U.S. patent application number 13/212916 was filed with the patent office on 2012-02-23 for compositions and methods to inhibit stem cell and progenitor cell binding to lymphoid tissue and for regenerating germinal centers in lymphatic tissues.
Invention is credited to Theresa Deisher.
Application Number | 20120045435 13/212916 |
Document ID | / |
Family ID | 45594254 |
Filed Date | 2012-02-23 |
United States Patent
Application |
20120045435 |
Kind Code |
A1 |
Deisher; Theresa |
February 23, 2012 |
COMPOSITIONS AND METHODS TO INHIBIT STEM CELL AND PROGENITOR CELL
BINDING TO LYMPHOID TISSUE AND FOR REGENERATING GERMINAL CENTERS IN
LYMPHATIC TISSUES
Abstract
The present invention relates to compositions and methods of
inhibiting stem cell binding to organs and tissues, including the
blocking of stem cell binding to germinal centers present in lymph
tissue. Disclosed are compositions and methods for regenerating
germinal centers in lymphatic tissue. Included in the compositions
are adjuvants, agonists to CD40, CD28 and the IL-21 receptor, and
antagonist to CD20.
Inventors: |
Deisher; Theresa; (Seattle,
WA) |
Family ID: |
45594254 |
Appl. No.: |
13/212916 |
Filed: |
August 18, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61374943 |
Aug 18, 2010 |
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61441485 |
Feb 10, 2011 |
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61449372 |
Mar 4, 2011 |
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Current U.S.
Class: |
424/133.1 ;
424/141.1; 424/93.7; 435/375 |
Current CPC
Class: |
A61K 39/3955 20130101;
C07K 2317/34 20130101; A61K 2039/505 20130101; A61K 31/573
20130101; C12N 5/0602 20130101; A61K 31/00 20130101; C07K 16/2896
20130101; C07K 16/2878 20130101; A61K 35/12 20130101; A61K 35/28
20130101; C07K 16/289 20130101; C07K 16/2809 20130101; A61P 1/16
20180101; C07K 16/2803 20130101; A61K 31/573 20130101; A61K 2300/00
20130101; A61K 35/28 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/133.1 ;
424/141.1; 424/93.7; 435/375 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12N 5/02 20060101 C12N005/02; A61P 19/10 20060101
A61P019/10; A61P 7/06 20060101 A61P007/06; A61P 9/00 20060101
A61P009/00; A61P 3/10 20060101 A61P003/10; A61P 25/00 20060101
A61P025/00; A61P 17/02 20060101 A61P017/02; A61P 19/02 20060101
A61P019/02; A61P 13/12 20060101 A61P013/12; A61P 25/16 20060101
A61P025/16; A61P 25/08 20060101 A61P025/08; A61P 37/00 20060101
A61P037/00; A61P 1/16 20060101 A61P001/16; A61P 25/28 20060101
A61P025/28; A61P 21/00 20060101 A61P021/00; A61P 27/02 20060101
A61P027/02; A61P 35/00 20060101 A61P035/00; A61P 35/02 20060101
A61P035/02; A61K 35/12 20060101 A61K035/12 |
Claims
1. A method to inhibit stem cells binding to lymphoid tissue
comprising: administering the stem cells in conjunction with a
therapeutic agent that inhibits binding of the stem cells to said
lymphoid tissue.
2. The method of claim 1 wherein the lymphoid tissue is comprised
of spleen, Peyer's patches and lymph nodes.
3. The method of claim 1 wherein the therapeutic agent inhibits
binding of the stem cells to germinal centers within the lymphoid
tissue.
4. The method of claim 3 wherein the germinal centers are present
in a lymph tissue selected from the group consisting of spleen,
Peyer's patches and lymph node tissue.
5. The method of claim 3 wherein the germinal center is active.
6. The method of claim 1, wherein the therapeutic agent is selected
from the group consisting of: agents that interfere with the
synthesis of purines, the anti-metabolites, radiation to the
spleen, chemotherapeutic agents, immunosuppressants,
glucocorticoids, anti-beta amyloid agents, anti-rhesus factor,
anti-TNF agents, anti-eotaxins, anti-T cell receptor (TCR) agents,
anti-interferons agents, anti-interferon alpha agents,
anti-interferon beta agents, anti-interferon gamma agents, anti-TGF
agents, anti-TGFalpha agents, anti-TGF beta agents, anti-Integrins
agents, anti-alpha 4 agents, anti-Interleukin agents,
anti-Interleukin 1 agents, anti-Interleukin 2 agents,
anti-Interleukin 4 agents, anti-Interleukin 5 agents,
anti-Interleukin 6 agents, anti-Interleukin 12 agents,
anti-Interleukin 13 agents, anti-Interleukin 23 agents, anti-IgE
agents, anti-Vascular Adhesion Protein (YAP) agents, anti-B7
agents, anti-Vascular Endothelial Growth Factor (VEGF) agents,
anti-BAFF (BLyS) agents, anti-CTLA4 agents, anti-complement agents,
anti-CD2 agents, anti-CD3 agents, anti-CD4 agents, anti-CD5 agents,
anti-CD20 agents, anti-CD23 agents, anti-CD25a agents, anti-CD40
agents, anti-CD154(CD40L) agents, anti-CD62L agents, anti-CD80
agents, anti-CD147 agents, anti-LFA1 agents, anti-(CD11a) agents,
anti-CD18 agents, inhibitors of purine synthesis, inhibitors of
pyrimidine synthesis, anti-proliferative agents, anti-metabolite
agents, anti-folate agents, and anti-mTOR agents.
7. The method of claim 1 wherein the therapeutic agents are
selected from the group consisting of: azathioprine, mycophenolic
acid, leflunomide, teriflunomide, methotrexate, tacrolimus,
ciclosporin, pimecrolimus, abetimus, gusperimus, thalidomide,
lenalidomide, anakinra, sirolimus, deforolimus, everolimus,
temsirolimus, zotarolimus, biolimus A9, eculizumab, infliximab,
adalimumab, certolizumab pegol, afelimomab, golimumab, mepolizumab,
omalizumab, nerelimomab, faralimomab, elsilimomab, lebrikizumab,
ustekinumab, muromonab-CD3, otelixizumab, teplizumab, visilizumab,
clenoliximab, keliximab, zanolimumab, efalizumab, erlizumab,
afutuzumab, ocrelizumab, pascolizumab, lumiliximab, teneliximab,
toralizumab, aselizumab, galiximab, gavilimomab, ruplizumab,
belimumab, ipilimumab, tremelimumab, bertilimumab, lerdelimumab,
metelimumab, natalizumab, tocilizumab, odulimomab, basiliximab,
daclizumab, inolimomab, zolimomab aritox, atorolimumab,
cedelizumab, dorlixizumab, fontolizumab, gantenerumab, gomiliximab,
maslimomab, morolimumab, pexelizumab, reslizumab, rovelizumab,
siplizumab, talizumab, telimomab aritox, vapaliximab, vepalimomab,
abatacept, belatacept, etanercept, pegsunercept, aflibercept,
alefacept, rilonacept, lymphotoxin alpha and beta inhibitors,
dacetuzumab SGN-40, HCD-32.
8. The method of claim 1, wherein the binding of stem cells to the
lymphoid tissue is inhibited by administering a therapeutic agent
that down-regulates or blocks a CD45 antigen.
9. The method of claim 1 wherein the therapeutic agent is
radiation.
10. The method of claim 1 wherein the stem cells are either
exogenous or endogenous stem cells.
11. The method of claim 1 wherein the therapeutic agent is a
chemotherapeutic agent.
12. The method of claim 1 wherein the stem cells are administered
to treat a disease selected from the group consisting of:
hematological malignancies, leukemias, lymphomas, cancers,
osteopetrosis, aplastic anemia and cytopenias, sickle cell disease
and thalassemia, limbal stem cell deficiency, breast cancer, acute
myocardial infarction, coronary artery disease, peripheral vascular
disease, heart failure, type I diabetes mellitus, type 2 diabetes
mellitus, stroke, spinal cord injury, neuroblastoma, multiple
sclerosis, systemic sclerosis, lupus erythematosus, chronic wound
healing, burns, fracture healing, cartilage repair, CNS tumors,
osteoarthritis, renal failure, Parkinson's Disease, myelomas,
diabetic foot, liver and biliary cirrhosis, dilated cardiomyopathy,
anemia, retinitis pigmentosa, Crohn's Disease, diabetic neuropathy,
mastocytosis, ovarian cancer, epilepsy, myasthenia gravis,
autoimmune diseases, granulomatous disease, osteonecrosis, liver
failure, PMD disease, lypodystrophy, demyelinating diseases,
cartilage defects, retinal disease, lupus nephritis, Alzheimer's
Disease, traumatic brain injury, sarcoma, myositis, hyperglycemia,
macular degeneration, ulcerative colitis, and muscle
degeneration.
13. A method for inhibiting binding of stem cells to lymph tissues
in an individual comprising inhibiting the formation of germinal
centers present in lymph tissues or destroying or ablating germinal
centers in lymph tissue.
14. The method of claim 13 wherein a therapeutic agent is
administered to the individual that inhibits the formation of the
germinal cells or promotes destruction or ablation of the germinal
cells wherein the therapeutic agent is selected from the group
consisting of agents that interfere with the synthesis of purines,
anti-metabolites, radiation, immunosuppressants, glucocorticoids,
anti-beta amyloid agents, anti-rhesus factor, anti-TNF agents,
anti-eotaxins, anti-T cell receptor (TCR) agents, anti-interferons
agents, anti-interferon alpha agents, anti-interferon beta agents,
anti-interferon gamma agents, anti-TGF agents, anti-TGFalpha
agents, anti-TGF beta agents. anti-Integrins agents, anti-alpha 4
agents, anti-Interleukin agents, anti-Interleukin 1 agents,
anti-Interleukin 2 agents, anti-Interleukin 4 agents,
anti-interleukin 5 agents, anti-interleukin 6 agents,
anti-Interleukin 12 agents, anti-Interleukin 13 agents,
anti-Interleukin 23 agents, anti-IgE agents, anti-Vascular Adhesion
Protein (VAP) agents, anti-B7 agents, anti-Vascular Endothelial
Growth Factor (VEGF) agents, anti-BAFF (BLyS) agents, anti-CTLA4
agents, anti-complement agents, anti-CD2 agents, anti-CD3 agents,
anti-CD4 agents, anti-CD5 agents, anti-CD20 agents, anti-CD23
agents, anti-CD25a agents, anti-CD40 agents, anti-CD J 54 (CD40L)
agents, anti-CD62L agents, anti-CD80 agents, anti-CD147 agents,
anti-LFA1 agents, anti-(CD11a) agents, anti-CD18 agents, inhibitors
of purine synthesis, inhibitors of pyrimidine synthesis,
anti-proliferative agents, anti-metabolite agents, anti-folate
agents, and anti-mTOR agents.
15. The method of claim 13 wherein the therapeutic agent is a
chemotherapeutic agent.
16. A method for regenerating germinal centers in lymphatic tissue,
wherein said germinal centers have been damaged by a chemical
agent, a biologic agent or by radiation comprising administering
one or more agents that stimulates regeneration of the germinal
centers.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application Ser. No. 61/374,943filed Aug. 18, 2010; U.S.
Provisional Patent Application Ser. No. 61/441,485 filed Feb. 10,
2011; and U.S. Provisional Patent Application Ser. No. 61/449,372
filed Mar. 4, 2011. All of the foregoing applications are hereby
incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] Subject matter of the disclosure concerns methods and
compositions to modulate stem cell binding to organs and tissues
and for regenerating germinal centers in lymphatic tissues.
BACKGROUND OF THE INVENTION
[0003] Regenerative Medicine is the process of creating living,
functional tissues to repair or replace tissue or organ function
lost due to damage, or congenital defects. This field holds the
promise of regenerating damaged tissues and organs in the body by
stimulating previously irreparable organs to heal themselves.
[0004] One method used to regenerate tissue or organ function is
the delivery of stem cells to the affected organ or tissue.
However, stem cells are not well retained in the organ targeted for
tissue regeneration even when the stem cells are directly injected
into the tissue of the injured organ. Imaging studies in humans and
animals have demonstrated that most of the delivered stem ceils can
be found within the spleen within an hour after stem cell
injection. Animal studies have also demonstrated that surgical
removal of the spleen prior to stem cell therapy after induced
myocardial infarction improved functional recovery of the damaged
hearts (Blood, Vol 84, No 5: 1482-1491, 1994; NATURE 410:701-705,
2001). Splenectomy has also been shown to improve engraftment in
human patients after bone marrow transplantation (Stem Cells Dev
13(1):51-62, 2004; Transplant Proc. 28(2):736-7, 1996; Am J
Hematol. 22(3):275-83, 1986). However, splenectomy is also
associated with surgical mortality, sepsis, and lifelong thrombotic
complications (Blood Rev. 14(3):121-129, 2000; Leukemia.
15(3):465-467, 2001; Pediatr Transplant 13(2):171-176, 2009)
[0005] Thus, there is a need to develop methods and compositions
that can be used to prevent localization of stem cells in the
spleen and other lymphoid tissues without removal of the
spleen.
DESCRIPTION OF THE INVENTION
Overview
[0006] The present invention fills this need by providing for
methods and compositions for inhibiting binding of stem cells to
lymphoid tissue comprising administering stem cells to an
individual in conjunction with a therapeutic agent or agents that
inhibit binding of stem cells to lymphoid tissue, in particular to
germinal centers in lymph nodes and germinal centers in the spleen.
The term `in conjunction with` means together, before or after the
stem cell treatment. `Stem cell treatment` means the act of
administering stem cells to the individual, mobilizing stem cells
from within the individual's endogenous stem cell stores, or
relying on spontaneous release of stem ceils from the individual's
endogenous stem cell stores.
[0007] For example, patients treated with stem cells to elicit
organ regeneration have demonstrated reductions in mortality and
improvements in function following stem cell therapy, although the
stem cell treatments do not generally restore the patient to their
functional status prior to organ injury. Reductions in stem cell
binding to the spleen and other lymphatics augment the numbers of
circulating stern cells that can be attracted to the injured organ
and thereby augment the degree of functional recovery induced by
steal cell treatment of that patient.
[0008] In administering the therapeutic agents that inhibit the
binding of stem ceils to lymphatic tissues it is preferred to
administer the therapeutic agents 1-14 days prior to treatment,
more preferably 3-7days and most preferably 3-4 days prior to
treatment with stem cells or mobilization of stem cells. In
administering the therapeutic agents that inhibit the binding of
stem cells to lymphatic tissues in conjunction with spontaneously
released stem cells from the individual's endogenous stem cell
stores it is preferred to administer the therapeutic agents over a
period of 1-60 days, more preferably 1-30 days, and most preferably
1-14 days.
[0009] Agents that inhibit the binding of stem ceils to lymphoid
tissues, particularly to the germinal centers of lymphoid tissues,
include radiation, chemotherapeutic agents, immune suppressants and
antagonists to CD45 and antagonists to CD26. Stem cells found
within the mononuclear fractions from whole blood or bone marrow,
or purified stem cells from whole blood or bone marrow, bind to the
white pulp regions in the spleen, more specifically to germinal
centers in the white pulp of lymphatic tissue including the spleen,
and even more specifically to active germinal centers in the white
pulp of the spleen. Antibodies to CD45, particularly to the epitope
identified by the 30-F11 rat IgG2b anti-mouse anti-CD45 monoclonal
antibody, reduce stem cell binding to the identified sites in the
spleen, making more stem cells available for biodistribution to the
targeted injured organ, and enhancing tissue regeneration and
functional recovery.
[0010] In another embodiment of the present invention therapeutic
agents are administered that reduce, destroy or ablate active
germinal centers in the lymphoid tissue thus resulting in the
reduction of the binding of stem cells to the lymph tissue. Another
embodiment of the present invention describes methods to reduce the
number of active germinal centers in the spleen to reduce stem cell
binding to the spleen, thereby increasing the numbers of
circulating stem cells available for delivery or homing to damaged
organs in need of repair. Agents that suppress the immune response
may reduce the numbers of active germinal centers in the spleen and
other lymphatic tissues. General categories of immune modulators
include agents that interfere with the synthesis of purines, the
anti-metabolites, radiation, radiation to the spleen,
immunosuppressants, glucocorticoids, anti-beta amyloid agents,
anti-rhesus factor, anti-TNF agents, anti-eotaxins, anti-T cell
receptor (TCR) agents, anti-interferons agents, anti-interferon
alpha agents, anti-interferon beta agents, anti-interferon gamma
agents, anti-TGF agents, anti-TGFalpha agents, anti-TGF beta
agents, anti-Integrins agents, anti-alpha 4 agents,
anti-Interleukin agents, anti-Interleukin 1 agents,
anti-interleukin 2 agents, anti-Interleukin 4 agents,
anti-Interleukin 5 agents, anti-interleukin 6 agents,
anti-Interleukin 12 agents, anti-Interleukin 13 agents,
anti-Interleukin 23 agents, anti-IgE agents, anti-Vascular Adhesion
Protein (VAP) agents, anti-B7 agents, anti-Vascular Endothelial
Growth Factor (VEGF) agents, anti-BAFF (BLyS) agents, anti-CTLA4
agents, anti-complement agents, anti-CD2 agents, anti-CD3 agents,
anti-CD4 agents, anti-CD5 agents. anti-CD20 agents, anti-CD23
agents, anti-CD25a agents, anti-CD40 agents, anti-CD 154 (CD40L)
agents, anti-CD62L agents, anti-CD80 agents, anti-CD 147 agents,
anti-LFA1 agents, anti-(CD11a) agents, anti-CD18 agents, inhibitors
of purine synthesis, inhibitors of pyrimidine synthesis,
anti-proliferative agents, anti-metabolite agents, anti-folate
agents, and anti-mTOR agents.
[0011] Adenosine deaminases deficiency will also lead to reduced
active germinal center formation as will agents which trigger the
accumulation of deoxyATP (J Immunol 171: 5562-5570, 2003).
Similarly, agents that enhance the expression of or activate CCR7
will lead to diminished active germinal center formation.
[0012] Chemotherapeutic agents can also be used to inhibit the
formation of the germinal centers of lymphoid tissue or to destroy
or ablate the germinal centers. Representative examples include
alkylating agents, anti-metabolites, plant alkaloids, topoisomerase
inhibitors, antineoplastics and arsenic trioxide.
[0013] Examples of alkylating agents include cisplatin and
carboplatin, as well as oxaliplatin, are alkylating agents. They
impair cell function by forming covalent bonds with the amino,
carboxyl, sulfhydryl, and phosphate groups in biologically
important molecules.
[0014] Examples of antimetabolites azathioprine, mercaptopurine,
capecitabinefluorouracil--which become the building blocks of DNA.
They prevent these substances from becoming incorporated in to DNA
during the "S" phase (of the cell cycle), stopping normal
development and division. They also affect RNA synthesis. Due to
their efficiency, these drugs are the most widely used
cytostatics.
[0015] Alkaloids include the vinca alkaloids and taxanes. Vinca
alkaloids include vincristin, vinblastin, vinorelbine, and
vindesine. Taxanes include taxol, paclitaxel and docetaxel.
[0016] Topoisomerases are essential enzymes that maintain the
topology of DNA. Inhibition of type I or type II topoisomerases
interferes with both transcription and replication of DNA by
upsetting proper DNA supercoiling. Some type I topoisomerase
inhibitors include camptothecins: irinotecan and topotecan.
Examples of type II inhibitors include amsacrine, etoposide,
etoposide phosphate, and teniposide.
[0017] Antineoplastic agents include dactinomycin, doxorubicin,
epirubicin, and bleomycin.
[0018] Definitions
[0019] Definitions used to describe the embodiments of the
invention:
[0020] The term agonist as used herein means any entity that
activates a specific receptor or downstream signaling pathway
essential to mediate the receptor's effect(s). Agonists may include
but are not limited to antibodies, antibody fragments, soluble
ligands, small molecules, cyclic peptides, cross-linking
agents.
[0021] The term antagonist as used herein means any entity that
interferes with the binding of a receptor's counter structure(s),
or with the activation of a specific receptor or downstream
signaling pathway essential to mediate the receptor's effect(s).
Antagonists may include but are not limited to antibodies, antibody
fragments, soluble ligands, Fc fusion receptors, chimeric
receptors, small molecules, cyclic peptides, peptides.
[0022] The term inhibitor as used herein means any entity that
diminishes the target effect of a specific ligand or its receptor.
Inhibitors may be small molecules, antisense agents, nucleic acids
including siRNA and microRNA.
SUMMARY OF THE INVENTION
[0023] Lymphatic Tissue is a specialized form of reticular
connective tissue in the lymphatic system that contains large
numbers of lymphocytes. This tissue type makes up the spleen, the
thymus, and the tonsils, as well as visceral nodes, peyer's patches
and lacteals that are ail associated with mucous membranes of the
gastro-intestinal tract.
[0024] A lymph node is a small ball-shaped organ of the immune
system, distributed widely throughout the body including the armpit
and stomach/gut and linked by lymphatic vessels. Lymph nodes are
garrisons of B-cells, T-cells, and cither immune cells. Lymph nodes
are found all through the body, and act as filters or traps for
foreign particles. The lymph node is surrounded by a fibrous
capsule, and inside the lymph node the fibrous capsule extends to
form trabeculae. The substance of the lymph node is divided into
the outer cortex and the inner medulla surrounded by the former ail
around except for at the hilum, where the medulla comes in direct
contact with the surface. The outer cortex consists mainly of the B
ceils arranged as follicles, which may develop a germinal center
when challenged with an antigen, and the deeper cortex mainly
consisting of the T cells. There is a zone known as the subcortical
zone where T-cells (or cells that are mainly red) mainly interact
with dendritic cells, and where the reticular network is dense.
[0025] The spleen is an intraperitoneal organ located on the left
side of the abdomen between the stomach and the diaphragm. This
organ is a major regulatory site of the immune system. It is a
vascular organ, having a large arterial blood supply. On entering
the spleen, the blood flow enters a meshwork of dilated blood
vessels, or "sinuses", which lie between large masses of
lymphocytes. The walls of the sinuses contain phagocytes that are
capable of engulfing dead cells and foreign particles in the blood
and removing them from the general circulation. Like the lymph
nodes, the spleen is an important source of antibodies, however, to
a greater extent than the lymph nodes, the spleen is concerned with
the removal of abnormal or normally worn out ("dying") red blood
ceils from the circulation by destroying them.
[0026] The spleen contains both a white pulp and a red pulp. The
red pulp of the spleen holds macrophages that normally filter and
remove senescent or defective red blood cells (RBCs) and
antibody-coated bacteria or red blood cells from the circulation.
The white pulp of the spleen contains the lymphoid compartments and
is crucial for immune surveillance and response: it synthesizes
antibodies against invading pathogens and releases platelets and
neutrophils in response to bleeding or infection. During
development the spleen is believed to have multiple roles including
being the first site of hematopoiesis (at six weeks of gestation).
While it was long believed that the spleen loses its hematopoietic
function during the early stages of development when bone marrow
hematopoiesis takes over, recent research has identified the adult
spleen as a site of stem cell generation, stem cell differentiation
into different lineages and stem cell storage (Trends Mol Med
11(6):271-276, 2005;) However, the sites within the spleen where
exogenous stem cells accumulate and the molecular mechanisms by
which exogenous stem cells bind to the spleen are not known.
[0027] The periarterial lymphoid sheaths (PALS) of the white pulp
of the spleen are populated mainly by T cells, while the lymphoid
portions are populated mainly by B cells. Germinal centers (GC) are
sites within lymph nodes or lymph nodules in peripheral lymph
tissues, and in the white pulp of the spleen where intense mature B
lymphocytes, otherwise known as Centrocytes rapidly proliferate,
differentiate, mutate through somatic hypermutation and class
switch during antibody responses. Germinal centers are an important
part of the B-cell humoral immune response. They develop
dynamically after the activation of B-cells by T-dependent antigen.
Histologically, the GCs describe microscopically distinguishable
parts in lymphoid tissues. Activated B-cells migrate from the
primary focus into the primary follicles follicular system and
begin monoclonal expansion in the environment of follicular
dendritic cells (FDC).
[0028] After several days of expansion the B cells mutate their
antibody-encoding DNA and thus generate a diversity of clones in
the germinal center. This involves random substitutions, deletions
and insertions due to somatic hypermutation. Upon some unidentified
stimulus from the FDC, the maturing B cells (Centroblasts) migrate
from the dark zone to the light zone and start to expose their
antibody to their surface and in this stage are referred to as
Centrocytes. The Centrocytes are in a state of activated apoptosis
and compete for survival signals from FDCs that present the
antigen. This rescue process is believed to be dependent on the
affinity of the antibody to the antigen. The functional B-cells
have then to interact with helper T cells to get final
differentiation signals. This also involves isotype switching for
example from IgM to IgG. The interaction with T cells is believed
to prevent the generation of autoreactive antibodies. The B cells
become either a plasma cell spreading antibodies or a memory B cell
that will be activated in subsequent contacts with the same
antigen. They may also restart the whole process of proliferation,
mutation and selection according to the recycling hypothesis.
[0029] The B cells contained within the white pulp region of the
spleen can be further divided into specific areas, identified by
staining with specific molecular markers. The marginal zone of the
spleen contains noncirculating mature B cells that border on the
white pulp creating a separation between the white and the red pulp
and express high levels of CD23 and IgM and CD24 and CD79a, and
measurable levels of CD9 and CD22. The mantle zone surrounds normal
germinal center follicles and expresses CD21, CD23 and CD38. The
follicular zone is contained within the germinal centers and
expresses high levels of IgD and CD23, intermediate levels of CD21
and CD24, and can also be identified by PNA staining. The germinal
center is best distinguished by PNA binding and expresses higher
levels of CD54 than the follicular zone. Germinal centers have a
special population of helper T cells that seem to distribute evenly
in ail germinal centers. Germinal centers are traditionally
associated with immune responses that require T helper cells,
although this is not absolute. Germinal centers are where
hypervariable gene mutation occurs and high affinity IgG producing
B cells are generated. Active germinal centers have tangible
macrophages and CD21 expressing dendritic cells. Follicular centers
can also be identified by the expression of CD45R (B220)
[Toxicologic Pathology, 35:366-375, 2007). CD45R follicular centers
are found surrounding germinal centers expressing Bc16 and Bc12.
BioEssays 29:166-177, 2007; Toxicol Pathol 34(5): 648-655,
(2006)]
[0030] CD45 is a common leukocyte antigen also known as PTPRC
(protein tyrosine phosphatase, receptor type C), found on all
differentiated hematopoietic cells except erythrocytes and plasma
cells. It is also expressed in lymphomas, B-cell chronic
lymphocytic leukemia, hairy cell leukemia, and acute
non-lymphocytic leukemia. It has shown to be essential in B- and
T-cell antigen receptor signaling. The CD45 family consists of
multiple members that are all products of a single complex gene.
This gene contains 34 exons and three exons of the primary
transcripts are alternatively spliced to generate up to eight
different mature mRNAs and after translation eight different
protein products. These three exons generate the RA, RB and RC
isoforms.
[0031] Various isoforms of the CD45 antigen exists. The CD45
antigen isoforms include CD45RA, CD45RB, CD45RC, CD45RAB, CD45RAC,
CD45RBC, CD45RO, CD45R (ABC). CD45A is located on naive T cells and
CD45RO is located on memory T cells. CD45 is also highly
glycosylated. CD45R is the longest protein and migrates at 200 kDa
when isolated from T cells. B cells also express CD45R with heavier
glycosylation, bringing the molecular weight to 220 kDa, hence the
name B220; B cell isoform of 220 kDa. B220 expression is not
restricted to B cells and can also be expressed on activated T
cells, on a subset of dendritic cells and other antigen presenting
cells. Naive T lymphocytes express large CD45 isoforms and are
usually positive for CD45RA. Activated and memory T lymphocytes
express the shortest CD45 isoform, CD45RO, which lacks RA, RB and
RC exons. This shortest isoform facilitates T cell activation. The
cytoplasmic domain of CD45 is one of the largest known and it has
an intrinsic phosphatase activity that removes an inhibitory
phosphate group on a tyrosine kinase called Lck (in T cells) or
Lyn/Fyn/Lck (in B cells) and activates it. CD45can exist in both
monomeric and dimeric forms, and dimerization may downregulate CD45
phosphatase activity (Blood v103(9):3440-3447, 2004).
[0032] As CD45 is expressed on all hematopoietic cells and is the
most broadly expressed of all hematopoietic antigens, it has been
used to isolate the population of cells that also contains
hematopoietic stem cells in transplant and other models of stem
cell reconstitution, however, mesenchymal stem cells, while derived
from a population of CD45+ earlier precursors, are generally found
to be CD45 negative (Stem Cells 28:140-151, 2010). A complete
absence of all isoforms of CD45 has been demonstrated in mice to
influence stem cell retention, motility and homing to the bone
marrow and to play a role in the generation of functional B cells
in the spleen from earlier stem cells (J. Exp. Med. 205:2381-2395,
2008). Interestingly, CD45 knock-out mice, lacking all isoforms of
CD45, had reduced numbers of cKit+Lin-hematopoietic progenitor
cells in the bone marrow, but increased numbers within the
spleen.
[0033] 30-F11 is a rat monoclonal IgG2b raised against thymus and
spleen of mouse origin. Clone 30-F11 reacts to both alloantigens
(CD45.1 and CD45.2) and all isoforms of CD45, 30-F11 and clone
30-F4 each block binding by the other to CD45, indicating that they
bind to the same or overlapping epitopes on CD45 (J Immunol
127(3):982-986, 1981). Likewise, both of these clones cross-block
with an antibody described by Dennert et. al., however, the
anti-CD45 antibody 55-6.1 does not cross-block with either 30-F11
or 30-F4 (Cell Immunol 53:350-364, 1980).
[0034] Radiolabeled 30-F11 antibody shows highest accumulation in
mouse spleen (60%), with only 20% accumulation in marrow (Blood
93(2):737-745, 1999), and has been used in mice to deliver targeted
hematopoietic irradiation. The 30-F11 antibody has been used to
identify, in conjunction with Sca1 antigen, stem cell fractions
from the muscle of mice, since CD45 is expressed on all
hematopoietic cells (PNAS 99 1341-1346, 2002). Radiolabeled 30-F11
and f(ab)'2 fragments of the 30-F11 have been evaluated as a method
to deliver radiotherapy. 30-F11 uptake was most dramatic in the
spleens of mice, followed by axillary lymph node (Cancer Res
52(5):1228-34, 1992).
[0035] The CD45 polypeptide can be produced by published
procedures. Methods for preparing anti-CD45 polyclonal and
monoclonal antibodies are well known in the art (see for example,
Sambrook et al., Molecular Cloning: A Laboratory Manual, Second
Edition (Cold Spring Harbor, N.Y., 1989); and Hurrell, J. G. R.,
Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications
(CRC Press, Inc., Boca Raton, Fla., 1982), which are incorporated
herein by reference). As would be evident to one of ordinary skill
in the art, polyclonal antibodies can be generated from a variety
of warm-blooded animals such as horses, cows, goats, sheep, dogs,
chickens, rabbits, mice, and rats. Production of humanized
antibodies is well known. See Riechmann L. Clark M, Waldmann H,
Winter G (1988). "Reshaping human antibodies for therapy". Nature
332 (6162): 332:323. .Queen C, Schneider W P, Selick H E, Payne P
W, Landolfi N F, Duncan J F, Avdalovic N M, Levitt M, Junghans R P,
Waldmann T A (December 1989). "A humanized antibody that binds to
the interleukin 2 receptor.". Proc Natl Acad Sci USA. 86 (24):
10029-33. Norderhaug L, Olafsen T. Michaelsen T E, Sandlie I. (May
1997). "Versatile vectors for transient and stable expression of
recombinant antibody molecules in mammalian cells.". J Immunol
Methods 204 (1): 77-87.
[0036] Other particular embodiments provide for a method to
moderate stem cell binding to a spleen by exposing the spleen to a
solution of antagonists to a cluster of differentiation 45 (CD45)
antigen. The solution of antagonist to the CD45 antigen may be
configured or formulated to bind to the 30-F11 epitope. The
solution of antagonists to the CD45 antigen may be formulated to
promote therapeutic regeneration by enhancing stem cell delivery to
the damaged tissues or organs. The solution containing the antibody
to the CD45 antigen may be formulated to bind to the 30-F11 epitope
and to a human equivalent of the 30F-11 epitope.
[0037] The present invention describes methods to reduce stem cell
binding to lymphatic tissue such as the lymph nodes and the spleen,
and uses of these methods to treat human patients. The invention
describes the specific site within the spleen where circulating
stem cells bind in the spleen, and methods to increase or decrease
stem cell binding to this site. Immunoflourescent and histological
analysis of fresh thick spleen sections demonstrated that stem
cells circulating in the vasculature bind to active germinal
centers in the spleen when administered either in vivo or ex vivo,
as shown in example 1, 2, 3, and 4. One method to decrease the
amount of stem cell binding to the spleen is to deliver agents that
block binding of the administered stem cells to the molecular
target on the active germinal centers of the spleen. According to
the process of the present invention, antibody 30-F11 binds to
mouse CD45 antigen and blocks stem cell binding to germinal centers
of the mouse spleen. Anti-human anti-CD45 antibodies that may be
the equivalent to the 30-F11 rat IgG2b anti-mouse CD45 include
YAML568 which recognizes epitope P of human CD45(J Nucl Med
47:1335-1341, 2006; In: Leucocyte Typing III: White Ceil
Differentiation Antigens pp 811-814, 1987; Transplantation
40:538-544, 1985), anti-CD45 clone HI30, or YTH-24 and YTH-54
anti-human anti-CD45 antibodies.
[0038] Another embodiment of the present invention is the use of
antagonists to CD26 such as antibodies to CD26 to inhibit stem cell
binding to germinal centers. As CD26 is expressed by stem cells and
is the antigen present on stem cells that adheres to lymph tissue
in particular to germinal centers in the lymph tissue. By blocking
the CD26 on stem cells, the stem cells are unable to bind to lymph
tissues.
Agents that Inhibit, Down-Regulate the Formation of Germinal
Centers or Destroy or Ablate Germinal Centers
[0039] Another embodiment of the present invention is to inhibit
stem cell binding to the lymph tissues by inhibiting or
down-regulating the proliferation of the germinal centers or to
destroy or ablate the germinal centers. Germinal centers (GCs)
develop dynamically after the activation of B-cells by T-dependent
antigen. The T-Cell antigen that activates the B-cells and thus
induces proliferation of the germinal centers is CD40L (also known
as CD154) that binds to the CD40 receptor present on the B-cells.
This binding of the CD40L to the CD40 receptor not only activates
the B-cells but also induces proliferation of the germinal centers.
Thus, another embodiment of the present invention is comprised of
administering to an individual an agent that inhibits the binding
of CD40L to CD40. Examples of such agents are antagonistic
antibodies to CD40 or to CD40L.
[0040] Another protein important for the development of germinal
centers is the `signaling lymphocyte activation molecule-associated
protein` (SAP). (Hai Qui, et al., Nature, 455:764-769 (2008). Thus,
an antibody against SAP would inhibit the formation of germinal
centers and thus inhibit binding of stem cells to lymph
tissues.
[0041] IL-21 is another polypeptide important for germinal center B
cell differentiation and proliferation through a B cell-intrinsic
mechanism. The absence of IL-21 signaling profoundly affects the B
cell response to protein antigen, reducing splenic and bone marrow
plasma cell formation and GC persistence and function, influencing
their proliferation, transition into memory B cells, and affinity
maturation. [Zotos, D., et al, JEM 207:365-378 (2010)]. Thus by
administering antagonists such as antibodies to IL-21 to someone
the germinal centers can be down-regulated and their formation
inhibited. This would inhibit the binding of stem cells to the
lymph tissue and spleen due to the lack of germinal centers in the
lymph tissue.
[0042] Chemotherapeutic agents can inhibit binding of stem cells to
germinal centers of lymphoid tissues including lymph nodes, Peyer's
patches, and the white pulp of the spleen. Also, agents that
suppress the immune response may reduce the numbers of active
germinal centers in the spleen. Such agents include:
[0043] Azathioprine, (IMURAN.RTM., Prometheus Laboratories, San
Diego, Calif.) administered in 3-5 mg/kg, daily, preferably 3-4
days prior to the administration of the stem cells. Azathioprine
interferes with the synthesis of purines (adenine and guanine),
which is required for DNA synthesis. Fast-growing cells, including
T-cells and B-cells, are particularly affected by the inhibition of
purine synthesis.
[0044] Corticosteroids such as dexamethasone, prednisolone,
methylprednisolone, dexamethasone sodium phosphate and
betamethsaone. Dexamethasone tablets (Merck) and Dexamethasone
sodium phosphate injections can be given 1-14 days prior to
treatment with stem cells more preferably 3-7 days and most
preferably 3-4 days prior to treatment with stem cells. The total
amount of dexamethasone that is administered is an amount
sufficient to down-regulate the germinal centers in the lymphatic
tissue so that stem cells cannot bind to the lymph tissue. The
total amount of dexamethasone over the period of time can range
from 2 mg to 3 g, preferably 27 mg in total. The daily dose of
dexamethasone can range from 0.75 mg to 700 mg per day preferably 7
mg per day. Dexamethasone, like the other glucocorticoid steroids
inhibits the formation and proliferation of germinal centers in the
lymph tissues.
[0045] Mycophenolic acid (Myfortic.RTM. delayed release capsules,
Novartis Pharmaceuticals Corporation East Hanover, N.J., 720 mg
administered twice daily (1440 mg total daily dose) on an empty
stomach, one hour before or two hours after food intake),
preferably 3-4 days prior to administration of stem cells.
(CellCept.RTM. Roche Labs, Nutley, N.J., mycophenolate mofetil)
Tablets and Capsule, Oral Suspension, mycopbenolate mofetil
hydrochloride) for Injection Intravenous, the 2-morpholinoethyl
ester of mycophenolic acid (MPA), administered IV 1 g twice a day
orally 1.5 g administered twice a day, preferably 3-4 days prior to
the administration of the stem cells. It inhibits inosine
monophosphate dehydrogenase, the enzyme that controls the rate of
synthesis of guanine monophosphate in the de novo pathway of purine
synthesis used in the proliferation of B and T lymphocytes.
Mycophenolate is potent and can be used in place of the older
anti-proliferative azathioprine. It is usually used as part of a
three-compound regimen of immunosuppressants, also including a
calcineurin inhibitor (ciclosporin or tacrolimus) and
prednisolone.
[0046] Leflunomide, Sanofi-Aventis U.S. LLC, Bridgewater, N.J. 100
mg per day for three days, 3-4days prior to administration of the
stem cells. Leflunomide is a pyrimidine synthesis inhibitor
belonging to the DMARD (disease-modifying antirheumatic drug) class
of drugs, which are chemically and pharmacologically very
heterogeneous. Leflunomide is an immunomodulatory drug inhibiting
dihydroorotate dehydrogenase (an enzyme involved in de novo
pyrimidine synthesis) (abbreviation DHODH).
[0047] Teriflunomide, the active metabolite of leflunomide,
Sanofi-Aventis U.S. LLC, Bridgewater, N.J. 100 mg per day for three
days, preferably 3-4 days prior to the administration of the stem
cells,
[0048] Methotrexate--is an antimetabolite and antifolate drug. It
acts by inhibiting the metabolism of folic acid. It is administered
orally or intramuscularly in doses of 15 to 30 mg daily for up to
five days, preferably 3-4 days prior to the administration of the
stem cells. Mylan Pharmaceuticals. Morgantown, W.Va.
Immunosuppresant Macrolides:
[0049] Tacrolimus reduces interleukin-2 (IL-2) production by
T-cells. in capsule or injection forms, 0.10-0.15 mg/kg/day,
preferably 3-4 days prior to the administration of the stem cells.
(Astellas Pharma US, Inc. Deerfield, Ill.).
[0050] Ciclosporine--Ciclosporin is thought to bind to the
cytosolic protein cyclophilin (immunophilin) of immunocompetent
lymphocytes, especially T-lymphocytes. Marketed as Sandimmune.RTM.,
in the form of capsules, oral solution or injection, and dosed at
14-18 mg/kg/day, preferably 3-4 days prior to the administration of
the stem cells. (Novartis Pharmaceuticals Corporation, East
Hanover, N.J.).
[0051] Pimecrolimus (Elidel) is an ascomycin macrolactam
derivative. It has been shown in vitro that pimecrolimus binds to
macrophilin-12 and inhibits calcineurin. Thus pimecrolimus inhibits
T-cell activation by inhibiting the synthesis and release of
cytokines from T-cells. Pimecrolimus also prevents the release of
inflammatory cytokines and mediators from mast cells. Pimecrolimus
is used as a topical 1% cream for up to 6 weeks, preferably prior
to the stem cell therapy.
[0052] Gusperimus is a derivative of the antitumor antibiotic
spergualin, and inhibits the interleukin-2-stimulated maturation of
T cells to the S and G2/M phases and the polarization of the T
cells into IFN-gamma-secreting Th1 effector T cells, resulting in
the inhibition of growth of activated naive CD4 T cells. It is
administered SC, 0.5 mg/kg/day for consecutive 21 days, preferably
completed 3-4 days prior to the administration of the stem cells.
Nippon Kayaku Co., Ltd.
[0053] Everolimus (RAD-001), administered orally at a dose of 10
mg/day, preferably 3-4 days prior to the administration of the stem
cells. Novartis, East Hanover, N.J., under the tradenames Zortress
(USA).
[0054] Thalidomide Thalidomide may reduce the levels of TNF.alpha.,
(THALOMID.RTM., Celgene Corporation, Summit, N.J.). Acceptable
dosing is 100 - 300 mg/day preferably at bedtime 1 hour after
evening meal, preferably 3-4 days prior to the administration of
the stem cells.
[0055] Lenalidomide is a derivative of thalidomide 50,000 times
more potent than thalidomide in inhibiting tumor necrosis
factor-alpha, and has less severe adverse drug reactions. Celgene
Corporation, Summit, N.J.) 25 mg once daily orally on Days 1-21,
preferably 3-4 days prior to the administration of the stem
cells.
[0056] Anakinra is a recombinant, non-glycosylated version of human
IL-1RA (RA for receptor antagonist) Kineret.RTM. Biovitrum,
Stockholm, Sweden delivered as injection concentrate containing 100
mg each single dose, within 7-14 days and preferably 3-4 days prior
to the administration of the stem cells.
[0057] Infliximab (trade name REMICADE.RTM.) is a monoclonal
antibody against tumour necrosis factor alpha (TNF.alpha.).
Centocor Ortho Biotech, Horsham, Pa. administered by intravenous
infusion at a dose of from 3 mg/kg up to 10 mg/kg, within 7-14 days
and preferably 3-4 days prior to the administration of the stem
cells.
[0058] Golimumab (CNTO 148) is a human monoclonal antibody and is
marketed under the brand name Simponi. Golimumab targets TNF-alpha.
Centocor Ortho Biotech, Horsham, Pa., administered by as a
subcutaneous injection of 50 mgs in 0.5 mls within 7-14 days and
preferably 3-4 days prior to the administration of the stem
cells.
[0059] Adalimumab (HUMIRA, Abbott Laboratories, North Chicago,
Ill.) is a TNF inhibitor, adalimumab binds to TNF.alpha.,
preventing it from activating TNF receptors; adalimumab was
constructed from a fully human monoclonal antibody, marketed in
both preloaded 0.8 mL syringes and also in preloaded pen devices
each containing 40 mg of adalimumab. To down-regulate the germinal
centers prior to stem cell administration of at least 40 mg of
adalimumab should be administered within 7-14 days and preferably
3-7 days prior to stem cell administration. Preferably two 40
mg-doses of adalimumab should be administered within 7-14 days and
preferably 3-7 days prior to administration of the stem cells.
[0060] Certolizumab pegol is a monoclonal antibody directed against
tumor necrosis factor alpha. More precisely, it is a PEGylated Fab'
fragment of a humanized TNF inhibitor monoclonal antibody. It is
administered as two subcutaneous injections of 200 mg, injections,
within 7-14 days and preferably 3-4 days prior to the
administration of the stem cells. (UCB Inc., Atlanta, Ga.).
[0061] Temsirolimus (Pfizer Corp.) is a specific inhibitor of mTOR
(mammalian target of rapamycin) and interferes with the synthesis
of proteins that regulate proliferation, growth, and survival of
tumor cells. The recommended dose of temsirolimus is 25 mg IV
infused over 30-60 minutes, within 7-14 days and preferably within
3-4 days prior to the administration of the stem cells.
[0062] Zotarolimus is a semi-synthetic derivative of rapamycin,
Abbot Laboratories, North Chicago, Ill.)
[0063] Biolimus A9 Biosensors International, Singapore
[0064] Eculizumab (trade name Soliris) is a monoclonal antibody
directed against the complement protein C5. This antibody blocks
the cleavage of C5 and halts the process of complement-mediated
cell destruction. Soliris is administered as an IV infusion
administered in 600-mg doses or 900-mg doses, within 7-14 days and
preferably 3-4 days prior to the administration of the stem cells.
(Alexion Pharmaceuticals Cheshire, Conn.)
[0065] Mepolizumab (proposed trade name Bosatria) is a humanized
monoclonal antibody that recognizes interleukin-5 (IL-5)
administered in as an infusions of 750 mg, within 7-14 days and
preferably 3-4days prior to the administration of the stem cells.
GlaxoSmithKline, King of Prussia, PA.
[0066] Omalizumab (trade name Xolair, Genentech/Novartis) is a
humanized antibody Omalizumab is a recombinant DNA-derived
humanized IgG1k monoclonal antibody that selectively binds to human
immunoglobulin E (IgE). Xolair (Omalizumab) 150 to 375 mg is
administered SC, within 7-14 days and preferably 3-4 days prior to
the administration of the stem cells.
[0067] Nerelimomab (BAYX) is a mouse anti-TNF antibody, and can be
administered at 10 mg/kg within 7-14 days and preferably 3-4 days
prior to the administration of the stem cells.
[0068] Faralimomab is a mouse anti-TNF antibody, and can be
administered at 10 mg/kg within 7-14 days and preferably 3-4 days
prior to the administration of the stem cells.
[0069] Elsilimomab (also known as B-E8) is a mouse monoclonal
antibody and an immunosuppressive drug. According to the present
invention, it can be administered at a dose of 10 mg/kg within 7-14
days and preferably 3-4 days prior to administration of stem
cells.
[0070] Lebrikizumab is a humanized monoclonal antibody that is
designed to bind specifically to EL-13 and can be administered at
10 mg/kg within 7-14 days and preferably 3-4 days prior to the
administration of the stem cells. Genentech, South San Francisco,
Calif.
[0071] Ustekinumab (experimental name CNTO 1275, proprietary
commercial name Stelara, Centocor) is a human monoclonal antibody.
It is directed against interleukin 12 and interleukin 23, naturally
occurring proteins that regulate the immune system and
immune-mediated inflammatory disorders. 2 injections, one-month
apart, of either 90 or 45 milligrams, or a single 45 mg injection,
completed within 7-14 days and preferably 3-4 days prior to
administration of stem cells.
[0072] Muromonab-CD3 (trade name Orthoclone OKT3, marketed by
Janssen-Cilag) is a monoclonal antibody targeted at the CD3
receptor, a membrane protein on the surface of T cells. It is
administered 5 mg/day in a single bolus intravenous injection for
10 to 14 days. The administration should be completed within 7-14
days and preferably 3-4 days prior to the administration of the
stem cells. Children weighing less than 30 lb should receive 2.5
mg/day (Ortho Biotech, Raritan, N.J.)
[0073] Otelixizumab is a monoclonal antibody that targets the
epsilon chain of CD3. It is administered 5 mg/day in a single bolus
intravenous injection for 10 to 14 days. The administration should
be completed preferably within 7-14 days and 3-4 days prior to the
administration of the stem cells. Children weighing less than 30 lb
should receive 2.5 mg/day. The antibody is being developed by
Tolerx, Inc. in collaboration with GlaxoSmithKline and is being
manufactured by Abbott Laboratories.
[0074] Teplizumab is a humanized Fc-engineered monoclonal antibody
also known as MGA031 and hQKT3.gamma.1 (Ala-Ala). It is an anti-CD3
antibody. It can be administered according to the present invention
at a dose of 5 mg/day in a single bolus intravenous injection for
10 to 14 days. The administration should be completed within 7-14
days and preferably 3-4 days prior to the administration of the
stem cells. Children weighing less than 30 lb should receive 2.5
mg/day (Eli Lilly. Indianapolis, Ind.
[0075] Visilizumab (tentative trade name Nuvion, PDL BioPharma
Inc.) is a humanized monoclonal antibody that targets CD3 on
activated T-Cells. It can be administered according to the present
invention at a dose of 5 mg/day in a single bolus intravenous
injection for 10 to 14 days. The administration should be completed
within 7-14 days and preferably 3-4 days prior to the
administration of the stem cells. Children weighing less than 30 lb
should receive 2.5 mg/day.
[0076] Clenoliximab is a monoclonal antibody against CD4. It can be
administered according to the present invention at a dose of 5
mg/day in a single bolus intravenous injection for 10 to 14 days.
The administration should be completed within 7-14 days and
preferably 3-4 days prior to the administration of the stem
cells.
[0077] Keliximab is a monoclonal antibody that binds to white blood
cells via the protein CD4. It is administered at a dose of 3 mg/kg
infusion within 7-14 days and preferably 3-4 days prior to the
administration of the stem cells.
[0078] Zanolimumab (expected trade name HuMax-CD4) is a human
monoclonal antibody that targets CD4 and is administered at a dose
20 mg/kg/day within 7-14 days and preferably 3-4 days prior to the
administration of the stem cells. (Genmab, A/S COPENHAGEN/TenX
Biopharma, Inc., Philadelphia, Pa.).
[0079] Efalizumab (trade name Raptiva, Genentech, Merck Serono) is
a recombinant humanized monoclonal antibody. Efalizumab binds to
the CD11a subunit of lymphocyte function-associated antigen 1.
According to the present invention, it can be administered once
weekly by subcutaneous injection at a dose of 20 mg/kg, within 7-14
days and preferably 3-4 days prior to the administration of the
stem cells.
[0080] Erlizumab, also known as rhuMAb, is a recombinant humanized
monoclonal antibody developed by Genentech under a partnership with
Roche. According to the present invention, it can be administered
once weekly by subcutaneous injection at a dose of 20 mg/kg, within
7-14 days and preferably 3-4 days prior to the administration of
the stem cells. The drug works by blocking a growth factor in blood
vessels. Specifically, erlizumab targets CD18 and an LFA-1
integrin.
[0081] Afutuzumab is an anti-CD20 monoclonal antibody. According to
the present invention, it can be administered once weekly by
subcutaneous injection at a dose of 20 mg/kg, within 7-14 days and
preferably 3-4 days prior to the administration of the stem cells.
(Hoffmann-La Roche Inc.)
[0082] Ocrelizumab is a humanized anti-CD20 monoclonal antibody. It
targets mature B lymphocytes. It is under development by
Hoffmann-La Roche's subsidiary Genentech, and Biogen Idec.
According to the present invention, it is dosed at 200 mg & 600
mg within 7-14 days and preferably 3-4 days prior to the
administration of the stem cells.
[0083] Pascolizumab is an anti-IL-4 humanized monoclonal antibody.
According to the present invention, it can be administered once
weekly by subcutaneous injection at a dose of 20 mg/kg, within 7-14
days and preferably 3-4 days prior to the administration of the
stem cells,
[0084] Lumiliximab is a monoclonal antibody that targets CD23.
According to the present invention, it can be dosed at 50 mg/m2, to
450 mg/m2, to 500 mg/m2 within 7-14 days and preferably 3-4 days
prior to the administration of the stem cells. The drug is a
chimeric antibody from Macaca irus and Homo sapiens. (Biogen
IDEC)
[0085] Teneliximab is a chimeric monoclonal antibody binding to the
immune stimulatory protein CD40. According to the present
invention, it can be administered once weekly by subcutaneous
injection at a dose of 20 mg/kg, within 7-14 days and preferably
3-4 days prior to the administration of the stem cells.
[0086] Toralizumab (IDEC 131) is a humanized monoclonal antibody.
According to the present invention, it can be administered once
weekly by subcutaneous injection at a dose of 20 mg/kg, within 7-14
days and preferably 3-4 days prior to the administration of the
stem cells. (IDEC Pharmaceuticals Corporation)
[0087] Aselizumab is an anti-CD62L administered by I.V. infusion at
doses ranging from 0.5-mg/kg, 1.0-mg/kg, and 2.0-mg/kg within 7-14
days and preferably 3-4 days prior to the administration of the
stem cells.
[0088] Galiximab is an anti-CD80 (Biogen Idec) monoclonal antibody
administered at a dose of 500 mg/m2 IV within 7-14 days and
preferably 3-4 days prior to the administration of the stem cells.
It is a chimeric antibody from Macaca irus and Homo sapiens
[0089] Gavilimomab is a mouse monoclonal antibody (also known as
ABX-CBL, developed by Abgenix. It binds to the antigen CD147.
According to the present invention it can be administered by I.V.
infusion at a dose of 20 mg/kg within 7-14 days and preferably 3-4
days prior to the administration of the stem cells.
[0090] BG9588, a humanized anti-CD40L administered at 20 mg/kg
within 7-14 days and 3-4 days prior to administration of the stem
cells. Administration of antibodies to CD 154, also called CD40
ligand or CD40L, is a protein that is primarily expressed on
activated T cells and is a member of the TNF superfamily of
molecules. It binds to CD40 on antigen-presenting cells (APC),
which leads to many effects depending on the target cell type. In
general, CD40L plays the role of a costimulatory molecule and
induces activation in APC in association with T cell receptor
stimulation by MHC molecules on the APC. In total CD40L has three
binding partners: CD40, .alpha.5.beta.1 integrin and
.alpha.IIb.beta.3.
[0091] (Hu5c8) 5c8, a monoclonal antibody that binds CD154 (CD40
ligand), thus blocking the interaction between CD40 and CD154,
administered at 20 mg/kg within 7-14 days and preferably 3-4 days
prior to administration of the stem cells.
[0092] Belimumab (registered name Benlysta previously known as
LymphoStat-B), is a fully human monoclonal antibody that
specifically recognizes and inhibits the biological activity of
B-Lymphocyte stimulator (BLyS), also known as B cell activation
factor of the TNF family (BAFF) Human Genome Sciences According to
the present invention, it can be administered at a dose of 10 mg/kg
within 7-14 days and preferably 3-4 days prior to administration of
stem cells.
[0093] Ipilimumab (also known as MDX-010 or MDX-101) is an
anti-CTLA-4 (cytotoxic T-Cell lymphocyte-associated) human
monoclonal antibody being developed by Bristol-Myers Squibb.
According to the present invention, it is administered 10 mg/kg
active drug within 7-14 days and preferably 3-4 days prior to
administration of stem cells.
[0094] Tremelimumab (formerly ticilimumab, CP-675,206) is a fully
human IgG2 monoclonal antibody produced by Pfizer. It binds to the
protein CTLA-4, which is expressed on the surface of activated T
lymphocytes. Tremelimumab blocks the binding of the
antigen-presenting cell ligands B7.1 and B7.2 to CTLA-4, resulting
in inhibition of B7-CTLA-4-mediated downregulation of T-cell
activation; subsequently, B7.1 or B7.2 may interact with another
T-cell surface receptor protein, CD28, resulting in a
B7-CD28-mediated T-cell activation unopposed by B7-CTLA-4-mediated
inhibition. Tremelimumab is administered by IV infusion at 3 mg/kg,
6 mg/kg, 10 mg/kg, 15 mg/kg within 7-14 days and preferably 3-4
days prior to administration of the stem cells.
[0095] Bertilimumab is a human monoclonal antibody that binds to
eotaxin-1. (iCo Therapeutics Inc. Vancouver, B.C.) According to the
present invention, it is administered at a dose of 10 mg/kg within
7-14 days and preferably 3-4 days prior to administration of stem
cells.
[0096] Lerdelimumab (CAT-152) is an anti-TGF beta-2 being developed
by Cambridge Antibody Technology. According to the present
invention, it can be administered at a dose of 10 mg/kg within 7-14
days and preferably 3-4 days prior to administration of stem
cells.
[0097] Metelimumab (CAT-192) is a human IgG4 monoclonal antibody
developed by Cambridge Antibody Technology that neutralizes TGF
beta 1. According to the present invention, it can be administered
at a dose of 10 mg/kg preferably 3-4 days prior to administration
of stem cells. Natalizumab is a humanized monoclonal antibody
against the cellular adhesion molecule .alpha.4-integrin. It is
co-marketed by Biogen Idec and Elan as Tysabri, and was previously
named Antegren. Natalizumab is administered at a dose of 300 mg
infused intravenously over approximately one hour within 7-14 days
and and preferably 3-4 days prior to administration of the stem
cells.
[0098] Tocilizumab or atlizumab, developed by Hoffmann-La Roche and
Chugai under the trade names Actemra and RoAetemra, is a humanized
monoclonal antibody against the interleukin-6 receptor (IL-6R).
According to the present invention, it can be administered by
intravenous infusions at 8 mg/kg, within 7-14 days and preferably
3-4 days prior to administration of stem cells.
[0099] Odulimomab is a mouse monoclonal antibody directed against
the alpha chain of the protein lymphocyte function-associated
antigen 1 which is involved in immune reactions. It is administered
10 mg/kg active drug within 7-14 days and 3-4 days prior to
administration of stem cells.
[0100] Basiliximab (trade name Simulect) is a chimeric mouse-human
monoclonal antibody to the a chain (CD25) of the IL-2 receptor of T
cells. Dose is 20 mg two times within 7-14 days and preferably 3-4
days prior to administration of the stem cells.
[0101] Daclizumab (trade name Zenapax) is a therapeutic humanized
monoclonal antibody to the alpha subunit of the IL-2 receptor of T
cells. Roche Pharmaceuticals, Hoffmann-La Roche Inc, 340 Kingsland
Street, Nutley, N.J. It is administered 10 mg/kg active drug within
7-14 days and preferably 3-4 days prior to administration of stem
cells.
[0102] Inolimomab is a mouse monoclonal antibody targeted against
the alpha chain of the interleukin-2 receptor.OPi (formerly Orphan
Pharma International). It is administered 10 mg/kg active drug
within 7-14 days and preferably 3-4 days prior to administration of
stem cells.
[0103] Zolimomab aritox is a mouse monoclonal antibody and is an
anti-CD5 antibody which is linked to the A chain of the ricin
protein (which is reflected by the aritox in the drug's name).
According to the present invention, it can be administered at a
dose of 10 mg/kg within 7-14 days and preferably 3-4 days prior to
administration of stem cells.
[0104] Atorolimumab is mouse monoclonal antibody directed against
the Rhesus factor. According to the present invention, it can be
administered at a dose of 10 mg/kg within 7-14 days and preferably
3-4 days prior to administration of stem cells.
[0105] Cedelizumab is an anti-CD4 monoclonal antibody. According to
the present invention, it can be administered at a dose of 10 mg/kg
within 7-14 days and preferably 3-4 days prior to administration of
stem cells.
[0106] Dorlixizumab is a chimeric/humanized monoclonal antibody and
an immunosuppressive drag. It is administered at a dose of 10 mg/kg
of active drug within 7-14 days and preferably 3-4 days prior to
administration of stem cells.
[0107] Fontolizumab (trade name HuZAF) is anti-interferon gamma
humanized monoclonal antibody. According to the present invention,
it can be administered at an I.V. dose of fontolizumab given as 4.0
mg/kg or 10.0 mg/kg within 7-14 days and preferably 3-4 days prior
to administration of the stem cells. (PDL Biopharma)
[0108] Gantenerumab is anti-beta amyloid monoclonal antibody
(Roche). It is administered at a dose of 10 mg/kg of active drug
within 7-14 days and preferably 3-4 days prior to administration of
stem cells.
[0109] Gomiliximab is a monoclonal antibody that targets the low
affinity IgE receptor (Fc.epsilon.RII or CD23). According to the
present invention, it can be administered at a dose of 10 mg/kg
within 7-14 days and preferably 3-4 days prior to administration of
stem cells.
[0110] Maslimomab is a mouse monoclonal antibody targets the T-Cell
receptor. According to the present invention, it can be
administered at a dose of 10 mg/kg within 7-14 days and preferably
3-4 days prior to administration of stem cells.
[0111] Morolimumab is a human monoclonal antibody against the human
Rhesus factor. According to the present invention, it can be
administered at a dose of 10 mg/kg within 7-14 days and preferably
3-4 days prior to administration of stem cells.
[0112] Pexelizumab is a single chain variable fragment of a
monoclonal antibody targeted against component 5 of the complement
system. According to the present invention, it can be administered
at a dose of 10 mg/kg within 7-14 days and preferably 3-4 days
prior to administration of stem cells.
[0113] Reslizumab is an anti-IL-5 humanized monoclonal antibody.
According to the present invention, It can administered as an
infusion at a preferred dose of reslizumab 3.0 mg/kg within 7-14
days and preferably 3-4 days prior to administration of the stem
cells. (Ception Therapeutics Inc).
[0114] Rovelizumab, also known as LeukArrest and Hu23F2G, is an
anti-CD11/CD18 humanized monoclonal antibody that suppresses white
blood cells. According to the present invention, it can be
administered at a dose of 10 mg/kg within 7-14 days and preferably
3-4 days prior to administration of stem cells.
[0115] Siplizumab (MEDI-507) is an anti-CD2 monoclonal antibody
with a human IgG1, kappa directed to CD2. The agent has shown
potent immunomodulatory effects, selectively suppressing the
function of T and NK cells. Siplizumab binds to CD2, a specific
receptor found in T cells and NK cells, thereby triggering a host
immune response that results in lysis of CD2+ cells, selective
suppression of the immune system, and control of activated T cell
growth. According to the present invention, Siplizumab can be
administered at a preferred dose of 0.04 mg/kg i.v. and 5 or 7
mg/kg s.c. within 7-14 days and preferably 3-4 days prior to
administration of stem cells. (Medimmune)
[0116] Talizumab (TNX-901) is a humanized monoclonal antibody being
developed by Tanox in Houston, Tex. It was designed to target
immunoglobulin E (or IgE) and IgE-expressing B lymphocytes
specifically, without binding to IgE already bound by the IgE
receptors on mast cells and basophils. According to the present
invention, it can be administered at a dose of 10 mg/kg within 7-14
days and preferably 3-4 days prior to administration of stem
cells.
[0117] Omalizumab is an anti-IgE monoclonal antibody, developed by
Tanox, Novartis, and Genentech. According to the present invention,
it can be administered at a dose of 10 mg/kg within 7-14 days and
preferably 3-4 days prior to administration of stem cells.
[0118] Telimomab aritox is a mouse monoclonal antibody. The
antibody is linked to the A chain of the ricin protein (which is
reflected by the aritox in the drug's name). According to the
present invention, it can be administered at a dose of 10 mg/kg
within 7-14 days and preferably 3-4 days prior to administration of
stem cells.
[0119] Vapaliximab is an anti-VAP-1 chimeric monoclonal antibody.
According to the present invention, it can be administered at a
dose of 10 mg/kg within 7-14 days and preferably 3-4 days prior to
administration of stem cells.
[0120] Vepalimomab is an anti-VAPI mouse monoclonal antibody
According to the present invention, it can be administered at a
dose of 10 mg/kg within 7-14 days and preferably 3-4 days prior to
administration of stem cells.
[0121] Abatacept (marketed as Orencia) is a fusion protein composed
of an immunoglobulin fused to the extracellular domain of CTLA-4, a
molecule capable of binding B7, thus preventing the full activation
of T cells. Abatacept should be administered as a 30-minute
intravenous infusion according to the specified dose schedule based
on weight. The doses should be preferably be 500 mg for <60 kg;
750 mg for 60 kg-100 kg; and 1 gram for >100 kg within 7-14 days
and preferably 3-4 days prior to the administration of the stem
cells.
[0122] Belatacept (Bristol-Myers-Squibb) is a fusion protein
composed of the Fc fragment of a human IgG1 immunoglobulin linked
to the extracellular domain of CTLA-4, which is a molecule crucial
for T-cell costimulation, selectively blocking the process of
T-cell activation. It was developed by. It differs from abatacept
(Orencia) by only 2 amino acids. According to the present
invention, it can be administered as a 30-minute intravenous
infusion according to the specified dose schedule based on weight
at preferable doses of 500 mg for <60 kg; 750 mg for 60 kg-100
kg; and 1 gram for >100 kg administered within 7-14 days and
preferably 3-4 days prior to administration of the stem cells.
[0123] Etanercept (trade name Enbrel, Amgen, Thousand Oaks, Calif.)
is a drug that treats autoimmune diseases by interfering with the
tumor necrosis factor (TNF, a part of the immune system) by acting
as a TNF inhibitor. Etanercept can be administered subcutaneously
(s.c.) at a dose 25 mg or 50 mg within 7-14 days and preferably 3-4
days prior to the administration of the stem cells.
[0124] Pegsunercept is a pegylated soluble tumor necrosis factor
receptor. According to the present invention, it can be
administered at a preferable dose of 9 mg/kg s.c, within 7-14 days
and preferably 3-4 days prior to administration of the stem
cells.
[0125] Aflibercept is protein comprised of segments of the
extracellular domains of human vascular endothelial growth factor
receptors 1 (VEGFR1) and 2 (VEGFR2) fused to the constant region
(Fc) of human IgG1 with potential antiangiogenic activity and is
being co-developed by Sanofi-Aventis and Regeneron Pharmaceuticals.
Aflibercept (VEGF Trap), an anti-angiogenic agent, is a fusion
protein specifically designed to bind all forms of Vascular
Endothelial Growth Factor-A (called VEGF-A). In addition,
aflibercept binds Placental Growth Factor (PLGF), which has also
been implicated in tumor angiogenesis. Aflibercept can be
administered by injection or IV infusion at preferable doses of 2
milligrams per kilogram (mg/kg) or 4 mg/kg, within 7-14 days and
preferably administered 3-4 days prior to the administration of the
stem cells.
[0126] Alefacept is a fusion protein: it combines part of an
antibody with a protein that blocks the growth of some types of T
cells. AMEVIVE.RTM. (alefacept) is an immunosuppressive dimeric
fusion protein that consists of the extracellular CD2-binding
portion of the human leukocyte function antigen-3 (LFA-3) linked to
the Fc (hinge, CH2 and CH3 domains) portion of human IgG1. The
preferred dosage is either 7.5 mg IV or 15 mg IM preferably
administered within 7-14 days and preferably 3-4 days prior to the
administration of the stem cells. Asteilas Pharma US, Inc.
Deerfield, Ill. 60015.
[0127] Rilonacept also known as IL-1 Trap (marketed under the trade
name Arcalyst), is a dimeric fusion protein consisting of the
extracellular domain of human interleukin-1 receptor and the FC
domain of human IgG1 that binds and neutralizes IL-1h. Treatment
should be initiated with a loading dose of 320 mg delivered as two,
2 mL, subcutaneous injections of 160 mg each given on the same day
at two different sites, within 7-14 days and preferably
administered 3-4 days prior to the administration of the stem
cells. Pediatric patients aged 12 to 17 years: Treatment should be
initiated with a loading dose of 4.4 mg/kg, up to amaximum of 320
mg, delivered as one or two subcutaneous injections with a maximum
single-injection volume of 2 mL, within 7-14 days and preferably
3-4 days prior to administration of the stem cells. Produced by
Regeneron.
[0128] Dacetuzumab (also known as SGN-40 or huS2C6) is an anti-CD40
humanized monoclonal antibody. The CD40 antigen is highly expressed
on most B-lineage hematologic malignancies including multiple
myeloma, non-Hodgkin lymphoma and chronic lymphocytic leukemia.
CD40 is also found on many types of solid tumors, including
bladder, renal and ovarian cancer and on cells that play a role in
immunologic disorders. It is administered at a preferred dose of 10
mg/kg of active drag within 7-14 days and preferably 3-4 days prior
to administration of stem cells. Seattle Genetics, Inc.
[0129] HCD122 is a fully human antagonist anti-CD40 monoclonal
antibody. CD40 is a cell-surface receptor that plays a pivotal role
in immune responses, as well as cell growth and survival signaling,
through its activation by CD40 ligand (CD40L). It is commonly
overexpressed and activated in B-cell malignancies. According to
the present invention, it can be administered at a dose of 10 mg/kg
of active drug within 7-14 days and preferably 3-4 days prior to
administration of stem cells. This is being developed by
XOMA/NOVARTIS ONCOLOGY.
[0130] Rituximab, sold under the trade names Rituxan and MabThera,
Genentech, Inc., San Francisco, Calif. is a chimeric monoclonal
antibody against the protein CD20. which is primarily found on the
surface of B cells. It can therefore destroy B cells. CD20 is
widely expressed on B cells, from early pre-B cells to later in
differentiation, but it is absent on terminally differentiated
plasma cells. Rituxan is supplied at a concentration of 10 mg/mL in
either 100 mg (10 mL) or 500 mg (50 mL) single-use vials. It can be
administered as an infusion at a rate of 50 mg/hr. In the absence
of infusion toxicity, increase infusion rate by 50 mg/hr increments
every 30 minutes, to a maximum of 400 mg/hr preferably administered
within 7-14 days and preferably 3-4 days prior to the
administration of the stem cells. The preferred recommended dose is
375 mg/m2 as an IV infusion preferably administered 3-4 days prior
to the administration of the stem cells.
[0131] Rituximab can also be administered as a Component of
Zevalin.RTM. by infusing rituximab at a preferable dose 250 mg/m2
within 4 hours prior to the administration of Indium-111-(In-111-)
Zevalin and within 4 hours prior to the administration of
Yttrium-90-(Y-90-) Zevalin, this should be done within 7-14 days
and preferably 3-4 days prior to the administration of the stem
cells. Rituxan can also be administered in combination with
methotrexate, preferable 3-4 days prior to the administration of
the stem cells. Biogen Idec Inc. and Genentech USA, Inc.
[0132] Ibritumomab tiuxetan, sold under the trade name Zevalin, is
a monoclonal antibody radioimmunotherapy targeting B-cells. The
drug uses the monoclonal mouse IgG1 antibody ibritumomab in
conjunction with the chelator tiuxetan, to which a radioactive
isotope (either yttrium-90 or indium-111) is added. Tiuxetan is a
modified version of DTPA whose carbon backbone contains an
isothiocyanatobenzyl and a methyl group.
[0133] Adenosine deaminase deficiency will also lead to reduced
active germinal center formation as will agents which trigger the
accumulation of deoxyATP (J Immunol 171:5562-5570, 2003).
Similarly, agents that enhance the expression of or activate CCR7
will lead to diminished active germinal center formation.
[0134] The term stem cell within the scope of the present invention
includes any cell capable of differentiating into a desired tissue.
Such cells include pluripotent stem cells, embryonic stem cells,
multipotent adult stem cells, and progenitor and precursor cells. A
"stem cell" is a cell from the embryo, fetus, or adult that has,
under certain conditions, the ability to reproduce itself for long
periods or. in the case of adult stem cells, throughout the life of
the organism. It also can give rise to specialized cells that make
up the tissues and organs of the body.
[0135] A "pluripotent stem cell" has the ability to give rise to
types of cells that develop from the three germ layers (mesoderm,
endoderm, and ectoderm) from which all the cells of the body arise.
Known natural sources of human pluripotent stem cells are those
isolated and cultured from early human embryos from fetal tissue
that was destined to be past of the gonads.
[0136] An "embryonic stem cell" is derived from a group of cells
called the inner cell mass, which is part of the early (4- to
5-day) embryo called the blastocyst. Once removed from the
blastocyst the cells of the inner cell mass can be cultured into
embryonic stem cells.
[0137] An "adult stem cell" is an undifferentiated (unspecialized)
cell that occurs in a differentiated (specialized) tissue, renews
itself, and becomes specialized to yield all of the specialized
cell types of the tissue in which it is placed when transferred to
the appropriate tissue. Adult stem cells are capable of making
identical copies of themselves for the lifetime of the organism.
This property is referred to as "self-renewal." Adult stem cells
usually divide to generate progenitor or precursor cells, which
then differentiate or develop into "mature" cell types that have
characteristic shapes and specialized functions, e.g., muscle cell
contraction or nerve cell signaling. Sources of adult stem cells
include but are not limited to bone marrow, blood, the cornea and
the retina of the eye, brain, skeletal muscle, dental pulp, liver,
skin, the lining of the gastrointestinal tract and pancreas.
[0138] The delivery or administration of stem cells to an
individual includes both the delivery or administration of
exogenous stem cells as well as the mobilization of endogenous stem
cells, as well as enhancing the bioavailability of spontaneously
released endogenous stem cells.
[0139] Stem cells from the bone marrow are the most-studied type of
adult stem cells. Currently, they are used clinically to restore
various blood and immune components to the bone marrow via
transplantation. There are currently identified two major types of
stem cells found in bone marrow: hematopoietic stem cells (HSC, or
CD34+ cells) which are typically considered to form blood and
immune cells, and stromal (mesenchymal) stem cells (MSG) that are
typically considered to form bone, cartilage, muscle and fat.
However, both types of marrow-derived stem cells recently have
demonstrated extensive plasticity and multipotency in their ability
to form the same tissues. The marrow, located in the medullary
cavity of bones, is the major site of hematopoiesis in adult
humans. It produces about six billion cells per kilogram of body
weight per day. Hematopoietically active (red) marrow regresses
after birth until late adolescence after which time it is focused
in the lower skull vertebrae, shoulder and pelvic girdles, ribs,
and sternum. Fat cells replace hematopoietic cells in the bones of
the hands, feet, legs and arms (yellow marrow). Fat comes to occupy
about fifty percent of the space of red marrow in the adult and
further fatty metamorphosis continues slowly with aging. In very
old individuals, a gelatinous transformation of fat to a mucoid
material may occur (white marrow). Yellow marrow can revert to
hematopoietically active marrow if prolonged demand is present such
as with hemolytic anemia. Thus hematopoiesis can be expanded by
increasing the volume of red marrow and decreasing the development
(transit) time from progenitor to mature cell.
[0140] The marrow stroma consists principally of a network of
sinuses that originate at the endosteum from cortical capillaries
and terminate in collecting vessels that enter the systemic venous
circulation. The trilaminar sinus wall is composed of endothelial
cells; an underdeveloped, thin basement membrane, and adventitial
reticular cells that are fibroblasts capable of transforming into
adipocytes. The endothelium and reticular cells are sources of
hematopoietic cytokines. Hematopoiesis takes place in the
intersinus spaces and is controlled by a complex array of
stimulatory and inhibitory cytokines, cell-to-cell contacts and the
effects of extracellular matrix components on proximate cells. In
this unique environment, lympbohematopoietic stem cells
differentiate into all of the blood cell types. Mature cells are
produced and released to maintain steady state blood cell levels.
The system may meet increased demands for additional cells as a
result of blood loss, hemolysis, inflammation, immune cytopenias,
and other causes.
[0141] A "progenitor or precursor" cell is partially specialized;
it self-renews and also gives rise to differentiated cells.
Researchers often distinguish precursor/progenitor cells from adult
stem cells in that when a stem cell divides, one of the two new
cells is often a stem cell capable of replicating itself again. In
contrast when a progenitor/precursor cell divides, it can form more
progenitor/precursor cells or it can form two specialized cells.
Progenitor/precursor cells can replace cells that are damaged or
dead, thus maintaining the integrity and functions of a tissue such
as liver or brain.
[0142] Means for isolating and culturing stem cells useful in the
present invention are well known. Umbilical cord blood is an
abundant source of hematopoietic stem cells. The stem cells
obtained from umbilical cord blood and those obtained from bone
marrow or peripheral blood appear to be very similar for
transplantation use. Placenta is an excellent readily available
source for mesenchymal stem cells. Moreover, mesenchymal stem cells
have been shown to be derivable from adipose tissue and bone marrow
stromal cells and speculated to be present in other tissues.
Amniotic fluid and tissue is another excellent source of stem
cells. While there are dramatic qualitative and quantitative
differences in the organs from which adult stem cells can be
derived, the initial differences between the cells may be
relatively superficial and balanced by the similar range of
plasticity they exhibit. For instance, adult stem cells both
hematopoietic and mesenchymal, under the appropriate conditions can
become cardiac muscle cells. Delineation of full range of potential
for adult stem cells has just begun. Stem cells may be isolated and
differentiated using known methods. For example, in mice, bone
marrow cells are isolated by sacrificing the mouse, cutting the leg
bones with a pair of scissors, and flushing the stem cells out.
Stem cells may also be isolated from bone marrow cells by panning
the bone marrow cells with antibodies which bind unwanted cells,
such as CD4+ and CD8+ (T cells), CD45+ (panB cells), GR-1
(granulocytes). For an example of this protocol see, Inaba et al.,
J. Exp. Med. 176:1693-1702(1992).
BRIEF DESCRIPTION OF THE DRAWINGS
[0143] FIG. 1 shows a typical view of layers resulting from
gradient centrifugation of whole blood. 1 shows the platelets; 2
the buffy coat with MNCs and stem cells; 3 the ficoll; and 4 the
RBC pellet and stem cells.
SUMMARY OF THE INVENTION
[0144] In humans, CD34+ hematopoietic stem cells can be obtained
from a variety of sources including cord blood, bone marrow, and
mobilized peripheral blood. Purification of CD34+ cells can be
accomplished by antibody affinity procedures. An affinity column
isolation procedure for isolating CD34+ cells is described by Ho et
al., Stem Cells 13 (suppl. 3): 100-105(1995). See also, Brenner,
Journal of Hematotherapy 2: 7-17 (1993). Methods for isolating,
purifying and culturally expanding mesenchymal stem cells are
known. Specific antigens for MSC are also known (see, U.S. Pat.
Nos. 5,486,359 and 5,837,539).
[0145] Stem cells are characterized by the ability to renew
themselves through mitotic cell division and to differentiate into
a diverse range of specialized cell types. Stem cells exist along a
range of potencies. Totipotent stem cells are cells such as a
fertilized egg that can generate ail tissues necessary for
development of a complete organism. Pluripotent stem cells are
cells that can give rise to stem cells for all 3 germ layers and
include cells such as embryonic stem cells, spermatogonial stem
cells (Cell. 119(7):1001-1012, 2009; NATURE 440:1199-1203, 2006),
or induced pluripotent stem cells that when injected into
tetraploid embryo can give rise to an entire organism (Stem Cell
Rev. 2010), but not to the extra-embryonic tissues needed, such as
the placenta. Very-small embryonic like stem cells are cells found
within the bone marrow, blood, heart and other tissues of the adult
that can give rise to cells of cells from all 3 germ layer
lineages, however, they have not yet been shown in tetraploid
complementation assays to generate an entire organism, so it is
unclear if they are true pluripotent stem cells for which somatic
imprinting prevents their activity in tetraploid complementation or
whether they are more restricted yet extremely plastic multipotent
stem cells (DEVELOPMENTAL DYNAMICS 236:3309-3320, 2007).
Multipotent stem cells are cells such as hematopoietic stem cells
(HSC) (J Exp Med. 207(6): 1127-1130, 2010), adipose stem cells
(ASC) (Stem Cells Dev. 2010 [Epub ahead of print]) (6) or
mesenchymal stem cells (MSC) (StemBook, Cambridge (Mass.): Harvard
Stem Cell Institute; 2008-2009) that can give rise to a variety of
functioning cells within restricted lineages.
[0146] Stem cells can be further characterized by the degree to
which they can differentiate and are distinguished by their
potency. Potency specifies the differentiation potential (the
potential to differentiate into different cell types) of the stem
cell.
[0147] Totipotent (a.k.a omnipotent) stem cells can differentiate
into embryonic and extraembryonic cell types. Such cells can
construct a complete, viable, organism. These cells are produced
from the fusion of an egg and sperm cell. Cells produced by the
first few divisions of the fertilized egg are also totipotent.
[0148] Pluripotent stem cells are the descendants of totipotent
cells and can differentiate into nearly all cells, i.e. cells
derived from any of the three germ layers.
[0149] Multipotent stem cells can differentiate into a number of
cells, but only those of a closely related family of cells.
[0150] Oligopotent stem cells can differentiate into only a few
cells, such as lymphoid or myeloid stem cells.
[0151] Unipotent cells can produce only one cell type, their own,
but have the properly of self-renewal that distinguishes them from
non-stem cells (e.g. muscle stem cells).
[0152] The two broad types of mammalian stem cells are: embryonic
stem cells that are isolated from the inner cell mass of
blastocysts, and adult stem cells that are found in adult
tissues.
[0153] Adult stem cells are undifferentiated cells, found
throughout the body after embryonic development, that multiply by
cell division to replenish dying cells and regenerate damaged
tissues. Also known as somatic stem cells, they can be found in
juvenile as well as adult animals and humans. The types of adult
stem cells include hematopoietic stem cells, mammary stem cells,
mesenchymal cells, endothelial stem cells, neural stem cells,
olfactory adult stem cells, adipose-derived stem cells, neural
crest stem cells, and testicular stem cells.
[0154] Progenitor cells have a tendency to differentiate into a
specific type of cell. In contrast to stem cells, however, they are
already far more specific: they are pushed to differentiate into
their "target" cell. The most important difference between stem
cells and progenitor cells is that stem cells can replicate
indefinitely, whereas progenitor cells can only divide a limited
number of times. Controversy about the exact definition remains and
the concept is still evolving.
[0155] The terms "progenitor cell" and "stem cell" are sometimes
equated.
[0156] Stem cells found within the mononuclear fraction of whole
blood, bone marrow, adipose tissue, umbilical cord blood, and
cither tissues, as well as isolated stem cells from these
mononuclear fractions have been demonstrated to provide a benefit
to human patients. A peripheral blood mononuclear cell (PBMC) is
any blood cell having a round nucleus. For example: a lymphocyte,
monocyte or a macrophage. These blood cells are a critical
component in the immune system to fight infection and adapt to
intruders. The lymphocyte population consists of T cells (CD4 and
CDS positive .about.75%), B cells and NK cells (.about.25%
combined). PBMCs are often extracted from whole blood using ficoll,
a hydrophilic polysaccharide that separates layers of blood, with
monocytes and lymphocytes forming a buffy coat under a layer of
plasma. This huffy coat contains the PBMCs. Additionally, PBMC can
be extracted from whole blood using a hypotonic lysis which will
preferentially lyse red blood cells. This method results in
neutrophils and other polymorphonuclear (PMN) cells, which are
important in innate immune defence being obtained. PBMC fractions
of bone marrow aspirates have been used to treat patients following
a myocardial infarction, and have been shown to reduce subsequent
mortality and to slightly improve cardiac function in these
patients (Eur Heart J 27:2775-2783, 2006). While mortality is
significantly reduced by these types of treatments, cardiac
function is only slightly improved. Nuclear imaging studies on
these patients have shown that the majority, up to 97%, of the
intra-coronary injected mononuclear fraction stem cells do not
remain in the heart, but can be found predominantly in the spleen
and liver within 60 to 90 minutes after injection (Circulation
111:2198-2202, 2005). Other imaging studies have likewise
demonstrated that stem cells found within the mononuclear fraction
of whole blood, bone marrow, adipose tissue, umbilical cord blood,
placenta, amniotic fluid, and other tissues, as well as isolated
stem cells from these mononuclear fractions, accumulate in the
spleen in many different species (STEM CELLS 24:2279-2283, 2006; J
Nucl Med 45:512-518, 2004; J Nucl Med 47:1212-1239, 2006; J Nucl
Med 47:1295-1301, 2006).
[0157] Animal studies have demonstrated that administration of
higher numbers of bone marrow mononuclear cells leads to improved
cardiac repair and functional recovery (Circulation 114:2163-2169,
2006). However, for the clinical patient, this would require
aspiration of large volumes of bone marrow, up to 200 mls, under
general anesthesia, and this is considered highly undesirable for
recent post-infarct patients whose heart function remains
depressed. Investigators have also tried to concentrate the stem
cells they are injecting in order to obtain better organ targeting
and retention. While enriching the number of CD34+ cells injected
has led to higher accumulation of CD34+ cells in the heart after
intra-coronary injection, this method to enhance stem cell delivery
is thought to be sub-optimal because it is not known at this time
which particular stem cells contained within the mononuclear cell
fraction are necessary for tissue regeneration (Circulation
111:2198-2202, 2005). Furthermore, purified human mesenchymal stem
cells (MSC) have been shown to augment the engraftment of human
umbilical cord blood CD34+ stem cells (Hematology VOL 14 NO
3:125-132, 2009).
[0158] Stem cells may be autologous or from an unrelated donor.
Stem cells may be contained within the mononuclear cell fraction
from bone marrow, whole blood, umbilical cord blood, adipose tissue
or other sources, or they may be purified by selection for CD34,
CD133, CD105, CD117, SSEA1-4, dye exclusion or other specific stem
cell antigens. Stem cells can be isolated from whole blood, bone
marrow, umbilical cord blood, adipose tissue, tissue scrapings from
the olfactory mucosa and other stem cell sources that can be
dissociated into single cell suspensions, such as umbilical cord
tissue, by density gradient centrifugation using Ficoll-Hypaque or
other commercially available gradients. Stem cells can be recovered
from the mononuclear cell fraction resulting from such procedures.
Alternatively, stem cells can be found within other fractions after
density gradient centrifugation (Stem Cells and Development 2011
Bhartiya et. al.) For instance, umbilical cord blood can be diluted
1:1 in PBS, carefully overlaid onto Histopaque 1077 (Sigma) and
centrifuged at 1500 rpms at room temperature for 30 minutes. The
resulting layers as depicted in Figure 1 cat) be further processed
for stem cell isolation. Layer 1 is the platelet layer, layer 2 is
the buffy coat containing mononuclear cells, layer 3 is the Ficoll
layer, and layer 4 is the red blood cell pellet layer. Layers 1, 2,
and 3 can be collected, diluted with appropriate media such as DMEM
F12 with or without FBS and centrifuged again to obtain the cell
pellet. Layer 4 can be diluted with appropriate media such as DMEM
F12 and centrifuged at 800 rpm for 15 minutes at room temperature
in a standard benchtop centrifuge. Stem cells can be recovered
predominantly from layer 2 (Buffy coat) and layer 4 (RBC
pellet).
[0159] Stem cells can be further characterized and isolated by
specific antigens expressed on their surface using cell sorters
such as the ARIA from BD, using magnetic columns such as those
available from Miltenyi, using magnetic beads and DYNAL magnets and
other antibody/antigen based separation methods known to those
skilled in the art. Stem cells can also be identified and isolated
by their ability to bind to other cells as described in this
disclosure.
[0160] Pluripotent stem cells can be characterized by the
expression of stage-specific embryonic antigen (SSEA), the
transcription factors Oct4 and Nanog and other markers.
Hematopoietic stem cells are characterized by expression of markers
such as CD34, CD133, ckit, Sca1, and are also CD45 positive. The
abbreviation CD refers to an antigen family and means "cluster of
differentiation".
[0161] Hematopoietic stem cells (HSCs) are multipotent stem cells
that give rise to all the blood cell types including myeloid
(monocytes and macrophages, neutrophils, basophils, eosinophils,
erythrocytes, megakaryocytes/platelets, dendritic cells), and
lymphoid lineages (T-cells, B-cells, NK-cells). The hematopoietic
tissue contains cells with long-term and short-term regeneration
capacities and committed multipotent, oligopotent, and unipotent
progenitors. HSCs constitute 1:10.000 of cells in myeloid tissue.
HSCs express the following antigens: CD34, CD90 (Thy1), CD45, CD41,
CD305, CD117 (c-kit), SCF (kit ligand), Ly6A/E (sca-1), CD127,
CD44, CD33, CD38, CD14, CD106, CD84, CD90, Flk-1, CD164, Notch1,
CD338 (ABCG2), CD202b, CD184, AC133 (=CD133), and CXCR4.
[0162] Mesenchymal stem cells, or MSCs, are multipotent stem cells
that can differentiate into a variety of cell types, including:
osteoblasts (bone cells), chondrocytes (cartilage cells) and
adipocytes (fat cells). Mesenchymal stem cells are characterized by
the expression of CD45, CD90, CD105, CD34, CD31, CD29, CD106, CD44,
CD51, CD166, Ly6A/E (sca-1), CD117, CD71, CD10, CD49d, CD49e, TNAP,
PTP LAR, W5C5 antigen, W3D5 antigen, W4A5 antigen, and CXCR4.
[0163] Endothelial stem cells (or endothelial progenitor or
precursor cells) are multipotent stem cells. They are one of the
three types of stem cells to be found in bone marrow and express
the following antigens: CD45, CD31, CD34, CD105, CD146, CD106,
CD54, CD117, CD102, CD120a, CD120b, CD14, CD29, CD49d, CD49e,
CD49f, CD62P, CD62L, and CXCR4.
[0164] Neural stem cell (NSCs) are the self-renewing, multipotent
cells that generate the main phenotypes of the nervous system.
Neural progenitor and stem cells have been isolated from the
striatal tissue, including the subventricular zone--one of the
neurogenic areas--of adult mice brain tissue and from various areas
of the adult brain, including non-neurogenic areas, such as the
spinal cord, and from various species including human. NSCs express
the following antigens: CD29, CD146, Notch1, Ki67, CD24, CD49f,
Vimentin, CD81 and CXCR4. Neural Progenitor Cells express the
following antigens: 57D2 antigen, W4A5 antigen and CXCR4.
[0165] Embryonic, Spermatogonial, Testicular and Pluripotent Stem
Cells such as from iPS, SCNT, ANT-OAR express the following
antigens: CD24, CD9, Nanog, Smad, Runx2, c myc, CD30, GSC, Oct3/4,
Sox2, SSEA 1 (CD15), SSEA 4, CD324, CD29, Tra-1-60, Tra-1-81, CD338
(ABCG2), CD49f, FoxD3, Stat3, Hox11, and CXCR4.
[0166] VSELs are positive for SSEA1, Oct4, Nanog, Rex1 and other
pluripotent stem cell markers, and for CD133, CD34, AP, cMet,
LIF-R, and CXCR4. (J Am Coll Cardiol 53(1): 10-20, 2009; Stem Cell
Rev 4:89-99, 2008). Additionally, novel stem cells are routinely
being identified that are characterized by distinct markers such as
the Hox11+ stem cells found in the adult spleen (Horm Metab Res 40:
137-146, 2008). A fetal stem cell remaining in the adult spleen has
been identified that is capable of regenerating pancreatic islet
cells, however, this cell is present in the CD45 negative fraction
of the spleen (Mol Cell Proteomics 4(10): 1459-1470, 2005). The
Hox11+ splenic cells, while negative for CD45, do express OCT3/4,
SOX2, KLF4, c-MYC and NANOG, making them potentially equivalent to
embryonic stem cells and induced pluripotent stern cells (iPS) (Int
J Biochem Cell Biol. 2009 Dec. 18.
[0167] Therapeutic Uses of Stem Cells
[0168] Stem cell therapy is being investigated and perfected for
the treatment of many diseases. Conditions which could benefit from
stem cell therapy include : ocular disease, neural disease. GI
diseases, musculoskeletal disease, metabolic diseases, endocrine
diseases, vascular diseases, pulmonary diseases, cardiac diseases,
cardiovascular diseases, immune mediated diseases, auto-immune
mediated diseases, cardiovascular diseases, and all diseases for
which regenerative therapy would be of benefit. Clinical trial
information contained on the NIH website www.clinicaltrials.gov
lists over 3000 stem cell investigations. Diseases under evaluation
include: vasculitis, rheumatic disorders using endothelial
progenitor cells, therapeutic neovascularization by the
implantation of autologous mononuclear cells in patients with
connective tissue diseases, repeated administrations of granulocyte
colony stimulating factor for blood stem cells mobilization in
patients with progressive supranuclear palsy, corticobasal
degeneration and multiple system atrophy; hematological
malignancies, leukemias, lymphomas, cancers, osteopetrosis,
aplastic anemia and cytopenias, sickle cell disease and
thalassemia, limbal stem cell deficiency, breast cancer, acute
myocardial infarction (See U.S. Pat. No. 7,862,810 isolating and
culturing cardiac stem cells that are c-kit positive) coronary
artery disease (See U.S. Pat. No. 7,470,538 isolating and
administering by infusion into the coronary artery enriched
CD133.sup.+/CD34.sup.+/CXCR4.sup.- cells isolated from umbilical
cord blood), peripheral vascular disease, heart failure, type I
diabetes mellitus (See U.S. Patent Application Publication No.
2011000830, Human Adipose Derived Insulin Making Mesenchymal Stem
Cells For Treating Diabetes Mellitus), type 2 diabetes mellitus,
stroke, spinal cord injury, neuroblastoma, multiple sclerosis (See
U.S. Patent Application Publication No. 20100166712, administering
autologous mesenchymal stem cell-derived neural precursors to treat
MS), systemic sclerosis, lupus erythematosus, chronic wound
healing, burns, fracture healing, cartilage repair, CNS tumors,
osteoarthritis, renal failure, Parkinson's Disease (See U.S. Patent
Application Publication No, 20100010087. Methods for Inducing Stem
Cell Migration and Specialization with EC-18), myelomas, diabetic
foot, liver and biliary cirrhosis, dilated cardiomyopathy, anemia,
retinitis pigmentosa, Crohn's Disease, diabetic neuropathy,
mastocytosis, ovarian cancer, epilepsy, myasthenia gravis,
autoimmune diseases, granulomatous disease, osteonecrosis, liver
failure, PMD disease, lypodystrophy, demyelinating diseases,
cartilage defects, retinal disease, lupus nephritis, Alzheimer's
Disease, traumatic brain injury, sarcoma, myositis, hyperglycemia,
macular degeneration, ulcerative colitis, muscle degeneration, and
others. Limitations to these stem cell therapies include an
inability to optimally deliver and engraft stem cells, whether to a
specific injured organ or to hematopoietic centers in the bone
marrow and spleen.
[0169] Delivery of Exogenous Stem Cells
[0170] Stem cells may be delivered to a patient by many routes. For
instance, stem cells, in an appropriate excipient that optimizes
stem cell viability and eliminates cell clumping, may be
administered by intravenous, intra-arterial, intramuscular,
intradermal, subcutaneous, intraperitoneal, intrapericardial,
intraocular, transvascularly, transendocardially,
transepicardially, transeptally, epicardially, by transcoronary
vein, by percutaneous transmyocardial revascularization,
intrathecal, intra-organ, intranasal, intraventricle, or
intra-epidural via needle, catheter or other minimally invasive
method. Stem cells may also be administered by these routes in a
`matrix` mixture or suspension mixture designed to help retain stem
cells at the site of injection, for instance a collagen,
fibrinogen, fibronectin, laminin, alginate, agarose,
methylcellulose, liposomal, nanoparticle, micelle, albumin bubble,
fatty acid, or other semi-solid suspension formulation.
[0171] Catheter based delivery systems that can be used to deliver
stem cells include standard balloon angioplasty infusion catheters,
percutaneous coronary artery delivery catheters, stop flow
inflations of over-the-wire balloon catheters, Swan Ganz type
catheters, Hickman type catheters, Foley type catheters, central
venous catheters, pigtail type catheters, SmartPort.TM. systems,
metal-tipped magnet guided catheters such as the Gentle Touch
Magnetic Navigation System developed by Stereotaxis Inc or the
Mitralign, the Accucinch System, and by catheters that inject
directly into an organ such as the HELIX.TM., the MyoCath.TM., NOGA
R-guided Myostar.TM., the Stiletto.TM., or the intravascular
ultrasound (IVUS) guided TransAccess Delivery System.TM., or
catheters that deliver via arterial routes such as the
OpenSail.TM., Concerto.TM., Microsyringe infusion catheter from
Mercator, and Maverick.TM., or via implantable device therapies
such left ventricular assist devices (LVADs), biventricular assist
devices (BiVADs), the Optimizer.TM., cell-delivery catheters such
as described in US 2009/0299269.
[0172] Stem cells may also be administered to a patient using
invasive surgical means, and then injected directly into the organ
or applied to the organ. Applications to apply the stem cell
composition to an organ include collagen matrices, extracellular
matrix compositions, biopolymer microthreads made of fibrin or
other extracellular matrix material, patches containing
extracellular matrix and biodegradable materials, fibrin patches,
alginateor agarose based patches, scaffolds composed of
extracellular matrix materials and biodegradable physiologically
inert material that could include components such as dextrans,
coating stem cells with organ specific antigens or binding
molecules, remnant extracellular matrices also known as scaffolds
or decellularized organs from ex vivo digested organ donors or
cadaveric organs, and contact lenses among others.
[0173] Mobilization of Endogenous Stent Cells
[0174] Another method to treat patients with stem cells involves
mobilizing their own body's stem cells to exit organs, such as the
bone marrow, and enter the circulation. For example, therapeutics
such as granulocyte colony stimulating factor (G-CSF; Filgrastim)
which is sold as Neupogen or in longer acting forms such as
Neulasta, granulocyte-macrophage colony stimulating factor (GM-CF8;
Sargramostim) which is sold as Leukine, AMD3100 which is sold as
Mozobil/Plerixafor by Genzyme Corporation, cause stem cell numbers
within the circulation to increase. Neupogen comes in single use
vials or single use syringes containing either 300 or 480
micrograms Filgrastim. The excipient is composed of acetate,
sorbitol, polysorbate 80, sodium and water for injection. Neupogen
is used clinically as an intravenous twice daily dose, a
subcutaneous once daily-dose or a chronic subcutaneous treatment.
Neupogen is approved to accelerate recovery of neutrophil counts in
cancer patients receiving myelosuppressive chemotherapy, in
patients with Acute Myeloid Leukemia receiving induction or
consolidation chemotherapy, in cancer patients receiving bone
marrow transplant, in patients with severe chronic neutropenia, and
in patients undergoing peripheral blood progenitor cell collection
and therapy. Neupogen is typically administered on a daily basis
between 3 and 69 micrograms per kilogram body weight starting 4
days after chemotherapy with treatment lasting for 2 to 20 days.
According to the package insert for Neupogen, G-CSF regulates the
production of neutrophils within the bone marrow and affects
neutrophil progenitor proliferation, differentiation, and selected
end-cell functional activation (including enhanced phagocytic
ability, priming of the cellular metabolism associated with
respiratory burst, antibody dependent killing, 7 and the increased
expression of some functions associated with cell surface
antigens). G-CSF has also been shown to mobilize stem cells into
the circulation by: its action to reduce CXCL12 expression on the
bone marrow stroma and to reduce CXCR4 expression, by leading to a
clipping of the N terminus of CXCR4 (1), by reducing VCAM
expression in the bone marrow (2) G-CSF has been shown to increase
CXCL2, the cognate ligand for CXCR2, Since it's approval for
clinical therapy, G-CSF has also been shown to increase the number
of very small embryonic-like stem cells in the circulation.
[0175] According to the package insert, LEUKINE is indicated for
the mobilization of hematopoietic progenitor cells into peripheral
blood for collection by leukapheresis. Mobilization allows for the
collection of increased numbers of progenitor cells capable of
engraftment as compared with collection without mobilization. After
myeloablative chemotherapy, the transplantation of an increased
number of progenitor cells can lead to more rapid engraftment.
which may result in a decreased need for supportive care. Myeloid
reconstitution is further accelerated by administration of LEUKINE
following peripheral blood progenitor cell transplantation. The
recommended dose of LEUKINE is 250 micrograms permeter square body
surface area per day, administered as a 24 hour intravenous
infusion or once daily subcutaneously. The optimal treatment time
with LEUKINE is apparently 5 days for stem cell mobilization into
the circulation. Leukine has also been shown to be effective in
combination with G-CSF in patients who were poor mobilizers in
response to G-CSF alone.
[0176] Mobozil has the chemical name 1, 1'-[1,4-phenylenebis
(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane. It has the
molecular formula C28H54N8. Mobozil is an inhibitor of the
chemokine receptor CXCR4, and blocks binding of its cognate ligand
SDF-1 (CXCL12). Mobozil causes circulating stem cell numbers to
increase by disrupting the binding of stem cell expressed CXCR4 to
SDF-1 (CXCL12) expressed by the stromal and other cells of the bone
marrow. Optimal mobilization after Mobozil treatment relies on
complement activation. Subcutaneous injection of Mobozil is
approved for use in combination with Neupogen to mobilize
hematopoietic stem cells to the peripheral blood for collection and
subsequent autologous transplantation in patients with non-Hodgkin
lymphoma (NHL) and multiple myeloma (MM), Mozobil is sold as a
single-use vial containing 1.2 mL of a 20 mg/mL solution. Patients
are treated with Mozobil according to the following recommended
schedule as detailed in the package insert: initiate Mozobil
treatment after the patient has received G-CSF once daily for 4
days; repeat Mozobil dose up to 4 consecutive days; select dose
based on 0.24 mg/kg actual body weight; administer by subcutaneous
injection approximately 11 hours prior to initiation of apheresis.
The combination of G-CSF and Mobozil has been shown to mobilize
more primitive stem cells into the circulation than the use of
G-CSF alone.
[0177] Other agents known to mobilize stem cells, including
hematopoietic stem cells, into the cirulcation include hepatocytre
growth factor (HGF) erythropoietin, parathyroid hormone,
Flt3-ligand, stem cell factor (SCF). Other agents known to or that
would be expected to result in increased mobilization of stem and
progenitor cells into the circulation include; agents that increase
stem cell proliferation such as colony stimulating factors, agents
that increase endogenous G-CSF production such as Maitake
beta-glucan, agents that reduce the expression of SDF-l or CXCR4
including CXCR4 down-regulating agonists, agents that reduce the
binding affinity of SDF-1 or CXCR4, agents that attenuate the
signaling of CXCR4, agents that block bioaccumulation of stem cells
away from the circulation, agents that enhance the egress of stem
cells into the circulation such as activation of complement or
increasing plasma sphingosine-1 -phosphate, agents that upregulate
the expression of CXCR2 in the bone marrow or its cognate ligand
CXCL2, agents that reduce VCAM expression in the bone marrow such
as the chemotherapeutic cyclophosphamide, retinoic receptor
agonists, small molecule inhibitors of VLA-4 such as BIOS 192 or
other blockers of VLA4, metalloproteinase or carboxypeptidase
activators that would degrade bone marrow expressed CXCL12, or
selected chemotherapy regimens, or regimens adding cyclophosphamide
or the topoisomerase inhibitor etoposide to G-CSF treatment,
ingestion of fucoidan, by the chemokine CXCL2, and colominic acid,
among others.
[0178] Spontaneously Released Endogenous Stem Cells
[0179] Another method to treat patients with stem cells involves
preventing spontaneously released endogenous stem cells from
sequestering in the lymphatic tissues. Stem and progenitor cells
are spontaneously released into the blood stream on a daily basis.
Experiments using parabiotic mice have demonstrated that the spleen
readily exchanges stem and progenitor cells with the circulation.
Additionally, disease states have been demonstrated to lead to
increased levels of circulating stem and progenitor cells, for
instance, hypercholesterolemia, heart attack, STEMI or CAD,
arterial ligation or transient ischemia, sojourn at moderate
altitude , primary hyperparathyroidism , and retinal pigment
epithelium damage, among others. Preventing spontaneously released
or disease-induced stem cells from sequestering in lymphatic
tissues will make more stem cells available for regeneration of
damaged tissues or organs.
[0180] Compositions and Methods for Regenerating Germinal Centers
in Lymphatic Tissues
[0181] The present invention fills this need by providing for
methods and compositions for regenerating, rejuvenating and
increasing the number of germinal centers in lymphatic tissues
after radiation or chemotherapy. Therapeutics that Rejuvenate or
Regenerate the Germinal Centers in Lymphatic Tissues according to
the present invention include immune activators, co-stimulatory
molecules, immune adjuvants and combinations thereof.
[0182] In one embodiment of the present invention germinal centers
in lymphatic tissues are regenerated by the administration of
adjuvants. Examples of such adjuvants include pathogen-associated
molecular patterns, liposome, lipopolysaccharides, molecular cages
for antigen, components of bacterial cell wails, endocytosed
nucleic acids such as double-stranded RNA (dsRNA). single-stranded
DNA (ssDNA), and unmethylated CpG dinucleotide-containing DNA,
mineral salts such aluminum hydroxide (alum), aluminum phosphate,
calcium phosphate, aluminum hydroxide, aluminum potassium
phosphate, aluminum sodium hydrogen phosphate, and aluminum
hydroxyphosphate sulfate. Other adjuvants include oil-in-water
emulsions such as squalene, montanide ISA720 (squalene) or ISA 51
(Drakeol), MF59 (Novartis) and SBAS2. Another class of adjuvants
which can be used according to the present invention is particulate
adjuvants such as virosomes, saponins and lipids including
microbial derivatives such as monophosphoryl lipid A CpG motifs,
BCG-primed immunity called BCG-CWS (Mycobacterium bovis).
[0183] Lipopolysaccharides and mitogens such as Concanavalin A.,
components of bacterial cell walls, archaeosomes (ether
glycerolipids of the archaeon Methanobrevibacter smithii), the TLR4
agonist GLA glucopyranosyl lipid adjuvant, LPS and BCG (Immune
Design) the TLR2 agonists BCG, peptidoglycan, and gram positive
bacteria, the TLR5 agonist flagellin, schistosome egg antigens
(SEAs), listeria monocytogenes (LM). Other adjuvants include
Toll-like receptor agonists and activators including CpG
oligonucleotides of lengths up to 100 bases, most preferably of
lengths of 20 bases, TLR1 agonists such as Pam3Cys, TLR2 agonists
such as Pam3Cys; TLR3 agonists such as dsRNA and poly PC, TLR7
agonists such as imidazolequinolenes for example Imiquimod (R-839)
and Resiquimod (R-848), TLR8 agonists such as Resiquimod (R-848);
and TLR9 agonists such as poly I:C and CpG. Also included within
the present invention are the use of plant derivative adjuvants,
beta-glucan, saponin based QS21, and concanavalin A.
[0184] Another embodiment of the present invention is comprised of
methods to augment the numbers of active germinal centers in the
spleen to enhance stem cell transplant engraftment and
hematological recovery in patients undergoing cancer therapy,
non-myeloablative therapy or myeloablative therapy, including
chemotherapy, radiation, and combination treatments. Increased
numbers of active germinal centers leads to enhanced stem cell
binding and engraftment in the spleen and subsequently accelerated
rates of hematopoietic recovery following chemotherapy conditioning
regimens and cancer treatments. In addition to treatment of cancer
and leukemias, non-myeloablative therapy is used for treatment of
autoimmune diseases (Pediatr Clin North Am. 57(1):239-71, 2010)
including type I diabetes (JAMA. 297(14):1568-1576, 2007), lupus
and multiple sclerosis (www.clinicaltrials.gov). The present
invention provides methods to enhance hematopoietic recovery in
cancer or autoimmune patients undergoing myeloablative or
non-myeloablative conditioning by increasing the numbers of splenic
germinal centers and thereby augmenting transplanted stem cell
binding, engraftment and proliferation. For example, patients
undergoing stem cell treatment following myeloablative or
non-myeloablative conditioning regimens are at risk of infection
and death during the time required for administered stem cell
engraftment and hematopoietic regeneration. Acceleration and
augmentation of stem cell engraftment by increasing the numbers of
germinal centers available for stem cell binding in the spleen can
reduce the time required for hematopoietic recovery and thereby
diminish risk of infection and death in these patients.
[0185] Stem cells may be autologous or from an unrelated donor.
Stem cells may be contained within the mononuclear cell fraction
from bone marrow, whole blood, umbilical cord blood, adipose tissue
or other sources, or they may be purified by selection for CD34,
CD133, CD 105, CD117, SSEA1-4, dye exclusion or other specific stem
cell antigens.
[0186] Splenic germinal centers can be specifically increased by
treatments which activate the CD40 receptor (Blood. 104:4088-4096,
2004; J. Clin. Invest. 112:1506-1520 2003) (50) (51). The
functional receptor is a CD40 trimer or multimer with TNFR1 or
TNFR2 components. Examples of treatments that activate the CD40
receptor include: Agonistic antibodies to CD40 activate the CD40
receptor, appropriate conformations of solCD40L may activate the
CD40 receptor, and agents which increase the expression of CD40
receptor by altering transcription rates such as via AT-hook
transcription factor AKNA, mRNA stability or protein stability can
also lead to increased activity and signaling. Alternatively,
members of the TRAF and TTRAP families interact with CD40 receptor
and mediate its signaling, leading to enhanced active germinal
center slumbers. Agents which activate germinal center B cell
cyclo-oxygenase 2 or the EP2 receptor replicate CD40 receptor
engagement and can lead to enhanced active germinal center
formation. Other means to activate germinal center formation and
persistence include inhibition or loss of CCR7 (J. Leukoc. Biol.
85: 409-417, 2009). Other means to activate germinal center
formation and persistence include inhibition or loss of CCR7 (J.
Leukoc. Biol. 85: 409-417, 2009).
[0187] In another embodiment of the present invention is comprised
of administering immunostimulatory molecules to promote
regeneration of the germinal centers in lymphatic tissue.
Immunostimulatory molecules may be antibodies, fusion proteins,
soluble ligands, small molecules, transcription regulators, mRNA or
protein stabilizers, and other immunostimulatory moieties. For
instance, the co-stimulatory CD28 pathway can be activated by
soluble B7 proteins and an antibody such as with TeGenero 1412
compound.
[0188] TGN1412 is a humanized monoclonal antibody designed as an
agonist of the CD28 receptor on T lymphocytes, which stimulates the
production and activation of T lymphocytes. Boerhinger Ingelheim
manufactured the TGN1412.
[0189] Additional co-stimulatory molecules and pathways include
OX40/OX40 ligand, 4-1BB/4-1BB ligand, the B7/CD28 family;
B7-1/CD80, CD28, B7-2/CD86, CTLA-4, B7-H1/PD-L1, ICOS, B7-H2, PD-1,
B7-H3, PD-L2/B7-DC, B7-H4, PDCD6, BTLA, the Co-stimulatory TNF
Superfamily Molecules; 4-1BB/TNFRSF9/CD137, 4-1BB Ligand/TNFSF9,
BAFF/BLyS/TNFSF13B, BAFF R/TNFRSF13C, CD27/TNFRSF7, CD27
Ligand/TNFSF7, CD30/TNFRSF8, CD30 Ligand/TNFSF8, CD40/TNFRSF5, CD40
Ligand/TNFSF5, GITR/TNFRSF18, GITR Ligand/TNFSF18, HVEM/TNFRSF14,
LIGHT/TNFSF14, OX40/TNFRSF4, OX40 Ligand/TNFSF4, and
TACI/TNFRSF13B, the SLAM Family; 2B4/CD244/SLAMF4, BLAME/SLAMF8,
CD2, CD2F-10/SLAMF9, CD48/SLAMF2, CD58/LFA-3, CD84/SLAMF5,
CD229/SLAMF3, CRACC/SLAMF7, NTB-A/SLAMF6, and SLAM/CD 150, and
other Co-stimulatory Molecules; CD2, CD53, CD82/Kai-1, CD90/Thy1,
CD96, CD160, Ikaros, Integrin alpha 4/CD49d, Integrin alpha 4 beta
1, Integrin alpha 4 beta 7/LPAM-1, LAG-3, LMIR1/CD300A, CRTAM,
DAP12, Dectin-1/CLEC7A, DPPIV/CD26, EphB6, TCL1A,
TIM-1/KIM-1/HAVCR, TIM-4, TSLP, TSLP R, HLA-DR, and ephrins.
[0190] Other agents that may enhance germinal center regeneration
are agents known to cause cytokine burst such as the Anti-CD20
(Rituximab).
[0191] Other immune-activating agents may include agonists to the
IL21 receptor, with agonistic antibodies.
[0192] Other adjuvants used preclinically or clinically
include:
[0193] Endocytosed nucleic acids such as double-stranded RNA
(dsRNA), single-stranded DNA (ssDNA), and unmethylated CpG
dinucleotide-containing DNA.
[0194] Adenovirus and adenovirus components such as adenovirus type
5 (Ad5).
[0195] Agonists and activators of retinoic acid-inducible gene
(RIG)-1-like receptors (RLR).
[0196] P2X1, P2X4, or P2X7 activators or agonists.
[0197] Agonists and activators of nucleotide-binding
oligomerization domain-like receptors (NLR).
[0198] Advax, liposomes, chitosan microspheres and
dimethyl-dioctyldecyl ammonium bromide (DDA).
[0199] Most newer human adjuvants in development are ISCOMS, QS21,
AS02, and AS04.
[0200] ASO.sub.4 is deacylated monophosphoryl lipid A MPL plus
aluminum.
[0201] CpG oligonucleotides can be of lengths up to 300 bases, most
preferably of lengths of 20 bases.
[0202] Anti-CD3 OKT3 can also be administered intravenously (i.v.)
to induce regeneration of germinal cells within lymphatic
tissues.
[0203] The present invention is also comprised of administering two
or more therapeutic agents to generate germinal centers within
lymphatic tissues. In another embodiment of the present invention,
stem cells are administered in conjunction with the therapeutic
agent that regenerates the germinal centers.
Dosing of Adjuvants
[0204] Aluminum potassium phosphate can be administered at a dose
of about 0.17 mgs.
[0205] Aluminum phosphate can be administered at a dose of about
1.5 mgs.
[0206] Aluminum hydroxide can be administered at a dose of about
0.15 mgs to about 0.3 mgs. Aluminum salts in general can be
administered at doses as high as 0.85 mgs.
[0207] Aluminum hydroxyphosphate can be administered at a dose of
about 0.225 mgs.
[0208] As a combination Aluminum hydroxide and Aluminum phosphate
can be administered at a combined dose of about 0.45 mgs.
Dosing of Oil Emulsions
[0209] SBAS-2 is an oil-in-water emulsion of MPL and QS21.
[0210] Oil-in-water emulsions such as montanide ISA720 (squalene)
or ISA 51 (Drakeol).
[0211] Squalene is in MF59 adjuvant used by Novartis and dosed at
1.95% or 2 parts per hundred or at 4 grams per 100 mls for an 0.5
to 1 ml injection for adjuvant activity,
[0212] MF59 adjuvant contains (Lipovant; 4-5% w/v squalene, 0.5%
w/v Tween 80, 0.5% Span 85, and optionally, varying amounts of
muramyl tripeptide phosphatidyl-ethanolamine (MTP-PE), which
activates non-TLR sensing receptors known as NOD-LRRs).
Dosing of Microbial Derivatives
[0213] BCG-primed immunity called BCG-CWS (Mycobacterium bovis) 1
to 8.times.10.sup.8 colony forming units (CFU) per vial for adults,
half the dosage for children.
[0214] Archaeosomes (ether glycerolipids of the archaeon
Methanobrevibacter smithii) dosed at 0.1 to 500 micrograms per gram
body weight and most preferably at 38 micrograms per mg body
weight.
[0215] Dosing of Toll-like Receptor Agonists and Activators
[0216] CpG oligonucleotides of lengths up to 100 bases, most
preferably of lengths of 20 bases, dosed at 1, 10, 100, 500
micrograms per 20-25grams body weight. The preferred dose is 10
.mu.g per 20-25 mgs of body-weight.
[0217] The co-stimulatory CD28 pathway can be activated by soluble
B7 proteins and an antibody such as with TeGenero 1412 compound.
TGN1412 is a humanized monoclonal antibody designed as an agonist
of the CD28 receptor on T lymphocytes, which stimulates the
production and activation of T lymphocytes. Boerhinger Ingelheim
manufactured the TGN1412, For the purposes of immune activation to
regenerate lymphatic germinal centers a preferred dose of TGN1412
would be less than 0.1 mg/kg body weight given over a 3-6 minute
infusion. Effective doses of TGN1412 for germinal center
regeneration would be between 0.001 mgs/kg to 0.1 mg/kg, preferably
0.01 mgs/kg.
[0218] Other agents that may enhance germinal center regeneration
are agents known to cause cytokine burst such as the anti-CD20
(Rituximab) dosed at 50 mg/mm2, or 150 mg/mm2 but below 375 mg/mm2;
the Anti-CD3 OKT3 administered intravenously (i.v.) at a preferred
dose of less than 5 mg/day for 10 to 14 days; the anti-CD52
(CAMPATH) antibodies administered at 30 mgs over a 2 hour infusion
given three times per week for up to 12 weeks.
[0219] Additional immune-activating agents may include agonists to
the IL21 receptor, with agonistic antibodies dosed between 0.001
mgs/kg to 50 mgs/kg, preferably between 0.01 and 0.1 mgs/kg.
Protein ligand agonists may be dosed daily between 0.0001 mgs/kg up
to 50 mgs/kg for up to 28 days after myeloablative or
non-myeloablative treatments. Protein ligand agonists will in
general be useful at 1/10.sup.th the dose of an antibody
therapeutic depending on the molecular weight and biodistribution
of the agonist.
[0220] Small molecule immune-activators may be delivered orally
between 1 mg to 1000 mgs daily, in a single oral dose, or at
specific intervals that may include every 2 hours, every 4-6 hours
or longer interval periods.
[0221] ASO.sub.4 is deacylated monophosphoryl lipd A MPL plus
aluminum dosed at 50 micrograms in 0.5 ml dose of Fendrix (GSK) in
combination with 0.5 mgs aluminum phosphate.
[0222] CpG oligonucleotides of lengths up to 100 bases, most
preferably of lengths of 20 bases, dosed at 1, 10, 100, 500
micrograms per 20-25 grams body weight plus alum. The preferred
dose is 10 .mu.g per 20-25 mgs body weight.
[0223] Administration
[0224] Administration of the therapeutic agents that induce
regeneration of germinal centers within lymphatic tissues can be by
any method including intravenous, intra-arterial, oral,
intramuscular, aerosolized, inhalable, intradermal, subcutaneous,
intraperitoneal, intrapericardial, intraocular, transvascularly,
transendocardially, transepicardially, transeptally, epicardially,
by transcoronary vein, by percutaneous transmyocardial
revascularization, intrathecal, intra-organ, intranasal,
intraventricle, or intra-epidural via needle, catheter or other
minimally invasive method.
[0225] Examples below illustrate experimental outcomes of methods
and compositions to regulate or moderate the number of binding
sites available to participate in stem cell binding.
Example 1
[0226] Stem Cell Enriched Bone Marrow and Whole Blood Mononuclear
Fractions Bind to B Cell Regions at the Edges of the White Pulp of
the Spleen when Administered to an Allogeniec Mouse
[0227] Bone marrow and whole blood mononuclear cell fractions from
a male 129S1/SvlmJ mouse were isolated using Histopaque and
combined. The cells were incubated at 37.degree. C., 5% CO2 for 4
days to allow differentiated somatic cells to die off, thereby
concentrating the stem cell fraction among the mononuclear cells.
The resulting cells were then labeled with cell tracker orange (CTO
Invitrogen) according to the manufacturer's instructions.
Approximately 10 million labeled cells were administered by
retro-orbital injection to a recipient litter mate, and 90 minutes
later the mouse was exsanguinated and blood collected, the
vasculature flushed of residual red blood cells, and the spleen
harvested. The spleen was fixed overnight in 1% PFA and then
embedded in low melting, low gelling temperature agarose and
sectioned at 200 microns thickness per section. MNC stem cell
binding to the spleen was visualized using immunoflourescence. The
labeled MNC containing stem cell fraction bound to the B cell
regions at the edges of the white pulp of the spleen.
Immunoflourescent examination of the MNC fraction from the whole
blood collected during exsanguinations demonstrated that
approximately 40,000 of the 10 million injected labeled cells
continued to be found in the circulation 90 minutes after
injection.
[0228] Results and Conclusions: CTO-labeled stem cell-enriched MNC
cells bound to the periphery of the white pulp region. Binding of
the cells is histologically evident on the B cell regions. This
shows that stem cell enriched bone marrow and whole blood
mononuclear fractions bind to B cell regions at the edges of the
white pulp of the spleen when administered to an allogeneic
mouse.
Example 2
CD34+, CD105+ and CD117+ Purified Stem Cells Bind to the Same
Splenic Region as the Labeled MNC Stem Cell Containing
Fractions.
[0229] Bone marrow and whole blood mononuclear cell fractions from
a male mouse were isolated using Histopaque and combined. MNC cells
labeled with cell tracker green (CTG, Invitrogen) were then
incubated with biotin-labeled antibodies to CD34, ckit and CD 105
and purified using Miltenyi magnetic separation columns according
to the manufacturer's recommendations. A portion of the isolated
MNC were labeled separately with CTO. One million CTO-labeled MNC
were co-incubated with 205,000 CTG labeled purified stem cells on
100-200 micron thick fresh spleen sections for 12-18 hours at
4.degree. C. The spleen sections were thoroughly washed to remove
unbound cells, fixed for one hour in 1% PFA and then wet mounted
for fluorescent imaging. MetaMorph software was used to capture and
overlay the resulting red (MNC) and green (stem cell) binding.
[0230] Results and Conclusions: The purified stem cell population
bound to the same splenic region as the MNC fractions. This
indicates that CD34+ CD105+ and CD117+ purified stem cells bind to
the same splenic region as the labeled MNC stem cell containing
fractions.
Example 3
Bone Marrow and Whole Blood Mononuclear Fraction Stem Cells Bind to
the PNA Positive Areas in the Germinal Centers of the Spleen
[0231] Bone marrow and whole blood mononuclear cells (MNC) are
isolated from adult mice using Histopaque. The resulting
mononuclear cells are stained with cell tracking dyes such as
CellTracker Orange, Green or Blue, Dil, or Calcein Orange or Blue
according to manufacturer's recommendations. FITC-labeled PNA (10
ugs), IgD (10 .mu.gs) or anti-CD21 (10 .mu.gs) are used to identify
specific B cell regions in the white pulp of the spleen. PNA labels
germinal centers, IgD labels follicular zones and anti-CD21 labels
marginal and mantle zones. FITC-labeled anti-CD3 (200 ng to 1
.mu.g) is used to identify T cell regions in the white pulp of the
spleen. After thorough washing the bound cells and antibodies are
fixed on the spleen sections using 1% PFA for 1 hour at 4.degree.
C. Wet mount sections are viewed for fluorescence and pictures
taken using MetaMorph software.
[0232] Results and Conclusions: The CTO-labeled MNC bound after a
15 hour 4.degree. C. incubation to active PNA+ germinal centers. No
CTO-labeled MNC binding was seen colocalized with IgD or anti-CD21.
This indicates that bone marrow and whole blood mononuclear
fraction stem cells bind to the PNA positive IgD negative CD21
negative areas in the germinal centers of the spleen.
Example 4
[0233] CD34+, CD105+, CD117+ Stem Cells Isolated from Whole Blood
and Bone Marrow Mononuclear Cell Fractions Bind to PNA+ Germinal
Centers of the White Pulp of the Spleen.
[0234] Mononuclear cell fractions from mouse whole blood and bone
marrow were combined, labeled with CTO, and then incubated with
biotinylated anti-CD34, anti-CD117 and anti-CD 105 and then
antibody bound cells were isolated using Miltenyi Magnetic Cell
Separation columns according to the manufacturer's instructions.
Numbers of CD34+ CD105+ CD117+ stem cells recovered ranged from
0.3% to 3% of the starting MNC fraction.
[0235] The resulting CD34+ CD 105+ CD117+ cells were co-incubated
for 15 hours with 10 .mu.gs PNA on fresh mouse spleen sections.
Positively selected cells were added at 100,000 cells per spleen
section (A), 50,000 (B), 25,000 (C), or 10.000 (D) cells per spleen
section. Similar to the MNC incubations, purified stem cells bound
to the PNA positive germinal centers of the white pulp of the
spleen. Stem cells bound in a concentration-dependent manner. The
cells bind to discrete niches in the germinal centers, and
quantities of added cells greater than approximately 100,000 result
in stronger binding signal, rather than expansion to additional
niches (see example 2).
[0236] Results and Conclusions--These results indicate that CD34+,
CD105+, CD117+ stem cells isolated from whole blood and bone marrow
mononuclear cell fractions bind to PNA+ germinal centers of the
white pulp of the spleen.
Example 5
The Binding of Stem Cells in the MNC Fraction is Blocked by
Anti-CD45 Antibody.
[0237] The binding of MNC and stem cells to spleen sections is
blocked by a rat monoclonal IgG2b anti-mouse anti-CD45 (800
nanograms to 4 micrograms), but not by anti-CD45R (10 micrograms)
or anti-CD3 antibodies (1 microgram). The 30-F11 rat anti-mouse
anti-CD45 antibody (Santa Cruz Biotechnology) or the I7A2 anti-CD3
antibody (Santa Cruz Biotechnology) was diluted 1:50 or 1:10 and
co-incubated with 250,000 CTO-labeled MNC for one hour. MNC binding
was counted visually as the number of binding niches and the size
of the niches. CTO-MNC control fresh spleen sections had between
3-6 MNC binding niches of medium to large size per section.
anti-CD3 antibody did not impact either the number or the size of
MNC binding niches. anti-CD45 30-F11 antibody at a 1:50 dilution
reduced both the number and the size of MNC binding niches by half.
anti-CD45 30-F11 antibody at a 1:10 dilution completely abolished
CTO-MNC binding to fresh spleen sections.
[0238] In another experiment, labeled MNC were incubated on spleen
sections for one hour in the presence of 30-F11 anti-CD45 (4
micrograms) or PC3/188A anti-CD3 (1 microgram). (Santa Cruz
Biotechnology).
[0239] In another experiment, cell tracker orange (CTO) labeled MNC
were incubated for 15 hours at 4.degree. C. with antibodies to
CD45R or 30-F11 anti-CD45. Antibody to CD45R (Miltenyi Biotec)
diluted 1:10 (5 .mu.gs) did not block CTO-MNC binding to fresh
mouse spleen sections. In contrast, 30-F11 anti-CD45 antibody
(Santa Cruz) at 3:10 (4 .mu.gs) reduced CTO-labeled MNC binding to
fresh spleen sections. CD45R binds follicular zone B cells but not
active germinal centers.
[0240] A 3:10 co-incubation with 30F-11 anti-CD45 reduced
CTO-labeled MNC binding to flesh spleen section by approximately
75%.
[0241] Results and Conclusions: These data indicate that the
binding of stem cells in the MNC fraction to the spleen is blocked
by anti-CD45 antibody.
Example 6
CD45 Epitope Bound by 30-F11 Rat IgG2b Anti-Mouse Anti-CD45
Antibody, 30-F11 Binds All Isoforms of Mouse CD45.
[0242] The exact binding of 30-F11 epitope has never been mapped.
30-F11 was generated by immunization with mouse spleen and thymus
cells. The extracellular domain of mouse CD45 isoform 1 is
comprised of amino acids 24 to 564. Isoform 2 is missing amino
acids 31 to 73, while isoform 3 is missing amino acids 31 to 169.
As 30-F 11 is reported to bind ail isoforms of mouse CD45, the
binding epitope therefore should be between amino acids 170 to 564
in isoform 1. Antigenic regions of proteins can be predicted using
hydrophobicity (Kyte Doolittle) and accessibility algorithms found
on the SwisProt web site. Antigenic regions are most likely to be
found in areas of both low hydrophobicity arid high accessibility.
Amino acid residues near 501 through 521 in the human sequence have
a low hydrophobicity prediction indicating this area of the protein
as a potential antigenic site. This region is also fairly well
conserved from mouse to human. (See Okumura M., et al. 1996 Aug 15;
157(4):1569-75).
Example 7
Immune Adjuvants Enhance Germinal Center Formation and Increase
Mononuclear Stem Cell Fraction Binding to the Spleen
[0243] Active germinal centers were elicited in normal mice by
purposeful immunization using Incomplete Freund's Adjuvant or Ribi.
Mice were intraperitoneally (IP) injected with 0.5 mls Incomplete
Freund's Adjuvant (FIA) mixed 1:1 with PBS or with 0.5 mls RIBI
adjuvant on day 0. On day 7 or day 14 after immunization, the mice
were heparinized with 100 U intraperitoneal heparin for 30 minutes
prior to avertin anesthesia. The mice were exsanguinated by
retroorbital eye bleed, obtaining a total of 1.5 to 1.8 mls whole
blood added to 200 .mu.l 5 U/ml heparin in a 15 ml conical tube.
Then, the abdominal aorta and vena cava were cut and the remaining
blood flushed completely out of the vasculature by slow push
infusion of 10 mls 5 U/mL heparin via the ascending thoracic vena
cava. The spleen was removed and dropped into growth media and
subsequently embedded in soft agarose and sectioned to obtain 200
micron thick uniform sections. The sternal and femur bone marrow
were flushed from the bones with Hanks Buffered Salt Solution, and
mononuclear stem cell fractions for both the whole blood and the
marrow were isolated using Histopaque and combined, FITC-labeled
PNA (10 .mu.gs) was used to identify germinal centers on the spleen
section by incubation at 4.degree. C. overnight. After the
overnight incubation with PNA, without washing, CTO-labeled
mononuclear stem cell fractions were added to the sections for one
hour at 4.degree. C. After thorough washing the bound cells and
labeled-PNA are fixed on the spleen sections using 1% PFA for 1
hour at 4.degree. C. Wet mount sections are viewed for fluorescence
and pictures taken using MetaMorph software.
[0244] PNA binding 7 days after immunization was similar in FIA and
RIBI treated mice, both almost double the binding of controls.
However, the FIA treated mice were healthier than the RIBI treated
mice so all subsequent experiments were conducted with FIA.
Comparing day 7 and day 14 after FIA treatment, PNA brightness was
higher on day 7, however, the day 14 active germinal centers
appeared to be more tightly organized and compact. Compared to
control, FIA immunization doubles the number of active germinal
centers in the spleens of the mice. Addition of CTO-labeled
mononuclear stem cell fractions, isolated from control mice, to the
spleens demonstrated that the enhanced active germinal center
formation observed in the FIA treated mice resulted in
significantly greater mononuclear stem cell binding to the spleens
as well. Mononuclear stem cell binding on day 7 was increased
approximately 3-5 fold over binding to control spleens, while
mononuclear stem cell fraction binding was increased up to and in
some cases beyond 10 fold higher than binding to control
spleens.
[0245] Results and Conclusions: These data indicate that Immune
Adjuvants Enhance Germinal Center Formation and Increase
Mononuclear Stem Cell Fraction Binding to the Spleen.
Example 8
Inhibition of Active Germinal Center Formation Reduces Ex Vivo Stem
Cell Binding to the Spleen
[0246] Mice were treated with 1 mg dexamethasone solubilized in
ethanol (1 part) and PBS (9 parts) by intraperitoneal injection 7
days prior to harvest of their spleen and stem cells for ex vivo
analysis of stem cell binding to splenic sections. Control mice
were treated with ethanol (1 part) and PBS (9 parts) only, in a
total volume of 1 ml. On day 7 the mice were harvested and
processed as detailed in Example 7. Mononuclear stem cell fractions
from the control mice were used for binding studies on both control
and dexamethasone treated spleen sections. Mononuclear stem cell
binding on day 7 was reduced by 30-40% following a single 1 mg
treatment with dexamethasone given 7 days prior to
experimentation.
[0247] A single 1 mg dexamethasone treatment 7 days prior to
harvest reduced spleen weights by an average of 22%, reduced
average circulating MNC number by 34%, and reduced PNA labeled
germinal centers by up to 24%, however, the percentage of stem
cells within the MNC fractions from whole blood or bone marrow were
not reduced and in fact increased by an average of 32% compared to
control.
[0248] Results and Conclusions: These data indicate that
immunosuppressants reduce the number of mononuclear cell fraction
binding cells in the spleens of treated mice.
Example 9
Inhibition of Active Germinal Center Formation Reduces In Vivo Stem
Cell Binding to the Spleen.
[0249] A naive control mouse was heparinized with 100 U
intraperitoneal heparin for 30 minutes prior to avertin anesthesia.
The mouse was exsanguinated by retroorbital eye bleed, obtaining a
total of 1.5 to 1.8 mls whole blood added to 200 .mu.l 5 U/ml
heparin in a 15 ml conical tube. Then, the abdominal aorta and vena
cava were cut and the remaining blood flushed completely out of the
vasculature by slow push infusion of 10 mls 5 U/mL heparin via the
ascending thoracic vena cava. The sternal and femur bone marrow
were flushed from the bones with Hanks Buffered Salt Solution, and
mononuclear stem cell fractions for both the whole blood and the
marrow were isolated using Histopaque and combined. The mononuclear
stem cell fractions were resuspended in growth media (DMEM plus 10%
FBS) and incubated at 37 deg C. 5% CO2 overnight. The next morning
the mononuclear stem cells were labeled with cell tracker green
(CTG) according to the manufacturer's instruction.
[0250] Immediately after staining the naive control mononuclear
stem cells (MNC) with CTG, recipient mice were each injected with
approximately 6 M CTG MNC in 100 uls by retroorbital sinus on day
7. Five control mice had received intraperitoneal injections of 100
uls ethanol and 900 uls PBS on day 0 and day 4; 3 mice had been
treated ip with 1 mg dexamethasone on day 0 and day 4; and two mice
had been treated ip with 1 mg dexamethasone on day 0, day 2 and day
5. One hour later the mice were exsanguinated by retroorbital eye
bleed. Then, the abdominal aorta and vena cava were cut and the
remaining blood flushed completely out of the vasculature by slow
push infusion of 10 mls 5 U/mL heparin via the ascending thoracic
vena cava. The spleens were taken and kept on ice in RPMI without
phenol red plus 1% BSA. The sternal and femur bone marrow were
flushed from the bones with Hanks Buffered Salt Solution, and
mononuclear stem cell fractions for both the whole blood and the
marrow were isolated using Histopaque.
[0251] Spleens were dissociated into single cell suspensions and
the degree of injected MNC CTG sequestration in the spleen was
determined using flow cytometry. Dexamethasone (total 2 mgs)
reduced MNC CTG total cell number accumulation in the spleen by
33-43%. while 3 mgs total dexamethasone dose reduced MNC CTG total
cell number accumulation in the spleen by approximately 70%.
[0252] Results and Conclusions: These data indicate that 7 day
immunosuppressant treatment reduces the numbers of exogenously
injected MNC stem cells that sequester in the spleen.
Example 10
Inhibition of Active Germinal Center Formation Reduces Stem Cell
Binding to the Spleen.
(Prophetic)
[0253] Human volunteers are treated prophylactically with
commercially available general immune suppressants such as
prednisone according to established clinical protocols, using doses
chosen to limit or completely avoid ail adverse effects of the
agents. Another human volunteer group is treated with antagonistic
antibodies to CD40 such as HCD-122 anti-CD40 mAb at doses between 5
and 100 mg/kg.
[0254] Prior to or on the final day of prophylactic immune
suppression autologous stem cells are isolated in the mononuclear
cell fraction from 50 mls ileac crest bone marrow or from whole
blood apharesis products. The resulting MNC fraction containing the
stem cells is labeled with 2-[18F]-fluoro-2-deoxy-D-glucose
(18F-FDG) for subsequent 3D -PET imaging or with an appropriate
nuclear imaging label for subsequent SPECT imaging. Labeled MNC
stem cell containing fractions, with between 2 and 10 million stem
cells, are injected intravenously and their biodistribution
determined by PET or SPECT imaging 60-90 minutes and up to 48 hours
after administration. Stem cells among the MNC accumulate
predominantly in the spleens of normal volunteers within 90 minutes
after injection. In contrast, immune suppressed and HCD-122 treated
volunteers have reduced splenic accumulation of MNC stem cell
fractions.
Example 11
Inhibition or Reduction of Active Germinal Center Formation
Enhances Stem Cell Delivery to the Heart and Promotes Functional
Recovery in Mice.
[0255] (Prophetic)
[0256] 3-month- and 12-month-old PN and 129S1/SvlmJ mice are
injected intravenously (i.v.; 250 mg/injection on days 0, 2, and 4)
with anti-CD40L mAb (PharMingen) or control hamster Ig (Pierce,
Rockford, Ill.). Whole blood and bone marrow mononuclear fraction
stem cells are collected from naive litter mate mice, labeled with
cell tracker dyes such as CTO, and then the stem cells are purified
using biotinylated anti-CD34, anti-CD105, anti-SSEA1 and anti-CD117
antibodies with Miltenyi magnetic separation columns. Experimental
mice are injected retroorbitally or intravenously with between
100,000 and 1M purified stem cells on day 5. 15to 24 hours later
the mice are exsanguinated and the blood collected, the vasculature
is rinsed of residual red blood cells and the spleens are collected
for fluorescent imaging of stem cell accumulation. Stem cell
accumulation is evident in active PNA+ germinal centers with
control IG treated PN mice demonstrating significantly higher
numbers of active germinal centers and consequent elevated stem
cell binding than 129S1 mice. anti-CD40L mAb treated 129S1 mice
have few if any active germinal centers evident and insignificant
to no stem cell binding. anti-CD40L mAb treated PN mice show
reduced numbers of active germinal centers compared to control Ig
treated PN mice, and consequently, parallel reductions in stem cell
binding.
[0257] To study cardiac regeneration in these mice, 3-month- and
12-month-old PN and 129S1/SvlmJ mice are injected intravenously
(i.v.; 250 mg/injection on days 0, 2, and 4) with anti-CD40L mAb
(PharMingen) or control hamster Ig (Pierce, Rockford, Ill.). On day
4 the mice are anesthetized, echocardiography is performed for
baseline cardiac function and volumes, and then the LAD coronary
artery is permanently ligated via thoracotomy to infarct
approximately 70% of the left ventricular free wall.
[0258] Whole blood and bone marrow mononuclear fraction stem cells
are collected from naive litter mate mice and the stem cells are
purified using biotinylated anti-CD34, anti-CD 105, anti-SSEA1 and
anti-CD117 antibodies with Miltenyi magnetic separation columns.
Experimental mice are injected retroorbitally or intravenously with
between 100,000 and 1M purified stem cells on day 7, three days
after permanent ligation of the LAD. Serial echocardiography is
perforated on the mice on day 14, day 21 and day 28.
[0259] Ig control treated mice who do not receive stem cell
injections show significant cardiac functional decline and
increasing end diastolic volumes, end systolic volumes and
increased non-infarct wall thicknesses, with 50-100% of the mice
succumbing to heart failure before day 28. Ig control treated 129S1
mice who receive stem cell injections show a reduction in heart
failure deaths and slightly improved cardiac function compared to
those not receiving stem cell injections. Ig control treated PN
mice who receive stem cell injections do not show improved survival
or function in comparison to 129S1 mice. In contrast, anti-CD40L
mAb treated stem cell injected I29S1 mice show a significant
improvement in survival and cardiac function compared to all other
groups of mice, while anti-CD40L mAb treated stem cell injected PN
mice have improved survival and cardiac function compared to
control Ig treated stem cell injected PN mice.
Example 12
Inhibition or Reduction of Active Germinal Center Formation
Enhances Stem Cell Delivery to the Heart and Promotes Functional
Recovery in Humans.
[0260] (Prophetic)
[0261] Patients with acute ST-segment elevation MI successfully
treated by percutaneous coronary intervention with stent
implantation in the acute phase of the infarction are eligible for
the study. Patients are treated with anti-CD40L mAb Replizumab at
5, 10, 20 or 100 mg/kg on day 1 by 30 minute IV infusion.
[0262] At 3 to 15 days after MI, mononuclear cells are recovered
from 50 mL bone marrow aspirate by Ficoll-Hypaque (Pharmacia,
Uppsala, Sweden). The entire process, from bone marrow aspiration
to the finished product, is performed according to Good
Manufacturing Practice guidelines.
[0263] Cardiac function is followed by MRI and additional outcomes
include death, recurrent MI, and subsequent revascularization or
hospitalization (event-free survival). Compared to placebo
patients, stem cell treated patients historically have one year
reductions in event-free survival rates of 80% versus 55-60%.
anti-CD40L and stem cell treated patients have improved event free
survival compared to stem cell only treated patients. Additionally,
anti-CD40L stem cell treated patients show further reductions in
ventricular volumes compared to stem cell only treated patients and
improved cardiac ejection parameters.
Example 13
Identification and Isolation of the Mononuclear Fraction Stem Cell
Binding Partner in the Mouse Spleen
[0264] Bone marrow and whole blood mononuclear cell fractions from
a male 129S1/SvlmJ mouse were isolated using Histopaque, combined
and labeled CTO according to the manufacturer's instructions.
Spleens were dissociated into single cell suspensions and labeled
with CTB. Stem cells were obtained by culturing MNC for 7 days in
growth media followed by 7 days in growth media without FBS
supplemented with 120 ng/ml stem cell factor and 25% horse serum
and harvesting the non-adherent cells. Typically, up to 40% of the
non-adherent cells express CD34, CD105, SSEA1, and/or CD117. Stem
cells were labeled with CTO.
[0265] CTB-labeled splenocytes were incubated on a rocker at 37 deg
C., 5% CO2 at a ratio of 1:3 (10 million CTB-labeled splenocytes
and 10M MNC in a 500 ul volume of PBS) with CTO-labeled MNC or a
ratio of 100:1 (10 million CTB-lab led splenocytes and 100,000
CTO-labeled stem cells in a 500 ul volume of PBS). Aliquots were
taken on run on a Beckman Coulter Gallios flow cytometer capturing
FL2 and FL9 fluorescence. Cell-cell binding was apparent as a
time-dependent increase in CTB+CTO+ co-positive signals in the
upper right (UR) quadrant of the scattergram. Background UR was
0.15%. Maximum binding of splenocytes and stem cells was 2.6% UR at
50 minutes, and maximum binding of splenocytes and MNC was 5% UR at
30 minutes.
[0266] Results and Conclusions: These data indicate that the spleen
cell involved in stem cell binding to the spleen can be identified
and isolated using flow cytometry.
[0267] While the preferred embodiment of the invention has been
illustrated and described, as noted above, many changes can be made
without departing from the spirit and scope of the invention.
Accordingly, the scope of the invention is not limited by the
disclosure of the preferred embodiment. Instead, the invention
should be determined entirely by reference to the claims that
follow.
* * * * *
References