide sequence with the expectation
that the resulting polypeptides would have a similar or improved
profile of the properties described above:
[0386] Substitution of alkyl-substituted hydrophobic amino acids:
including alanine, leucine, isoleucine, valine, norleucine,
S-2-aminobutyric acid, S-cyclohexylalanine or other simple
alpha-amino acids substituted by an aliphatic side chain from C1-10
carbons including branched, cyclic and straight chain alkyl,
alkenyl or alkynyl substitutions.
[0387] Substitution of aromatic-substituted hydrophobic amino
acids: including phenylalanine, tryptophan, tyrosine,
biphenylalanine, 1-naphthylalanine, 2-naphthylalanine,
2-benzothienylalanine, 3-benzothienylalanine, histidine, amino,
alkylamino, dialkylamino, aza, halogenated (fluoro, chloro, bromo,
or iodo) or alkoxy-substituted forms of the previous listed
aromatic amino acids, illustrative examples of which are: 2-, 3- or
4-aminophenylalanine, 2-, 3- or 4-chlorophenylalanine, 2-, 3- or
4-methylphenylalanine, 2-, 3- or 4-methoxyphenylalanine, 5-amino-,
5-chloro-, 5-methyl- or 5-methoxytryptophan, 2'-, 3'-, or
4'-amino-, 2'-, 3'-, or 4'-chloro-, 2, 3, or 4-biphenylalanine, 2',
-3',- or 4'-methyl-2, 3 or 4-biphenylalanine, and 2- or
3-pyridylalanine.
[0388] Substitution of amino acids containing basic functions:
including arginine, lysine, histidine, ornithine,
2,3-diaminopropionic acid, homoarginine, alkyl, alkenyl, or
aryl-substituted (from C.sub.1-C.sub.10 branched, linear, or
cyclic) derivatives of the previous amino acids, whether the
substituent is on the heteroatoms (such as the alpha nitrogen, or
the distal nitrogen or nitrogens, or on the alpha carbon, in the
pro-R position for example. Compounds that serve as illustrative
examples include: N-epsilon-isopropyl-lysine,
3-(4-tetrahydropyridyl)-glycine, 3-(4-tetrahydropyridyl)-alanine,
N,N-gamma, gamma'-diethyl-homoarginine. Included also are compounds
such as alpha methyl arginine, alpha methyl 2,3-diaminopropionic
acid, alpha methyl histidine, alpha methyl ornithine where alkyl
group occupies the pro-R position of the alpha carbon. Also
included are the amides formed from alkyl, aromatic, heteroaromatic
(where the heteroaromatic group has one or more nitrogens, oxygens,
or sulfur atoms singly or in combination) carboxylic acids or any
of the many well-known activated derivatives such as acid
chlorides, active esters, active azolides and related derivatives)
and lysine, ornithine, or 2,3-diaminopropionic acid.
[0389] Substitution of acidic amino acids: including aspartic acid,
glutamic acid, homoglutamic acid, tyrosine, alkyl, aryl, arylalkyl,
and heteroaryl sulfonamides of 2,4-diaminopropionic acid, ornithine
or lysine and tetrazole-substituted alkyl amino acids.
[0390] Substitution of side chain amide residues: including
asparagine, glutamine, and alkyl or aromatic substituted
derivatives of asparagine or glutamine.
[0391] Substitution of hydroxyl containing amino acids: including
serine, threonine, homoserine, 2,3-diaminopropionic acid, and alkyl
or aromatic substituted derivatives of serine or threonine. It is
also understood that the amino acids within each of the categories
listed above can be substituted for another of the same group.
[0392] For example, the hydropathic index of amino acids may be
considered (Kyte & Doolittle, 1982, J. Mol. Biol.,
157:105-132). The relative hydropathic character of the amino acid
contributes to the secondary structure of the resultant protein,
which in turn defines the interaction of the protein with other
molecules. Each amino acid has been assigned a hydropathic index on
the basis of its hydrophobicity and charge characteristics (Kyte
& Doolittle, 1982), these are: isoleucine (+4.5); valine
(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine
(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4);
threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine
(-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5);
glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine
(-3.9); and arginine (-4.5). In making conservative substitutions,
the use of amino acids can include various hydropathic indices. In
one aspect, the hydropathic indices are within +/-2, in another
they are within +/-1, and in one aspect, they are within
+/-0.5.
[0393] Amino acid substitution may also take into account the
hydrophilicity of the amino acid residue (e.g., U.S. Pat. No.
4,554,101). Hydrophilicity values have been assigned to amino acid
residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0);
glutamate (+3.0); serine (+0.3); asparagine (+0.2); glutamine
(+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-0.1);
alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine
(-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine
(-2.3); phenylalanine (-2.5); tryptophan (-3.4) In one aspect, the
replacement of amino acids with others of similar hydrophilicity is
provided by the invention.
[0394] Other considerations include the size of the amino acid side
chain. For example, it would generally not be preferable to replace
an amino acid with a compact side chain, such as glycine or serine,
with an amino acid with a bulky side chain, e.g., tryptophan or
tyrosine. The effect of various amino acid residues on protein
secondary structure is also a consideration. Through empirical
study, the effect of different amino acid residues on the tendency
of protein domains to adopt an alpha-helical, beta-sheet or reverse
turn secondary structure has been determined and is known in the
art (see, e.g., Chou & Fasman, 1974, Biochemistry, 13:222-245;
1978, Ann. Rev. Biochem., 47: 251-276; 1979, Biophys. J.,
26:367-384).
[0395] Based on such considerations and extensive empirical study,
tables of conservative amino acid substitutions have been
constructed and are known in the art. For example: arginine and
lysine; glutamate and aspartate; serine and threonine; glutamine
and asparagine; and valine, leucine and isoleucine. Alternatively:
Ala (A) leu, ile, val; Arg (R) gln, asn, lys; Asn (N) his, asp,
lys, arg, gln; Asp (D) asn, glu; Cys (C) ala, ser; Gln (Q) glu,
asn; Glu (E) gln, asp; Gly (G) ala; His (H) asn, gln, lys, arg; Ile
(I) val, met, ala, phe, leu; Leu (L) val, met, ala, phe, ile; Lys
(K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F) leu, val, ile,
ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W) phe, tyr;
Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.
[0396] Other considerations for amino acid substitutions include
whether or not the residue is located in the interior of a protein
or is solvent exposed. For interior residues, conservative
substitutions would include: Asp and Asn; Ser and Thr; Ser and Ala;
Thr and Ala; Ala and Gly; Ile and Val; Val and Leu; Leu and Ile;
Leu and Met; Phe and Tyr; Tyr and Trp. (See, e.g., PROWL
Rockefeller University website). For solvent exposed residues,
conservative substitutions would include: Asp and Asn; Asp and Glu;
Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala and Gly;
Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu;
Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Various matrices have
been constructed to assist in selection of amino acid
substitutions, such as the PAM250 scoring matrix, Dayhoff matrix,
Grantham matrix, McLachlan matrix, Doolittle matrix, Henikoff
matrix, Miyata matrix, Fitch matrix, Jones matrix, Rao matrix,
Levin matrix and Risler matrix (Idem.)
[0397] In determining amino acid substitutions, one may also
consider the existence of intermolecular or intramolecular bonds,
such as formation of ionic bonds (salt bridges) between positively
charged residues (e.g., His, Arg, Lys) and negatively charged
residues (e.g., Asp, Glu) or disulfide bonds between nearby
cysteine residues.
[0398] Methods of substituting any amino acid for any other amino
acid in an encoded peptide sequence are well known and a matter of
routine experimentation for the skilled artisan, for example by the
technique of site-directed mutagenesis or by synthesis and assembly
of oligonucleotides encoding an amino acid substitution and
splicing into an expression vector construct.
[0399] Linkers
[0400] Additionally, modifications encompassed by the invention
include introduction of linkers or spacers between the targeting
sequence of the binding moiety or binding polypeptide and a
detectable label or therapeutic agent. For example, use of such
linkers/spacers can improve the relevant properties of the binding
peptides (e.g., increase serum stability, etc.). These linkers can
include, but are not restricted to, substituted or unsubstituted
alkyl chains, polyethylene glycol derivatives, amino acid spacers,
sugars, or aliphatic or aromatic spacers common in the art.
[0401] In other embodiments, therapeutic agents, including, but not
limited to, cytotoxic agents, anti-angiogenic agents, pro-apoptotic
agents, antibiotics, hormones, hormone antagonists, chemokines,
drugs, prodrugs, toxins, enzymes or other agents may be used as
adjunct therapies when using the antibody/peptide ligand complexes
described herein.
[0402] Nucleic acids useful in the present invention include, by
way of example and not limitation, oligonucleotides and
polynucleotides such as antisense DNAs and/or RNAs; ribozymes; DNA
for gene therapy; viral fragments including viral DNA and/or RNA;
DNA and/or RNA chimeras; mRNA; plasmids; cosmids; genomic DNA;
cDNA; gene fragments; various structural forms of DNA including
single-stranded DNA, double-stranded DNA, supercoiled DNA and/or
triple-helical DNA; Z-DNA; and the like. The nucleic acids may be
prepared by any conventional means typically used to prepare
nucleic acids in large quantity. For example, DNAs and RNAs may be
chemically synthesized using commercially available reagents and
synthesizers by methods that are well-known in the art (see, e.g.,
Gait, 1985, OLIGONUCLEOTIDE SYNTHESIS: A PRACTICAL APPROACH (IRL
Press, Oxford, England)). RNAs may be produce in high yield via in
vitro transcription using plasmids such as SP65 (Promega
Corporation, Madison, Wis.).
[0403] The invention further provides cells transfected with the
nucleic acid containing an enhancer/promoter combination of the
invention.
[0404] Promoters may be coupled with other regulatory
sequences/elements which, when bound to appropriate intracellular
regulatory factors, enhance ("enhancers") or repress ("repressors")
promoter-dependent transcription. A promoter, enhancer, or
repressor, is said to be "operably linked" to a transgene when such
element(s) control(s) or affect(s) transgene transcription rate or
efficiency. For example, a promoter sequence located proximally to
the 5' end of a transgene coding sequence is usually operably
linked with the transgene. As used herein, term "regulatory
elements" is used interchangeably with "regulatory sequences" and
refers to promoters, enhancers, and other expression control
elements, or any combination of such elements.
[0405] Promoters are positioned 5' (upstream) to the genes that
they control. Many eukaryotic promoters contain two types of
recognition sequences: TATA box and the upstream promoter elements.
The TATA box, located 25-30 bp upstream of the transcription
initiation site, is thought to be involved in directing RNA
polymerase II to begin RNA synthesis as the correct site. In
contrast, the upstream promoter elements determine the rate at
which transcription is initiated. These elements can act regardless
of their orientation, but they must be located within 100 to 200 bp
upstream of the TATA box.
[0406] Enhancer elements can stimulate transcription up to
1000-fold from linked homologous or heterologous promoters.
Enhancer elements often remain active even if their orientation is
reversed (Li et al., J. Bio. Chem. 1990, 266: 6562-6570).
Furthermore, unlike promoter elements, enhancers can be active when
placed downstream from the transcription initiation site, e.g.,
within an intron, or even at a considerable distance from the
promoter (Yutzey et al., Mol. and Cell. Bio. 1989,
9:1397-1405).
[0407] It is known in the art that some variation in this distance
can be accommodated without loss of promoter function. Similarly,
the positioning of regulatory elements with respect to the
transgene may vary significantly without loss of function. Multiple
copies of regulatory elements can act in concert. Typically, an
expression vector comprises one or more enhancer sequences followed
by, in the 5' to 3' direction, a promoter sequence, all operably
linked to a transgene followed by a polyadenylation sequence.
[0408] The present invention further relies on the fact that many
enhancers of cellular genes work exclusively in a particular tissue
or cell type. In addition, some enhancers become active only under
specific conditions that are generated by the presence of an
inducer such as a hormone or metal ion. Because of these
differences in the specificities of cellular enhancers, the choice
of promoter and enhancer elements to be incorporated into a
eukaryotic expression vector is determined by the cell type(s) in
which the recombinant gene is to be expressed.
[0409] In one aspect, the regulatory elements of the invention may
be heterologous with regard to each other or to a transgene, that
is, they may be from different species. Furthermore, they may be
from species other than the host, or they also may be derived from
the same species but from different genes, or they may be derived
from a single gene.
[0410] The present invention further encompasses kits.
[0411] Compositions of the present invention may be presented in a
pack or dispenser device, such as an FDA approved kit, which may
contain one or more unit dosage forms containing the therapeutic
compound as described herein.
[0412] In some embodiments, the kit may include a therapeutic
compound (as described herein), metal or plastic foil, such as a
blister pack, a dispenser device or an applicator, tubes, buffers,
and instructions for administration. The various reagent components
of the kits may be present in separate containers, or some or all
of them may be pre-combined into a reagent mixture in a single
container, as desired. The dispenser device or applicator may also
be accommodated by a notice associated with the container in a form
prescribed by a governmental agency regulating the manufacture, use
or sale of pharmaceuticals, which notice is reflective of approval
by the agency of the form of the compositions or human or
veterinary administration. Such notice, for example, may be of
labeling approved by the U.S. Food and Drug Administration for
prescription drugs or of an approved product insert.
[0413] In some cases, the kit includes at least one dose of
monoclonal antibody (mAb) to CD24.
[0414] In some cases, the kit includes at least one dose of a
fragment of monoclonal antibody (mAb) to CD24.
[0415] In some cases, the kit includes at least one dose of an
expression vector comprising a nucleic acid sequence encoding the
full length or segments of CD24 protein.
[0416] In some cases, the kit includes at least one dose of an
expression vector comprising a nucleic acid sequence encoding the
full length or segments of SCF protein.
[0417] The invention is now described with reference to the
following Examples and Embodiments. Without further description, it
is believed that one of ordinary skill in the art can, using the
preceding description and the following illustrative examples, make
and utilize the present invention and practice the claimed methods.
The following working examples therefore, are provided for the
purpose of illustration only and specifically point out some
embodiments of the present invention, and are not to be construed
as limiting in any way the remainder of the disclosure. Therefore,
the examples should be construed to encompass any and all
variations which become evident as a result of the teaching
provided herein.
EXAMPLES
Example 1
[0418] Administration of the anti-CD24 mAb induces stress
erythropoiesis in mice, and expression of CD 24 by cells of
hematopoietic origin is required for induction of extra medullary
hematopoiesis. Mice were infused intraperitoneally (i.p.) with 150
.mu.g control rat IgG or .alpha.CD24 (clone M1/69) and necropsied
on days 1, 3 or 5 after Ab treatment. (A) Representative
macroscopic appearance of the spleens over time after .alpha.CD24
mAb infusion in wt B6 mice (n>20). Mice that received M1/69
exhibited marked splenomegaly first demonstrable at d3 post
treatment. (B) M1/69 treatment promotes stress erythropoiesis. As
mice and likely men typically respond to anemia and other hypoxic
stresses by inducing extramedullary stress erythropoiesis in the
spleen, we examined spleens of M1/69-treated mice in more detail.
Single cell suspensions prepared from spleens at d5 post treatment
were analyzed for cell types expressed by flow cytometry and
enumerated (n.gtoreq.5). Interestingly, this increase in spleen
size cannot be attributed to leukocyte (CD45.sup.+Ter119.sup.-,
group I) expansion, as leukocyte numbers were unaffected by
M1/69-treatment (B) as well as the distribution of lymphocytic- and
myeloid-lineage cells (data not shown). Therefore, we turned into
examining the splenic erythroid compartment, based on the
expression of the erythroid lineage cell surface marker Ter119. We
found that the spleens of M1/69-treated mice have a dramatic
increase in erythrocytes (CD45.sup.+Ter119.sup.+, group II) and
erythroid progenitor cells (CD45.sup.-Ter119.sup.+, group III),
respectively, with the most dramatic expansion observed within the
spleen in the erythroid progenitor cell (CD45.sup.-Ter119.sup.+,
group III) compartment. (C) We further examined for the erythroid
compartment, based on the differential expression of Ter119 and the
transferrin receptor, CD71. We found that the M1/69 treatment
results in increase in all erythroblast subsets, consisting of
basophilic (Ter119.sup.+CD71.sup.hi, group I--least mature),
polychromatophilic (Ter119.sup.+CD71.sup.med, group ii), and
orthochromatic (Ter119.sup.+CD71.sup.lo, group iii) erythroblasts
in spleen. Of note, consistent with this notion, these erythroid
progenitors underwent proliferation as determined by active uptake
of BrdU and by staining for a proliferation-associated nuclear
antigen, Ki-67 (data not shown). (D) Erythropoiesis induced by
M1/69 treatment requires CD24 expression. To investigate if these
effects are specific to M1/69 mAb interaction with CD24, mice
deficient in CD24 expression were given Ig or .alpha.CD24 mAb and
examined at d5 p.t. for gross appearance of spleens shown in (D)
and absolute number of CD45.sup.-Ter119.sup.+ (data not shown,
n=3-5). Therefore, M1/69 treatment did not have any off target
effects (E) CD24 expression by the cells of hematopoietic origin,
but not the cells in stromal compartment, is required for
M1/69-induced stress erythropoiesis. CD24 (also known as heat
stable antigen) is ubiquitously expressed on many cell types of
hematopoietic (bone marrow-derived) and of non-hematopoietic
origin. To assess the contribution of CD24-expressing cell type
from the respective compartment, BM chimeric mice were established
by transferring 2.times.10.sup.6 donor BM cells derived from either
WT or CD24.sup.-/- mice into lethally irradiated either WT or
CD24.sup.-/- recipient mice. At 6 weeks after BM cell
reconstitution, these chimeric mice were infused with Ig or M1/69.
At d5, mice were necropsied and evaluated for gross appearance of
spleens (right panel) and for erythroid progenitor cells
frequency/numbers (left panel) (n=3/group). CD24 expression on
cells of bone marrow/hematopoietic origin was necessary and
sufficient for stress-induced hematopoiesis mediated by M1/69
treatment. It is also noteworthy that erythropoiesis induced by
.alpha.CD24 mAb depends on F(ab).sub.2, but not Fc, fragments (data
not shown). Data represent mean.+-.SD. ***P<0.001.
[0419] Stress erythropoiesis in the peripheral blood after
.alpha.CD24 mAb treatment. The effect of M1/69 treatment in
erythropoiesis was also observed in peripheral blood. WT mice were
infused i.p. with 150 .mu.g control Ig or .alpha.CD24 and examined
at the indicated time points. Peripheral blood smears were prepared
at d5 p.t. and stained by methylene blue. Typical staining of
residual RNA in reticulocytes--immature RBC for about a day in the
blood stream before developing into mature RBCs--is examined by
microscope (data now shown). (A) Percentages of reticulocytes in
the peripheral blood were also assessed by flow cytometry using
thiazole orange staining (to selectively stain nucleic acid, in
this case RNA) for about an 1 hr at room temperature. A
representative flow cytometric analysis of examining reticulocytes
in blood at d5 is shown. Data are representative of >20
independently repeated experiments. Numbers denote the percentage
of reticulocytes staining with thiazole orange dye in peripheral
blood. (B) Kinetic analyses of percent reticulocytes circulating in
the blood of mice treated with Ig or .alpha.CD24 at the indicated
dates (n=4-7/day).
[0420] Stress erythropoiesis induced by .alpha.CD24 mAb treatment
depends on the spleen and, to a lesser extent, bone marrow.
Increased erythropoiesis is a key component of a physiological
stress response to increase oxygen delivery to tissues. While
earlier works have identified the murine spleen as the primary site
of erythropoiesis in response to hypoxic conditions, stress
erythropoiesis is also viewed as an intensified version of steady
state erythropoiesis, which for the most part is restricted to the
bone marrow. To study the extent of CD24-induced erythropoiesis in
the spleen as well as BM, respectively, WT mice were injected i.p.
with 150-.mu.g control Ig or .alpha.CD24. (A) Representative of the
frequency of CD45.sup.-Ter119.sup.+ erythroid progenitors in
spleens (top panels in A) and BM (bottom panels in A), as measured
by flow cytometry at d5 after .alpha.CD24 treatment. (B) Total
erythroid progenitors per spleen and BM, respectively, at d5 are
depicted (n=3-5). (C-E) To evaluate the impact of spleen on stress
erythropoiesis, mice were splenectomized prior to antibody
administration. WT B6 mice that had undergone splenectomy 5 weeks
earlier were treated as in A. At d5, percentage of reticulocytes in
the blood (C and D) and absolute cell number of
CD45.sup.-Ter119.sup.+ cells in BM (E) was assessed by flow
cytometric analyses (n=3-5). Mean.+-.SD is shown (***P<0.001,
**P<0.01 and **P<0.05). These data indicate that spleen is
the major site for stress erythropoiesis induced by .alpha.CD24
mAb. However, while the bone marrow is a minor contributor to
.alpha.CD24-induced erythropoiesis when the spleen is present, bone
marrow displays a more vigorous stress erythropoiesis response in
the absence of spleen, presumably as a compensatory mechanism.
[0421] A novel role of conventional dendritic cells, in particular
CD8.alpha.+ DC, in CD24-mediated stress erythropoiesis in vivo.
Maintenance of the number of circulating RBCs is a tightly
regulated process balancing between the production of RBCs derived
from erythroid progenitors and the removal of the senescent RBCs by
the hemophagocytic system. The cell types that are critically
involved in regulating stress erythropoiesis in vivo are unclear.
Our data shown in FIG. 1E indicate that cells originating from
hematopoietic compartment are required for stress erythropoiesis
induced by .alpha.CD24 mAb treatment. To investigate the specific
cell type(s) governing stress erythropoiesis in vivo, we employed
mice deficient in or depleted of various cell types (T, B, and
inflammatory monocytes, neutrophils and NK cells) (See FIG. 4). Our
analyses found that none of these hematopoietic cell lineages were
important regulators of stress erythropoiesis induced by M1/69
treatment (data not shown). Next, we assessed the impact of
dendritic cells (DCs) on the stress erythropoiesis. DCs are well
recognized as professional antigen presenting cells, composed of
heterogeneous subpopulations. In lymphoid organs such as spleens,
three major subsets of DCs are classified; two conventional DC
(cDC) subsets including CD8.alpha..sup.+CD11b.sup.-
(CD8.alpha..sup.+ cDC, group I) and CD8.alpha..sup.-CD11b.sup.+
(CD11b.sup.+ cDC, group II) (top panels in A) and plasmacytoid DC
(pDC) (data not shown). Notably, it is the CD8.alpha..sup.+ cDC
among DC subsets in the spleen which express cell surface CD24 at
the highest level (lower panel in A). To study the role of
CD24.sup.hi CD8.alpha..sup.+ cDC in .alpha.CD24 mAb-induced stress
erythropoiesis, we obtained mice lacking transcriptional factor
Batf3 gene (Batf3 KO mice), which are developmentally devoid of
CD8.alpha..sup.+ cDC in the spleen and of nodes and a related
lineage of DC which populate certain non-lymphoid tissues such as
lung and kidney etc. (B). WT and Batf3 KO mice were injected i.p.
with 150 .mu.g control Ig or .alpha.CD24. After 5 days, gross
appearance of spleens (data not shown), a representative of the
percentage of circulating reticulocytes in the blood (C) and
absolute number of CD45.sup.-Ter119.sup.+ erythroid progenitors in
the spleens (D) were assessed by flow cytometry (n=4-6). The
results of these analyses indicate that CD8.alpha..sup.+ cDC in the
spleen and possibly a related tissue-specific DC subset play a
critical role in promoting stress erythropoiesis. To exclude a
possibility of an additional unanticipated role of the Batf3 gene
on erythropoiesis, i.e., other than its role in the generation of a
specific DC lineage, we employed CD11c-DTR mice, which are
engineered to express non-human primate diphtheria toxin receptor
(DTR) driven off of murine CD11c (a conventional marker for murine
DC) promoter. Upon diphtheria toxin (DTx) administration (100
ng/mouse via i.p.), DTR expressing CD11c.sup.+ cells (that is cDC)
are conditionally selectively ablated within 24 hrs (E). These
cDC-ablated mice were infused with Ig or .alpha.CD24 mAb 1 day
after first DTx administration. With second dose of DTx at d1 post
treatment, mice were necropsied at d5 for gross appearance of the
spleens (data not shown). The percentage of circulating
reticulocytes in the blood (data not shown) and absolute number of
CD45.sup.-Ter119.sup.+ erythroid progenitors in the spleens (F)
were assessed by flow cytometry (n=3-5). The analysis demonstrates
that CD11c.sup.+ cells, i.e., cDC, play a critical role in
regulating the development of stress hematopoiesis mediated by CD
24 engagement. Data represent mean.+-.SD.
[0422] Conventional DCs are required for the expansion of c-Kit
expressing erythroid progenitors in the spleen during
extramedullary stress erythropoiesis. Our data thus far are
consistent with the prevailing view that the spleen is a major
organ for extramedullary stress erythropoiesis and that this
process can be triggered by engagement of CD24, and finally that
cDC are essential for the physiological process of the
extramedullary erythropoiesis in vivo. Recent studies show that
stress erythropoiesis depends on a population of stress erythroid
progenitor cells that are distinct from the counterpart present in
BM. The development, expansion, and differentiation of these
progenitors are regulated in part by a complex of less understood
signals. We initially attempted to identify these progenitors based
on c-Kit (CD117) expression as engagement of this receptor has been
demonstrated to be essential for stress erythropoiesis. (FIG. 5A-C)
WT mice were injected i.p. with 150 .mu.g control Ig or
.alpha.CD24. (5A) Anti-CD24 mAb treatment promotes the expansion of
cKit.sup.+CD45.sup.+/- cells in the spleen. At d3 and 5 post
treatments, single cells suspensions prepared from the necropsied
spleens were stained with fluorochrome-conjugated mAb recognizing
c-Kit (left panel). In contrast to Ig treated mice, mice undergoing
M1/69 treatment have a dramatic increase in the number of
c-Kit.sup.+ cells, peaking at d3 (n>6), with little to no
expression of the standard hematopoietic lineage marker CD45 (i.e.,
CD45.sup.+/-c-Kit.sup.+ cells (right panel). (5B)
CD45.sup.+/-c-Kit.sup.+ progenitors exhibited greater (compared to
DC and B cells) forward (FSC) and side scatter (SSC) plot by flow
cytometry analyses and expressed different levels of Ter119 as well
as CD71, indicative of progenitor cells with erythroid lineage
commitment such as proerythroblasts. (C) To determine if
c-Kit.sup.+ proerythroblasts undergo proliferative expansion, the
M1/69-treated mice were fed with nucleic acid analog, BrdU, at d5
to label proliferating cells. After 24 hr BrdU injection,
c-Kit.sup.+ erythroid progenitors were examined for active uptake
of BrdU in combination with staining for a proliferation-associated
nuclear antigen, Ki-67. In contrast to c-Kit.sup.+ cells in the
Ig-treated spleen, c-Kit.sup.+ erythroid progenitors isolated from
the mice treated with M1/69 underwent active proliferation. (D)
Furthermore, in support of the importance of splenic cDC in
orchestrating stress erythropoiesis, ablation of cDC by treatment
of CD11c-DTR mice with diphtheria toxin resulted in minimal
expansion of these progenitors when stimulated by .alpha.CD24 mAb
infusion (n=3-5). Collectively, our data strongly support the view
that cDCs, particularly lymph node-resident CD8.alpha.+ cDC subset
and/or a developmentally related tissue-resident cDC subset, play
an essential role in regulating extramedullary stress
erythropoiesis.
[0423] Stem cell factor produced by cDC in the spleen is required
for extramedullary stress erythropoiesis. We next investigated the
molecular mechanism underlying cDC-dependent proerythroblast
proliferation (FIG. 6). Soluble mediators--i.e., Erythropoietin
(Epo), stem cell factor (SCF) and bone morphogenetic protein 4 (BMP
4), IL-3 and GM-CSF--have each been implicated as necessary to
expand erythroid progenitors during erythropoiesis. We first
examined the profile of gene expression by cDC and non-DC
subpopulations after magnetically sorting out the cell types from
total splenic cells prepared at d1 (data now shown) or d2 (A) or
after M1/69 infusion. The splenic cells were sorted into DCs (based
on CD11c.sup.+), T cells (CD90.sup.+), B cells (B220.sup.+) and
remaining cell types, i.e., both CD45.sup.+ hematopoietic origin
cells and CD45.sup.- splenic stromal cells. We measured expression
levels of mRNA encoding for SCF, Epo, BMP4, IL-3, and GM-CSF,
respectively. Our data revealed that M1/69 treatment induced the
expression of SCF exclusively by cDC (6A). In contrast, cDC in the
spleen did not upregulate mRNA for Epo, BMP4, IL-3 and GM-CSF (data
now shown). (6B-D) This suggests that SCF produced by splenic cDC
via CD24 signaling contributes to M1/69-induced erythropoiesis. To
investigate the role of SCF-c-Kit signaling in stress
erythropoiesis, c-Kit KO mice were infused with .alpha.CD24 mAb.
c-Kit deficiency resulted in an a markedly reduced percentage of
reticulocytes in peripheral blood of mAb treated KO mice compared
to treated wild type control mice when analyzed at d5 after mAb
infusion (B). The importance of c-kit-mediated signaling in this
process was further validated by the analysis of the gross
appearance of spleens (C) and the accumulation of c-Kit.sup.-
erythroid progenitors (CD45.sup.-Ter119.sup.+, left panel in D) and
c-Kit.sup.+ proerythroblasts (right panel in D) in the spleens at
d5 after mAb infusion. As shown in C and D, the absence of
c-Kit-SCF signaling axis resulted in the almost complete abrogation
of M1/69-stimulated erythropoiesis. (6E and F) We complemented the
findings in c-Kit KO mice on the extramedullary stress
erythropoiesis, by analyzing the impact of the pharmacological
inhibitor of c-Kit signaling, imatinib (Gleevec). The drug (1
mg/kg) was administrated i.p. daily for 4 days into M1/69-treated
WT mice. Consistent with the observations in c-Kit KO mice,
administration of Imatinib into mAb treated WT B6 mice
significantly reduced the reticulocytes in the blood (E) and
inhibited the expansion/accumulation of CD45.sup.-Ter119.sup.+
erythroid progenitors (left panel in F) and c-Kit.sup.+
proerythroblasts (right panel in F) in the spleen. In summary, our
data suggest that SCF produced by splenic cDC stimulated by
engagement of the CD24 molecule on the cells with the M1/69 mAb
promotes the expansion of c-Kit.sup.+ erythroid progenitors in the
spleen through a engagement of the c-Kit receptor on the early
splenic erythroid progenitors.
[0424] FIG. 7. Stress erythropoiesis triggered by M1/69 treatment
requires Epo production by the kidneys, and Epo production is in
turn dependent on the presence of cDC. Epo is a glycoprotein
hormone that serves as a primary regulator of differentiation,
proliferation, and survival of erythroid progenitor cells, and is
made mainly by stromal cells within the kidneys. Epo and c-Kit
signaling are both necessary for efficient erythropoiesis and work
synergistically in this process. To fully account for the robust
expansion of erythroid lineage progenitor cells in the spleen and
the subsequent reticulocytosis following mAb treatment, we
postulated that engagement of CD24 stimulates directly or
indirectly Epo production by stromal cells in the kidney, which
acts in concert with SCF produced locally in spleen to orchestrate
extramedullary hematopoiesis. (FIG. 7A) When serum Epo levels were
measured after M1/69 injection, we indeed detected a robust
increase in Epo, peaking d3 after injection and then gradually
decreasing over the several days. This burst of Epo production, as
expected, proceeds the appearance of reticulocytes in the
peripheral blood (see FIG. 2). (7B) In FIG. 6, we demonstrated that
cDCs are critical in stress erythropoiesis induced by M1/69
treatment at least via the provision of SCF to stimulate the
expansion of c-Kit receptor-expressing erythroid progenitors in the
spleen. We reasoned that in parallel to splenic cDC, the
counterpart of cDC subsets in the kidney may play a crucial role in
promoting the production of Epo by epo-producing renal stromal
cells. To test this hypothesis, we first measured Epo levels in the
circulation of mice deficient in cDC, either genetically (i.e.,
Batf3 KO mice) or following cDC ablation (i.e., DTx-treated
CD11c-DTR mice). We found a significant decrease in Epo production
in Batf3 KO mice (7C) and nearly complete ablation of Epo
production in DTx-treated CD11c-DTR mice (7D). These data
demonstrate that cDCs localized in respective tissues play a
crucial role in CD24-mediated stress erythropoiesis via at least
stimulation of Epo induction in the kidneys and SCF production in
the spleen. To dissect the contribution of Epo and SCF,
respectively, we measured Epo concentration in c-Kit KO mice, which
have an intact cDC compartment in the kidney (data not shown). In
stark contrast to the absence of the expansion of c-Kit.sup.+
erythroid progenitors in the spleen, M1/69 treated c-Kit KO mice
have demonstrated an elevated Epo level in the circulation. This is
in keeping with the failure of expansion of c-Kit.sup.+ erythroid
progenitors in the spleens of c-Kit KO mice which would be the
primary consumers of Epo through binding of Epo to its receptor on
the cells. This result indicates that the production of Epo alone,
although necessary, is not sufficient to induce extramedullary
stress erythropoiesis after CD24 engagement.
[0425] Model for CD24-mediated stress erythropoiesis in vivo.
Without wishing to be bound by any particular theory, hypothesized
herein is a novel model for extramedullary stress erythropoiesis in
vivo after M1/69 treatment (FIG. 8). As described above, the
present application discloses a previously unknown function of CD24
expressed by a distinct subset of splenic DCs; that is, engagement
of CD24 on this spleen resident cell type stimulates EPO production
and concomitant vigorous production of RBCs in the spleen and bone
marrow. The current invention encompasses a novel strategy to
enhance stress-mediated erythropoiesis. Hypoxic stresses such as
anemia, blood loss, aging and so on release host-derived danger
associated-pattern molecules (i.e., HMGB1, Hsp70, etc.), which are
recognized by the CD24 receptor expressed on DCs residing in spleen
and kidney, respectively. Ligation of CD24 on splenic DCs
transduces intracellular signals to produce SCF, which act on
neighboring c-Kit.sup.+ progenitors with potential to commit toward
erythroid lineage to undergo expansion. Concomitantly, CD24
interaction on DCs in the kidney results in stimulating in trans
renal epithelial and/or tubular cells to produce growth factors,
including mainly Epo and possibly BMP4. The proliferating
proerythroblasts and erythroid progenitor cells in the spleen
receive the second signals through Epo receptors and undergo
further differentiation, proliferation, and maturation in the
spleen, ultimately releasing reticulocytes into the circulation to
deliver oxygen.
Example 2
[0426] The present example provides: 1.) analysis in the mouse of
erythropoiesis following administration of other monoclonal
antibodies to CD24; 2.) an analysis of the reticulocyte response
following a second administration of the CD24 agonistic mAb; and
3.) an analysis of the response of the human DC subset
corresponding to the murine CD8.alpha..sup.+ DC subset found in the
murine spleen to stimulation by human anti-CD24 mAbs on the
expression of human SCF.
[0427] I. Along with the prototype M1/69 mAb to CD24 employed in
Example 1, the present example includes the analysis of the
stimulation of splenic erythrocyte progenitor
(Ter119.sup.+CD45.sup.- cells) expansion in the spleen following
administration of M1/69 or 1 of 3 other monoclonal anti-CD24
antibodies, 91, 30-F 1, and J11 d, respectively. These data are
included in Supplemental FIGS. 1A and 1B. The results demonstrate
that M1/69 and 91 are potent stimulators of erythropoiesis while
30-F1 antibody has an immediate potency and the J11d has a weak or
no activity.
[0428] II. Supplemental FIG. 2 shows the results of an analysis of
the reticulocyte response to repeated administration of monoclonal
antibody. In this instance, mice were first injected with M1/69 and
the reticulocyte count in the blood was evaluated over time. At day
8 after the injection of the M1/69 antibody, the mice received
either a second injection of M1/69 antibody or an injection of 1 of
the 3 additional anti-CD24 monoclonal antibodies and the
reticulocyte response to a second exposure to anti-CD24 antibody
was monitored over time. The 3 monoclonal antibodies with
stimulatory capacity in primary treatment (i.e., M1/69, 91 and 30-F
1) could each stimulate a second wave of reticulocytosis.
[0429] III. In Supplemental FIG. 3 we have isolated conventional
dendritic cells from the spleens of mice in the usual fashion and
were treated with anti-CD24 monoclonal antibody overnight in
cultures. We monitored the expression of the murine stem cell
factor (mSCF) on the splenic CD8.alpha..sup.+ DC subset and the
more abundant CD8.alpha..sup.-CD11b.sup.+ splenic DCs. As the
figure indicates, mSCF is abundantly expressed on the surface of
CD8.alpha..sup.+ DC subset.
[0430] The human dendritic cell subset corresponding to the murine
CD8.alpha..sup.+ DC expresses the cell surface molecule BDCA3
(CD141) and is present in the human spleen. Therefore, in humans,
the primary targeted DC cell subset are those cells expressing the
cell surface molecule BDCA3 (CD141).
[0431] Human spleen will be tested as described above for mouse
cells. We're currently in the process of obtaining human spleen for
the isolation of this DC subset and for the analysis of the impact
of stimulation of CD24 displayed by these dendritic cells on the
up-regulation of expression of human stem cell factor (hSCF) on the
surface of the cells.
[0432] IV. In the interim we have employed a strategy for
generating BDCA3.sup.+ DC from human peripheral blood mononuclear
cells in vitro using a cocktail of growth factors consisting of
GM-CSF, IL-4, SCF, and Flt3L. This cocktail can also be used in
conjunction with the other agents of the invention to stimulate
erythropoiesis in a subject in need thereof. These growth factors
are highly potent stimulators of proliferation and differentiation
of circulating mononuclear stem cells into a variety of cell types.
Nonetheless, as Supplemental FIG. 4A demonstrates we can detect a
small population of BDCA3.sup.+ DC-like cells in in vitro culture
with a larger population of BDCA3.sup.- cells. As Supplemental FIG.
4B demonstrates we can detect up-regulation of hSCF in response to
treatment with 2 different monoclonal antibodies to human CD24:
eBioSN3 and ML5.
[0433] As demonstrated in Supplemental FIG. 4B, the BioSN3
monoclonal antibody regulates hSCF expression selectively on
BDCA3.sup.+ DC. Note that the background expression of cell surface
hSCF is high in both DC subsets. This may be due to the strong
growth promoting activity of the growth factor cocktails. When this
analysis is repeated with resting BDCA3.sup.+ DC isolated from
human spleen, the background might be lower. Nevertheless, these
findings demonstrate that the corresponding up-regulation of cell
surface hSCF by engagement of the CD24 receptor occurs as well in
the corresponding population of human dendritic cells.
[0434] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
by reference herein in their entirety.
[0435] Headings are included herein for reference and to aid in
locating certain sections. These headings are not intended to limit
the scope of the concepts described therein under, and these
concepts may have applicability in other sections throughout the
entire specification.
[0436] While this invention has been disclosed with reference to
specific embodiments, it is apparent that other embodiments and
variations of this invention may be devised by others skilled in
the art without departing from the true spirit and scope of the
invention.
BIBLIOGRAPHY
[0437] 1. Hunte B E, Capone M, Zlotnik A, Rennick D, Moore T A.
1998. Acquisition of CD24 expression by Lin-CD43+B220(low)ckit(hi)
cells coincides with commitment to the B cell lineage. Eur J
Immunol. 28(11):3850-6.
[0438] 2. Wilson, A., L. M. Day, et al. 1988. Subpopulations of
mature murine thymocytes: properties of CD4-CD8+ and CD4+CD8-
thymocytes lacking the heat-stable antigen. Cell Immunol 117(2):
312-26.
[0439] 3. Alterman, L. A., I. N. Crispe, et al. 1990.
Characterization of the murine heat-stable antigen: an
hematolymphoid differentiation antigen defined by the J11d, M1/69
and B2A2 antibodies. Eur J Immunol 20(7): 1597-602.
[0440] 4. Springer T, Galfre G, Secher D S, Milstein C. 1978.
Monoclonal xenogeneic antibodies to murine cell surface antigens:
identification of novel leukocyte differentiation antigens. Eur J
Immunol. 8(8):539-51.
[0441] 5. Thomas et al., epub. Aug. 27, 2012, Cancer Research, CD24
is an effector of HIF-1 driven primary tumor growth and
metastasis.
[0442] 6. Zhou, Q., et al., "CD24 is a genetic modifier for risk
and progression of multiple sclerosis", PNAS, 2003, Vol. 100, No.
25, 15041-15046.
[0443] 7. Jaggupilli, A., et al., "Significance of CD44 and CD24 as
Cancer Stem Cell Markers: an Enduring Ambiguity", Clinical and
Developmental Immunology", Vol. 2012, Article ID 708036, 1-11.
[0444] 8. Fischer, G., et al., "Signal Transduction in Lymphocytic
and Myeloid Cells via CDE 24, A New Member of
Phosphoinositol-Anchored Membrane Molecules.sup.1". Journal of
Immunology, Vol. 144, No. 2, 1990, 638-641.
[0445] 9. Wu, D., et al., "Antibody-Directed Lentiviral Gene
Transduction for Live-Cell Monitoring and Selection of Human iPS
and hES Cells", PLos ONE, April, 2012, Vol. 7, Issue 4, 1-10.
[0446] 10. Kume, A., et al., "Long-term tracking of murine
hematopoietic cells transduced with a bicistronic retrovirus
containing CD24 and EGFP genes", Gene Therapy (2000), 7,
1193-1199.
[0447] 11. Cao, X., et al., "Upregulation of VEGF-A and CD24 Gene
Expression by the tGLI1 Transcription Factor Contributes to the
Aggressive Behavior or Breast Cancer Cells", Oncogene, January,
2012, 31 (1): 104-115.
[0448] 12. Williams, L., et al., "Identification of a novel
dendritic cell surface antigen defined by carbohydrate specific
CD24 antibody cross-reactivity", Immunology 1996, 89, 120-125.
[0449] 13. Zhu, J., et al., "Identification of Glycoprotein Markers
for Pancreatic Cancer CD24.sup.+CD44.sup.+ Stem-like Cells Using
Nano-LC-MS/MS and Tissue Microarray", J. Proteome Research 2012,
11, 2272-2281.
[0450] 14. Fang, X., et al., "CD24: from A to Z", Cellular &
Molecular Immunology" (2010), 7, 100-103.
[0451] 15. Kay, F., et al., "CD24, A Signal Transducer Modulating B
Cell Activation Responses, is a Very Short Peptide with a Glycosyl
Phosphatidylinositol Membrane Anchor.sup.1", J. of Immunology, Vol.
147, 1412-1416, No. 4, Aug. 15, 1991.
[0452] 16. Salamone, M., et al., "Antibodies recognizing CD24 LAP
epitope on human T cells enhance CD28 and IL-2 T cell
proliferation", J. of Leukocyte Biology, Vol. 69, February 2001,
215-223.
Sequence CWU 1
1
412194DNAHomo sapiens 1gggtctcgcc ggctcgccgc gctccccacc ttgcctgcgc
ccgcccggag ccagcggttc 60tccaagcacc cagcatcctg ctagacgcgc cgcgcaccga
cggaggggac atgggcagag 120caatggtggc caggctcggg ctggggctgc
tgctgctggc actgctccta cccacgcaga 180tttattccag tgaaacaaca
actggaactt caagtaactc ctcccagagt acttccaact 240ctgggttggc
cccaaatcca actaatgcca ccaccaaggc ggctggtggt gccctgcagt
300caacagccag tctcttcgtg gtctcactct ctcttctgca tctctactct
taagagactc 360aggccaagaa acgtcttcta aatttcccca tcttctaaac
ccaatccaaa tggcgtctgg 420aagtccaatg tggcaaggaa aaacaggtct
tcatcgaatc tactaattcc acacctttta 480ttgacacaga aaatgttgag
aatcccaaat ttgattgatt tgaagaacat gtgagaggtt 540tgactagatg
atggatgcca atattaaatc tgctggagtt tcatgtacaa gatgaaggag
600aggcaacatc caaaatagtt aagacatgat ttccttgaat gtggcttgag
aaatatggac 660acttaatact accttgaaaa taagaataga aataaaggat
gggattgtgg aatggagatt 720cagttttcat ttggttcatt aattctataa
ggccataaaa caggtaatat aaaaagcttc 780catgattcta tttatatgta
catgagaagg aacttccagg tgttactgta attcctcaac 840gtattgtttc
gacagcacta atttaatgcc gatatactct agatgaagtt ttacattgtt
900gagctattgc tgttctcttg ggaactgaac tcactttcct cctgaggctt
tggatttgac 960attgcatttg accttttatg tagtaattga catgtgccag
ggcaatgatg aatgagaatc 1020tacccccaga tccaagcatc ctgagcaact
cttgattatc catattgagt caaatggtag 1080gcatttccta tcacctgttt
ccattcaaca agagcactac attcatttag ctaaacggat 1140tccaaagagt
agaattgcat tgaccacgac taatttcaaa atgcttttta ttattattat
1200tttttagaca gtctcacttt gtcgcccagg ccggagtgca gtggtgcgat
ctcagatcag 1260tgtaccattt gcctcccggg ctcaagcgat tctcctgcct
cagcctccca agtagctggg 1320attacaggca cctgccacca tgcccggcta
atttttgtaa ttttagtaga gacagggttt 1380caccatgttg cccaggctgg
tttcgaactc ctgacctcag gtgatccacc cgcctcggcc 1440tcccaaagtg
ctgggattac aggcttgagc ccccgcgccc agccatcaaa atgcttttta
1500tttctgcata tgttgaatac tttttacaat ttaaaaaaat gatctgtttt
gaaggcaaaa 1560ttgcaaatct tgaaattaag aaggcaaaaa tgtaaaggag
tcaaaactat aaatcaagta 1620tttgggaagt gaagactgga agctaatttg
cattaaattc acaaactttt atactctttc 1680tgtatataca ttttttttct
ttaaaaaaca actatggatc agaatagcca catttagaac 1740actttttgtt
atcagtcaat atttttagat agttagaacc tggtcctaag cctaaaagtg
1800ggcttgattc tgcagtaaat cttttacaac tgcctcgaca cacataaacc
tttttaaaaa 1860tagacactcc ccgaagtctt ttgttcgcat ggtcacacac
tgatgcttag atgttccagt 1920aatctaatat ggccacagta gtcttgatga
ccaaagtcct ttttttccat ctttagaaaa 1980ctacatggga acaaacagat
cgaacagttt tgaagctact gtgtgtgtga atgaacactc 2040ttgctttatt
ccagaatgct gtacatctat tttggattgt atattgtgtt tgtgtattta
2100cgctttgatt catagtaact tcttatggaa ttgatttgca ttgaacacaa
actgtaaata 2160aaaagaaatg gctgaaagag caaaaaaaaa aaaa 2194280PRTHomo
sapiens 2Met Gly Arg Ala Met Val Ala Arg Leu Gly Leu Gly Leu Leu
Leu Leu1 5 10 15Ala Leu Leu Leu Pro Thr Gln Ile Tyr Ser Ser Glu Thr
Thr Thr Gly 20 25 30Thr Ser Ser Asn Ser Ser Gln Ser Thr Ser Asn Ser
Gly Leu Ala Pro 35 40 45Asn Pro Thr Asn Ala Thr Thr Lys Ala Ala Gly
Gly Ala Leu Gln Ser 50 55 60Thr Ala Ser Leu Phe Val Val Ser Leu Ser
Leu Leu His Leu Tyr Ser65 70 75 8031825DNAMus musculus 3ccaccttgcc
tgcgcccgcg cgagcttagc agatctccac ttaccgaaca tctagagagt 60cgcgccgcgc
gccgacggag cggacatggg cagagcgatg gtggccaggc tagggctggg
120gttgctgctt ctggcactgc tcctacccac gcagatttac tgcaaccaaa
catctgttgc 180accgtttccc ggtaaccaga atatttctgc ttccccaaat
ccaagtaacg ctaccaccag 240agggggtggc agctccctgc agtccacagc
tggtctcctg gctctctctc tctctcttct 300acatctctac tgttagagac
tcaggccagg aaacgtctct acttccccat cttctacacc 360taccccaaat
ggcaaccaca agtccaatgt gatcaggaag aaacaggtcc acctcgaatt
420ggctgttacc atatctcaac agaaaacacg gagaattcga aattcgacgg
gattaaagga 480cgcgtgaaag gtttgagaga agagagatgc cgctattgaa
tctgctggag ttttacatcc 540caagatgaag acagcattca gaattgatgt
gatttccttg aatgtggctt aggaaatgtg 600gacacttaaa actctcactt
gaaattgggc acaggtttga tgtagagata aggacggggt 660gcggaatgga
gacccatttt gtcattgatt catctgaccg ataaggccat agtgcagtta
720ggtgatattc gaaagcttct ttgatgctct ttatgtatat gttggaagga
actaccaggc 780gttgctttaa attcccaatg tgttgtttcg ttactactaa
tttaataccg taagctctag 840gtaaagttcc atgttgttga actctgactg
ttctctttgg aattgaacgt tttgcatcct 900cctcctgtgg ctttaggtct
gacattgtat ttgaccttta ctagtaatta acatgtgcca 960ggcaatggtg
gattggaacc catccccaag tccagccacc actgaataaa tctgatttca
1020aaagtcaaac agtagacatt tcccattgtc gtttctcact caccacaagc
accaaattca 1080ctagagtaca ctggttccag agagcagaat catgttggcc
ttggctaatt tcaaaatgct 1140gtcttttact ttggtatatg ttgagggctt
ttcataattt aaagtgtgtt ctgttagcaa 1200ggcaaaaatt atgagtctta
attctacagg caaatatgca aaggagccaa aactgtaaac 1260ccagcatttg
ggatgtgaag actggaagct aactctcatt gaattcacaa agtcttttat
1320acaatttctg tacatacttt tttttttttt aagagaaaaa caaacggtgg
atcagaatag 1380ccacgtttgg aatactttgg ttatccattc atatttttag
atagttattg gtcctgtgcc 1440tgaaaggggg cttggttcta ccgtaagttt
ttccaatttc cttgatatac acataccttc 1500taaaacctag acatttcctg
aaaaaaatct tttgttcgca tggtcacaca ctgatgctta 1560cccgtacagt
agtcttgata accagagtca ttttctccat ctttagaaac cttcctggga
1620agaaggagag ctcacagacc cgaagctact gtgtgtgtga atgaacactc
cccttgcctc 1680acacctgaat gctgtacatc tatttgattg taaattgtgt
ttgtgtattt atgctttgat 1740tcatagtaac ttctcatgtt atggaattga
tttgcattga acacaaactg taaataaaag 1800aaagaaatgg cggagaaaaa aaaaa
1825476PRTMus musculus 4Met Gly Arg Ala Met Val Ala Arg Leu Gly Leu
Gly Leu Leu Leu Leu1 5 10 15Ala Leu Leu Leu Pro Thr Gln Ile Tyr Cys
Asn Gln Thr Ser Val Ala 20 25 30Pro Phe Pro Gly Asn Gln Asn Ile Ser
Ala Ser Pro Asn Pro Ser Asn 35 40 45Ala Thr Thr Arg Gly Gly Gly Ser
Ser Leu Gln Ser Thr Ala Gly Leu 50 55 60Leu Ala Leu Ser Leu Ser Leu
Leu His Leu Tyr Cys65 70 75
* * * * *