U.S. patent application number 15/804555 was filed with the patent office on 2018-05-24 for compositions and methods for enhanced generation of hematopoietic stem/progenitor cells.
The applicant listed for this patent is Fred Hutchinson Cancer Research Center, NOVARTIS INSTITUTE FOR FUNCTIONAL GENOMICS, INC., DBA THE GENOMICS INSTITUTE OF THE NOVARTIS. Invention is credited to Irwin D. Bernstein, Anthony E. Boitano, Michael Cooke, Colleen Delaney.
Application Number | 20180142212 15/804555 |
Document ID | / |
Family ID | 47436230 |
Filed Date | 2018-05-24 |
United States Patent
Application |
20180142212 |
Kind Code |
A1 |
Bernstein; Irwin D. ; et
al. |
May 24, 2018 |
COMPOSITIONS AND METHODS FOR ENHANCED GENERATION OF HEMATOPOIETIC
STEM/PROGENITOR CELLS
Abstract
The present invention relates to methods, kits and compositions
for expansion of hematopoietic stem/progenitor cells and providing
hematopoietic function to human patients in need thereof. In one
aspect, it relates to kits and compositions comprising a Notch
agonist and an aryl hydrocarbon receptor antagonist. Also provided
herein are methods for expanding the hematopoietic stem/progenitor
cells using kits and compositions comprising a Notch agonist and an
aryl hydrocarbon receptor antagonist. The hematopoietic
stem/progenitor cells expanded using the disclosed kits,
compositions and methods include human umbilical cord blood
stem/progenitor cells, placental cord blood stem/progenitor cells
and peripheral blood stem cells. The present invention also relates
to administering hematopoietic stem/progenitor cells expanded using
a combination of a Notch agonist and an aryl hydrocarbon receptor
antagonist to a patient for short-term and/or long-term in vivo
repopulation benefits.
Inventors: |
Bernstein; Irwin D.;
(Seattle, WA) ; Boitano; Anthony E.; (San Diego,
CA) ; Cooke; Michael; (San Diego, CA) ;
Delaney; Colleen; (Seattle, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Fred Hutchinson Cancer Research Center
NOVARTIS INSTITUTE FOR FUNCTIONAL GENOMICS, INC., DBA THE GENOMICS
INSTITUTE OF THE NOVARTIS |
Seattle
San Diego |
WA
CA |
US
US |
|
|
Family ID: |
47436230 |
Appl. No.: |
15/804555 |
Filed: |
November 6, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14363748 |
Jun 6, 2014 |
9834755 |
|
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PCT/US2012/068599 |
Dec 7, 2012 |
|
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15804555 |
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61568573 |
Dec 8, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2501/26 20130101;
A61K 2035/124 20130101; C12N 2501/38 20130101; C12N 5/0647
20130101; C12N 2501/2303 20130101; C12N 2501/145 20130101; A61K
35/28 20130101; C12N 2501/999 20130101; C12N 2501/2306 20130101;
C12N 2501/60 20130101; C12N 2501/125 20130101; C12N 2501/42
20130101 |
International
Class: |
C12N 5/0789 20060101
C12N005/0789; A61K 35/28 20060101 A61K035/28 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
HL080245, CA009351, CA076930, and HL100395, awarded by the National
Institutes of Health. The government has certain rights in the
invention.
Claims
1-55. (canceled)
56. A kit comprising, in one or more containers: (a) a Notch
agonist, and (b)
4-(2-(2-(benzothiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phe-
nol (SR1).
57. The kit of claim 56, wherein the Notch agonist and the SR1 are
in separate containers.
58. The kit of claim 56, wherein the Notch agonist is an
extracellular domain of a Delta protein or a Jagged protein, or a
Notch-binding portion of any of the foregoing.
59. The kit of claim 58, wherein the domain or portion is fused to
a fusion partner.
60. The kit of claim 59, wherein the fusion partner is an Fc region
of an IgG.
61. The kit of claim 58, wherein the Notch agonist is an
extracellular domain of a Delta protein.
62. The kit of claim 58, wherein the Notch agonist is
Delta.sup.ext-IgG.
63. The kit of claim 62, wherein the Delta.sup.ext-IgG is present
in the container at a concentration between about 0.2 and 20
.mu.g/ml.
64. The kit of claim 62, wherein the Delta.sup.ext-IgG is present
in the container at a concentration between about 1.25 and 10
.mu.g/ml.
65. (canceled)
66. The kit of claim 56, wherein the kit comprises a solid phase on
which the Notch agonist is immobilized.
67. (canceled)
68. The kit of claim 62, wherein the kit comprises a solid phase on
which Delta.sup.ext-IgG is immobilized.
69. The kit of claim 68, wherein the Delta.sup.ext-IgG has been
applied to the solid phase at a concentration between about 0.2 and
20 .mu.g/ml.
70. The kit of claim 68, wherein the Delta.sup.ext-IgG has been
applied to the solid phase at a concentration between about 1.25
and 10 .mu.g/ml.
71. (canceled)
72. The kit of claim 56, further comprising a fibronectin or a
fragment thereof.
73. The kit of claim 72, wherein the kit comprises a fragment of
fibronectin, wherein the fragment of fibronectin is CH-296,
immobilized on the solid phase.
74. (canceled)
75. (canceled)
76. The kit of claim 56, further comprising one or more growth
factors.
77. The kit of claim 76, wherein the growth factors are selected
from stem cell factor (SCF), Flt-3 ligand (Flt-3), Interleukin-6
(IL-6), Interleukin-3 (IL-3), Interleukin-11 (IL-11),
thrombopoietin (TPO), Granulocyte-macrophage colony stimulating
factor (GM-CSF), and granulocyte colony stimulating factor
(G-CSF).
78. The kit of claim 76, comprising three or more growth
factors.
79. The kit of claim 78, comprising IL-6, Flt-3, SCF and TPO.
80. The kit of claim 78, comprising IL-6, Flt-3, SCF, TPO and
IL-3.
81-103. (canceled)
Description
PRIORITY BENEFIT
[0001] This application is a divisional application based on U.S.
patent application Ser. No. 14/363,748, filed on Jun. 6, 2014,
which is a U.S. National Phase application based on International
Patent Application No. PCT/US2012/068599, filed on Dec. 7, 2012,
which claims priority to U.S. provisional application No.
61/568,573, filed on Dec. 8, 2011, each of which is incorporated
herein by reference in their entirety.
STATEMENT REGARDING SEQUENCE LISTING
[0003] The Sequence Listing associated with this application is
provided in text format in lieu of a paper copy, and is hereby
incorporated by reference into the specification. The name of the
text file containing the Sequence Listing is "1QM7625.txt". The
text file is about 1.84 KB, was created on Dec. 6, 2017, and is
being submitted electronically via EFS-Web.
FIELD OF THE INVENTION
[0004] The present invention relates to methods, kits and
compositions for expansion of hematopoietic stem/progenitor cells
and providing hematopoietic function to human patients in need
thereof. In one aspect, it relates to kits and compositions
comprising a Notch agonist and an aryl hydrocarbon receptor
antagonist. Also provided herein are methods for expanding the
hematopoietic stem/progenitor cells using kits and compositions
comprising a Notch agonist and an aryl hydrocarbon receptor
antagonist. The hematopoietic stem/progenitor cells expanded using
the disclosed kits, compositions and methods include human
umbilical cord blood stem/progenitor cells, placental cord blood
stem/progenitor cells and peripheral blood stem cells. The present
invention also relates to administering hematopoietic
stem/progenitor cells expanded using a combination of a Notch
agonist and an aryl hydrocarbon receptor antagonist to a patient
for short-term and/or long-term in vivo repopulation benefits.
BACKGROUND OF THE INVENTION
[0005] Hematopoietic stem cells (HSC) have therapeutic potential as
a result of their capacity to restore blood and immune cells in
transplant recipients. Specifically, autologous or allogeneic
transplantation of HSC can be used for the treatment of patients
with inherited immunodeficient and autoimmune diseases and diverse
hematopoietic disorders to reconstitute the hematopoietic cell
lineages and immune system defense. Human bone marrow
transplantation methods are currently used as therapies for
leukemia, lymphoma, and other life-threatening diseases. For these
procedures, a large number of stem cells must be isolated to ensure
that there are enough HSC for engraftment. The number of HSC
available for treatment is a clinical limitation. See U.S. Patent
Publication No. 2010/0183564.
[0006] Prolonged pancytopenia is common following intensive
chemotherapy regimens, myeloablative and reduced intensity regimens
for hematopoietic cell transplantation (HCT), and exposure to acute
ionizing radiation. Of particular concern is prolonged neutropenia,
which results in a significant risk of infection despite improved
antimicrobial therapy and increases morbidity and mortality. Thus,
novel therapies that can abrogate prolonged
pancytopenia/neutropenia following high dose chemotherapy and/or
radiation, and potentially facilitate more rapid hematopoietic
recovery, are needed.
[0007] 1.1 Hematopoietic Stem Cells
[0008] The hematopoietic stem cell is pluripotent and ultimately
gives rise to all types of terminally differentiated blood cells.
The hematopoietic stem cell can self-renew, or it can differentiate
into more committed progenitor cells, which progenitor cells are
irreversibly determined to be ancestors of only a few types of
blood cell. For instance, the hematopoietic stem cell can
differentiate into (i) myeloid progenitor cells, which myeloid
progenitor cells ultimately give rise to monocytes and macrophages,
neutrophils, basophils, eosinophils, erythrocytes,
megakaryocytes/platelets, dendritic cells, or (ii) lymphoid
progenitor cells, which lymphoid progenitor cells ultimately give
rise to T-cells, B-cells, and lymphocyte-like cells called natural
killer cells (NK-cells). Once the stem cell differentiates into a
myeloid progenitor cell, its progeny cannot give rise to cells of
the lymphoid lineage, and, similarly, lymphoid progenitor cells
cannot give rise to cells of the myeloid lineage. For a general
discussion of hematopoiesis and hematopoietic stem cell
differentiation, see Chapter 17, Differentiated Cells and the
Maintenance of Tissues, Alberts et al., 1989, Molecular Biology of
the Cell, 2nd Ed., Garland Publishing, New York, N.Y.; Chapter 2 of
Regenerative Medicine, Department of Health and Human Services,
August 2006, and Chapter 5 of Hematopoietic Stem Cells, 2009, Stem
Cell Information, Department of Health and Human Services.
[0009] In vitro and in vivo assays have been developed to
characterize hematopoietic stem cells, for example, the spleen
colony forming (CFU-S) assay and reconstitution assays in
immune-deficient mice. Further, presence or absence of cell surface
protein markers defined by monoclonal antibody recognition have
been used to recognize and isolate hematopoietic stem cells. Such
markers include, but are not limited to, Lin, CD34, CD38, CD43,
CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD133, CD166, and HLA DR,
and combinations thereof. See Chapter 2 of Regenerative Medicine,
Department of Health and Human Services, August 2006, and the
references cited therein.
[0010] 1.2 Notch Pathway
[0011] Members of the Notch family encode large transmembrane
proteins that play central roles in cell-cell interactions and
cell-fate decisions during early development in a number of
invertebrate systems (Simpson, 1995, Nature 375:736-7;
Artavanis-Tsakonis et al., 1995, Science. 268:225-232; Simpson,
1998, Semin. Cell Dev. Biol. 9:581-2; Go et al., 1998, Development.
125:2031-2040; Artavanis-Tsakonas and Simpson, 1991, Trends Genet.
7:403-408). The Notch receptor is part of a highly conserved
pathway that enables a variety of cell types to choose between
alternative differentiation pathways based on those taken by
immediately neighboring cells. This receptor appears to act through
an undefined common step that controls the progression of
uncommitted cells toward the differentiated state by inhibiting
their competence to adopt one of two alternative fates, thereby
allowing the cell either to delay differentiation, or in the
presence of the appropriate developmental signal, to commit to
differentiate along the non-inhibited pathway.
[0012] Genetic and molecular studies have led to the identification
of a group of genes which define distinct elements of the Notch
signaling pathway. While the identification of these various
elements has come exclusively from Drosophila using genetic tools
as the initial guide, subsequent analyses have lead to the
identification of homologous proteins in vertebrate species
including humans. The molecular relationships between the known
Notch pathway elements as well as their subcellular localization
are depicted in Artavanis-Tsakonas et al., 1995, Science
268:225-232; Artavanis-Tsakonas et al., 1999, Science 284:770-776;
and in Kopan et al., 2009, Cell 137:216-233. Proteins of the Delta
family and proteins of the Serrate (including Jagged, the mammalian
homolog of Serrate) family are extracellular ligands of Notch. The
portion of Delta and Serrate responsible for binding to Notch is
called the DSL domain, which domain is located in the extracellular
domain of the protein. Epidermal growth factor-like repeats (ELRs)
11 and 12 in the extracellular domain of Notch are responsible for
binding to Delta, Serrate and Jagged. See Artavanis-Tsakonas et
al., 1995, Science 268:225-232 and Kopan et al., 2009, Cell
137:216-233.
[0013] 1.3 Notch Pathway in Hematopoiesis
[0014] Evidence of Notch-1 mRNA expression in human CD34+
precursors has led to speculation for a role for Notch signaling in
hematopoiesis (Milner et al., 1994, Blood 3:2057-62). This is
further supported by the demonstration that Notch-1 and -2 proteins
are present in hematopoietic precursors, and, in higher amounts, in
T cells, B cells, and monocytes, and by the demonstration of
Jagged-1 protein in hematopoietic stroma (Ohishi et al., 2000,
Blood 95:2847-2854; Varnum-Finney et al., 1998, Blood 91:4084-91;
Li et al., 1998, Immunity 8:43-55). The clearest evidence for a
physiologic role of Notch signaling has come from studies of T cell
development which showed that activated Notch-1 inhibited B cell
maturation but permitted T cell maturation (Pui et al., 1999,
Immunity 11:299-308). In contrast, inactivation of Notch-1 or
inhibition of Notch-mediated signaling by knocking out HES-1
inhibited T cell development but permitted B cell maturation
(Radtke et al., 1999, Immunity 10: 47-58; Tomita et al., 1999,
Genes Dev. 13:1203-10). These opposing effects of Notch-1 on B and
T cell development raise the possibility that Notch-1 regulates
fate decisions by a common lymphoid progenitor cell.
[0015] Other studies in transgenic mice have shown that activated
Notch-1 affects the proportion of cells assuming a CD4 vs. CD8
phenotype as well as an .alpha..beta. vs. .gamma..delta. cell-fate
(Robey et al., 1996, Cell 87:483-92; Washburn et al., 1997, Cell
88:833-43). Although this may reflect an effect on fate decisions
by a common precursor, more recent studies have suggested that
these effects may result from an anti-apoptotic effect of Notch-1
that enables the survival of differentiating T cells that would
otherwise die (Deftos et al., 1998, Immunity 9:777-86; Jehn et al.,
1999, J Immunol. 162:635-8).
[0016] Studies have also shown that the differentiation of isolated
hematopoietic precursor cells can be inhibited by ligand-induced
Notch signaling. Co-culture of murine marrow precursor cells
(Lin-Sca-1+c-kit+) with 3T3 cells expressing human Jagged-1 led to
a 2 to 3 fold increase in the formation of primitive precursor cell
populations (Varnum-Finney et al., 1998, Blood 91:4084-4991; Jones
et al., 1998, Blood 92:1505-11). Incubation of sorted precursors
with beads coated with the purified extracellular domain of human
Jagged-1 also led to enhanced generation of precursor cells
(Varnum-Finney et al., 1998, Blood 91:4084-91).
[0017] In a study of human CD34+ cells, expression of the
intracellular domain of Notch-1 or exposure to cells that
overexpressed Jagged-2 also led to enhanced generation of precursor
cells and prolonged maintenance of CD34 expression (Carlesso et
al., 1999, Blood 93:838-48). In another study, the effects of
Jagged-1-expressing cells on CD34.sup.+ cells were influenced by
the cytokines present in the cultures; in the absence of added
growth factors, the interaction with cell-bound Jagged-1 led to
maintenance of CD34.sup.+ cells in a non-proliferating,
undifferentiated state, whereas the addition of c-kit ligand led to
a 2-fold increase in erythroid colony-forming cells (Walker et al.,
1999, Stem Cells 17:162-71).
[0018] 1.4 Expansion and Engraftment of Hematopoietic
Stem/Progenitor Cells
[0019] There is a need for successful expansion of human
stem/progenitor cells (HSPC). This has particular immediate
relevance for cord blood (CB) transplants where the stem cell dose
in a single cord blood unit is often inadequate for a larger child
or adult recipient and double cord blood transplantation (dCBT) is
required. Despite dCBT for these individuals, engraftment is often
delayed for more than 3 weeks leaving the recipient susceptible to
infection resulting in increased morbidity and mortality (see
Barker et al., 2005, Blood. 105 (3): 1343-1347). Thus successful ex
vivo generation of both short- and long-term repopulating HSPC,
including CB HSPC, has both biological relevance for better
understanding HSPC self-renewal and clear clinical impact.
[0020] Past efforts have attempted to expand HSPC using soluble
cytokine mediated methodologies; however, these attempts have
demonstrated limited clinical efficacy (see Shpall et al., 2002,
Biol Blood Marrow Transplant. 8(7): 368-376; de Lima et al., 2008,
Blood. 112: Abstract 154; Jaroscak et al., 2003, Blood. 101(12):
5061-5067).
[0021] Varnum-Finney et al., 1993, Blood 101:1784-1789 demonstrated
that activation of endogenous Notch receptors in mouse marrow
precursor cells by an immobilized Notch ligand revealed profound
effects on the growth and differentiation of the precursor cells,
and that a multilog increase in the number of precursor cells with
short-term lymphoid and myeloid repopulating ability was
observed.
[0022] Delaney et al., 2005, Blood 106:2693-2699 and Ohishi et al.,
2002, J. Clin. Invest. 110:1165-1174 demonstrated that incubation
of human cord blood progenitors in the presence of an immobilized
Notch ligand generated an approximate 100-fold increase in the
number of CD34+ cells with enhanced repopulating ability as
determined in an immunodeficient mouse model. See also U.S. Pat.
No. 7,399,633 B2.
[0023] Delaney et al., 2010, Nature Med. 16(2): 232-236
demonstrated that a population of CD34+ cells obtained from a
frozen cord blood sample, which population had been cultured in the
presence of a Notch ligand (resulting in a greater than 100 fold
increase in the number of CD34+ cells), repopulated immunodeficient
mice with markedly enhanced kinetics and magnitude, and provided
more rapid myeloid engraftment in humans in a clinical phase 1
myeloablative cord blood transplant trial.
[0024] Expansion techniques for cord blood stem cells have been
described. See, e.g., U.S. Pat. No. 7,399,633 B2 to Bernstein et
al., and Delaney et al., 2010, Nature Med. 16(2): 232-236. Delaney
et al. reported rapid engraftment after infusion of previously
cryopreserved cord blood stem cells which had been selected on the
basis of HLA matching, and which had been expanded ex vivo.
[0025] International Patent Publication No. WO 2006/047569 A2
discloses methods for expanding myeloid progenitor cells that do
not typically differentiate into cells of the lymphoid lineage, and
which can be MHC-mismatched with respect to the recipient of the
cells.
[0026] International Patent Publication No. WO 2007/095594 A2
discloses methods for facilitating engraftment of hematopoietic
stem cells by administering myeloid progenitor cells in conjunction
with the hematopoietic stem cell graft, for example, where the
hematopoietic stem cell graft is suboptimal because it has more
than one MHC mismatch with respect to the cells of the recipient
patient.
[0027] U.S. Pat. No. 5,004,681 to Boyse et al. discloses the use of
human cord blood stem cells for hematopoietic reconstitution.
[0028] U.S. Patent Publication No. 2010/0183564 to Boitano et al.
discloses methods and compositions for expanding HSPC populations
using an agent capable of down-regulating the activity and/or
expression of aryl hydrocarbon receptor and/or a downstream
effector of aryl hydrocarbon receptor pathway.
[0029] International Patent Publication No. WO 2011/127470 A1
discloses methods and compositions for providing hematopoietic
function to a human patient, by selecting an expanded human
umbilical cord blood stem/progenitor cell sample without taking
into account the HLA-type of the expanded human cord blood stem
cell/progenitor sample or the HLA-type of the patient, and
administering the selected expanded human cord blood
stem/progenitor cell sample to the patient; as well as methods for
obtaining the expanded human cord blood stem cell/progenitor cell
samples; and banks of frozen expanded human cord blood stem
cell/progenitor cell samples, and methods for producing such
banks.
[0030] International Patent Publication No. WO 2011/127472 A1
discloses methods and compositions for providing hematopoietic
function to a human patient, by selecting a pool of expanded human
umbilical cord blood stem/progenitor cell samples for
administration to a patient, wherein the samples in the pool
collectively do not mismatch the patient at more than 2 of the HLA
antigens or alleles typed in the patient, and administering the
selected pool of expanded human cord blood stem/progenitor cell
samples to the patient; as well as methods for obtaining the pools
of expanded human cord blood stem cell/progenitor cell samples; and
banks of frozen pools of expanded human umbilical cord blood stem
cell/progenitor cell samples, and methods for producing such
banks.
[0031] 1.5 Effects of Aryl Hydrocarbon Receptor Antagonist on Cell
Expansion and Engraftment
[0032] Boitano et al. described enhanced ex vivo expansion of CD34+
cord blood HSPC with StemRegenin1 (SR1), an aryl hydrocarbon
receptor (AhR) antagonist that acts at least in part by preventing
cellular differentiation resulting in HSC expansion (see Boitano et
al., 2010, Science 329(5997): 1345-1348). Specifically, Boitano et
al. demonstrated that culturing HSPC with SR1 leads to a 50-fold
increase in CD34+ cells and a 17-fold increase in the number of
cells with the ability to repopulate immunodeficient mice. Boitano
et al further demonstrated that these SR1-mediated effects are
direct result of inhibition of AhR present on HSPC.
[0033] Citation or identification of any reference in Section 2 or
any other section of this application shall not be construed as an
admission that such reference is available as prior art to the
present invention.
SUMMARY OF THE INVENTION
[0034] The present invention relates to methods and compositions
for expansion of hematopoietic stem/progenitor cells ex vivo by
methods comprising using a Notch agonist, such as any of the Notch
agonists as described in U.S. Pat. No. 7,399,633 incorporated by
reference herein in its entirety (e.g., an extracellular domain of
a Notch ligand (such as Delta or Serrate) or a Notch-binding
fragment thereof), in combination with using an aryl hydrocarbon
receptor antagonist, such as any of the aryl hydrocarbon receptor
antagonists as described in U.S. Patent Publication No.
2010/0183564 incorporated by reference herein in its entirety
(e.g., a compound of Formula I as depicted below, such as
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol (StemRegenin1, "SR1")). The expanded hematopoietic
stem/progenitor cells can be administered to a patient in need
thereof to provide hematopoietic function to the patient.
[0035] Compositions and methods for expansion of precursor cells
such as hematopoietic stem/progenitor cells, said method comprising
culturing the precursor cells in the presence of a Notch agonist,
as well as uses of such expanded cells, are described in U.S. Pat.
No. 7,399,633 to Bernstein et al. In a specific embodiment, as
described in U.S. Pat. No. 7,399,633 to Bernstein et al., the Notch
agonist is an extracellular domain of a Notch ligand (e.g.,
Delta.sup.ext-IgG) immobilized on a solid support for expansion of
hematopoietic stem/progenitor cells. The entire disclosure of U.S.
Pat. No. 7,399,633 is hereby incorporated by reference herein in
its entirety.
[0036] Compositions and methods for expansion of hematopoietic
stem/progenitor cells, said method comprising culturing the
hematopoietic stem/progenitor cells in the presence of an agent
capable of antagonizing the activity and/or expression of aryl
hydrocarbon receptor and/or downstream effector of aryl hydrocarbon
receptor pathway, as well as uses of such expanded cells, are
described in U.S. Patent Publication No. 2010/0183564 to Boitano et
al. In a specific embodiment, as described in U.S. Patent
Publication No. 2010/0183564 to Boitano et al.,
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol (Stem Regenin1, "SR1") is used for expansion of hematopoietic
stem/progenitor cells. The entire disclosure of U.S. Patent
Publication No. 2010/0183564 is hereby incorporated by reference
herein in its entirety.
[0037] The present invention describes methods, kits and
compositions comprising a combination of a Notch agonist and an
aryl hydrocarbon receptor antagonist, and uses of such compositions
and combinations. In certain embodiments, the Notch agonist is an
extracellular domain of a Notch ligand (e.g., Delta or Serrate) or
a Notch-binding fragment thereof. In preferred embodiments, the
Notch agonist is an extracellular domain of a human or rodent
(e.g., rat) Notch ligand (e.g., human or rodent Delta, or human or
rodent Jagged) or a Notch-binding fragment thereof. Preferably, the
Notch agonist is the extracellular domain of Delta or
Serrate/Jagged (or a Notch-binding portion thereof) fused to a
fusion partner. The fusion partners can be, but are not limited to,
an Fc domain of IgG or tags that contain an antigenic determinant
such as a myc tag. In a preferred embodiment, the Notch agonist is
Delta.sup.ext-IgG.
[0038] In some embodiments, the Notch agonist is immobilized on a
solid phase in contact with the HSPC. In specific embodiments, SR1
is in a fluid medium contacting the HSPC.
[0039] In specific embodiments, described herein are kits and
compositions comprising HSPC, an immobilized extracellular domain
of a Notch ligand (preferably fused to a fusion partner) in contact
with the HSPC, and SR1 in contact with the HSPC. In specific
embodiments, the extracellular domain of a Notch ligand (e.g.,
Delta.sup.ext-IgG) is immobilized on a solid phase, on which HSPC
are cultured, whereas SR1 is in a fluid medium contacting the
HSPC.
[0040] In one aspect, disclosed herein are methods for expansion of
hematopoietic stem/progenitor cells using the described kits and
compositions comprising a Notch agonist and an aryl hydrocarbon
receptor antagonist. In preferred embodiments, disclosed herein are
methods for expansion of hematopoietic stem/progenitor cells using
a combination of an extracellular domain of a Notch ligand (e.g.,
Delta.sup.ext-IgG) and SR1. Hematopoietic stem/progenitor cells
that may be expanded using the disclosed compositions include, but
are not limited to, human umbilical cord blood stem/progenitor
cells, placental cord blood stem/progenitor cells, peripheral blood
stem/progenitor cells (e.g., mobilized peripheral blood
stem/progenitor cells) and bone marrow stem/progenitor cells.
[0041] In another aspect, disclosed herein are methods of treatment
comprising administering the expanded hematopoietic stem/progenitor
cells disclosed herein to a patient in need thereof. In one
embodiment, the patient is a human. The hematopoietic
stem/progenitor cells expanded using the methods described herein
can be effectively used for short term in vivo
repopulation/engraftment. In particular, the hematopoietic
stem/progenitor cells expanded using the methods described herein
can be effectively used for early myeloid repopulation and
neutrophil engraftment in treated patients. Further, the
hematopoietic stem/progenitor cells expanded using the methods
described herein can be effectively used for long term in vivo
repopulation/engraftment. In particular, the hematopoietic
stem/progenitor cells expanded using the methods described herein
can be effectively used for multi-lineage, sustained in vivo
repopulation. In one aspect, disclosed herein are methods of
treatment comprising administering the expanded hematopoietic
stem/progenitor cells disclosed herein to a patient in need of
short-term and/or long-term in vivo repopulation.
[0042] Preferably, the combination of a Notch agonist and an aryl
hydrocarbon receptor antagonist (e.g., the combination of an
immobilized extracellular domain of a Delta, a Serrate, or a Jagged
protein with SR1) has synergistic or additive activities upon HSPC
engraftment and/or expansion.
[0043] In certain embodiments, described herein are methods of
expanding hematopoietic stem/progenitor cells comprising culturing
isolated hematopoietic stem/progenitor cells ex vivo in the
presence of a composition comprising a Notch agonist and an aryl
hydrocarbon receptor antagonist, thereby producing an expanded
hematopoietic stem/progenitor cell sample. In one embodiment, the
isolated hematopoietic stem/progenitor cells are human. In some
embodiments, during the culturing step, hematopoietic
stem/progenitor cells are cultured in the presence of an
immobilized Notch agonist that is the extracellular domain of a
Notch ligand, preferably fused to a fusion partner, and in the
presence of an aryl hydrocarbon receptor antagonist (e.g., in a
fluid contacting the cells). In specific embodiments, during the
culturing step, hematopoietic stem/progenitor cells are cultured on
a solid phase coated with the Notch agonist, and the aryl
hydrocarbon receptor antagonist is in a fluid contacting the cells.
In some embodiments, the isolated HSPC are expanded in the presence
of one or more growth factors, two or more growth factors, three or
more growth factors, or four or more growth factors (e.g., in a
fluid medium). For example, the growth factors can be selected from
stem cell factor (SCF), Flt-3 ligand (Flt-3), Interleukin-6 (IL-6),
Interleukin-3 (IL-3), Interleukin-11 (IL-11), thrombopoietin (TPO),
Granulocyte-macrophage colony stimulating factor (GM-CSF), or
granulocyte colony stimulating factor (G-CSF). In one embodiment,
the isolated HSPC are expanded in the presence of IL-6, Flt-3, SCF
and TPO (e.g., in a fluid medium). In another embodiment, the
isolated HSPC are expanded in the presence of IL-6, Flt-3, SCF, TPO
and IL-3 (e.g., in a fluid medium).
[0044] In specific embodiments, the Notch agonist used in the
methods of expanding HSPC described herein is the extracellular
domain of a Delta, a Jagged or a Serrate protein, fused to an Fc
region of an IgG (or fused to another fusion partner such as a myc
or other epitope). In one embodiment, the Notch agonist is Delta
1.sup.ext-IgG. In some embodiments, Delta.sup.ext-IgG is applied to
the solid phase at a concentration between about 0.2 and 20
.mu.g/ml, between about 1.25 and 10 .mu.g/ml, or between about 2
and 6 .mu.g/ml. In some embodiments of the compositions for
expansion of hematopoietic stem/progenitor cells in which (e.g.,
Delta1.sup.ext-IgG) Delta.sup.ext-G (e.g., Delta1.sup.ext-IgG) is
immobilized on a solid phase, Delta.sup.ext-IgG (e.g.,
Delta1.sup.ext-IgG) has been applied to the solid phase at a
concentration between about 0.2 and 20 .mu.g/ml, between about 1.25
and 10 .mu.g/ml, or between about 2 and 6 .mu.g/ml.
[0045] In specific embodiments, the aryl hydrocarbon receptor
antagonist used in the methods of expanding HSPC described herein
is a compound of Formula I:
##STR00001##
in which: G.sub.1 is selected from N and CR.sub.3; G.sub.2, G.sub.3
and G.sub.4 are independently selected from CH and N; with the
proviso that at least 1 of G.sub.3 and G.sub.4 is N; with the
proviso that G.sub.1 and G.sub.2 are not both N; L is selected from
--NR.sub.5a(CH.sub.2).sub.0-3-,
--NR.sub.5aCH(C(O)OCH.sub.3)CH.sub.2--,
--NR.sub.5a(CH.sub.2).sub.2NR.sub.5b--,
--NR.sub.5a(CH.sub.2).sub.2S--,
--NR.sub.5aCH.sub.2CH(CH.sub.3)CH.sub.2--,
--NR.sub.5aCH.sub.2CH(OH)-- and --NR.sub.5aCH(CH.sub.3)CH.sub.2--;
wherein R.sub.5a and R.sub.5b are independently selected from
hydrogen and C.sub.1-4alkyl; R.sub.1 is selected from hydrogen,
phenyl, thiophenyl, furanyl, 1H-benzoimidazolyl, isoquinolinyl,
1H-imidazopyridinyl, benzothiophenyl, pyrimidinyl, 1H-pyrazolyl,
pyridinyl, 1H-imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl,
1H-pyrrolyl and thiazolyl; wherein said phenyl, thiophenyl,
furanyl, 1H-benzoimidazolyl, isoquinolinyl, 1H-imidazopyridinyl,
benzothiophenyl, pyrimidinyl, 1H-pyrazolyl, pyridinyl,
1H-imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, 1H-pyrrolyl or
thiazolyl of R.sub.1 can be optionally substituted by 1 to 3
radicals independently selected from cyano, hydroxy,
C.sub.1-4alkyl, C.sub.1-4alkoxy, halo,
halo-substituted-C.sub.1-4alkyl, halo-substituted-C.sub.1-4alkoxy,
hydroxy, amino, --C(O)R.sub.5a, --S(O).sub.0-2R.sub.8a,
--C(O)OR.sub.5a and --C(O)NR.sub.8aR.sub.8b; wherein R.sub.8a and
R.sub.8b are independently selected from hydrogen and
C.sub.1-4alkyl; with the proviso that R.sub.1 and R.sub.3 are not
both hydrogen; R.sub.2 is selected from
--S(O).sub.2NR.sub.6aR.sub.6b, --NR.sub.6aC(O)R.sub.6b,
--NR.sub.6aC(O)NR.sub.6bR.sub.6c, phenyl, 1H-pyrrolopyridin-3-yl,
1H-indolyl, thiophenyl, pyridinyl, 1H-1,2,4-triazolyl,
2-oxoimidazolidinyl, 1H-pyrazolyl,
2-oxo-2,3-dihydro-1H-benzoimidazolyl and 1H-indazolyl; wherein
R.sub.6a, R.sub.6b and R.sub.6c are independently selected from
hydrogen and C.sub.1-4alkyl; wherein said phenyl,
1H-pyrrolopyridin-3-yl, 1H-indolyl, thiophenyl, pyridinyl,
1H-1,2,4-triazolyl, 2-oxoimidazolidinyl, 1H-pyrazolyl,
2-oxo-2,3-dihydro-1H-benzoimidazolyl or 1H-indazolyl of R.sub.2 is
optionally substituted with 1 to 3 radicals independently selected
from hydroxy, halo, methyl, methoxy, amino,
--O(CH.sub.2).sub.nNR.sub.7aR.sub.7b,
--S(O).sub.2NR.sub.7aR.sub.7b, --OS(O).sub.2NR.sub.7aR.sub.7b and
--NR.sub.7aS(O).sub.2R.sub.7b; wherein R.sub.7a and R.sub.7b are
independently selected from hydrogen and C.sub.1-4alkyl; R.sub.3 is
selected from hydrogen, C.sub.1-4alkyl and biphenyl; and R.sub.4 is
selected from C.sub.1-10alkyl, prop-1-en-2-yl, cyclohexyl,
cyclopropyl, 2-(2-oxopyrrolidin-1-yl)ethyl, oxetan-3-yl,
benzhydryl, tetrahydro-2H-pyran-3-yl, tetrahydro-2H-pyran-4-yl,
phenyl, tetrahydrofuran-3-yl, benzyl,
(4-pentylphenyl)(phenyl)methyl and
1-(1-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-1H-1,2,3-triazol-4-yl)et-
hyl; wherein said alkyl, cyclopropyl, cyclohexyl,
2-(2-oxopyrrolidin-1-yl)ethyl, oxetan-3-yl, oxetan-2-yl,
benzhydryl, tetrahydro-2H-pyran-2-yl, tetrahydro-2H-pyran-3-yl,
tetrahydro-2H-pyran-4-yl, phenyl, tetrahydrofuran-3-yl,
tetrahydrofuran-2-yl, benzyl, (4-pentylphenyl)(phenyl)methyl or
1-(1-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-1H-1,2,3-triazol-4-yl)et-
hyl can be optionally substituted with 1 to 3 radicals
independently selected from hydroxy, C.sub.1-4alkyl and
halo-substituted-C.sub.1-4alkyl; or a salt thereof. In one
embodiment, the aryl hydrocarbon receptor antagonist used in the
methods of expanding HSPC described herein is
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol (i.e., "SR1"). In particular embodiments, the aryl hydrocarbon
receptor antagonist used in the methods of expanding HSPC described
herein is SR1, and the Notch agonist is an immobilized
extracellular domain of Delta1 (e.g., Delta1.sup.ext-IgG).
[0046] In certain embodiments, the isolated HSPC are expanded in
the presence of a fibronectin or a fragment thereof (e.g., CH-296).
For example, the isolated HSPC can be expanded in the presence of
an immobilized fibronectin or a fragment thereof (e.g., immobilized
on the same solid phase as the Notch agonist, or immobilized on a
solid phase that is different from the solid phase on which the
Notch agonist is immobilized).
[0047] In some embodiments, the isolated hematopoietic
stem/progenitor cells used in the methods of the invention are
derived from umbilical cord blood and/or placental cord blood
(e.g., form a single human collected at birth of said human, or
from a pool of two or more different humans at birth). In other
embodiments, the isolated hematopoietic stem/progenitor cells are
derived from peripheral blood (e.g., mobilized peripheral blood
stem cells). In yet other embodiments, the isolated hematopoietic
stem/progenitor cells are derived from bone marrow.
[0048] In some embodiments, the isolated hematopoietic
stem/progenitor cells are derived from a single human, while in
other embodiments, the isolated HSPC are derived from two or more
humans (where the two or more humans can be, but are not limited
to, humans of the same race or humans of the same ethnicity).
[0049] In certain embodiments, the percentage of CD34+ cells in the
expanded hematopoietic stem/progenitor cell sample, obtained using
the methods described herein, is higher than the percentage of
CD34+ cells in the isolated hematopoietic stem/progenitor cells
prior to expansion. In particular embodiments, the percentage of
CD34+ cells in the expanded hematopoietic stem/progenitor cell
sample, obtained using the methods described herein, is higher than
the percentage of CD34+ cells in a sample of the hematopoietic
stem/progenitor cells expanded using a Notch agonist alone and/or
an aryl hydrocarbon receptor antagonist alone.
[0050] In particular embodiments, the percentage of CD34+CD90+
cells in the expanded hematopoietic stem/progenitor cell sample,
obtained using the methods described herein, is higher than the
percentage of CD34+CD90+ cells in a sample of the hematopoietic
stem/progenitor cells expanded using a Notch agonist alone and/or
an aryl hydrocarbon receptor antagonist alone.
[0051] In particular embodiments, the percentage of
Lin-CD34+CD38-CD45RA-CD90+ cells in the expanded hematopoietic
stem/progenitor cell sample, obtained using the methods described
herein, is higher than the percentage of Lin-CD34+CD38-CD45RA-CD90+
cells in a sample of the hematopoietic stem/progenitor cells
expanded using a Notch agonist alone and/or an aryl hydrocarbon
receptor antagonist alone.
[0052] In particular embodiments, the percentage of CD34+ cells in
the expanded hematopoietic stem/progenitor cell sample obtained
using the methods described herein is either the same or lower than
the percentage of CD34.sup.+ cells in a sample of the hematopoietic
stem/progenitor cells expanded using an aryl hydrocarbon receptor
antagonist alone, but the percentage of CD34.sup.+CD90.sup.+ (e.g.,
Lin.sup.-CD34.sup.+CD38.sup.-CD45RA.sup.-CD90.sup.+) cells in the
expanded hematopoietic stem/progenitor cell sample obtained using
the methods described herein is higher than the percentage of
CD34.sup.+90.sup.+ (e.g.,
Lin.sup.-CD34.sup.+CD38.sup.-CD45RA.sup.-CD90.sup.+) cells in a
sample of the hematopoietic stem/progenitor cells expanded using a
Notch agonist alone and/or an aryl hydrocarbon receptor antagonist
alone. In a specific embodiment, the ratio of the total number of
CD34.sup.+CD90.sup.+ (e.g.,
Lin.sup.-CD34.sup.+CD38.sup.-CD45RA.sup.-CD90.sup.+) cells to the
total number of CD34.sup.+ cells in the expanded hematopoietic
stem/progenitor cell sample, obtained using the methods described
herein, is higher than such ratio in the isolated hematopoietic
stem/progenitor cells prior to expansion. In another specific
embodiment, the ratio of the total number of CD34.sup.+CD90.sup.+
(e.g., Lin.sup.-CD34.sup.+CD38.sup.-CD45RA.sup.-CD90.sup.+) cells
to the total number of CD34.sup.+ cells in the expanded
hematopoietic stem/progenitor cell sample, obtained using the
methods described herein, is higher than such ratio in a sample of
the hematopoietic stem/progenitor cells expanded using a Notch
agonist alone and/or an aryl hydrocarbon receptor antagonist
alone.
[0053] In specific embodiments, the percentage of CD34-CD14+ cells
in the expanded hematopoietic stem/progenitor cell sample, obtained
using the methods described herein, is lower than the percentage of
CD34-CD14+ cells in the isolated hematopoietic stem/progenitor
cells prior to expansion. In particular embodiments, the percentage
of CD34-CD14+ cells in the expanded hematopoietic stem/progenitor
cell sample, obtained using the methods described herein, is lower
than the percentage of CD34-CD14+ cells in a sample of the
hematopoietic stem/progenitor cells expanded using a Notch agonist
alone and/or an aryl hydrocarbon receptor antagonist alone.
[0054] In some embodiments, the percentage of mature myeloid CD14+
and/or CD15+ cells in the expanded hematopoietic stem/progenitor
cell sample, obtained using the methods described herein, is lower
than the percentage of mature myeloid CD14+ and/or CD15+ cells in a
sample of the hematopoietic stem/progenitor cells expanded using an
aryl hydrocarbon receptor antagonist alone.
[0055] In certain embodiments, the expanded hematopoietic
stem/progenitor cell sample, obtained using the methods described
herein, has an improved in vivo repopulating ability relative to a
sample of the hematopoietic stem/progenitor cells expanded using a
Notch agonist alone and/or an aryl hydrocarbon receptor antagonist
alone. In one embodiment, the expanded hematopoietic
stem/progenitor cell sample is capable of enhanced generation of
short-term in vivo repopulating cells relative to a sample of the
hematopoietic stem/progenitor cells expanded using a Notch agonist
alone and/or an aryl hydrocarbon receptor antagonist alone. In
another embodiment, the expanded hematopoietic stem/progenitor cell
sample is capable of enhanced generation of multi-lineage long-term
in vivo repopulating cells relative to a sample of the
hematopoietic stem/progenitor cells expanded using a Notch agonist
alone and/or an aryl hydrocarbon receptor antagonist alone. In yet
another embodiment, the expanded hematopoietic stem/progenitor cell
sample is capable of improved engraftment of CD45.sup.+ cells
relative to a sample of the hematopoietic stem/progenitor cells
expanded using a Notch agonist alone and/or an aryl hydrocarbon
receptor antagonist alone. In one embodiment, the expanded
hematopoietic stem/progenitor cell sample is capable of improved
engraftment of CD45.sup.+CD33.sup.+ cells relative to a sample of
the hematopoietic stem/progenitor cells expanded using a Notch
agonist alone and/or an aryl hydrocarbon receptor antagonist alone.
In another embodiment, the expanded hematopoietic stem/progenitor
cell sample is capable of improved engraftment of
CD45.sup.+CD34.sup.+ cells relative to a sample of the
hematopoietic stem/progenitor cells expanded using a Notch agonist
alone and/or an aryl hydrocarbon receptor antagonist alone. In a
specific embodiment, the expanded hematopoietic stem/progenitor
cell sample is capable of improved early or short-term engraftment
of CD45.sup.+CD34.sup.+ cells and/or CD45.sup.+CD33.sup.+ cells
relative to a sample of the hematopoietic stem/progenitor cells
expanded using a Notch agonist alone and/or an aryl hydrocarbon
receptor antagonist alone. In specific embodiments, the expanded
hematopoietic stem/progenitor cell sample is capable of improved
early or short-term engraftment of CD45.sup.+CD34.sup.+CD33.sup.-
cells and/or CD45.sup.+CD34.sup.+CD33.sup.+ cells relative to a
sample of the hematopoietic stem/progenitor cells expanded using a
Notch agonist alone and/or an aryl hydrocarbon receptor antagonist
alone. In a particular embodiment, the expanded hematopoietic
stem/progenitor cell sample is capable of improved early or
short-term engraftment of CD45.sup.+CD14.sup.+CD15.sup.+ cells
relative to a sample of the hematopoietic stem/progenitor cells
expanded using a Notch agonist alone and/or an aryl hydrocarbon
receptor antagonist alone. In certain embodiments, the expanded
hematopoietic stem/progenitor cell sample is capable of improved
long-term total human engraftment, such as improved long-term
engraftment of CD45.sup.+ cells, relative to a sample of the
hematopoietic stem/progenitor cells expanded using a Notch agonist
alone and/or an aryl hydrocarbon receptor antagonist alone. In yet
another embodiment, the expanded hematopoietic stem/progenitor cell
sample is capable of improved long-term engraftment of
CD45.sup.+CD19.sup.+ cells relative to a sample of the
hematopoietic stem/progenitor cells expanded using a Notch agonist
alone and/or an aryl hydrocarbon receptor antagonist alone. In one
embodiment, the expanded hematopoietic stem/progenitor cell sample
is capable of improved long-term engraftment of
CD45.sup.+CD19.sup.+CD33.sup.- cells relative to a sample of the
hematopoietic stem/progenitor cells expanded using a Notch agonist
alone and/or an aryl hydrocarbon receptor antagonist alone. In one
embodiment, the expanded hematopoietic stem/progenitor cell sample
is capable of improved long-term engraftment due to lymphocyte
repopulation relative to a sample of the hematopoietic
stem/progenitor cells expanded using a Notch agonist alone and/or
an aryl hydrocarbon receptor antagonist alone.
[0056] In a specific embodiment, described herein are methods of
expanding human hematopoietic stem/progenitor cells comprising
culturing isolated hematopoietic stem/progenitor cells ex vivo on a
solid phase coated with Delta1.sup.ext-IgG and CH-296, and further
in the presence of a medium comprising an aryl hydrocarbon receptor
antagonist and four or more growth factors; wherein the aryl
hydrocarbon antagonist is
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol; and wherein the four or more growth factors are selected from
IL6, TPO, Flt-3 ligand, CSF and IL3; thereby producing an expanded
hematopoietic stem/progenitor cell sample.
[0057] Also described herein are hematopoietic stem/progenitor cell
samples expanded using any of the methods disclosed herein.
[0058] Further embodiments of the invention include methods for
providing hematopoietic function to a patient in need thereof,
comprising administering to a patient the expanded hematopoietic
stem/progenitor cell sample obtained using any of the methods
described herein. Other embodiments of the invention include
methods for providing hematopoietic function to a patient in need
thereof, comprising carrying out any of the methods for expansion
of hematopoietic stem/progenitor cells described herein, and
administering to a patient the expanded hematopoietic
stem/progenitor cells (the expanded hematopoietic stem/progenitor
cell sample) thereby obtained. In preferred embodiments, the
patient treated in accordance with the invention is human. In some
embodiments, the expanded hematopoietic stem/progenitor cell
sample, obtained using the methods described herein, is derived
from hematopoietic stem/progenitor cells isolated from one or two
patients (e.g., human patients). In some embodiments, the expanded
hematopoietic stem/progenitor cell sample, obtained using the
methods described herein, is derived from hematopoietic
stem/progenitor cells isolated from a single human at birth. In
other embodiments, the expanded hematopoietic stem/progenitor cell
sample, obtained using the methods described herein, is a pool of
two or more different expanded hematopoietic stem/progenitor cell
samples, each different sample in the pool derived from
hematopoietic stem/progenitor cells isolated from a different human
at birth. In one embodiment, all the samples in the pool are
derived from the hematopoietic stem/progenitor cells of humans of
the same race. In another embodiment, all the samples in the pool
are derived from the hematopoietic stem/progenitor cells of humans
of the same ethnicity. In some embodiments, the expanded
hematopoietic stem/progenitor cell sample, obtained using the
methods described herein, is frozen prior to administering such
sample to the patient, and the method further includes a step of
thawing the sample prior to administering it to the patient. In yet
other embodiments, the expanded hematopoietic stem/progenitor cell
sample, obtained using the methods described herein, has not been
frozen prior to administering to the patient. In some embodiments,
the expanded hematopoietic stem/progenitor cell sample, obtained
using the methods described herein and administered to a patient,
is not HLA-matched to the patient. In specific embodiments, the
expanded hematopoietic stem/progenitor cell sample is administered
to a patient without taking into account the HLA-type of the
expanded human cord blood stem cell/progenitor sample or the
HLA-type of the patient. Patients that can be treated in accordance
with the invention include, but are not limited to, patients with
pancytopenia or neutropenia, any of which may be caused by an
intensive chemotherapy regimen, a myeloablative regimen for
hematopoietic cell transplantation, or exposure to acute ionizing
radiation.
[0059] Further described herein are kits comprising in one or more
containers: (a) a Notch agonist, and (b) an aryl hydrocarbon
receptor antagonist. Preferably, the Notch agonist is a human or
rodent protein or a fragment or derivative thereof. In one
embodiment, the Notch agonist and the aryl hydrocarbon receptor
antagonist are in separate containers. In some embodiments, the
Notch agonist is an extracellular domain of a Delta, a Jagged, or a
Serrate protein. In specific embodiments, an immobilized Notch
agonist, e.g., an extracellular domain of a Notch ligand (such as
an immobilized extracellular domain of a Delta, a Jagged, or a
Serrate protein), is used in the methods of the invention. In
particular embodiments, the Notch agonist is an extracellular
domain of a Delta protein, e.g., Delta-1, Delta-3 or Delta-4. In
other embodiments, the Notch agonist is an extracellular domain of
a Jagged protein, e.g., Jagged-1 or Jagged-2. In one specific
embodiment, the Notch agonist is Delta.sup.ext-IgG. In some
embodiments, Delta.sup.ext-IgG (e.g., Delta.sup.ext-IgG) is present
in the container at a concentration between about 0.2 and 20
.mu.g/ml, between about 1.25 and 10 .mu.g/ml, or between about 2
and 6 .mu.g/ml. In other embodiments, the Notch agonist is
Delta.sup.ext-myc. In some embodiments, the kits of the invention
include a solid phase coated with a Notch agonist (e.g., an
extracellular domain of a Notch ligand), which may be fused to a
fusion partner. In one embodiment, the kits of the invention
include a solid phase coated with Delta.sup.ext-IgG. In certain
embodiments, the solid phase comprises a surface of a cell culture
dish (e.g., an inside plastic surface of the cell culture dish). In
other embodiments, the solid phase comprises beads (e.g., Sepharose
beads). In some embodiments, Delta.sup.ext-IgG is (or has been)
applied to the solid phase at a concentration between about 0.2 and
20 .mu.g/ml, between about 1.25 and 10 .mu.g/ml, or between about 2
and 6 .mu.g/ml. In certain embodiments, the kits of the invention
further include a fibronectin or a fragment thereof (e.g., CH-296),
which can be immobilized on a solid phase (e.g., on the solid phase
coated with a Notch agonist, e.g., an extracellular domain of a
Notch ligand). In some embodiments of the kits described herein,
the aryl hydrocarbon receptor antagonist is a compound of Formula
I:
##STR00002##
in which: G.sub.1 is selected from N and CR.sub.3; G.sub.2, G.sub.3
and G.sub.4 are independently selected from CH and N; with the
proviso that at least 1 of G.sub.3 and G.sub.4 is N; with the
proviso that G.sub.1 and G.sub.2 are not both N; L is selected from
--NR.sub.5a(CH.sub.2).sub.0-3-,
--NR.sub.5aCH(C(O)OCH.sub.3)CH.sub.2--,
--NR.sub.5a(CH.sub.2).sub.2NR.sub.5b--,
--NR.sub.5a(CH.sub.2).sub.2S--,
--NR.sub.5aCH.sub.2CH(CH.sub.3)CH.sub.2--,
--NR.sub.5aCH.sub.2CH(OH)-- and --NR.sub.5aCH(CH.sub.3)CH.sub.2--;
wherein R.sub.5a and R.sub.5b are independently selected from
hydrogen and C.sub.1-4alkyl; R.sub.1 is selected from hydrogen,
phenyl, thiophenyl, furanyl, 1H-benzoimidazolyl, isoquinolinyl,
1H-imidazopyridinyl, benzothiophenyl, pyrimidinyl, 1H-pyrazolyl,
pyridinyl, 1H-imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl,
1H-pyrrolyl and thiazolyl; wherein said phenyl, thiophenyl,
furanyl, 1H-benzoimidazolyl, isoquinolinyl, 1H-imidazopyridinyl,
benzothiophenyl, pyrimidinyl, 1H-pyrazolyl, pyridinyl,
1H-imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, 1H-pyrrolyl or
thiazolyl of R.sub.1 can be optionally substituted by 1 to 3
radicals independently selected from cyano, hydroxy,
C.sub.1-4alkyl, C.sub.1-4alkoxy, halo,
halo-substituted-C.sub.1-4alkyl, halo-substituted-C.sub.1-4alkoxy,
hydroxy, amino, --C(O)R.sub.5a, --S(O).sub.0-2R.sub.8a,
--C(O)OR.sub.5a and --C(O)NR.sub.8aR.sub.8b; wherein R.sub.8a and
R.sub.8b are independently selected from hydrogen and
C.sub.1-4alkyl; with the proviso that R.sub.1 and R.sub.3 are not
both hydrogen; R.sub.2 is selected from
--S(O).sub.2NR.sub.6aR.sub.6b, --NR.sub.9aC(O)R.sub.9b,
--NR.sub.6aC(O)NR.sub.6bR.sub.6c, phenyl, 1H-pyrrolopyridin-3-yl,
1H-indolyl, thiophenyl, pyridinyl, 1H-1,2,4-triazolyl,
2-oxoimidazolidinyl, 1H-pyrazolyl,
2-oxo-2,3-dihydro-1H-benzoimidazolyl and 1H-indazolyl; wherein
R.sub.6a, R.sub.6b and R.sub.6c are independently selected from
hydrogen and C.sub.1-4alkyl; wherein said phenyl,
1H-pyrrolopyridin-3-yl, 1H-indolyl, thiophenyl, pyridinyl,
1H-1,2,4-triazolyl, 2-oxoimidazolidinyl, 1H-pyrazolyl,
2-oxo-2,3-dihydro-1H-benzoimidazolyl or 1H-indazolyl of R.sub.2 is
optionally substituted with 1 to 3 radicals independently selected
from hydroxy, halo, methyl, methoxy, amino,
--O(CH.sub.2).sub.nNR.sub.7aR.sub.7b,
--S(O).sub.2NR.sub.7aR.sub.7b, --OS(O).sub.2NR.sub.7aR.sub.7b and
--NR.sub.7aS(O).sub.2R.sub.7b; wherein R.sub.7a and R.sub.7b are
independently selected from hydrogen and C.sub.1-4alkyl; R.sub.3 is
selected from hydrogen, C.sub.1-4alkyl and biphenyl; and R.sub.4 is
selected from C.sub.1-10alkyl, prop-1-en-2-yl, cyclohexyl,
cyclopropyl, 2-(2-oxopyrrolidin-1-yl)ethyl, oxetan-3-yl,
benzhydryl, tetrahydro-2H-pyran-3-yl, tetrahydro-2H-pyran-4-yl,
phenyl, tetrahydrofuran-3-yl, benzyl,
(4-pentylphenyl)(phenyl)methyl and
1-(1-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-1H-1,2,3-triazol-4-yl)et-
hyl; wherein said alkyl, cyclopropyl, cyclohexyl,
2-(2-oxopyrrolidin-1-yl)ethyl, oxetan-3-yl, oxetan-2-yl,
benzhydryl, tetrahydro-2H-pyran-2-yl, tetrahydro-2H-pyran-3-yl,
tetrahydro-2H-pyran-4-yl, phenyl, tetrahydrofuran-3-yl,
tetrahydrofuran-2-yl, benzyl, (4-pentylphenyl)(phenyl)methyl or
1-(1-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-1H-1,2,3-triazol-4-yl)et-
hyl can be optionally substituted with 1 to 3 radicals
independently selected from hydroxy, C.sub.1-4alkyl and
halo-substituted-C.sub.1-4alkyl; or a salt thereof. In some
embodiments, the kits of the invention include
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol (i.e., "SR1"), which is one of the compounds of Formula I.
[0060] In certain embodiments, the kits described herein further
include one or more growth factors, two or more growth factors, or
three or more growth factors (e.g., wherein one or more growth
factors are in a container separate from the container(s)
comprising the Notch agonist and/or the aryl hydrocarbon receptor
antagonist). In specific embodiments, the growth factors are
selected from stem cell factor (SCF), Flt-3 ligand (Flt-3),
Interleukin-6 (IL-6), Interleukin-3 (IL-3), Interleukin-11 (IL-11),
thrombopoietin (TPO), Granulocyte-macrophage colony stimulating
factor (GM-CSF), and granulocyte colony stimulating factor (G-CSF).
In one embodiment, the kits described herein include IL-6, Flt-3,
SCF and TPO. In another embodiment, the kits described herein
include IL-6, Flt-3, SCF, TPO and IL-3.
[0061] Also described herein is a solid phase comprising a surface
on which a Notch agonist is immobilized, wherein the solid phase is
in contact with the hematopoietic stem/progenitor cells, and the
hematopoietic stem/progenitor cells are in contact with a fluid
medium comprising an aryl hydrocarbon receptor antagonist in
contact with the cells. In some embodiments, the solid phase is a
cell culture container comprising (a) a Notch agonist immobilized
on an inside surface of the container, and (b) hematopoietic
stem/progenitor cells cultured on the inside surface, wherein the
cells are in contact with a fluid comprising an aryl hydrocarbon
receptor antagonist. Also described herein are cell culture
containers comprising hematopoietic stem/progenitor cells, an
immobilized Notch agonist in contact with the cells, and an aryl
hydrocarbon receptor antagonist in contact with the cells (e.g., in
a fluid medium). For example, described herein are cell culture
containers comprising a Notch agonist immobilized on a solid phase
surface of the container (e.g., on an inside surface of the
container) on which hematopoietic stem/progenitor cells are
cultured, which cells are in contact with a fluid medium containing
an aryl hydrocarbon receptor antagonist. In some embodiments of the
cell culture containers or the solid phase, the Notch agonist
comprises an extracellular domain of a Delta, a Jagged, or a
Serrate protein. In one embodiment, the Notch agonist is
Delta.sup.ext-IgG. In another embodiment, the Notch agonist is
Delta.sup.ext-myc. In some embodiments of the containers or the
solid phase, Delta.sup.ext-IgG is (or has been) applied to the
solid phase at a concentration between about 0.2 and 20 .mu.g/ml,
between about 1.25 and 10 .mu.g/ml, or between about 2 and 6
.mu.g/ml. In some embodiments of the cell culture containers or the
solid phase, the aryl hydrocarbon receptor antagonist is a compound
of Formula I:
##STR00003##
in which: G.sub.1 is selected from N and CR.sub.3; G.sub.2, G.sub.3
and G.sub.4 are independently selected from CH and N; with the
proviso that at least 1 of G.sub.3 and G.sub.4 is N; with the
proviso that G.sub.1 and G.sub.2 are not both N; L is selected from
--NR.sub.5a(CH.sub.2).sub.0-3-,
--NR.sub.5aCH(C(O)OCH.sub.3)CH.sub.2--,
--NR.sub.5a(CH.sub.2).sub.2NR.sub.5b--,
--NR.sub.5a(CH.sub.2).sub.2S--,
--NR.sub.5aCH.sub.2CH(CH.sub.3)CH.sub.2--,
--NR.sub.5aCH.sub.2CH(OH)-- and --NR.sub.5aCH(CH.sub.3)CH.sub.2--;
wherein R.sub.5a and R.sub.5b are independently selected from
hydrogen and C.sub.1-4alkyl; R.sub.1 is selected from hydrogen,
phenyl, thiophenyl, furanyl, 1H-benzoimidazolyl, isoquinolinyl,
1H-imidazopyridinyl, benzothiophenyl, pyrimidinyl, 1H-pyrazolyl,
pyridinyl, 1H-imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl,
1H-pyrrolyl and thiazolyl; wherein said phenyl, thiophenyl,
furanyl, 1H-benzoimidazolyl, isoquinolinyl, 1H-imidazopyridinyl,
benzothiophenyl, pyrimidinyl, 1H-pyrazolyl, pyridinyl,
1H-imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, 1H-pyrrolyl or
thiazolyl of R.sub.1 can be optionally substituted by 1 to 3
radicals independently selected from cyano, hydroxy,
C.sub.1-4alkyl, C.sub.1-4alkoxy, halo,
halo-substituted-C.sub.1-4alkyl, halo-substituted-C.sub.1-4alkoxy,
hydroxy, amino, --C(O)R.sub.8a, --S(O).sub.0-2R.sub.8a,
--C(O)OR.sub.8a and --C(O)NR.sub.8aR.sub.8b; wherein R.sub.8a and
R.sub.8b are independently selected from hydrogen and
C.sub.1-4alkyl; with the proviso that R.sub.1 and R.sub.3 are not
both hydrogen; R.sub.2 is selected from
--S(O).sub.2NR.sub.6aR.sub.6b, --NR.sub.9aC(O)R.sub.9b,
--NR.sub.6aC(O)NR.sub.6bR.sub.6c, phenyl, 1H-pyrrolopyridin-3-yl,
1H-indolyl, thiophenyl, pyridinyl, 1H-1,2,4-triazolyl,
2-oxoimidazolidinyl, 1H-pyrazolyl,
2-oxo-2,3-dihydro-1H-benzoimidazolyl and 1H-indazolyl; wherein
R.sub.6a, R.sub.6b and R.sub.6c are independently selected from
hydrogen and C.sub.1-4alkyl; wherein said phenyl,
1H-pyrrolopyridin-3-yl, 1H-indolyl, thiophenyl, pyridinyl,
1H-1,2,4-triazolyl, 2-oxoimidazolidinyl, 1H-pyrazolyl,
2-oxo-2,3-dihydro-1H-benzoimidazolyl or 1H-indazolyl of R.sub.2 is
optionally substituted with 1 to 3 radicals independently selected
from hydroxy, halo, methyl, methoxy, amino,
--O(CH.sub.2).sub.nNR.sub.7aR.sub.7b,
--S(O).sub.2NR.sub.7aR.sub.7b, --OS(O).sub.2NR.sub.7aR.sub.7b and
--NR.sub.7aS(O).sub.2R.sub.7b; wherein R.sub.7a and R.sub.7b are
independently selected from hydrogen and C.sub.1-4alkyl; R.sub.3 is
selected from hydrogen, C.sub.1-4alkyl and biphenyl; and R.sub.4 is
selected from C.sub.1-10alkyl, prop-1-en-2-yl, cyclohexyl,
cyclopropyl, 2-(2-oxopyrrolidin-1-yl)ethyl, oxetan-3-yl,
benzhydryl, tetrahydro-2H-pyran-3-yl, tetrahydro-2H-pyran-4-yl,
phenyl, tetrahydrofuran-3-yl, benzyl,
(4-pentylphenyl)(phenyl)methyl and
1-(1-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-1H-1,2,3-triazol-4-yl)et-
hyl; wherein said alkyl, cyclopropyl, cyclohexyl,
2-(2-oxopyrrolidin-1-yl)ethyl, oxetan-3-yl, oxetan-2-yl,
benzhydryl, tetrahydro-2H-pyran-2-yl, tetrahydro-2H-pyran-3-yl,
tetrahydro-2H-pyran-4-yl, phenyl, tetrahydrofuran-3-yl,
tetrahydrofuran-2-yl, benzyl, (4-pentylphenyl)(phenyl)methyl or
1-(1-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-1H-1,2,3-triazol-4-yl)et-
hyl can be optionally substituted with 1 to 3 radicals
independently selected from hydroxy, C.sub.1-4alkyl and
halo-substituted-C.sub.1-4alkyl; or a salt thereof. In specific
embodiments, described herein are cell culture containers or the
solid phase, wherein the aryl hydrocarbon receptor antagonist is
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol (i.e., SR1).
[0062] In certain embodiments, the cell culture containers or the
solid phase described herein further comprise one or more growth
factors, two or more growth factors, or three or more growth
factors (such as any of the growth factors described herein) in
contact with the cells (e.g., in a fluid medium). In one
embodiment, the cell culture container or the solid phase of the
invention contains IL-6, Flt-3, SCF and TPO (e.g., in a fluid
medium). In another embodiment, the cell culture container or the
solid phase of the invention contains IL-6, Flt-3, SCF, TPO and
IL-3 (e.g., in a fluid medium). The cell culture containers or the
solid phase described herein, in some embodiments, further comprise
a fibronectin or a fragment thereof (e.g., CH-296) immobilized on
the solid phase (e.g., on the inside surface of the container).
2. Definitions
[0063] Although any methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present invention, the preferred methods and materials are
described. For purposes of the present invention, the following
terms are defined below.
[0064] As used herein, the terms "hematopoietic stem/progenitor
cells" or "HSPC" mean hematopoietic stem cells and/or hematopoietic
progenitor cells. The hematopoietic stem/progenitor cells can be
positive for a specific marker expressed in increased levels on
hematopoietic stem/progenitor cells relative to other types of
hematopoietic cells. For example, such markers can be, but are not
limited to CD34, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117,
CD133, CD166, HLA DR, or a combination thereof. Also, the
hematopoietic stem/progenitor cells can be negative for an
expressed marker relative to other types of hematopoietic cells.
For example, such markers can be, but are not limited to Lin, CD38,
or a combination thereof. Preferably, the hematopoietic
stem/progenitor cells are CD34.sup.+ cells. The HSPC are preferably
human. The HSPC can be derived from umbilical cord blood and/or
placental blood collected at birth, peripheral blood, bone marrow
or another source.
[0065] As used herein, the term "Enriched HSPC" refers to a cell
population enriched in hematopoietic stem/progenitor cells. The
Enriched HSPC are preferably human.
[0066] As used herein, the term "CB" refers to cord blood.
[0067] As used herein, the term "CB Stem Cells" refers to herein
interchangeably as "a CB Stem Cell Sample," refers to a cell
population enriched in hematopoietic stem/progenitor cells derived
from umbilical cord blood and/or placental blood collected at
birth. The CB Stem Cells are preferably human.
[0068] As used herein, the term "Expanded HSPC" refers to HSPC that
have been expanded in cell number by use of a combination of a
Notch agonist and an aryl hydrocarbon receptor antagonist according
to a method of the invention as disclosed herein. Preferably, such
method results in (i) an increase in the number of HSPC in an
aliquot of the sample thus expanded, or (ii) an increased number of
SCID repopulating cells determined by limiting-dilution analysis as
shown by enhanced engraftment in NOD/SCID mice infused with an
aliquot of the sample thus expanded; relative to that seen with an
aliquot of the sample that is not subjected to the expansion
method. In a specific embodiment, the enhanced engraftment in
NOD/SCID mice can be detected by detecting an increased percentage
of human CD45.sup.+ cells in the bone marrow of mice infused with
an aliquot of the expanded sample relative to mice infused with an
aliquot of the sample prior to expansion, at, e.g., 10 days, 3
weeks or 9 weeks post-infusion (see Delaney et al., 2010, Nature
Med. 16(2): 232-236). In a specific embodiment, the expansion
method results in an at least 50-, 75-, 100-, 150-, 200-, 250-,
300-, 350-, 400-, 450-, or 500-fold increase in the number of HSPC
in an aliquot of the sample expanded, and preferably is at least
100-, 200-, 300- or 500-fold increase.
[0069] As used herein, the term "Delta" refers to any of the
proteins or genes, as the case may be, of the Drosophila Delta
family or its mammalian homolog Delta (also known as "Delta-like")
family. Proteins or genes of the Delta family, as the case may be,
include, but are not limited to, Delta-1 (where mammalian Delta-1
is also known as Delta-like 1), Delta-3 (where mammalian Delta-3 is
also known as Delta-like 3), and Delta-4 (where mammalian Delta-4
is also known as Delta-like 4).
[0070] As used herein, the term "Serrate" refers to any of the
proteins or genes, as the case may be, of the Drosophila Serrate
family or its mammalian homolog, Jagged, family.
[0071] As used herein, the term "Jagged" refers to any of the
proteins or genes, as the case may be, of the Jagged family such
as, but not limited to, Jagged-1 and Jagged-2.
[0072] As used herein, the term "Alkyl" as a group and as a
structural element of other groups, for example
halo-substituted-alkyl and alkoxy, can be either straight-chained
or branched. For example, alkyl includes methyl, ethyl, propyl,
isopropyl, butyl, isobutyl, sec-butyl, t-butyl, etc. C1-4-alkoxy
includes methoxy, ethoxy, and the like. Halo-substituted alkyl
includes trifluoromethyl, pentafluoroethyl, and the like.
[0073] As used herein, the term "Aryl" refers to a monocyclic or
fused bicyclic aromatic ring assembly containing six to ten ring
carbon atoms. For example, aryl can be phenyl or naphthyl,
preferably phenyl. "Arylene" refers to a divalent radical derived
from an aryl group.
[0074] As used herein, the term "Heteroaryl" is as defined for aryl
where one or more of the ring members are a heteroatom or moiety
selected from --O--, --N, --NR--, --C(O)--, --S--, --S(O)-- or
--S(O)2-, wherein R is hydrogen, C1-4alkyl or a nitrogen protecting
group. For example, heteroaryl includes pyridyl, indolyl,
indazolyl, quinoxalinyl, quinolinyl, benzofuranyl, benzopyranyl,
benzothiopyranyl, benzo[1,3]dioxole, imidazolyl, benzo-imidazolyl,
pyrimidinyl, furanyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl,
pyrazolyl, thienyl, etc.
[0075] As used herein, the term "Cycloalkyl" refers to a saturated
or partially unsaturated, monocyclic, fused bicyclic or bridged
polycyclic ring assembly containing the number of ring atoms
indicated. For example, C.sub.3-10cycloalkyl includes cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, etc. "Heterocycloalkyl" refers
to cycloalkyl, as defined herein, provided that one or more of the
ring carbons indicated, are replaced by a moiety selected from
--O--, --N.dbd., --NR--, --C(O)--, --S--, --S(O)-- or
--S(O).sub.2--, wherein R is hydrogen, C.sub.1-4alkyl or a nitrogen
protecting group. For example, C.sub.3-8heterocycloalkyl as used in
this application to describe compounds of the invention includes
morpholino, pyrrolidinyl, piperazinyl, piperidinyl, piperidinylone,
2-Oxo-pyrrolidin-1-yl, 1,4-dioxa-8-aza-spiro[4.5]dec-8-yl, etc.
[0076] As used herein, the term "Halogen" (or halo) preferably
refers to chloro or fluoro, but can also be bromo or iodo.
[0077] Delta.sup.ext-IgG and Delta.sup.Ext-IgG are used
interchangeably herein.
[0078] Delta1.sup.ext-IgG and Delta1.sup.Ext-IgG are used
interchangeably herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0079] FIG. 1. Combination of Delta1.sup.Ext-IgG and SR-1 expand CB
HSPC in vitro compared to control. Cells were cultured in the
presence of IgG at 2.5 .mu.g/ml or Delta1.sup.Ext-IgG (2 dose
densities) and SR1. CD34 fold expansion (CD34 cell number at given
time point/CD34 cell number at day 0) calculated at 7, 14, 18, and
21 days in culture. Hatched lines represent combination groups,
solid line represents IgG control.
[0080] FIGS. 2A-2C. Combination of Delta1.sup.ext-IgG and SR-1
maintain CB HSPC in culture. CD34+CB HSPC were incubated with
Delta1.sup.ext-IgG, SR1 or the combination for 16 days prior to
transplant into immunodeficient mice. Percent (2A) CD34+ cells,
(2B) CD34.sup.+CD90+, or (2C) CD34-14+ cells after 16 days in
culture. Results are the mean of 4 combined experiments.+-.SEM. *,
** Significantly different from Delta1.sup.ext-IgG control, p=0.02
and 0.01. #, ## Significantly different from IgG+SR1 control,
p=0.01 and 0.01. All other comparisons did not achieve significant.
Non-parametric, two-tailed t-test.
[0081] FIGS. 3A, 3B. Maintenance of CD34.sup.+CD90+ cells
correlates with engraftment. CD34+ selected CB HSPC were incubated
with Delta1.sup.ext-IgG at 2.5 .mu.g/ml (circle), SR1 (light
squares) or the combination (dark squares) with Delta1.sup.ext-IgG
densities of 2.5, 5, or 10 .mu.g/ml for 16 days and transplanted
into NSG mice. SR1 and combination groups were performed in
duplicate. Percent CD34+CD90+ cells at time of transplant v. (3A)
total human engraftment at 2 weeks or (3B) total myeloid
engraftment at 2 weeks. Pearson correlation coefficient and
associated p-values displayed on graphs.
[0082] FIGS. 4A-4E. SR-1 promotes cellular expansion and
Delta1.sup.Ext-IgG blocks differentiation. CD34+ selected CB HSPC
were cultured for 16 days in the presence of SR1 and increasing
densities of Delta1.sup.Ext-IgG (0, 0.5, 2.5, or 5 .mu.g/ml) or an
IgG control. (4A) Total nucleated cell (TNC) fold expansion at 7,
10, 13, and 16 days in culture. (4B-4E) Total nucleated,
CD34.sup.-CD14.sup.+, CD34.sup.+CD90.sup.+, or CD34+CD7.sup.+ cell
numbers at day 16 of culture for SR1 with increasing densities of
Delta1.sup.Ext-IG.
[0083] FIGS. 5A-5C. Higher densities of Delta1.sup.ext-IgG result
in enhanced generation of CB HSPC. Representative experiment
demonstrating (5A) ex vivo CD34+ cell expansion and (5B, 5C) in
vivo repopulating of these expanded cells in immunodeficient mice.
All cultures were initiated with 8.5.times.10.sup.4 CD34+ cells
from the same pool of CB HSPC. Panels B and C demonstrate total
human CD45 engraftment and total marrow CD33 engraftment at 2 weeks
post-transplant. (.circle-solid.) represent individual mice with
horizontal lines demonstrating means. *,** Significantly different
from Delta1.sup.Ext-IgG 0.5, p-value=0.0144, 0.0099. #,##
Significantly different from Delta1.sup.Ext-IgG 0.5,
p-value=0.0284, 0.0165. Non-parametric, two-tailed t-test.
[0084] FIGS. 6A-6C. Delta1.sup.ext-IgG and SR-1 combined enhance
early myeloid engraftment while maintaining progenitor cells.
CD34+CB HSPC were cultured for 16 days in the presence of
Delta1.sup.ext-IgG, SR1 or the combination and transplanted into
NSG mice. Data represent results of 4 independent experiments.
(.circle-solid.) represent individual mice with horizontal lines
demonstrating means. (6A) shows total human engraftment (CD45) at 2
weeks post-transplant. *, ** Significantly different from
Delta1.sup.ext-IgG 2.5 control, p-value=0.0056, 0.0026. #, ##, Not
significantly different from IgG+SR1, p-value=0.1624, 0.1699. (6B)
shows total myeloid engraftment (CD33) at 2 weeks post-transplant.
! Significantly different from Delta1.sup.ext-IgG 2.5 control,
p-value=0.0196, !! Not significantly different from
Delta1.sup.ext-IgG 2.5 control, p-value=0.0686. &, &&
Significantly different from IgG+SR1, p-value=0.008, 0.0338. (6C)
shows total progenitor cell (CD34) engraftment 2 weeks
post-transplant. %, %% Significantly different from
Delta1.sup.ext-IgG 2.5 control, p-value=0.0012, 0.0002. +,++
Significantly different from IgG+SR1, p-value=0.0.0068, 0.0038. All
statistics represent non-parametric, two-tailed t-test.
[0085] FIGS. 7A-7D. Delta1.sup.ext-IgG and SR-1 combined
demonstrate multi-lineage repopulation of NSG mice. CD34+CB HSPC
were cultured for 16 days in the presence of Delta1.sup.ext-IgG,
IgG+SR-1, or the combination (with two densities of
Delta1.sup.ext-IgG) and the progeny of 10,000 cells transplanted
into sublethally irradiated NSG mice. (7A) Percent overall human
engraftment (CD45), (7B) Percent progenitor cell engraftment
(CD34), (7C) Percent total myeloid engraftment (CD33), (7D) Percent
total lymphoid engraftment at 2, 8, and 13 weeks post transplant.
Data are the results of a single transplant into 10 mice per
condition, with mean.+-.SEM.
[0086] FIGS. 8A, 8B. Delta1.sup.ext-IgG and SR-1 in combination may
enhance maintenance of long-term repopulating HSPC. CD34+CB HSPC
were cultured for 16 days in the presence of Delta1.sup.ext-IgG,
IgG+SR-1, or the combination (with two densities of
Delta1.sup.ext-IgG) and the progeny of 10,000 cells transplanted
into sublethally irradiated NSG mice. (.circle-solid.) represent
individual mice with mean engraftment indicated by horizontal line.
Long-term total human (8A) and progenitor cell (8B) engraftment 13
weeks post-transplant are shown. **,!, !! Significantly different
from Delta1.sup.ext-IgG 2.5 control, p-values=0.0411, 0.0417,
0.0317. * Not significantly different from Delta1.sup.ext-IgG 2.5
control, p-value=0.2436. #, ##,&,&& Not significantly
different from IgG+SR1, p-values=0.4601, 0.0638, 0.9670,
0.0952.
[0087] FIGS. 9A, 9B. Delta1.sup.Ext-IgG and SR1 in combination
enhance generation of mPBSC HSPC. CD34+ mPSBC HSPC were cultured
for 16 days in the presence of SR1 or Delta1.sup.ext-IgG (1.25 or 5
.mu.g/ml) and the progeny of 20,000 cells transplanted into NSG
mice. Week 3 total human and myeloid engraftment is shown here.
P-values are shown on the graphs. Non-expanded cells were also
transplanted from the same donor. Comparisons between non-expanded
cells at the 20,000 cell dose and Delta1.sup.ext-IG/SR1 combination
groups all achieved significance (p-values <0.05).
[0088] FIGS. 10A-10D. Delta1.sup.Ext-IgG delays differentiation of
cord blood HSPC cultured with SR1. CD34+CB HSPC were cultured for
14 days in the presence of Delta1.sup.Ext-IgG (2.5 .mu.g/ml), SR1
(750 nM), or the combination of Delta1.sup.Ext-IgG and SR1 with
increasing doses of Delta1.sup.Ext-IgG (0.5, 2.5, or 5 .mu.g/ml).
Linear regression analysis models were used to test differences in
number of cells generated across culture conditions. Base 2
logarithm transformations were applied to cell numbers to
accommodate modeling assumptions. Two-sided P values from
regression models were derived from the Wald test. No adjustments
were made for multiple comparisons. Analyses were performed using
SAS software, version 9.3 (SAS Institute, Cary, N.C.). Total
nucleated cell (TNC) number was significantly decreased with
Delta1.sup.Ext-IgG or the combination with Delta1.sup.Ext-IgG at 5
.mu.g/ml as compared to SR1 alone (p<0.001, p=0.04).
Significantly fewer TNC and CD34+ cells were generated with
increasing Delta1.sup.Ext-IgG dose: 0.5, 2.5, and 5 .mu.g/ml
(p=0.02, p=0.04). Although not significant,
Lin-CD34+CD38-CD45RA-CD90+ numbers tended to increase over
Delta1.sup.Ext-IgG dose (p=0.07) (10A). Similar numbers of common
myeloid progenitors (CMP) were generated across all
Delta1.sup.Ext-IgG doses in combination with SR1 as compared to SR1
alone (p=0.63); however, although not statistically significant,
greater percentages of these cells were maintained with higher
Delta1.sup.Ext-IgG doses (p=0.18) (10B). Although not significant,
generation of granulocyte-monocyte progenitors (GMP) and
megakaryocyte-erythrocyte progenitors (MEP) cells decreased with
higher Delta1.sup.Ext-IgG doses (10C, 10D). Significantly fewer
CD14/15+ mature myeloid cells were generated with increasing
Delta1.sup.Ext-IgG dose (p=0.005) (10C). Megakaryocyte generation
was comparable across all groups with suggestion of greater
percentage of these cells in Delta1.sup.Ext-IgG containing groups
(10D). Results shown are means.+-.s.e.m. from 5 independent
experiments.
[0089] FIGS. 11A-11F. Delta1.sup.Ext-IgG and SR1 in combination
enhance generation of early progenitor and myeloid repopulating
cells. CD34+ selected CB HSPC were cultured for 16 days in the
presence of Delta1.sup.Ext-IgG 2.5 .mu.g/ml, IgG 2.5 .mu.g/ml with
SR1 or the combination of SR1 with Delta1.sup.Ext-IgG 5 .mu.g/ml.
The cultured progeny of 10,000 starting CD34+ cells were
transplanted into NOD-SCID IL-2R.gamma.-null mice (NSG) and early
and late repopulating capability assessed at 2 weeks by bone marrow
aspirates and 12-14 weeks by bone marrow harvests. Engraftment was
assessed by immunofluorescence analysis for early myeloid
repopulation (percent CD45+CD33+) and progenitor repopulation
(percent CD45+CD34+) (11A, 11B). Progressive maturation of myeloid
precursors was assessed in 3 cell populations: percent
CD45+CD34+CD33-, CD45+CD34+CD33+ and CD45+CD34-CD33+(11C). Early
monocyte/granulocyte (percent human CD45+CD14+CD15+) (11D) and
early B-lymphoid repopulation (CD45+CD19+CD33-) were assessed
(11E). All groups demonstrated multi-lineage engraftment with
longer-term primary transplantation (11F) with enhanced total human
and B-lymphoid engraftment in the combination group (11F). All
p-values represent non-parametric two-tailed student t-tests,
GraphPad software and are displayed on graphs with lines denoting
comparisons. Results shown represent 4 independent experiments.
DETAILED DESCRIPTION OF THE INVENTION
[0090] The present invention provides kits, compositions and
methods for expanding precursor cells, such as hematopoietic
stem/progenitor cells, and providing hematopoietic function to a
human patient in need thereof by administering the expanded
hematopoietic stem/progenitor cells to the patient. In particular,
the present invention relates to methods, kits and compositions for
expanding hematopoietic stem/progenitor cells and providing
hematopoietic function to human patients in need thereof, that use
a Notch agonist, such as any of the Notch agonists described in
U.S. Pat. No. 7,399,633 (which is incorporated herein by reference
in its entirety), in combination with an aryl hydrocarbon receptor
antagonist, such as any of the aryl hydrocarbon receptor
antagonists described in U.S. Patent Publication No. 2010/0183564
(which is incorporated by reference herein in its entirety).
[0091] The inventors of the present invention found that combining
the compositions and methodology described in U.S. Patent
Publication No. 2004/0067583 (now U.S. Pat. No. 7,399,633) with the
compositions and methodology described in U.S. Patent Publication
No. 2010/0183564 results in an additive and/or synergistic effect.
Specifically, the inventors combined a Notch agonist and an aryl
hydrocarbon receptor antagonist for use in expansion of HSPC and
demonstrated that this combination leads to an additive and/or
synergistic effect on HSPC ex vivo expansion and in vivo
repopulation/engraftment. The compositions and methodology for HSPC
expansion described in U.S. Patent Publication No. 2004/0067583,
U.S. Pat. No. 7,399,633 and U.S. Patent Publication No.
2010/0183564 are incorporated by reference herein in their
entireties.
Hematopoietic stem/progenitor cells are fundamental for stem cell
transplantation and gene therapy uses; however, historically ex
vivo expansion attempts of HSPC have been insufficient for
appreciable clinical application. Prior to the present invention,
it was not appreciated that a combination of a Notch agonist and an
aryl hydrocarbon receptor antagonist could be used for HSPC
expansion and for subsequent administration of such expanded HSPC
to patients in need thereof. In particular, it was not appreciated
that the disclosed combination of agents results in synergistic or
additive effects on ex vivo expansion and in vivo repopulation. The
present invention takes advantage of the prompt short-term and
long-term hematopoietic benefit provided by HSPC expanded using a
combination of a Notch agonist and an aryl hydrocarbon receptor
antagonist.
[0092] Specifically, and without being limited by any particular
mechanism of action, the inventors of the present invention have
discovered that use of a Notch agonist capable of blocking cellular
differentiation and use of an aryl hydrocarbon receptor antagonist
capable of promoting cellular expansion, at least in part by
blocking cellular differentiation, leads to generation of greater
numbers of HSPC capable of in vivo repopulation in NOD-SCID
IL-2R.gamma.-null mice (NSG) compared to either approach alone.
These findings suggest a novel approach to ex vivo expansion of
HSPC by targeting these different aspects of stem cell self-renewal
for additive and/or synergistic effect.
[0093] A potential model of enhancing HSPC expansion by targeting
different pathways in HSPC self-renewal and differentiation, is
that Notch2 affects HSPC self-renewal by blocking differentiation
into multi-potent progenitors (MPP) and myeloid/monocytic (M) cell
lineage, while SR1 promotes HSPC self-renewal likely, at least in
part, by preventing cellular differentiation. Notch1 promotes T
cell (T) differentiation versus B cell (B) differentiation.
[0094] Recent culture strategies have led to ex vivo expansion of
CB HSPC with enhanced in vivo short-term repopulating abilities
(see Delaney C, Heimfeld S, Brashem-Stein C, Voorhies H, Manger R
L, Bernstein I D. Nat Med. 2010; 16(20): 232-237; and Boitano A E,
Wang J, Romeo R, et al. Science. 2010; 329(5997): 1345-1348).
However, by combining two culture approaches, Delta.sup.ext-IgG, a
Notch pathway ligand capable of blocking cellular differentiation,
and StemRegenin1 (SR1), an aryl hydrocarbon receptor antagonist
capable of promoting cellular expansion also, at least in part, by
blocking cellular differentiation, the inventors have been able to
generate greater numbers of HSPC capable of in vivo short-term
repopulation in NOD-SCID IL-2R.gamma.-null mice (NSG) compared to
either approach alone.
[0095] Delayed neutrophil engraftment remains a challenge in CB
transplantation with time to engraftment of 3 weeks even in the
setting of double cord blood transplantation (Barker et al., 2005,
Blood. 105 (3): 1343-1347). Recent approaches in phase I trials
expanding CB HSPC on the immobilized Notch ligand
Delta1.sup.Ext-IgG or on a layer of mesenchymal stem cells have
demonstrated over one week reduction in time to engraftment when
expanded cells are infused with non-manipulated CB units (de Lima
et al., 2010, Blood. 116, Abstract 362; Delaney et al., 2010, Nat
Med. 16(20): 232-237). The inventors of this invention have
observed increased generation of repopulating cells capable of
early myeloid repopulation in NSG mice as compared to
Delta1.sup.Ext-IgG or SR1 alone, suggesting this combination may
further enhance generation of short-term repopulating cells. HSPC
expanded using the compositions and methods described herein could
have clear clinical benefit since they can further reduce time to
neutrophil engraftment in transplant recipients.
[0096] Further, the inventors have found that culturing of HSPC in
the presence of both a Notch agonist and an aryl hydrocarbon
receptor antagonist results in greater maintenance of immature
hematopoietic precursors both in vitro and in vivo. In particular,
the inventors have observed enhanced maintenance of
CD34.sup.+CD90.sup.+ cells when CD34.sup.+ CB HSPC were cultured in
the presence of Delta.sup.Ext-IgG and SR1. This effect was
particularly pronounced with higher densities of the Notch agonist,
Delta.sup.ext-IgG, such as 2.5 and 5 .mu.g/ml Delta.sup.ext-IgG.
This is in notable contrast to the results using Delta.sup.ext-IgG
alone, which showed maximal generation of long-term repopulating
cells with lower Delta.sup.ext-IgG densities (see Delaney C,
Varnum-Finney B, Aoyama K, Brashem-Stein C, Bernstein I D. Blood.
2005; 106(9): 2693-2699). It is possible that induction of more
Notch signaling to block differentiation is required in the
combination approach given the significant cellular expansion
induced by SR1.
[0097] Further, the inventors have observed that expansion of HSPC
in the presence of both a Notch agonist and an aryl hydrocarbon
receptor antagonist results in greater maintenance of immature
hematopoietic precursors in vitro, while decreasing total nucleated
cell (TNC) and total CD34.sup.+ cell generation, relative to the
HSPC expanded in the presence of an aryl hydrocarbon antagonist
alone. In particular, the inventors have observed that culturing of
CB HSPC in the presence of SR1 and increasing concentrations of
Delta.sup.Ext-IgG (0.5, 2.5 and 5 .mu.g/ml Delta.sup.ext-IgG)
significantly decreases TNC and total CD34.sup.+ cell number while
maintaining or enhancing
Lin.sup.-CD34.sup.+CD38.sup.-CD45RA.sup.-CD90.sup.+ cell number
relative to CB HSPC cultured in the presence of an aryl hydrocarbon
antagonist alone. In addition, the inventors have found that
culturing of CB HSPC in the presence of SR1 and increasing
concentrations of Delta.sup.Ext-IgG (0.5, 2.5 and 5 .mu.g/ml
Delta.sup.ext-IgG) leads to decreased generation of more mature
myeloid cell populations (CD14.sup.+ and CD15.sup.+ cells) relative
to CB HSPC cultured in the presence of an aryl hydrocarbon
antagonist alone.
[0098] Unexpectedly, the inventors have found that expansion of
HSPC in the presence of both a Notch agonist and an aryl
hydrocarbon receptor antagonist results in generation of more cells
capable of greater early engraftment (such as cells capable of
rapidly repopulating bone marrow with early myeloid and progenitor
cells) than expansion of HSPC in the presence of an aryl
hydrocarbon receptor antagonist alone, despite the lesser number of
total CD34.sup.+ cells generated in the presence of both a Notch
agonist and an aryl hydrocarbon receptor antagonist relative to the
number generated in the presence of an aryl hydrocarbon receptor
antagonist alone.
[0099] Furthermore, the inventors have found that expansion of HSPC
in the presence of the combination of a Notch agonist and an aryl
hydrocarbon receptor antagonist results in generation of more cells
capable of short-term in vivo repopulation than expansion of HSPC
in the presence of a Notch agonist alone or an aryl hydrocarbon
receptor antagonist alone. In particular, the inventors have found
that expansion of HSPC in the presence of the combination of a
Notch agonist (specifically, Delta.sup.ext-IgG) and an aryl
hydrocarbon receptor antagonist (specifically, SR1) results in
early generation of a higher percentage of early myeloid cells
(CD45+CD33+), early progenitor cell (CD45+CD34+), CD45+CD34+CD33-
cells, CD45+CD34+CD33+ cells, and early monocyte/granulocyte cells
(CD45+CD14+CD15+), for short-term in vivo repopulation, than
expansion of HSPC in the presence of a Notch agonist alone and/or
an aryl hydrocarbon receptor antagonist alone.
[0100] Furthermore, the inventors have found that expansion of HSPC
in the presence of the combination of a Notch agonist and an aryl
hydrocarbon receptor antagonist results in generation of cells with
multi-lineage potential capable of long-term in vivo repopulation
(in addition to generation of cells capable of short-term in vivo
repopulation). As detailed in the examples hereinafter, the
inventors have discovered that culturing HSPC in the presence of a
combination of a Notch agonist and an aryl hydrocarbon receptor
antagonist leads to generation of greater numbers of HSPC capable
of in vivo long-term, multi-lineage repopulation in NOD-SCID
IL-2R.gamma.-null mice (NSG) than either approach alone. As
described herein, the inventors have found that culturing HSPC in
the presence of a combination of a Notch agonist (specifically,
Delta.sup.ext-IgG) and an aryl hydrocarbon receptor antagonist
(specifically, SR1) leads to increase in long-term B-lymphocyte
engraftment (i.e., engraftment of CD45.sup.+CD19.sup.+CD33.sup.-
cells) in NOD-SCID IL-2R.gamma.-null mice (NSG) than either
approach alone. Thus, the described combination, can be used for
both short-term and/or long-term clinical benefits. This is
significant because, despite advances in generation of short-term
repopulating cells capable of enhanced early myeloid repopulation,
significant generation of HSPC capable of sustained long-term in
vivo repopulation in immunodeficient mice or humans remained
elusive. Current expansion approaches in early clinical trials
require administration of non-manipulated CB cells along with the
cultured product. While some patients have demonstrated longer-term
repopulation with expanded cells, most sustained long-term
engraftment in these patients is from the non-manipulated CB unit.
The expansion approach described herein, capable of generating both
short- and long-term repopulating cells, can provide a significant
benefit by allowing administration of a single, expanded HSPC unit
(e.g., a single, expanded CB unit) to recipients (e.g., without
administration of non-manipulated HSPC such as non-manipulated CB
cells). However, administration of the non-manipulated CB cells
along with the Expanded HSPC of the invention is also contemplated
for additional benefits.
[0101] In addition, the inventors of this application have
discovered that the combination of a Notch agonist and an aryl
hydrocarbon receptor antagonist is effective to expand peripheral
blood stem cells (e.g., mobilized peripheral blood stem cells) and
to generate cells capable of enhanced in vivo repopulation. This
effect was significantly more pronounced when the combination of
agents described herein was used than when an aryl hydrocarbon
receptor antagonist alone was used. The inventors demonstrated a
similar effect on maintenance of immature progenitor cells and
enhanced engraftment when mPBSC (i.e., mobilized peripheral blood
stem cells) were cultured in the presence of SR1 and
Delta.sup.Ext-IgG, as that obtained in CB HSPC in the presence of
SR1 and Delta.sup.Ext-IgG. This is a significant finding
considering that previous attempts to expand mPBSC ex vivo had
generated no difference in engraftment. This data show that
peripheral blood stem cells can be used as a source of HSPC for ex
vivo expansion and subsequent therapeutic use for short-term and
long-term in vivo repopulation in patients. It also suggests that
not only CB HSPC but multiple sources of stem/progenitor cells can
be used for effective expansion and engraftment using the
combination of agents described herein.
[0102] Infusion of the Expanded HSPC of the invention can provide a
therapeutic benefit for patients with immunodeficient and
autoimmune diseases, diverse hematopoietic disorders, or those who
had undergone chemotherapy. The use of chemotherapeutic agents can
be immunosuppressive and/or highly myelosuppressive, leading to
prolonged neutropenia, often resulting in frequent infections in
treated patients. In some aspects of the invention, the infusion of
HSPC expanded in accordance with the methods described herein
abrogate or ameliorate neutropenia in a patient. In one aspect, the
Expanded HSPC of the invention abrogate or ameliorate neutropenia
resulting from chemotherapy, preventing infectious complications,
and facilitating host hematopoietic recovery post-chemotherapy.
[0103] 2.1 HSPC Culture/Expansion
[0104] In a preferred embodiment of the present invention, HSPC are
expanded by culturing the cells in the presence of an agonist of
Notch function and an aryl hydrocarbon receptor antagonist for a
given period of time. One of more growth factors or cytokines can
also be added during cell culture for a given period of time.
Culturing HSPC can take place under any suitable culture
medium/conditions described in U.S. Patent Publication No.
2004/0067583, U.S. Pat. No. 7,399,633, or U.S. Patent Publication
No. 2010/0183564 (each of which is incorporated by reference herein
in their entireties), or known in the art (see, e.g., Freshney
Culture of Animal Cells, Wiley-Liss, Inc., New York, N.Y. (1994)).
The time in culture is a time sufficient to produce an Expanded
HSPC population, as defined herein. For example, HSPC can be
cultured in a serum-free medium in the presence of an agonist of
Notch function, an aryl hydrocarbon receptor antagonist, and,
optionally, one or more growth factors or cytokines for 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 32, or 35 days; or, preferably, for at
least 10 or at least 15 days or at least 16 days. Optionally, at
any point during the culturing period, the culture medium can be
replaced with fresh medium or fresh medium can be added. In one
embodiment, the fresh culture medium is added every 3 or 4
days.
[0105] 2.2 Notch Agonists
[0106] The present invention contemplates use of a Notch agonist.
Contemplated for use in the present invention are any of the Notch
agonists disclosed in U.S. Pat. No. 7,399,633, incorporated by
reference herein in its entirety, or any other Notch agonists known
in the art (also, the disclosure of Notch agonists in sec. 5.1 of
U.S. Pat. No. 7,399,633 is specifically incorporated herein by
reference in its entirety). The description of Notch agonists
provided herein is largely found in sec. 5.1 of U.S. Pat. No.
7,399,633.
[0107] A Notch agonist is an agent that promotes, i.e., causes or
increases, activation of Notch pathway function. As used herein,
"Notch pathway function" shall mean a function mediated by the
Notch signaling (signal transduction) pathway, including but not
limited to nuclear translocation of the intracellular domain of
Notch, nuclear translocation of RBP-JK or its Drosophila homolog
Suppressor of Hairless; activation of bHLH genes of the Enhancer of
Split complex, e.g., Mastermind; activation of the HES-1 gene or
the KBF2 (also called CBF1) gene; inhibition of Drosophila
neuroblast segregation; and binding of Notch to a Delta protein, a
Jagged/Serrate protein, Fringe, Deltex or RBP-J.kappa./Suppressor
of Hairless, or homologs or analogs thereof. See generally the
review article by Kopan et al., 2009, Cell 137:216-233 for a
discussion of the Notch signal transduction pathway and its effects
upon activation; see also Jarriault et al., 1998, Mol. Cell. Biol.
18:7423-7431.
[0108] Notch activation is carried out by exposing a cell to a
Notch agonist. The agonist of Notch can be but is not limited to a
soluble molecule, a molecule that is recombinantly expressed on a
cell-surface, a molecule on a cell monolayer to which the HSPC are
exposed, or a molecule immobilized on a solid phase. Exemplary
Notch agonists are the extracellular binding ligands Delta and
Serrate (e.g., Jagged) which bind to the extracellular domain of
Notch and activate Notch signal transduction, or a fragment (e.g.,
the extracellular domain) of Delta or Serrate (e.g., Jagged) that
binds to the extracellular domain of Notch and activates Notch
signal transduction. Nucleic acid and amino acid sequences of Delta
family members and Serrate family members (e.g., Jagged family
members) have been isolated from several species, including human,
are known in the art, and are disclosed in International Patent
Publication Nos. WO 93/12141, WO 96/27610, WO 97/01571, Gray et
al., 1999, Am. J. Path. 154:785-794. Jagged is a mammalian
homologue of Serrate. As used in this application, Serrate shall
encompass Jagged unless the context indicates otherwise.
[0109] In a specific embodiment, the Notch agonist is an
extracellular domain of a Delta protein or a Serrate (e.g., Jagged)
protein, or a Notch-binding region thereof, fused to a different
protein (a fusion partner). The Notch agonist is preferably
immobilized on a solid support. In certain embodiments, the Notch
agonist is an immobilized fragment of a Delta or a Serrate (e.g.,
Jagged) protein consisting of the extracellular domain of the
protein fused to a myc epitope tag (Deltaext-myc or Serrateext-myc,
respectively) or an immobilized fragment of a Delta or a Serrate
(e.g., Jagged) protein consisting of the extracellular domain of
the protein fused to the Fc portion of IgG (Delta.sup.ext-IgG or
Serrate.sup.ext-IgG, respectively). In preferred embodiments, the
Notch agonist is an immobilized fragment of a Delta or a Serrate
(e.g., Jagged) protein consisting of the extracellular domain of
the Delta or Serrate fused to the Fc domain of human IgG1. In
preferred embodiments, a Delta protein is a human or rodent Delta
protein, and a Serrate or Jagged protein is a human or rodent
Jagged protein.
[0110] Notch agonists of the present invention include but are not
limited to Notch proteins and analogs and derivatives (including
fragments) thereof; proteins that are other elements of the Notch
pathway and analogs and derivatives (including fragments) thereof;
activating antibodies thereto and fragments or other derivatives of
such antibodies containing the binding region thereof; nucleic
acids encoding the proteins and derivatives or analogs; as well as
proteins and derivatives and analogs thereof which bind to or
otherwise interact with Notch proteins or other proteins in the
Notch pathway such that Notch pathway activity is promoted. Such
agonists include but are not limited to Notch proteins and
derivatives thereof comprising the intracellular domain, Notch
nucleic acids encoding the foregoing, and proteins comprising the
Notch-interacting domain of Notch ligands (e.g., the extracellular
domain of Delta or Serrate). Other agonists include but are not
limited to RBPJ.kappa./Suppressor of Hairless or Deltex. Fringe can
be used to enhance Notch activity, for example in conjunction with
Delta protein. These proteins, fragments and derivatives thereof
can be recombinantly expressed and isolated or can be chemically
synthesized.
[0111] In another specific embodiment, the Notch agonist is a cell
which recombinantly expresses a protein or fragment or derivative
thereof, which agonizes Notch. The cell expresses the Notch agonist
in such a manner that it is made available to HSPC in which Notch
signal transduction is to be activated, e.g., it is secreted,
expressed on the cell surface, etc.
[0112] In yet another specific embodiment, the agonist of Notch is
a peptidomimetic or peptide analog or organic molecule that binds
to a member of the Notch signaling pathway. Such an agonist can be
identified by binding assays selected from those known in the art,
for example the cell aggregation assays described in Rebay et al.,
1991, Cell 67:687-699 and in International Patent Publication No.
WO 92/19734.
[0113] In a preferred embodiment the agonist is a protein
consisting of at least a fragment of a protein encoded by a
Notch-interacting gene which mediates binding to a Notch protein or
a fragment of Notch, which fragment of Notch contains the region of
Notch responsible for binding to the agonist protein, e.g.,
epidermal growth factor-like repeats 11 and 12 of Notch. Notch
interacting genes, as used herein, shall mean the genes Notch,
Delta, Serrate, Jagged, RBPJ.kappa., Suppressor of Hairless and
Deltex, as well as other members of the Delta/Serrate family or
Deltex family which may be identified by virtue of sequence
homology or genetic interaction and more generally, members of the
"Notch cascade" or the "Notch group" of genes, which are identified
by molecular interactions (e.g., binding in vitro, or genetic
interactions (as depicted phenotypically, e.g., in Drosophila).
Exemplary fragments of Notch-binding proteins containing the region
responsible for binding to Notch are described in U.S. Pat. Nos.
5,648,464; 5,849,869; and 5,856,441.
[0114] The Notch agonists utilized by the methods of the invention
can be obtained commercially, produced by recombinant expression,
or chemically synthesized.
[0115] In a specific embodiment, the Notch agonist is a dominant
active mutant of a Notch protein (e.g., a Notch receptor lacking
the extracellular, ligand binding domain). In another embodiment,
the Notch agonist is not a dominant active mutant of a Notch
protein.
[0116] In some embodiments, the Notch agonist is recombinantly
expressed from a nucleic acid introduced into the HSPC. Methods
that can be used for recombinantly expressing a Notch agonist are
described in sec. 5.3 of U.S. Pat. No. 7,399,633, which is
specifically incorporated by reference herein in its entirety. In
particular embodiments, the Notch agonist is a Notch protein (e.g.,
human or murine Notch-1, Notch-2, Notch-3 or Notch-4) consisting
essentially of the intracellular domain of the Notch protein
expressed recombinantly in HSPC. In specific embodiments, the
recombinantly expressed Notch agonist is a chimeric Notch protein
which comprises the intracellular domain of Notch receptor and the
extracellular domain of another ligand-binding surface receptor
(e.g., the EGF receptor). In such embodiments, the Notch pathway
can be activated by exposure to a ligand of such another
ligand-binding surface receptor (e.g., EGF). The recombinantly
expressed Notch agonist can be expressed by HSPC from an inducible
promoter. In certain embodiments, the expression of the nucleic
acid encoding the Notch agonist is under the control of Cre/Lox
system or FLP/FRT system. In one embodiment, the Notch agonist is
flanked by Cre sites.
[0117] In a specific embodiment, exposure of the cells to a Notch
agonist is not done by incubation with other cells recombinantly
expressing a Notch ligand on the cell surface (although in other
embodiments, this method can be used), but rather is by exposure to
a cell-free Notch ligand, e.g., incubation with a cell-free ligand
of Notch, which ligand is immobilized on the surface of a solid
phase, e.g., immobilized on the surface of a tissue culture
dish.
[0118] In specific embodiments, Notch activity is promoted by the
binding of Notch ligands (e.g., Delta ligands, Serrate ligands) to
the extracellular portion of the Notch receptor. Notch signaling
appears to be triggered by the physical interaction between the
extracellular domains of Notch and its ligands that are either
membrane-bound on adjacent cells or immobilized on a solid surface.
Full length ligands are agonists of Notch, as their expression on
one cell triggers the activation of the pathway in the neighboring
cell which expresses the Notch receptor. Soluble truncated Delta or
Serrate (e.g., Jagged) molecules, comprising the extracellular
domains of the proteins or Notch-binding portions thereof,
preferably fused to a different protein, that have been immobilized
on a solid surface, such as a tissue culture plate, are
particularly preferred Notch pathway agonists. Such soluble
proteins can be immobilized on a solid surface by an antibody or
interacting protein, for example an antibody directed to an epitope
tag with which a Delta or a Serrate is expressed as a fusion
protein (e.g., a myc epitope tag, which is recognized by the
antibody 9E10) or a protein which interacts with an epitope tag
with which a Delta or a Serrate is expressed as a fusion protein
(e.g., an immunoglobulin epitope tag, which is bound by Protein A).
Immobilization can be by any method known in the art (see, e.g.,
Section 6.8).
[0119] In another specific embodiment, and as described in U.S.
Pat. No. 5,780,300 to Artavanis-Tsakonas et al., Notch agonists
include reagents that promote or activate cellular processes that
mediate the maturation or processing steps required for the
activation of Notch or a member of the Notch signaling pathway,
such as the furin-like convertase required for Notch processing,
Kuzbanian, the metalloprotease-disintegrin (ADAM) thought to be
required for the activation of the Notch pathway upstream or
parallel to Notch (Schlondorff and Blobel, 1999, J. Cell Sci.
112:3603-3617), or, more generally, cellular trafficking and
processing proteins such as the rab family of GTPases required for
movement between cellular compartments (for a review on Rab
GTPases, see Olkkonen and Stenmark, 1997, Int. Rev. Cytol.
176:1-85). The agonist can be any molecule that increases the
activity of one of the above processes, such as a nucleic acid
encoding a furin, Kuzbanian or rab protein, or a fragment or
derivative or dominant active mutant thereof, or a peptidomimetic
or peptide analog or organic molecule that binds to and activates
the function of the above proteins.
[0120] U.S. Pat. No. 5,780,300 further discloses classes of Notch
agonist molecules (and methods of their identification) which can
be used to activate the Notch pathway in the practice of the
present invention, for example molecules that trigger the
dissociation of the Notch ankyrin repeats with RBP-J.kappa.,
thereby promoting the translocation of RBP-JK from the cytoplasm to
the nucleus.
[0121] 2.3 Aryl Hydrocarbon Receptor Antagonists
[0122] In addition to the Notch agonist, the present invention
contemplates use of an aryl hydrocarbon receptor antagonist. Such
agent may include any compound capable of down-regulating the
activity and/or expression of aryl hydrocarbon receptor and/or a
downstream effector of aryl hydrocarbon receptor pathway (e.g., an
agent capable of down-regulating the protein expression of aryl
hydrocarbon receptor and/or the protein expression of one or more
downstream effectors of aryl hydrocarbon receptor). Contemplated
for use in the present invention are any of the compounds disclosed
in U.S. Patent Application No. 2010/0183564, which is incorporated
by reference herein in its entirety (also, the disclosure of
compounds at pages 1-9 and 21-67 of U.S. Patent Application No.
2010/0183564 is specifically incorporated by reference herein in
its entirety). The description of aryl hydrocarbon receptor
antagonists provided herein is largely found in U.S. Patent
Publication No. 2010/0183564.
[0123] In certain embodiments, an aryl hydrocarbon receptor
antagonist is an organic compound, a small interference RNA (siRNA)
molecule capable of down-regulating the expression of aryl
hydrocarbon receptor, or an antisense oligonucleotide capable of
down-regulating the expression of aryl hydrocarbon receptor (see
U.S. Patent Publication No. 2010/0183564).
[0124] In certain embodiments, an aryl hydrocarbon receptor
antagonist is a compound of Formula I:
##STR00004##
in which: G.sub.1 is selected from N and CR3; G.sub.2, G.sub.3 and
G.sub.4 are independently selected from CH and N; with the proviso
that at least 1 of G.sub.3 and G.sub.4 is N; with the proviso that
G.sub.1 and G.sub.2 are not both N; L is selected from
--NR.sub.5a(CH.sub.2).sub.0-3-- (0-3 herein means 0, 1, 2 or 3),
--NR.sub.5aCH(C(O)OCH.sub.3)CH.sub.2--,
--NR.sub.5a(CH.sub.2).sub.2NR.sub.5b--,
--NR.sub.5a(CH.sub.2).sub.2S--,
--NR.sub.5aCH.sub.2CH(CH.sub.3)CH.sub.2--,
--NR.sub.5aCH.sub.2CH(OH)-- and --NR.sub.5aCH(CH.sub.3)CH.sub.2--;
wherein R.sub.5a and R.sub.5b are independently selected from
hydrogen and C1-4alkyl; R.sub.1 is selected from hydrogen, phenyl,
thiophenyl, furanyl, 1H-benzoimidazolyl, isoquinolinyl,
1H-imidazopyridinyl, benzothiophenyl, pyrimidinyl, 1H-pyrazolyl,
pyridinyl, 1H-imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl,
1H-pyrrolyl and thiazolyl; wherein said phenyl, thiophenyl,
furanyl, 1H-benzoimidazolyl, isoquinolinyl, 1H-imidazopyridinyl,
benzothiophenyl, pyrimidinyl, 1H-pyrazolyl, pyridinyl,
1H-imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, 1H-pyrrolyl or
thiazolyl of R.sub.1 can be optionally substituted by 1 to 3
radicals independently selected from cyano, hydroxy,
C.sub.1-4alkyl, C.sub.1-4alkoxy, halo,
halo-substituted-C.sub.1-4alkyl, halo-substituted-C.sub.1-4alkoxy,
hydroxy, amino, --C(O)R.sub.8a, --S(O).sub.0-2R.sub.8a,
--C(O)OR.sub.8a and --C(O)NR.sub.8aR.sub.8b; wherein R.sub.8a and
R.sub.8b are independently selected from hydrogen and
C.sub.1-4alkyl; with the proviso that R.sub.1 and R.sub.3 are not
both hydrogen; R.sub.2 is selected from
--S(O).sub.2NR.sub.6aR.sub.6b, --NR.sub.9aC(O)R.sub.9b,
--NR.sub.6aC(O)NR.sub.6bR.sub.6c, phenyl, 1H-pyrrolopyridin-3-yl,
1H-indolyl, thiophenyl, pyridinyl, 1H-1,2,4-triazolyl,
2-oxoimidazolidinyl, 1H-pyrazolyl,
2-oxo-2,3-dihydro-1H-benzoimidazolyl and 1H-indazolyl; wherein
R.sub.6a, R.sub.6b and R.sub.6c are independently selected from
hydrogen and C.sub.1-4alkyl; wherein said phenyl,
1H-pyrrolopyridin-3-yl, 1H-indolyl, thiophenyl, pyridinyl,
1H-1,2,4-triazolyl, 2-oxoimidazolidinyl, 1H-pyrazolyl,
2-oxo-2,3-dihydro-1H-benzoimidazolyl or 1H-indazolyl of R.sub.2 is
optionally substituted with 1 to 3 radicals independently selected
from hydroxy, halo, methyl, methoxy, amino,
--O(CH.sub.2).sub.nNR.sub.7aR.sub.7b,
--S(O).sub.2NR.sub.7aR.sub.7b, --OS(O).sub.2NR.sub.7aR.sub.7b and
--NR.sub.7aS(O).sub.2R.sub.7b; wherein R.sub.7a and R.sub.7b are
independently selected from hydrogen and C.sub.1-4alkyl; R.sub.3 is
selected from hydrogen, C.sub.1-4alkyl and biphenyl; and R.sub.4 is
selected from C.sub.1-10alkyl, prop-1-en-2-yl, cyclohexyl,
cyclopropyl, 2-(2-oxopyrrolidin-1-yl)ethyl, oxetan-3-yl,
benzhydryl, tetrahydro-2H-pyran-3-yl, tetrahydro-2H-pyran-4-yl,
phenyl, tetrahydrofuran-3-yl, benzyl,
(4-pentylphenyl)(phenyl)methyl and
1-(1-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-1H-1,2,3-triazol-4-yl)et-
hyl; wherein said alkyl, cyclopropyl, cyclohexyl,
2-(2-oxopyrrolidin-1-yl)ethyl, oxetan-3-yl, oxetan-2-yl,
benzhydryl, tetrahydro-2H-pyran-2-yl, tetrahydro-2H-pyran-3-yl,
tetrahydro-2H-pyran-4-yl, phenyl, tetrahydrofuran-3-yl,
tetrahydrofuran-2-yl, benzyl, (4-pentylphenyl)(phenyl)methyl or
1-(1-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-1H-1,2,3-triazol-4-yl)et-
hyl can be optionally substituted with 1 to 3 radicals
independently selected from hydroxy, C.sub.1-4alkyl and
halo-substituted-C.sub.1-4alkyl; or an N-oxide derivative, prodrug
derivative, protected derivative, individual isomer or mixture of
isomers thereof; or a salt (preferably a pharmaceutically
acceptable salt) or solvate (e.g. hydrate) of such compound.
[0125] Examples of the compounds of Formula I are depicted in
Tables 1 below.
[0126] In a specific embodiment, an aryl hydrocarbon receptor
antagonist is a pharmaceutically acceptable salt of a compound of
Formula I.
[0127] In one embodiment, an aryl hydrocarbon receptor antagonist
is
4-2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)pheno-
l ("SR1"). The structure of SR1 is provided below:
##STR00005##
[0128] Examples of aryl hydrocarbon receptor antagonists that can
be used in the compositions and methods of the present invention
include, but are not limited to: SR1,
4-(2-(Pyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol,
4-(2-(9-Isopropyl-2-(2-methyl-1H-imidazol-1-yl)-9H-purin-6-ylamino)ethyl)-
phenol,
4-(2-(2-(5-Chloropyridine-3-yl)-9-isopropyl-9H-purin-6-ylamino)eth-
yl)phenol,
4-(2-(6-(5-Fluoropyridin-3-yl)-1-isopropyl-1H-pyrazolo[3,4-d]py-
rimidin-4-ylamino)ethyl)phenol,
4-(2-(2-(5-Fluoropyridin-3-yl)-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-4-y-
lamino)ethyl)phenol,
(R)-4-2-(2-(benzo[b]thiophen-3-yl)-9-tetrahydrofuran-3-yl)-9H-purin-6-yla-
mino)ethyl)phenol,
2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-6-yl-
amino)ethyl)phenol,
(R)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin--
9-yl)propan-1-ol,
(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin--
9-yl)propan-1-ol,
4-(2-(6-(5-Fluoropyridin-3-yl)-1-isopropyl-1H-imidazol[4,5-c]pyridin-4-yl-
amino)ethyl)phenol,
4-(2-(5-(5-Fluoropyridin-3-yl)-3-isopropyl-3H-imidazo[4,5-c]pyridin-7-yla-
mino)ethyl)phenol,
3-2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-1H-i-
ndol-5-yl
6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thienol{3,4-d}imidazol-4-yl-
)pentanamido)hexonoate, and
3-2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-1H-i-
ndol-5-yl 6-(tert-butoxycarbonylamino)hexonoate. U.S. Patent
Publication 2010/0183564 describes and exemplifies these and other
aryl hydrocarbon receptor antagonists that can be used in the
compositions and methods of this invention (see, e.g., "Description
of the Preferred Embodiments" section, and in particular,
"Examples" section, Table 1 and Table 2, the disclosures of which
are specifically incorporated by reference herein in their
entireties).
In certain embodiments, an aryl hydrocarbon receptor antagonist is
one or more of SR1,
4-(2-(Pyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol,
4-(2-(9-Isopropyl-2-(2-methyl-1H-imidazol-1-yl)-9H-purin-6-ylamino)ethyl)-
phenol,
4-(2-(2-(5-Chloropyridine-3-yl)-9-isopropyl-9H-purin-6-ylamino)eth-
yl)phenol,
4-(2-(6-(5-Fluoropyridin-3-yl)-1-isopropyl-1H-pyrazolo[3,4-d]py-
rimidin-4-ylamino)ethyl)phenol,
4-(2-(2-(5-Fluoropyridin-3-yl)-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-4-y-
lamino)ethyl)phenol,
(R)-4-2-(2-(benzo[b]thiophen-3-yl)-9-tetrahydrofuran-3-yl)-9H-purin-6-yla-
mino)ethyl)phenol,
2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-6-yl-
amino)ethyl)phenol,
(R)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin--
9-yl)propan-1-ol,
(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin--
9-yl)propan-1-ol,
4-(2-(6-(5-Fluoropyridin-3-yl)-1-isopropyl-1H-imidazol[4,5-c]pyridin-4-yl-
amino)ethyl)phenol,
4-(2-(5-(5-Fluoropyridin-3-yl)-3-isopropyl-3H-imidazo[4,5-c]pyridin-7-yla-
mino)ethyl)phenol,
3-2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-1H-i-
ndol-5-yl
6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thienol{3,4-d}imidazol-4-yl-
)pentanamido)thexonoate, and
3-2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-1H-i-
ndol-5-yl 6-(tert-butoxycarbonylamino)thexonoate.
[0129] In another embodiment, an aryl hydrocarbon receptor
antagonist is a compound of Formula Ia, Ib, Ic, Id or Ie disclosed
at pages 2-9 of U.S. Patent Application No. 2010/0183564, described
below. In yet another embodiment, an aryl hydrocarbon receptor
antagonist is any one or more of the compounds disclosed in Table I
of U.S. Patent Application No. 2010/0183564, described below.
[0130] In certain embodiments, an aryl hydrocarbon receptor
antagonist is a compound of Formula Ia, Ib, Ic, Id or Ie:
##STR00006##
in which: L is selected from --NR.sub.5a(CH.sub.2).sub.0-3-,
--NR.sub.5aCH (C(O)OCH.sub.3)CH.sub.2--,
--NR.sub.5a(CH.sub.2).sub.2NR.sub.5b--,
--NR.sub.5a(CH.sub.2).sub.2S--,
--NR.sub.5aCH.sub.2CH(CH.sub.3)CH.sub.2--,
--NR.sub.5aCH.sub.2CH(OH)-- and --NR.sub.5aCH(CH.sub.3)CH.sub.2--;
wherein R.sub.5a and R.sub.5b are independently selected from
hydrogen and C.sub.1-4alkyl; wherein the right side of the L moiety
as shown is attached to R.sub.2, for example:
--NR.sub.5a(CH.sub.2).sub.0-3--R.sub.2, --NR.sub.5aCH
(C(O)OCH.sub.3)CH.sub.2--R.sub.2,
--NR.sub.5a(CH.sub.2).sub.2NR.sub.5b--R.sub.2,
--NR.sub.5a(CH.sub.2).sub.2S--R.sub.2,
--NR.sub.5aCH.sub.2CH(CH.sub.3)CH.sub.2--R.sub.2,
--NR.sub.5aCH.sub.2CH(OH)--R.sub.2 and
--NR.sub.5aCH(CH.sub.3)CH.sub.2--R.sub.2. R.sub.1 is selected from
hydrogen, phenyl, thiophen-2-yl, thiophen-3-yl, furan-3-yl,
1H-benzo[d]imidazol-1-yl, isoquinolin-4-yl,
1H-imidazo[4,5-b]pyridin-1-yl, benzo[b]thiophen-3-yl,
pyrimidin-5-yl, 1H-pyrazol-4-yl, pyridin-2-yl, pyridin-4-yl,
1H-imidazol-1-yl, pyrrolidin-1-yl, pyrazin2-yl, pyridin-3-yl,
pyridazin-4-yl, 1H-pyrrol-2-yl and thiazol-5-yl; wherein said
phenyl, thiophen-2-yl, thiophen-3-yl, furan-3-yl,
1H-benzo[d]imidazol-1-yl, isoquinolin-4-yl,
1H-imidazo[4,5-b]pyridin-1-yl, benzo[b]thiophen-3-yl,
pyrimidin-5-yl, pyridin-2-yl, pyridin-4-yl, 1H-imidazol-1-yl,
pyrrolidin-1-yl, pyrazin-2-yl, pyridin-3-yl, pyridazin-4-yl,
1H-pyrrol-2-yl or thiazol-5-yl of R.sub.1 can be optionally
substituted by 1 to 3 radicals independently selected from cyano,
hydroxy, C.sub.1-4alkyl, C.sub.1-4alkoxy, halo,
halo-substituted-C.sub.1-4alkyl, --S(O).sub.0-2R.sub.8a and
--C(O)OR.sub.8a; wherein R.sub.8a and R.sub.8b are independently
selected from hydrogen and C.sub.1-4alkyl; with the proviso that
R.sub.1 and R.sub.3 are not both hydrogen; R.sub.2 is selected from
--NR.sub.6aC(O)NR.sub.6bR.sub.6c, phenyl,
1H-pyrrolo[2,3-b]pyridin-3-yl, 1H-pyrrolo[2,3-b]pyridin-5-yl,
1H-indol-3-yl, thiophen-3-yl, pyridin-2-yl, pyridin-3-yl,
pyridin-4-yl, 1H-1,2,4-triazol-5-yl, 2-oxoimidazolidin-1-yl,
1H-pyrazol-3-yl, 1H-pyrazol-4-yl,
2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl and 1H-indazol-3-yl;
wherein R.sub.6a, R.sub.6b and R.sub.6c are independently selected
from hydrogen and C.sub.1-4alkyl; wherein said phenyl,
1H-pyrrolo[2,3-b]pyridin-3-yl, 1H-pyrrolo[2,3-b]pyridin-5-yl,
1H-indol-3-yl, thiophen-3-yl pyridin-2-yl, pyridin-3-yl,
pyridin-4-yl, 1H-1,2,4-triazol-5-yl, 2-oxoimidazolidin-1-yl,
1H-pyrazol-3-yl, 1H-pyrazol-4-yl,
2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl or 1H-indazol-3-yl of
R.sub.2 is optionally substituted with 1 to 3 radicals
independently selected from hydroxy, halo, methoxy, amino,
--OS(O).sub.2NR.sub.7aR.sub.7b and --NR.sub.7aS(O).sub.2R.sub.7b;
wherein R.sub.7a and R.sub.7b are independently selected from
hydrogen and C.sub.1-4alkyl; R.sub.3 is selected from hydrogen,
C.sub.1-4alkyl and biphenyl; and R.sub.4 is selected from
isopropyl, methyl, ethyl, prop-1-en-2-yl, isobutyl, cyclohexyl,
sec-butyl, (S)-sec-butyl, (R-sec-butyl, 1-hydroxypropan-2-yl,
(S)-1-hydroxypropan-2-yl, (R)-1-hydroxypropan-2-yl, nonan-2-yl,
2-(2-oxopyrrolidin-1-yl)ethyl, oxetan-3-yl, oxetan-2-yl,
benzhydryl, tetrahydro-2H-pyran-2-yl, phenyl, tetrahydrofuran-3-yl
and benzyl; wherein said cyclohexyl, 2-(2-oxopyrrolidin-1-yl)ethyl,
oxetan-3-yl, oxetan-2-yl, benzhydryl, tetrahydro-2H-pyran-4-yl,
phenyl, tetrahydrofuran-3-yl or benzyl can be optionally
substituted with 1 to 3 radicals independently selected from
C.sub.1-4alkyl and halo-substituted C.sub.1-4alkyl.
[0131] In another embodiment, L is selected from --NR.sub.5a
(CH.sub.2).sub.0-3-, --NR.sub.5aCH(C(O)OCH.sub.3)CH.sub.2--,
--NR.sub.5a(CH.sub.2).sub.2NR.sub.5b--,
--NR.sub.5a(CH.sub.2).sub.2S--,
--NR.sub.5aCH.sub.2CH(CH.sub.3)CH.sub.2--,
--NR.sub.5aCH(CH.sub.3)CH.sub.2-- and --NR.sub.5a CH.sub.2CH(OH)--;
wherein R.sub.5a and R.sub.5b are independently selected from
hydrogen and methyl; and R.sub.1 is selected from hydrogen, phenyl,
thiophen-2-yl, thiophen-3-yl, furan-3-yl, 1H-benzo[d]imidazol-1-yl,
isoquinolin-4-yl, 1H-imidazo[4,5-b]pyridin-1-yl,
benzo[b]thiophen-3-yl, pyrimidin-5-yl, 1H-pyrazol-4-yl,
pyridin-2-yl, pyridin-4-yl, 1H-imidazol-1-yl, pyrrolidin-1-yl,
pyrazin-2-yl, pyridin-3-yl, pyridazin-4-yl, 1H-pyrrol-2-yl and
thiazol-5-yl; wherein said phenyl, thiophen-2-yl, thiophen-3-yl,
furan-3-yl, 1H-benzo[d]imidazol-1-yl, isoquinolin-4-yl,
1H-imidazo[4,5-b]pyridin-1-yl, benzo[b]thiophen-3-yl,
pyrimidin-5-yl, pyridin-2-yl, pyridin-4-yl, 1H-imidazol-1-yl,
pyrrolidin-1-yl, pyrazin-2-yl, pyridin-3-yl, pyridazin-4-yl,
1H-pyrrol-2-yl orthiazol-5-yl of R.sub.1 can be optionally
substituted by 1 to 3 radicals independently selected from cyano,
hydroxy, C.sub.1-4alkyl, C.sub.1-4alkoxy, halo,
halo-substituted-C.sub.1-4alkyl, --S(O).sub.0-2R.sub.8a and
--C(O)OR.sub.8a; wherein R.sub.8a and R.sub.8b are independently
selected from hydrogen and C.sub.1-4alkyl; with the proviso that
R.sub.1 and R.sub.3 are not both hydrogen.
[0132] In another embodiment, when L is
--NR.sub.5a(CH.sub.2).sub.0-3, it is preferably
--NR.sub.5a(CH.sub.2).sub.1-3 (where 1-3 herein 1, 2 or 3).
[0133] In another embodiment, R.sub.2 is selected from urea,
phenyl, 1H-indol-2-yl, 1H-indol-3-yl, thiophen-3-yl,
piperidin-1-yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl,
1H-1,2,4-triazol-3-yl, 1H-1,2,4-triazol-5-yl,
2-oxoimidazolidin-1-yl, 1H-pyrazol-3-yl, 1H-pyrazol-4-yl,
2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl,
1H-benzo[d]imidazol-5-yl and 1H-imidazol-4-yl; wherein said phenyl,
1H-indol-2-yl, 1H-indol-3-yl, thiophen-3-yl, piperidin-1-yl,
pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, 1H-1,2,4-triazol-3-yl,
1H-1,2,4-triazol-5-yl, 2-oxoimidazolidin-1-yl, 1H-pyrazol-3-yl,
1H-pyrazol-4-yl, 2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl or
1H-benzo[d]imidazol-5-yl of R2 is optionally substituted with
hydroxy, methoxy, methyl, halo, amino and aminosulfonyl.
[0134] In another embodiment, R.sub.3 is selected from hydrogen,
methyl and biphenyl; and R.sub.4 is selected from isopropyl,
methyl, ethyl, prop-1-en-2-yl, isobutyl, cyclohexyl, sec-butyl,
(S)-sec-butyl, (R)-sec-butyl, 1-hydroxypropan-2-yl,
(S)-1-hydroxypropan-2-yl, (R)-1-hydroxypropan-2-yl, nonan-2-yl,
2-(2-oxopyrrolidin-1-yl)ethyl, oxetan-3-yl, oxetan-2-yl,
benzhydryl, tetrahydro-2H-pyran-2-yl, phenyl, tetrahydrofuran-3-yl
and benzyl; wherein said cyclohexyl, 2-(2-oxopyrrolidin-1-yl)ethyl,
oxetan-3-yl, oxetan-2-yl, benzhydryl, tetrahydro-2H-pyran-4-yl,
phenyl, tetrahydrofuran-3-yl or benzyl can be optionally
substituted with 1 to 3 radicals independently selected from methyl
and trifluoromethyl.
[0135] In another embodiment are compounds selected from:
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-ylamino)ethyl)p-
henol;
4-(2-(9-benzhydryl-2-(benzo[b]thiophen-3-yl)-9H-purin-6-ylamino)eth-
yl)phenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(tetrahydro-2H-pyran-3-yl)-9H-
-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(thiophen-2-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(4-(trifluoromethyl)benzyl)-9H-purin-6--
ylamino)ethyl) phenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isobutyl-9H-purin-6-ylamino)ethyl)
phenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-methyl-9H-purin-6-ylamino)ethyl-
)phenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(4-methylbenzyl)-9H-purin-6-yla-
mino)ethyl)phenol;
N-(2-(1H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-
-6-amine;
2-(benzo[b]thiophen-3-yl)-9-isopropyl-N-(2-(thiophen-3-yl)ethyl)-
-9H-purin-6-amine;
3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol;
2-(benzo[b]thiophen-3-yl)-N-(4-fluorophenethyl)-9-isopropyl-9H-purin-6-
-amine;
N-(4-aminophenethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropyl-98-puri-
n-6-amine;
4-(2-(9-isopropyl-2-(pyrimidin-5-yl)-9H-purin-6-ylamino)ethyl)p-
henol;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-phenyl-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(thiophen-3-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(2-(furan-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol;
2-(benzo[b]thiophen-3-yl)-N-(4-fluorophenethyl)-9-phenyl-9H-purin-6-amine-
; N-benzyl-8-(biphenyl-4-yl)-9-isopropyl-9H-purin-6-amine;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(nonan-2-yl)-9H-purin-6-ylamino)ethyl)p-
henol;
N-(2-(I-Hindol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-
-purin-6-amine;
3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-1H--
indol-5-yl,
5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate;
N-(2-(2-(2-(2-(4-(1-(2-(benzo[b]thiophen-3-yl)-6-(4-hydroxyphenethylamino-
)-9H-purin-9-yl)ethyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)ac-
etamide;
4-(2-(9-isopropyl-2-(pyridin-4-yl)-9H-purin-6-ylamino)ethyl)pheno-
l;
ethyl5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)nicotinat-
e;
ethyl5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)nicotinat-
e;
4-(2-(2-(6-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phe-
nol;
4-(2-(9-isopropyl-2-(4-methylpyridin-3-yl)-9H-purin-6-ylamino)ethyl)p-
henol;
5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)nicotinoni-
trile;
4-(2-(9-isopropyl-2-(pyrrolidin-1-yl)-9H-purin-6-ylamino)ethyl)phen-
ol;
4-(2-(2-(1H-imidazol-1-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol-
;
4-(2-(9-isopropyl-2-(pyridazin-4-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(pyrazin-2-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(pyridin-2-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(5-(methylsulfonyl)pyridin-3-yl)-9H-purin-6-ylamino)e-
thyl)phenol;
4-(2-(9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(2-(4-chloropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phe-
nol;
4-(2-(2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)
phenol;
4-(2-(9-isopropyl-2-(1-methyl-1H-pyrazol-4-yl)-9H-purin-6-ylamino-
)ethyl)phenol;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-2-methoxyphe-
nol;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-2-methox-
yphenol;
N-[2-(6-methoxy-1H-indol-3-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3-
-yl)-9H-purin-6-amine;
N-[2-(5-methyl-1H-indol-3-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3-yl)-9H-p-
urin-6-amine;
1-(2-{[9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-yl]amino}ethyl)imidazo-
lidin-2-one;
N-(2-{[9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-yl]amino}ethyl)pyridin-
-2-amine;
9-(propan-2-yl)-N-[3-(1H-pyrazol-4-yl)propyl]-2-(pyridin-3-yl)-9-
H-purin-6-amine;
N-{2-[(3-methyl-1H-1,2,4-triazol-5-yl)sulfanyl]ethyl}-9-(propan-2-yl)-2-(-
pyridin-3-yl)-9H-purin-6-amine;
1-(2-{[2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}ethyl-
)imidazolidin-2-one;
N-[2-(5-amino-1H-1,2,4-triazol-3-yl)ethyl]-2-(1-benzothiophen-3-yl)-9-(pr-
opan-2-yl)-9H-purin-6-amine;
N-(2-{[2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}ethyl-
)pyridin-2-amine;
2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-N-[3-(1H-pyrazol-4-yl)propyl]-9H-
-purin-6-amine;
2-(1-benzothiophen-3-yl)-N-[3-(3,5-dimethyl-1H-pyrazol-4-yl)propyl]-9-(pr-
opan-2-yl)-9H-purin-6-amine;
(2-{[2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}ethyl)u-
rea;
5-({[2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}met-
hyl)-2,3-dihydro-1H-1,3-benzodiazol-2-one;
N-[2-(1H-indol-3-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-am-
ine;
N-(4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenyl-
)methane-sulfonamide;
4-(2-(2-(pyridin-3-yl)-9-(tetrahydrofuran-3-yl)-9H-purin-6-ylamino)ethyl)-
phenol; 4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)
propyl)phenol;
4-(2-(9-(oxetan-3-yl)-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol;
5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)-N-methylnicotin-
amide;
4-(2-(9-(1-hydroxypropan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-ylamino)-
ethyl)phenol;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenylsulfama-
te;
4-(2-(2-(2-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)ph-
enol;
4-(2-(9-isopropyl-2-(1-methyl-1H-pyrrol-2-yl)-9H-purin-6-ylamino)eth-
yl)phenol;
4-(2-(9-isopropyl-2-(thiazol-5-yl)-9H-purin-6-ylamino)ethyl)phe-
nol;
4-(2-(2-(1H-benzo[d]imidazol-1-yl)-9-isopropyl-9H-purin-6-ylamino)eth-
yl)phenol;
4-(2-(2-(2,4-dimethyl-1H-imidazol-1-yl)-9-isopropyl-9H-purin-6--
ylamino)ethyl) phenol;
4-(2-(9-isopropyl-2-(2-methyl-1H-imidazol-1-yl)-9H-purin-6-ylamino)ethyl)-
phenol;
5-(9-sec-butyl-6-(4-hydroxy-3-methylphenethylamino)-9H-purin-2-yl)-
nicotinonitrile;
N-(2-(1H-pyrrolo[2,3-b]pyridin-5-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9-
H-purin-6-amine;
9-isopropyl-N-(2-(5-methyl-1H-pyrazol-3-yl)ethyl)-2-(pyridin-3-yl)-9H-pur-
in-6-amine;
4-(2-(2-(5-fluoropyridin-3-yl)-9-(oxetan-3-yl)-9H-purin-6-ylamino)ethyl)p-
henol;
4-(2-(2-(5-chloropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl-
)phenol;
4-(2-(9-isopropyl-2-(5-(trifluoromethyl)pyridin-3-yl)-9H-purin-6--
ylamino)ethyl)phenol;
5-(6-(2-(1H-indol-3-yl)ethylamino)-9-sec-butyl-9H-purin-2-yl)nicotinonitr-
ile;
N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H-pu-
rin-6-amine;
(R)--N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-p-
urin-6-amine;
(S)--N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-p-
urin-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-purin--
6-amine;
(R)--N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3--
yl)-9H-purin-6-amine;
(S)--N-(2-(1H-indol-3-yl)ethyl)-9-secbutyl-2-(5-methylpyridin-3-yl)-9H-pu-
rin-6-amine;
5-(6-(4-hydroxyphenethylamino)-9-(oxetan-3-yl)-9H-purin-2-yl)nicotinonitr-
ile;
4-(2-(6-(5-fluoropyridin-3-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidi-
n-4-ylamino)ethyl)phenol;
4-(2-(6-(benzo[b]thiophen-3-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-
-ylamino)ethyl)phenol;
(R)-4-(2-(2-(5-fluoropyridin-3-yl)-9-(tetrahydrofuran-3-yl)-9H-purin-6-yl-
amino)ethyl)phenol;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-3-methylphen-
ol;
5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)picolinonitri-
le;
3-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)isonicotinoni-
trile;
4-(2-(2-(5-fluoropyridin-3-yl)-7-isopropyl-7H-pyrrolo[2,3-d]pyrimid-
in-4-ylamino)ethyl)phenol;
3-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)picolinonitrile;
4-(2-(9-isopropyl-2-(6-methylpyridin-3-yl)-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(9-isopropyl-2-(isoquinolin-4-yl)-9H-purin-6-ylamino)ethyl)phenol;
2-chloro-4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phen-
ol;
3-fluoro-4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)p-
henol;
N-(2-(5-chloro-1H-indol-3-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-
-purin-6-amine;
N-(2-(5-fluoro-1H-indol-3-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-purin-
-6-amine;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-2-m-
ethylphenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(oxetan-3-yl)-9H-purin-6-ylamino)ethyl)-
phenol;
(S)-4-(2-(2-(benzo[b]thiophen-3-yl)-9-(tetrahydrofuran-3-yl)-9H-pu-
rin-6-ylamino)ethyl)phenol;
(R)-4-(2-(2-(benzo[b]thiophen-3-yl)-9-(tetrahydrofuran-3-yl)-9H-purin-6-y-
lamino)ethyl)phenol;
2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl-
)propan-1-ol;
(R)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin--
9-yl)pro-pan-I-ol;
(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin--
9-yl)propan-I-ol;
(R)--N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-(tetrahydrofur-
an-3-yl)-9H-purin-6-amine;
4-(2-(2-(3H-imidazo[4,5-b]pyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)et-
hyl)phenol;
4-(2-(2-(1H-imidazo[4,5-b]pyridine-1-yl)-9-isopropyl-9H-purin-6-ylamino)e-
thyl)phenol;
4-(2-(6-(5-fluoropyridin-3-yl)-1-isopropyl-1H-imidazo[4,5-c]pyridin-4-yla-
mino)ethyl)phenol;
4-(2-(2-(4,5-dimethyl-1H-imidazol-1-yl)-9-isopropyl-9H-purin-6-ylamino)et-
hyl)phenol;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(pyridin-3-yl)ethyl)-9H-purin-6-
-amine; 4-(2-(2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-yl
amino)-1-hydroxyethyl)phenol;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(6-methoxy-1H-indol-3-yl)ethyl)-
-9H-purin-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin--
6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(5-methoxy-1H-indol-3-y-
l)ethyl)-9H-purin-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-(prop-1-en-2-yl)-9H-
-purin-6-amine;
5-(2-(2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)pyrid-
in-2-ol;
N-(2-(1H-pyrrolo[2,3-b]pyridin-3-yl)ethyl)-2-(5-fluoropyridin-3-y-
l)-9-isopropyl-9H-purin-6-amine;
N-(2-(6-(2-(diethylamino)ethoxy)-1H-indol-3-yl)ethyl)-2-(5-fluoropyridin--
3-yl)-9-isopropyl-9H-purin-6-amine;
4-(2-(5-t5-fluoropyridin-3-yl)-3-isopropyl-3H-imidazo[4,5-b]pyridin-7-yla-
mino)ethyl)phenol;
N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(2-methyl-1H-imidazol-1-yl)-9H-p-
urin-6-amine;
4-(2-(2-(2-ethyl-1H-imidazol-1-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)p-
henol;
4-(2-(9-isopropyl-2-(2-propyl-1H-imidazol-1-yl)-9H-purin-6-ylamino)-
ethyl)phenol;
3-(2-(2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-1H-i-
ndol-6-ol;
N-(2-(1H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-
-9H-purin-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-9-isopropyl-2-(2-methyl-1H-imidazol-1-yl)-9H-p-
urin-6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(7-methyl-1H-indol-3-yl)ethyl)--
9H-purin-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-(oxetan-3-yl)-9H-pu-
rin-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-2-(5-methylpyridin-3-yl)-9-(oxetan-3-yl)-9H-pu-
rin-6-amine;
N-(2-(6-fluoro-1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl--
9H-purin-6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(6-methyl-1H-indol-3-yl)ethyl)--
9H-purin-6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(2-methyl-1H-indol-3-yl)ethyl)--
9H-purin-6-amine;
N-(2-(4-fluoro-1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl--
9H-purin-6-amine;
N-(2-(7-fluoro-1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl--
9H-purin-6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(4-methyl-1H-indol-3-yl)ethyl)--
9H-purin-6-amine;
4-(2-(2-(benzo[b]thiophen-3-yl)-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-4--
ylamino)ethyl)phenol;
9-isopropyl-2-(pyridin-3-yl)-N-(2-(pyridin-4-yl)ethyl)-9H-purin-6-amine;
N-(2-(1H-pyrrolo[2,3-b]pyridin-5-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9-
H-purin-6-amine;
4-(2-(2-(5-fluoropyridin-3-yl)-9-(1-hydroxypropan-2-yl)-9H-purin-6-ylamin-
o)ethyl)-2-methylphenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-cyclohexyl-9H-purin-6-ylamino)ethyl)phe-
nol;
4-(2-(9-isopropyl-2-(thiophen-3-yl)-9H-purin-6-ylamino)ethyl)phenol;
and
1-(2-(2-(benzo[b]thiophen-3-yl)-6-(4-hydroxyphenethylamino)-9H-purin--
9-yl)ethyl)pyrrolidin-2-one.
[0136] In another embodiment, an aryl hydrocarbon receptor
antagonist is a compound of Formula Ia:
##STR00007##
in which: L is selected from --NR.sub.5a(CH.sub.2).sub.0-3-,
--NR.sub.5aCH(C(O)OCH.sub.3)CH.sub.2--,
--NR.sub.5a(CH.sub.2).sub.2NR.sub.5b--,
--NR.sub.5a(CH.sub.2).sub.2S--,
--NR.sub.5aCH.sub.2CH(CH.sub.3)(CH.sub.2--, --NR.sub.5a
CH(CH.sub.3) CH.sub.2--, --(CH.sub.2).sub.3--,
--CH.sub.2OCH.sub.2--, --CH.sub.2NR.sub.5aCH.sub.2--,
--NR.sub.5aC(O)CH.sub.2-- and --NR.sub.5aY--; wherein R.sub.5a and
R.sub.5b are independently selected from hydrogen and
C.sub.1-4alkyl; and Y is a 5 member heteroaryl ring containing up
to 3 heteroatoms selected from O, N and S; R.sub.1 is selected from
hydrogen, phenyl, thiophen-2-yl, thiophen-3-yl, furan-2-yl,
furan-3-yl, benzo[b]thiophen-2-yl, benzo[b]thiophen-3-yl,
benzofuran-2-yl, benzofuran-3-yl, pyrimidin-4-yl, pyrimidin-5-yl,
1H-pyrazol-4-yl, 1H-pyrazol-3-yl, pyridin-2-yl, pyridazin-3-yl,
pyridin-4-yl, 1H-imidazol-1-yl, pyrrolidin-1-yl, pyrazin-2-yl,
pyridin-3-yl, 1H-pyrazol-1-yl, pyridazin-4-yl, 1H-indol-2-yl,
thiazol-4-yl, 1H-indol-3-yl, 1H-pyrrol-2-yl and thiazol-5-yl;
wherein said phenyl, thiophen-2-yl, thiophen-3-yl, furan-2-yl,
furan-3-yl, benzo[b]thiophen-2-yl, benzo[b]thiophen-3-yl,
benzofuran-2-yl, benzofuran-3-yl, pyrimidin-4-yl, pyrimidin-5-yl,
1H-pyrazol-4-yl, 1H-pyrazol-3-yl, pyridin-2-yl, pyridazin-3-yl,
pyridin-4-yl, 1H-imidazol-1-yl, pyrrolidin-1-yl, pyrazin-2-yl,
pyridin-3-yl, 1H-pyrazol-1-yl, pyridazin-4-yl, 1H-indol-2-yl,
thiazol-4-yl, 1H-indol-3-yl, 1H-pyrrol-2-yl or thiazol-5-yl of
R.sub.1 can be optionally substituted by 1 to 3 radicals
independently selected from cyano, hydroxy, C.sub.1-4alkyl,
C.sub.1-4alkoxy, halo, halo-substituted-C.sub.1-4alkyl,
halo-substituted-C.sub.1-4alkoxy, hydroxy, amino, --C(O)R.sub.8a,
--S(O).sub.0-2R.sub.8a, --C(O)OR.sub.8a and
--C(O)NR.sub.8aR.sub.8b; wherein R.sub.8a and R.sub.8b are
independently selected from hydrogen and C.sub.1-4alkyl; with the
proviso that R.sub.1 and R.sub.3 are not both hydrogen; R.sub.2 is
selected from --S(O).sub.2NR.sub.6aR.sub.6b,
--NR.sub.9aC(O)R.sub.9b, --NR.sub.6aC(O)NR.sub.6bR.sub.6c, phenyl,
1H-indol-2-yl, 1H-indol-3-yl, benzo[b]thiophen-2-yl,
benzo[b]thiophen-3-yl, benzofuran-2-yl, benzofuran-3-yl,
thiophen-2-yl, thiophen-3-yl, furan-2-yl, furan-3-yl,
piperidin-4-yl, piperidin-3-yl, piperidin-2-yl, piperidin-1-yl,
pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, 1H-1,2,4-triazol-3-yl,
1H-1,2,4-triazol-5-yl, 2-oxoimidazolidin-1-yl, 1H-pyrazol-3-yl,
1H-pyrazol-4-yl, 3-oxopiperazin-1-yl,
2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl,
1,2,3,4-tetrahydronaphthalen-2-yl, indolin-5-yl, 2-oxoindolin-5-yl,
1H-benzo[d]imidazol-5-yl, 1H-indazol-5-yl and 1H-imidazol-4-yl;
wherein R.sub.6a, R.sub.6b and R.sub.6c are independently selected
from hydrogen and C.sub.1-4alkyl; wherein said phenyl,
1H-indol-2-yl, 1H-indol-3-yl, benzo[b]thiophen-2-yl,
benzo[b]thiophen-3-yl, benzofuran-2-yl, benzofuran-3-yl,
thiophen-2-yl, thiophen-3-yl or furan-2-yl, furan-3-yl,
piperidin-4-yl, piperidin-3-yl, piperidin-2-yl, piperidin-1-yl,
pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, 1H-1,2,4-triazol-3-yl,
1H-1,2,4-triazol-5-yl, 2-oxoimidazolidin-1-yl, 1H-pyrazol-3-yl,
3-oxopiperazin-1-yl, 2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl,
1,2,3,4-tetrahydronaphthalen-2-yl, indolin-5-yl, 2-oxoindolin-5-yl,
1H-benzo[d]imidazol-5-yl, 1H-indazol-5-yl or 1H-imidazol-4-yl of
R.sub.2 is optionally substituted with 1 to 3 radicals
independently selected from hydroxy, halo, methyl, methoxy, amino,
--S(O).sub.2NR.sub.7aR.sub.7b, --OS(O).sub.2NR.sub.7aR.sub.7b and
--NR.sub.7aS(O).sub.2R.sub.7b; wherein R.sub.7a and R.sub.7b are
independently selected from hydrogen and C.sub.1-4alkyl; or a
single radical selected from
5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyloxy-
,
2-(2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)penta-
namido)ethoxy)ethoxy and
2-(4-(4-hex-5-ynamidobenzoyl)phenylamino)-2-oxoethoxy; R.sub.3 is
selected from hydrogen, C.sub.1-4alkyl and biphenyl; and R.sub.4 is
selected from isopropyl, isobutyl, sec-butyl, 1-hydroxypropan-2-yl,
cyclopropyl, oxetan-3-yl, oxetan-2-yl, benzhydryl, piperidin-4-yl,
piperidin-3-yl, piperidin-2-yl, tetrahydro-2H-pyran-2-yl,
tetrahydro-2H-pyran-3-yl, tetrahydro-2H-pyran-4-yl, phenyl,
tetrahydrofuran-3-yl, tetrahydrofuran-2-yl, benzyl,
(4-pentylphenyl)(phenyl)methyl and
1-(1-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-1H-1,2,3-triazol-4-yl)et-
hyl; wherein said cyclopropyl, oxetan-3-yl, oxetan-2-yl,
benzhydryl, piperidin-4-yl, piperidin-3-yl, piperidin-2-yl,
tetrahydro-2H-pyran-2-yl, tetrahydro-2H-pyran-3-yl,
tetrahydro-2H-pyran-4-yl, phenyl, tetrahydrofuran-3-yl,
tetrahydrofuran-2-yl, benzyl, (4-pentylphenyl)(phenyl)methyl or
1-(1-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-1H-1,2,3-triazol-4-yl)et-
hyl can be optionally substituted with 1 to 3 radicals
independently selected from C.sub.1-4alkyl and
halo-substituted-C.sub.1-4alkyl; or an N-oxide derivative, prodrug
derivative, protected derivative, individual isomer or mixture of
isomers thereof; or a pharmaceutically acceptable salt or solvate
(e.g. hydrate) of such compound.
[0137] In another embodiment, with reference to compounds of
Formula Ia, L is selected from --NR.sub.5a(CH.sub.2).sub.0-3-,
--NR.sub.5aCH(C(O)OCH.sub.3)CH.sub.2--,
--NR.sub.5a(CH.sub.2).sub.2NR.sub.5b--,
--NR.sub.5a(CH.sub.2).sub.2S--,
--NR.sub.5aCH.sub.2CH(CH.sub.3)CH.sub.2--,
--NR.sub.5aCH(CH.sub.3)CH.sub.2--, --(CH.sub.2).sub.3--,
--CH.sub.2OCH.sub.2--, --CH.sub.2NR.sub.5aCH.sub.2--,
--NR.sub.5aC(O)CH.sub.2-- and --NR.sub.5aY--; wherein R.sub.5a and
R.sub.5b are independently selected from hydrogen and methyl; Y is
selected from isoxazole and 1,3,4-oxadiazole.
[0138] In another embodiment, when L is
--NR.sub.5a(CH.sub.2).sub.0-3, it is preferably
--NR.sub.5a(CH.sub.2).sub.1-3 (where 1-3 herein means 1, 2 or
3).
[0139] In another embodiment, R.sub.1 is selected from hydrogen,
phenyl, thiophen-3-yl, thiophen-2-yl, furan-3-yl, furan-2-yl,
benzo[b]thiophen-3-yl, pyrimidin-5-yl, pyridin-4-yl, pyridin-2-yl,
pyrrolidin-1-yl, 1H-pyrazol-4-yl, pyrazin-2-yl, pyridazin-3-yl,
pyridazin-4-yl, 1H-pyrazol-1-yl, 1H-pyrazol-3-yl, 1H-imidazol-1-yl,
thiazol-4-yl, 1H-pyrrol-2-yl, thiazol-5-yl, and pyridin-3-yl;
wherein said phenyl, thiophen-3-yl, thiophen-2-yl, furan-3-yl,
furan-2-yl, benzo[b]thiophen-3-yl, pyrimidin-5-yl, pyridin-4-yl,
pyridin-2-yl, pyrrolidin-1-yl, 1H-pyrazol-4-yl, pyrazin-2-yl,
pyridazin-3-yl, pyridazin-4-yl, 1H-pyrazol-1-yl, 1H-pyrazol-3-yl,
1H-imidazol-1-yl, thiazol-4-yl, 1H-pyrrol-2-yl, thiazol-5-yl or
pyridin-3-yl of R.sub.1 is optionally substituted with 1 to 3
radicals independently selected from cyano, methyl,
methyl-sulfonyl, methoxy, halo, hydroxy, carboxyl, ethoxy-carbonyl,
methyl-amino-carbonyl and amino; with the proviso that R.sub.1 and
R.sub.3 are not both hydrogen.
[0140] In another embodiment, R.sub.2 is selected from
amino-sulfonyl, methyl-carbonyl-amino, methyl-sulfonyl-amino,
amino-sulfonyl-oxy, urea, phenyl, 1H-indol-2-yl, 1H-indol-3-yl,
benzo[b]thiophen-2-yl, benzo[b]thiophen-3-yl, benzofuran-2-yl,
benzofuran-3-yl, thiophen-2-yl, thiophen-3-yl, furan-2-yl,
furan-3-yl, piperidin-4-yl, piperidin-3-yl, piperidin-2-yl,
piperidin-1-yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl,
1H-1,2,4-triazol-3-yl, 1H-1,2,4-triazol-5-yl,
2-oxoimidazolidin-1-yl, 1H-pyrazol-3-yl, 1H-pyrazol-4-yl,
3-oxopiperazin-1-yl, 2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl,
1,2,3,4-tetrahydronaphthalen-2-yl, indolin-5-yl, 2-oxoindolin-5-yl,
1H-benzo[d]imidazol-5-yl, 1H-indazol-5-yl and 1H-imidazol-4-yl;
wherein said phenyl, 1H-indol-2-yl, 1H-indol-3-yl,
benzo[b]thiophen-2-yl, benzo[b]thiophen-3-yl, benzofuran-2-yl,
benzofuran-3-yl, thiophen-2-yl, thiophen-3-yl, furan-2-yl,
furan-3-yl, piperidin-4-yl, piperidin-3-yl, piperidin-2-yl,
piperidin-1-yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl,
1H-1,2,4-triazol-3-yl, 1H-1,2,4-triazol-5-yl,
2-oxoimidazolidin-1-yl, 1H-pyrazol-3-yl, 1H-pyrazol-4-yl,
3-oxopiperazin-1-yl, 2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl,
1,2,3,4-tetrahydronaphthalen-2-yl, indolin-5-yl, 2-oxoindolin-5-yl,
1H-benzo[d]imidazol-5-yl, 1H-indazol-5-yl and 1H-imidazol-4-yl of
R.sub.2 is optionally substituted with hydroxy, methoxy, methyl,
halo, amino, amino-sulfonyl,
5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyloxy-
,
2-(2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)penta-
namido)ethoxy)ethoxy and
2-(4-(4-hex-5-ynamidobenzoyl)phenylamino)-2-oxoethoxy.
[0141] In another embodiment, R.sub.3 is selected from hydrogen,
methyl, and biphenyl; and R.sub.4 is selected from isopropyl,
isobutyl, sec-butyl, 1-hydroxypropan-2-yl, cyclopropyl,
oxetan-3-yl, oxetan-2-yl, benzhydryl, piperidin-4-yl,
piperidin-3-yl, piperidin-2-yl, tetrahydro-2H-pyran-2-yl,
tetrahydro-2H-pyran-3-yl, tetrahydro-2H-pyran-4-yl, phenyl,
tetrahydrofuran-3-yl, tetrahydrofuran-2-yl, benzyl,
(4-pentylphenyl)(phenyl)methyl and
1-(1-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-1H-1,2,3-triazol-4-yl)et-
hyl; wherein said cyclopropyl, oxetan-3-yl, oxetan-2-yl,
benzhydryl, piperidin-4-yl, piperidin-3-yl, piperidin-2-yl,
tetrahydro-2H-pyran-2-yl, tetrahydro-2H-pyran-3-yl,
tetrahydro-2H-pyran-4-yl, phenyl, tetrahydrofuran-3-yl,
tetrahydrofuran-2-yl, benzyl, (4-pentylphenyl)(phenyl)methyl or
1-(1-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-1H-1,2,3-triazol-4-yl)et-
hyl can be optionally substituted with 1 to 3 radicals
independently selected from methyl and trifluoromethyl.
[0142] In another embodiment are compounds selected from:
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-ylamino)ethyl)phe-
nol;
4-(2-(9-benzhydryl-2-(benzo[b]thiophen-3-yl)-9H-purin-6-ylamino)ethyl-
)phenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(tetrahydro-2H-pyran-3-yl)-9H-p-
urin-6-ylamino)ethyl)phenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(4-(trifluoromethyl)benzyl)-9H-purin-6--
ylamino)ethyl)phenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isobutyl-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-methyl-9H-purin-6-ylamino)ethyl)
phenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(4-methylbenzyl)-9H-purin-6-yla-
mino)ethyl)phenol;
N-(2-(1H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-
-6-amine;
2-(benzo[b]thiophen-3-yl)-9-isopropyl-N-(2-(thiophen-3-yl)ethyl)-
-9H-purin-6-amine;
3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol;
2-(benzo[b]thiophen-3-yl)-N-(4-fluorophenethyl)-9-isopropyl-9H-purin-6-
-amine;
N-(4-aminophenethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-puri-
n-6-amine;
4-(2-(9-isopropyl-2-(pyrimidin-5-yl)-9H-purin-6-ylamino)ethyl)p-
henol;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-phenyl-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(thiophen-3-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(2-(furan-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol;
2-(benzo[b]thiophen-3-yl)-N-(4-fluorophenethyl)-9-phenyl-9H-purin-6-amine-
; N-benzyl-8-(biphenyl-4-yl)-9-isopropyl-9H-purin-6-amine;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(nonan-2-yl)-9H-purin-6-ylamino)ethyl)p-
henol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-((4-pentylphenyl)(phenyl)methyl)--
9H-purin-6-ylamino)ethyl)phenol;
N-(2-(1H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-
-6-amine;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-ylamino)e-
thyl) phenol;
3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-1H--
indol-5-ol;
3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-1H--
indol-5-yl,
5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate;
N-(2-(2-(3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)e-
thyl)-1H-indol-5-yloxy)ethoxy)ethyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thi-
eno[3,4-d]imidazol-4-yl)pentanamide;
N-(4-(4-(2-(3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamin-
o)ethyl)-1H-indol-5-yloxy)acetamido)benzoyl)phenyl)hex-5-ynamide;
N-(2-(2-(2-(2-(4-(1-(2-(benzo[b]thiophen-3-yl)-6-(4-hydroxyphenethylamino-
-)-9H-purin-9-yl)ethyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)a-
cetamide;
4-(2-(9-isopropyl-2-(pyridin-4-yl)-9H-purin-6-ylamino)ethyl)phen-
ol; ethyl
5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)nicotin-
ate; ethyl
5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)nicoti-
nate;
4-(2-(2-(6-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)
phenol;
4-(2-(9-isopropyl-2-(4-methylpyridin-3-yl)-9H-purin-6-ylamino)eth-
yl)phenol;
4-(2-(9-isopropyl-2-(2-methoxypyridin-3-yl)-9H-purin-6-ylamino)-
ethyl)phenol;
5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)nicotinonitrile;
4-(2-(9-isopropyl-2-(pyrrolidin-1-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(1H-pyrazol-1-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(2-(1H-imidazol-1-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(pyridazin-3-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(pyridazin-4-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(pyrazin-2-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(pyridin-2-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(5-(methylsulfonyl)pyridin-3-yl)-9H-purin-6-ylamino)e-
thyl)phenol;
4-(2-(9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(2-(2-chloropyridin-3-yl)-6-isopropyl-2,6-dihydroimidazo[4,5-c]pyr-
azol-3-ylamino)ethyl)phenol;
4-(2-(2-(4-chloropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(9-isopropyl-2-(4-methoxypyridin-3-yl)-9H-purin-6-ylamino)ethyl)ph-
enol;
4-(2-(2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-
phenol;
4-(2-(9-isopropyl-2-(thiazol-4-yl)-9H-purin-6-ylamino)ethyl)phenol-
;
4-(2-(9-isopropyl-2-(1-methyl-1H-pyrazol-4-yl)-9H-purin-6-ylamino)ethyl)-
phenol;
4-(2-(9-isopropyl-2-(1H-pyrazol-3-yl)-9H-purin-6-ylamino)ethyl)phe-
nol;
4-(2-(9-isopropyl-2-(1H-pyrazol-4-yl)-9H-purin-6-ylamino)ethyl)phenol-
;
4-(2-(9-isopropyl-2-(thiophen-2-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)thiophene-2-carb-
oxylic acid;
4-(2-(2-(furan-2-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(4-methylthiophen-3-yl)-9H-purin-6-ylamino)ethyl)phen-
ol;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-2-methoxy-
phenol;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-2-met-
hoxyphenol;
N-[2-(6-methoxy-1H-indol-3-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3-yl)-9H--
purin-6-amine;
N-[2-(5-methyl-1H-indol-3-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3-yl)-9H-p-
urin-6-amine;
N-[2-(piperidin-4-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-a-
mine;
1-(2-{[9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-yl]amino}ethyl)pi-
peridin-4-ol; methyl
(2S)-3-(4-hydroxyphenyl)-2-{[9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6--
yl]amino}propanoate;
4-(2-{[9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-yl]amino}ethyl)benzene-
-1-sulfonamide;
2-{[2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}ethane-1-
-sulfonamide;
4-(2-{[9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-yl]amino}ethyl)benzene-
-1,2-diol;
N-[2-(1H-imidazol-4-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3-yl)--
9H-purin-6-amine;
1-(2-{[9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-yl]amino}ethyl)imidazo-
lidin-2-one;
N-[2-(5-amino-1H-1,2,4-triazol-3-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3-y-
l)-9H-purin-6-amine;
N-(2-{[9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-yl]amino}ethyl)pyridin-
-2-amine;
9-(propan-2-yl)-N-[3-(1H-pyrazol-4-yl)propyl]-2-(pyridin-3-yl)-9-
H-purin-6-amine;
N-[2-({[9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-yl]amino}methyl)propy-
l]acetamide;
4-(2-{[9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-yl]amino}ethyl)piperaz-
in-2-one;
N-{2-[(3-methyl-1H-1,2,4-triazol-5-yl)sulfanyl]ethyl}-9-(propan--
2-yl)-2-(pyridin-3-yl)-9H-purin-6-amine;
N-[3-(3,5-dimethyl-1H-pyrazol-4-yl)propyl]-9-(propan-2-yl)-2-(pyridin-3-y-
l)-9H-purin-6-amine;
(2-{[9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-yl]amino}ethyl)urea;
5-({[9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-yl]amino}methyl)-2,3-dih-
ydro-1H-1,3-benzodiazol-2-one;
2-(1-benzothiophen-3-yl)-N-[2-(1H-imidazol-4-yl)ethyl]-9-(propan-2-yl)-9H-
-purin-6-amine;
1-(2-{[2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}ethyl-
)imidazolidin-2-one;
N-[2-(5-amino-1H-1,2,4-triazol-3-yl)ethyl]-2-(1-benzothiophen-3-yl)-9-(pr-
opan-2-yl)-9H-purin-6-amine;
N-(2-{[2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}ethyl-
)pyridin-2-amine;
2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-N-[3-(1H-pyrazol-4-yl)propyl]-9H-
-purin-6-amine;
N-[2-({[2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}meth-
yl)propyl]acetamide;
4-(2-{[2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}ethyl-
)piperazin-2-one;
2-(1-benzothiophen-3-yl)-N-{2-[(3-methyl-1H-1,2,4-triazol-5-yl)sulfanyl]e-
thyl}-9-(propan-2-yl)-9H-purin-6-amine;
2-(1-benzothiophen-3-yl)-N-[3-(3,5-dimethyl-1H-pyrazol-4-yl)propyl]-9-(pr-
opan-2-yl)-9H-purin-6-amine;
(2-{[2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}ethyl)u-
rea;
5-({[2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}met-
hyl)-2,3-dihydro-1H-1,3-benzodiazol-2-one;
N-[2-(1H-indol-3-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-am-
ine;
N-(4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenyl-
)methanesulfonamide;
4-(2-(2-(pyridin-3-yl)-9-(tetrahydrofuran-3-yl)-9H-purin-6-ylamino)ethyl)-
phenol;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)propyl)pheno-
l;
4-(2-(9-(oxetan-3-yl)-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol;
5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)-N-methylnicotin-
amide;
6-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)-5,6,7,8-tetrahy-
dronaphthalen-2-ol;
N-(2-(1H-indazol-3-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-amin-
e;
4-(2-((9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-yl)(methyl)amino)ethyl)p-
henol;
4-(2-(9-isopropyl-8-methyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethy-
l)phenol;
1-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-1H--
benzo[d]imidazol-2(3H)-one;
4-(3-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-yl)propyl)phenol;
4-((((9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-yl)methyl)(methyl)amino)met-
hyl)phenol;
4-(((9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-yl)methylamino)methyl)phenol-
;
4-(((9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-yl)methoxy)methyl)phenol;
N-(2-(indolin-5-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-amine;
4-(2-(9-(1-methylpiperidin-4-yl)-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethy-
l)phenol;
4-(2-(9-(piperidin-4-yl)-2-(pyridin-3-yl)-9H-purin-6-ylamino)eth-
yl) phenol;
N-(2-(1H-indazol-5-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-amin-
e;
N-(2-(1H-benzo[d]imidazol-5-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-p-
urin-6-amine;
5-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)indolin-2-one-
;
4-(2-(9-cyclopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-(1-hydroxypropan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-
phenol;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenyl
sulfamate;
2-(4-hydroxyphenyl)-N-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-yl)acetami-
de;
4-(5-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)isoxazol-3-yl)ph-
enol;
4-(5-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)-1,3,4-oxadiaz-
ol-2-yl)phenol;
4-(2-(2-(2-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(9-isopropyl-2-(1-methyl-1H-pyrrol-2-yl)-9H-purin-6-ylamino)ethyl)-
phenol; and
4-(2-(9-isopropyl-2-(thiazol-5-yl)-9H-purin-6-ylamino)ethyl)phenol.
[0143] In another embodiment, an aryl hydrocarbon receptor
antagonist is a compound of Formula If:
##STR00008##
in which: R.sub.2 is selected from 1H-indol-3-yl and phenyl
optionally substituted with hydroxy; and R.sub.4 is selected from
isopropyl, sec-butyl, benzhydryl, nonan-2-yl, oxetan-3-yl and
tetrahydrofuran-3-yl.
[0144] In a further embodiment are compounds selected from:
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-ylamino)ethyl)p-
henol;
4-(2-(9-benzhydryl-2-(benzo[b]thiophen-3-yl)-9H-purin-6-ylamino)eth-
yl)phenol;
N-(2-(1H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropy-
l-9H-purin-6-amine;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(nonan-2-yl)-9H-purin-6-ylamino)ethyl)p-
henol;
N-(2-(1H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-
-purin-6-amine;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(oxetan-3-yl)-9H-purin-6-ylamino)ethyl)-
-phenol;
(S)-4-(2-(2-(benzo[b]thiophen-3-yl)-9-(tetrahydrofuran-3-yl)-9H-p-
urin-6-ylamino)ethyl)phenol; and
(R)-4-(2-(2-(benzo[b]thiophen-3-yl)-9-(tetrahydrofuran-3-yl)-9H-purin-6-y-
lamino)ethyl)phenol.
[0145] In another embodiment, an aryl hydrocarbon receptor
antagonist is a compound of Formula Ig:
##STR00009##
in which: R.sub.2 is selected from: 1H-pyrrolo[2,3-b] pyridin-3-yl;
1H-indol-3-yl optionally substituted with 1 to 2 radicals
independently selected from halo, methyl and methoxy; and phenyl
optionally substituted with 1 to 2 radicals independently selected
from methyl, halo and hydroxy; R.sub.4 is selected from isopropyl,
sec-butyl, 1-hydroxypropan-2-yl, prop-1-en-2-yl, benzhydryl,
nonan-2-yl, oxetan-3-yl and tetrahydrofuran-3-yl; and Ra, Rb and Rc
are independently selected from hydrogen, cyano, methyl, halo,
--SO.sub.2CH.sub.3 and trifluoromethyl.
[0146] In a further embodiment are compounds selected from:
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(2-(6-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(9-isopropyl-2-(4-methylpyridin-3-yl)-9H-purin-6-ylamino)ethyl)phe-
nol;
5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)nicotinonitr-
ile;
4-(2-(9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-ylamino)ethyl)p-
henol;
4-(2-(9-isopropyl-2-(5-(methylsulfonyl)pyridin-3-yl)-9H-purin-6-yla-
mino)ethyl)phenol;
4-(2-(2-(4-chloropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phe-
nol;
9-isopropyl-N-(2-(6-methoxy-1H-indol-3-yl)ethyl)-2-(pyridin-3-yl)-9H--
purin-6-amine;
9-isopropyl-N-(2-(5-methyl-1H-indol-3-yl)ethyl)-2-(pyridin-3-yl)-9H-purin-
-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-purin-
-6-amine;
4-(2-(9-(oxetan-3-yl)-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-
phenol;
4-(2-(9-(1-hydroxypropan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-ylamino-
)ethyl)phenol;
4-(2-(2-(2-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)pheno-
l;
5-(9-sec-butyl-6-(4-hydroxy-3-methylphenethylamino)-9H-purin-2-yl)nicot-
inonitrile;
4-(2-(2-(5-fluoropyridin-3-yl)-9-(oxetan-3-yl)-9H-purin-6-ylamino)ethyl)
phenol;
4-(2-(2-(5-chloropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)eth-
yl)phenol;
4-(2-(9-isopropyl-2-(5-(trifluoromethyl)pyridin-3-yl)-9H-purin--
6-ylamino)ethyl)phenol;
5-(6-(2-(1H-indol-3-yl)ethylamino)-9-sec-butyl-9H-purin-2-yl)nicotinonitr-
ile;
N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H-pu-
rin-6-amine;
(R)--N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-p-
urin-6-amine;
(S)--N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-p-
urin-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-purin--
6-amine;
(R)--N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3--
yl)-9H-purin-6-amine;
(S)--N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H-p-
urin-6-amine;
5-(6-(4-hydroxyphenethylamino)-9-(oxetan-3-yl)-9H-purin-2-yl)nicotinonitr-
ile;
4-(2-(6-(5-fluoropyridin-3-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidi-
n-4-ylamino)ethyl)phenol;
3-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)isonicotinonitri-
le; 4-(2-(2-(5-fluoropyridin-3-yl)-7-isopropyl-7H-pyrrolo[2,3-d]
pyrimidin-4-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(6-methylpyridin-3-yl)-9H-purin-6-ylamino)ethyl)pheno-
l;
2-chloro-4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)ph-
enol;
3-fluoro-4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl-
)phenol;
N-(2-(5-fluoro-1H-indol-3-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)--
9H-purin-6-amine;
N-(2-(5-chloro-1H-indol-3-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-purin-
-6-amine;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-2-m-
ethylphenol;
2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl-
)propan-1-ol;
(R)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin--
9-yl)propan-1-ol;
(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin--
9-yl)propan-1-ol;
(R)--N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-(tetrahydrofur-
an-3-yl)-9H-purin-6-amine;
4-(2-(6-(5-fluoropyridin-3-yl)-1-isopropyl-1H-imidazo[4,5-c]pyridin-4-yla-
mino)ethyl)phenol;
N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin--
6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(6-methoxy-1H-indol-3-y-
l)ethyl)-9H-purin-6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-
-9H-purin-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-(prop-1-en-2-yl)-9H-
-purin-6-amine; N-(2-(1H-pyrrolo[2,3-b]
pyridin-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-amine-
;
4-(2-(5-(5-fluoropyridin-3-yl)-3-isopropyl-3H-imidazo[4,5-b]pyridin-7-yl-
amino)ethyl)phenol;
N-(2-(1H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin--
6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(7-methyl-1H-indol-3-yl-
)ethyl)-9H-purin-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-(oxetan-3-yl)-9H-pu-
rin-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-2-(5-methylpyridin-3-yl)-9-(oxetan-3-yl)-9H-pu-
rin-6-amine;
N-(2-(6-fluoro-1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl--
9H-purin-6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(2-methyl-1H-indol-3-yl)ethyl)--
9H-purin-6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(6-methyl-1H-indol-3-yl)ethyl)--
9H-purin-6-amine;
N-(2-(4-fluoro-1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl--
9H-purin-6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(4-methyl-1H-indol-3-yl)ethyl)--
9H-purin-6-amine;
N-(2-(7-fluoro-1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl--
9H-purin-6-amine; and
4-(2-(2-(5-fluoropyridin-3-yl)-9-(1-hydroxypropan-2-yl)-9H-purin-6-ylamin-
o)ethyl)-2-methylphenol.
In specific embodiments, an aryl hydrocarbon receptor antagonist is
salt (e.g., a pharmaceutically acceptable salt) of a compound of
Formula Ia, Ib, Ic, Id, Ie, If or Ig. In specific embodiments, an
aryl hydrocarbon receptor antagonist is one of the following
compounds (the methods of making of which are described at pages
21-27 of U.S. Patent Publication No. 2010/0183564, which are
specifically incorporated by reference herein):
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)pheno-
l
##STR00010##
[0147] 4-(2-(Pyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)
phenol
##STR00011##
[0148]
4-(2-(9-Isopropyl-2-(2-methyl-1H-imidazol-1-yl)-9H-purin-6-ylamino)-
ethyl)phenol
##STR00012##
[0149]
4-(2-(2-(5-Chloropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl-
)phenol
##STR00013##
[0150]
4-(2-(6-(5-Fluoropyridin-3-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimi-
din-4-ylamino)ethyl)phenol
##STR00014##
[0151]
4-(2-(2-(5-Fluoropyridin-3-yl)-7-isopropyl-7H-pyrrolo[2,3-d]pyrimid-
in-4-ylamino)ethyl)phenol
##STR00015##
[0152]
(R)-4-(2-(2-(benzo[b]thiophen-3-yl)-9-(tetrahydrofuran-3-yl)-9H-pur-
in-6-ylamino)ethyl)phenol
##STR00016##
[0153]
2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-puri-
n-9-yl)propan-1-ol
##STR00017##
[0154]
(R)-2-(6(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridine-3-yl)-9H--
purin-9-yl)propan-1-ol &
(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin--
9-yl)propan-1-ol
##STR00018##
##STR00019##
[0155]
(R)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H--
purin-9-yl)propan-1-ol
##STR00020##
[0156]
(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H--
purin-9-yl)propan-1-ol
##STR00021##
[0157]
4-(2-(6-(5-Fluoropyridin-3-yl)-1-isopropyl-1H-imidazo[4,5-c]pyridin-
-4-ylamino)ethyl)phenol
##STR00022##
[0158]
4-(2-(5-(5-Fluoropyridin-3-yl)-3-isopropyl-3H-imidazo[4,5-b]pyridin-
-7-ylamino)ethyl)phenol
##STR00023##
[0160] In certain embodiments, an aryl hydrocarbon receptor
antagonist is one of the compounds depicted in Table 1
(corresponding to Table 1 of U.S. Patent Publication No.
2010/0183564).
TABLE-US-00001 TABLE 1 EC.sub.50 (% Example Physical Data CD34+)
Number Structure .sup.1H NMR and/or MS .mu.M 1 ##STR00024## .sup.1H
NMR (500 MHz, CDCl.sub.3): .delta. = 9.20 (d, 1H), 8.58 (s, 1H),
8.00-7.80 (m, 2H), 7.55-7.38 (m, 3H), 7.11 (d, 2H), 6.72 (d, 2H),
6.18 (br, 1H), 5.01-4.68 (m, 1H), 4.02 (br, 2H), 3.00 (t, 2H), 1.68
(d, 6H); HRMS (El) m/z 430.1698 (M + 1) 0.12 2 ##STR00025## .sup.1H
NMR (500 MHz, CDCl.sub.3): .delta. = 9.22 (d, 1H), 8.53 (s, 1H),
7.92 (d, 1H), 7.80 (s, 1H), 7.52-7.33 (m, 3H), 7.13 (d, 2H), 6.74
(d, 2H), 6.08 (br, 1H), 4.80-4.62 (m, 1H), 4.02 (br, 2H), 3.01 (t,
2H), 2.20-1.90 (m, 2H), 1.77 (d, 3H), 0.92 (t, 3H); HRMS (El) m/z
444.1857 (M + 1) 0.03 3 ##STR00026## HRMS (El) m/z 554.2005 (M + 1)
0.15 4 ##STR00027## HRMS (El) m/z 472.1807 (M + 1) 1.49 5
##STR00028## HRMS (El) m/z 546.1571 (M + 1) 2.08 6 ##STR00029##
HRMS (El) m/z 444.1857 (M + 1) 2.53 7 ##STR00030## HRMS (El) m/z
402.1385 (M + 1) 7.2 8 ##STR00031## HRMS (El) m/z 492.1856 (M + 1)
6.03 9 ##STR00032## .sup.1H NMR (500 MHz, CDCl.sub.3): .delta. =
9.21 (d, 1H), 8.48 (s, 1H), 8.02 (br, 1H), 7.89 (d, 1H), 7.79 (s,
1H), 7.70 (d, 1H), 7.50-7.07 (m, 6H), 5.82 (br, 1H), 5.00-4.88 (m,
1H), 4.13 (br, 2H), 3.22 (t, 2H), 1.69 (d, 6H); HRMS (El) m/z
453.1857 (M + 1) 0.02 10 ##STR00033## HRMS (El) m/z 420.1315 (M +
1) 1.38 11 ##STR00034## HRMS (El) m/z 430.1697 (M + 1) 1.45 12
##STR00035## HRMS (El) m/z 432.1853 (M + 1) 1.76 13 ##STR00036##
HRMS (El) m/z 429.1853 (M + 1) 5.75 14 ##STR00037## HRMS (El) m/z
376.1881 (M + 1) 0.17 15 ##STR00038## .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 9.57 (d, 1H), 8.85-8.83 (m, 1H), 8.59 (q, 1H),
8.16 (s, 1H), 7.57 (q, 1H), 7.13 (d, 2H), 6.72 (d, 2H), 4.98-4.91
(m, 1H), 3.91 (bs, 2H), 2.98 (t, 2H), 1.68 (d, 6H); HRMS (El) m/z
375.1928 (M + 1) 0.19 16 ##STR00039## HRMS (El) m/z 374.1976 (M +
1) 0.46 17 ##STR00040## HRMS (El) m/z 380.1544 (M + 1) 0.97 18
##STR00041## HRMS (El) m/z 364.1769 (M + 1) 3.9 19 ##STR00042##
HRMS (El) m/z 466.1493 (M + 1) 1.1 20 ##STR00043## HRMS (El) m/z
420.2184 (M + 1) 7.8 21 ##STR00044## HRMS (El) m/z 514.2638 (M + 1)
0.13 23 ##STR00045## HRMS (El) m/z 467.2013 (M + 1) 0.019 31
##STR00046## MS m/z 375.2 (M + 1) 0.66 32 ##STR00047## MS m/z 447.2
(M + 1) 5.6 33 ##STR00048## MS m/z 405.2 (M + 1) 0.27 34
##STR00049## MS m/z 393.2 (M + 1) 0.16 35 ##STR00050## MS m/z 389.2
(M + 1) 0.34 37 ##STR00051## MS m/z 400.2 (M + 1) 0.024 38
##STR00052## MS m/z 367.2 (M + 1) 1.6 40 ##STR00053## MS m/z 364.2
(M + 1) 0.26 42 ##STR00054## MS m/z 376.2 (M + 1) 0.64 43
##STR00055## MS m/z 376.2 (M + 1) 2.4 44 ##STR00056## MS m/z 375.2
(M + 1) 1.7 45 ##STR00057## MS m/z 389.2 (M + 1) 0.063 46
##STR00058## MS m/z 453.2 (M + 1) 0.65 48 ##STR00059## MS m/z 409.2
(M + 1) 0.51 50 ##STR00060## MS m/z 393.2 (M + 1) 0.034 52
##STR00061## MS m/z 378.2 (M + 1) 55 ##STR00062## MS m/z 380.2 (M +
1) 1.3 58 ##STR00063## MS m/z 394.2 (M + 1) 0.24 60 ##STR00064## MS
m/z 405.1 (M + 1) 3.2 61 ##STR00065## MS m/z 428.1 (M + 1) 0.13 62
##STR00066## MS m/z 412.1 (M + 1) 0.72 70 ##STR00067## MS m/z 367.2
(M + 1) 2.7 72 ##STR00068## MS m/z 375.2 (M + 1) 6.3 73
##STR00069## MS m/z 363.2 (M + 1) 8.2 76 ##STR00070## MS m/z 396.2
(M + 1) 6.0 81 ##STR00071## MS m/z 422.1 (M + 1) 2.7 82
##STR00072## MS m/z 420.1 (M + 1) 7.9 83 ##STR00073## MS m/z 430.1
(M + 1) 7.1 84 ##STR00074## MS m/z 418.1 (M + 1) 5.4 88
##STR00075## MS m/z 446.10 (M + 1) 2.6 89 ##STR00076## MS m/z
396.10 (M + 1) 1.4 90 ##STR00077## MS m/z 456.2 (M + 1) 3.3 91
##STR00078## MS m/z 398.1 (M + 1) 0.029 92 ##STR00079## MS m/z
452.2 (M + 1) 7.1 93S ##STR00080## MS m/z 403.1 (M + 1) 1.1 93R
##STR00081## MS m/z 403.1 (M + 1) 0.52 94 ##STR00082## MS m/z 389.1
(M + 1) 0.97 95 ##STR00083## MS m/z 389.1 (M + 1) 2.3 98
##STR00084## MS m/z 399.2 (M + 1) 8.2 99 ##STR00085## MS m/z 389.2
(M + 1) 7.5 113 ##STR00086## MS m/z 391.2 (M + 1) 0.54 114
##STR00087## MS m/z 454.1 (M + 1) 1.1 118 ##STR00088## MS m/z 393.2
(M + 1) 0.45 119 ##STR00089## MS m/z 377.2 (M + 1) 1.4 120
##STR00090## MS m/z 381.2 (M + 1) 1.4 121 ##STR00091## MS m/z 414.2
(M + 1) 0.086 122 ##STR00092## MS m/z 414.2 (M + 1) 0.42 123
##STR00093## .sup.1H NMR (400 MHz, DMSO): .delta. = 9.21 (br, 1H),
8.57 (t, 1H), 8.36 (s, 1H), 8.23 (d, 1H), 7.70 (d, 1H), 7.04 (d,
2H), 6.66 (d, 2H), 4.84-4.72 (m, 1H), 3.67 (q, 2H), 2.99 (s, 3H),
2.83 (t, 2H), 1.56 (d, 6H); MS m/z 378.2 (M + 1) 0.066 124
##STR00094## MS m/z 428.2 (M + 1) 0.003 125 ##STR00095## MS m/z
399.2 (M + 1) 126 ##STR00096## MS m/z 363.2 (M + 1) 5.0 127
##STR00097## MS m/z 407.3 (M + 1) 0.47 128 ##STR00098## .sup.1H NMR
(400 MHz, DMSO): .delta. = 9.47 (s, 1H), 8.71 (s, 1H), 8.67 (s,
1H), 8.32 (s, 1H), 8.04 (t, 1H), 7.10 (d, 2H), 6.69 (d, 2H),
4.91-4.81 (m, 1H), 3.80-3.70 (m, 2H), 2.86 (t, 2H), 1.58 (d, 6H);
MS m/z 409.2 (M + 1) 0.019 129 ##STR00099## MS m/z 443.2 (M + 1)
0.12 130 ##STR00100## .sup.1H NMR (400 MHz, DMSO): .delta. = 10.82
(s, 1H), 9.74 (s, 1H), 9.10 (s, 1H), 8.99 (s, 1H), 8.32 (s, 1H),
8.13 (t, 1H), 7.65 (d, 1H), 7.32 (d, 1H), 7.22 (s, 1H), 7.06 (t,
1H), 6.99 (t, 1H), 4.72-4.60 (m, 1H), 3.96-3.85 (m, 2H), 3.08 (t,
2H), 2.08-1.88 (m, 2H), 1.58 (d, 3H), 0.77 (t, 3H); MS m/z 437.2 (M
+ 1) 0.001 131 ##STR00101## .sup.1H NMR (400 MHz, DMSO): .delta. =
10.83 (s, 1H), 9.40 (s, 1H), 8.97 (s, 1H), 8.76 (s, 1H), 8.35 (s,
1H), 8.18 (t, 1H), 7.62 (d, 1H), 7.33 (d, 1H), 7.23 (s, 1H), 7.06
(t, 1H), 6.97 (t, 1H), 4.72-4.60 (m, 1H), 3.96-3.82 (m, 2H), 3.10
(t, 2H), 2.53 (s, 3H), 2.09-1.89 (m, 2H), 1.58 (d, 3H), 0.77 (t,
3H); MS m/z 426.2 (M + 1) 0.004 131R ##STR00102## MS m/z 430.2 (M +
1) 0.001 131S ##STR00103## MS m/z 430.2 (M + 1) 0.002 132
##STR00104## .sup.1H NMR (400 MHz, DMSO): .delta. = 10.83 (s, 1H),
9.42 (s, 1H), 8.66 (s, 1H), 8.41 (d, 1H), 8.31 (s, 1H), 8.09 (t,
1H), 7.64 (d, 1H), 7.34 (d, 1H), 7.22 (s, 1H), 7.07 (t, 1H), 6.97
(t, 1H), 4.68-4.60 (m, 1H), 3.92-3.84 (m, 2H), 3.08 (t, 2H),
2.08-1.90 (m, 2H), 1.58 (d, 3H), 0.77 (t, 3H); MS m/z 430.2 (M + 1)
0.003 132R ##STR00105## MS m/z 426.2 (M + 1) 0.003 132S
##STR00106## MS m/z 426.2 (M + 1) 0.003 133 ##STR00107## MS m/z
414.2 (M + 1) 0.18 134 ##STR00108## .sup.1H NMR (400 MHz, DMSO):
.delta. = 9.44 (s, 1H), 9.21 (s, 1H), 8.69 (d, 1H), 8.56 (t, 1H),
8.47 (d, 1H), 8.14 (s, 1H), 7.09 (d, 2H), 6.69 (d, 2H), 5.17-5.09
(m, 1H), 3.80-3.75 (m, 2H), 2.87 (t, 2H), 1.48 (d, 6H); MS m/z
393.2 (M + 1) 0.20 135 ##STR00109## MS m/z 430.2 (M + 1) 0.38 137
##STR00110## MS m/z 421.1 (M + 1) 138 ##STR00111## MS m/z 389.2 (M
+ 1) 0.40 139 ##STR00112## MS m/z 400.2 (M + 1) 1.3 140
##STR00113## MS m/z 400.2 (M + 1) 0.091 141 ##STR00114## .sup.1H
NMR (400 MHz, DMSO): .delta. = 9.42 (s, 1H), 8.63 (d, 1H), 8.42 (d,
1H), 7.79 (t, 1H), 7.35 (d, 1H), 7.09 (d, 2H), 6.70 (d, 2H), 6.61
(d, 1H), 5.08-5.00 (m, 1H), 3.76-3.70 (m, 2H), 2.87 (t, 2H), 1.47
(d, 6H); MS m/z 392.2 (M + 1) 0.16 143 ##STR00115## MS m/z 400.2 (M
+ 1) 4.3 144 ##STR00116## MS m/z 389.2 (M + 1) 0.16 145
##STR00117## MS m/z 425.2 (M + 1) 5.4 146 ##STR00118## MS m/z 409.1
(M + 1) 0.24 147 ##STR00119## MS m/z 393.2 (M + 1) 0.092 148
##STR00120## MS m/z 432.2 (M + 1) 0.75 149 ##STR00121## MS m/z
416.2 (M + 1) 0.52 150 ##STR00122## MS m/z 389.2 (M + 1) 0.057 151
##STR00123## MS m/z 444.1 (M + 1) 0.17 152 ##STR00124## MS m/z
458.2 (M + 1) 0.35 153 ##STR00125## .sup.1H NMR (400 MHz, CD3OD):
.delta. = 9.14 (s, 1H), 8.55 (S, 1H), 8.33 (s, 1H), 7.96 (d, 1H),
7.14 (t, 1H), 7.15 (d, 2H),
6.73 (d, 2H), 5.46-5.43 (m, 1H), 4.27-3.94 (m, 6H), 2.98 (t, 2H),
2.73-2.64 (m, 1H), 2.46-2.39 (m, 1H); MS m/z 458.2 (M + 1) 0.22 157
##STR00126## .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. = 9.40 (s,
1H), 8.53-8.48 (m, 2H), 8.23 (s, 1H), 7.65 (d, 1H), 7.31 (d, 1H),
7.11 (s, 1H), 7.08- 7.04 (m, 1H), 7.01- 6.97 (m, 1H), 4.08- 4.03
(m, 3H), 3.94 (dd, 1H), 3.35-3.30 (m, 1H), 3.19 (t, 2H), 1.68 (d,
3H); MS m/z 432.2 (M + 1) 0.005 157R ##STR00127## MS m/z 432.2 (M +
1) 0.008 157S ##STR00128## MS m/z 432.2 (M + 1) 0.003 158
##STR00129## MS m/z 444.2 (M + 1) 0.012 159 ##STR00130## MS m/z
415.2 (M + 1) 0.59 160 ##STR00131## MS m/z 415.2 (M + 1) 1.9 161
##STR00132## .sup.1H NMR (400 MHz, DMSO): .delta. = 9.11 (s, 1H),
8.64 (s, 1H), 8.51 (s, 1H), 8.30 (d, 1H), 7.74 (s, 1H), 7.09 (d,
2H), 6.69 (d, 2H), 4.88-4.76 (m, 1H), 3.88-3.78 (m, 2H), 2.88 (t,
2H), 1.56 (d, 6H); MS m/z 392.2 (M + 1) 0.17 162 ##STR00133## MS
m/z 392.2 (M + 1) 0.14 166 ##STR00134## MS m/z 378.1 (M + 1) 7.5
167 ##STR00135## MS m/z 409.2 (M + 1) 0.29 169 ##STR00136## MS m/z
446.2 (M + 1) 0.044 170 ##STR00137## MS m/z 416.2 (M + 1) 0.006 172
##STR00138## MS m/z 446.2 (M + 1) 0.42 173 ##STR00139## MS m/z
414.1 (M + 1) 0.012 174 ##STR00140## MS m/z 394.2 (M + 1) 2.2 175
##STR00141## MS m/z 417.2 (M + 1) 0.42 176 ##STR00142## MS m/z
513.3 (M + 1) 1.1 177 ##STR00143## .sup.1H NMR (400 MHz, DMSO):
.delta. = 9.18 (s, 1H), 9.15 (s, 1H), 8.57 (d, 1H), 8.29 (d, 1H),
8.26 (s, 1H), 7.11 (d, 2H), 7.01 (s, 1H), 6.79 (t, 1H), 6.95 (d,
2H), 4.92-4.84 (m, 1H), 3.72-3.62 (m, 2H), 2.83 (t, 2H), 1.56 (d,
6H); MS m/z 392.2 (M + 1) 0.14 178 ##STR00144## .sup.1H NMR (400
MHz, DMSO): .delta. = 10.83 (s, 1H), 8.67 (t, 1H), 8.37 (s, 1H),
8.15 (d, 1H), 7.71 (d, 1H), 7.57 (d, 1H), 7.33 (d, 1H), 7.20 (s,
1H), 7.06 (t, 1H), 6.96 (t, 1H), 4.60-4.48 (m, 1H), 3.86-3.76 (m,
2H), 3.06 (t, 2H), 2.96 (s, 3H), 2.05-1.85 (m, 2H), 1.56 (d, 3H),
0.76 (t, 3H); MS m/z 415.2 (M + 1) 0.003 180 ##STR00145## MS m/z
392.2 (M + 1) 0.13 181 ##STR00146## MS m/z 406.2 (M + 1) 2.5 182
##STR00147## MS m/z 432.2 (M + 1) 5.1 183 ##STR00148## .sup.1H NMR
(400 MHz, DMSO): .delta. = 10.84 (s, 1H), 9.37 (s, 1H), 8.52 (s,
1H), 8.50 (s, 1H), 8.29 (s, 1H), 8.01 (t, 1H), 7.66 (d, 1H), 7.34
(d, 1H), 7.23 (m, 1H), 7.07 (t, 1H), 6.98 (t, 1H), 4.89-4.83 (m,
1H), 3.95-3.85 (m, 2H), 3.09 (t, 2H), 2.41 (s, 3H), 1.58 (d, 6H);
MS m/z 412.2 (M + 1) 0.01 184 ##STR00149## MS m/z 401.2 (M + 1)
0.008 185 ##STR00150## MS m/z 430.2 (M + 1) 0.024 186 ##STR00151##
MS m/z 430.2 (M + 1) 0.007 187 ##STR00152## .sup.1H NMR (400 MHz,
DMSO): .delta. = 10.84 (s, 1H), 9.38 (s, 1H), 8.49 (m, 1H), 8.47
(s, 1H), 8.10 (t, 1H), 7.67 (d, 1H), 7.35 (d, 1H), 7.22 (m, 1H),
7.07 (t, 1H), 6.98 (t, 1H), 5.85-5.78 (m, 1H), 5.17 (t, 2H), 5.03
(t 2H), 3.84-3.84 (m, 2H), 3.09 (t, 2H), 2.40 (s. 3H); MS m/z 426.2
(M + 1) 0.034 188 ##STR00153## MS m/z 434.2 (M + 1) 0.005 189
##STR00154## .sup.1H NMR (400 MHz, DMSO): .delta. = 10.65 (s, 1H),
9.42 (s, 1H), 8.68 (m, 1H), 8.41 (d, 1H), 8.34 (s, 1H), 8.08 (t,
1H), 7.53 (d, 1H), 7.12 (m, 2H), 6.81 (d, 1H), 4.90-4.81 (m, 1H),
3.93-3.80 (m, 2H), 3.05 (t, 2H), 2.38 (s, 3H), 1.58 (d, 6H); MS m/z
432.0 (M + 1) 0.026 190 ##STR00155## .sup.1H NMR (400 MHz, DMSO):
.delta. = 10.71 (s, 1H), 9.42 (s, 1H), 8.67 (d, 1H), 8.38 (dd, 1H).
8.32 (s, 1H), 8.05 (t, 1H), 7.55 (d, 1H), 7.21 (d, 1H), 6.98 (t,
1H), 6.93 (t, 1H), 4.92-4.83 (m, 1H), 3.78-3.71 (m, 2H), 2.99 (t,
2H), 2.33 (s, 3H), 1.59 (d, 6H); MS m/z 430.2 (M + 1) 0.005 195
##STR00156## HPLC-MS calculated MS m/z 434.2 (M + 1) 0.003 196
##STR00157## MS m/z 434.2 (M + 1) 0.002 197 ##STR00158## .sup.1H
NMR (400 MHz, DMSO): .delta. = 10.79 (s, 1H), 9.37 (s, 1H), 8.64
(d, 1H), 8.39 (d, 1H), 8.31 (s, 1H), 8.06 (t, 1H), 7.15 (s, 1H),
7.13 (d, 1H), 6.90 (t, 1H), 6.69 (d, 1H), 4.90- 4.83 (m, 1H), 3.83-
3.87 (m, 2H), 3.24 (t, 2H), 2.65 (s, 3H), 1.57 (d, 6H); MS m/z
430.2 (M + 1) 0.011 198 ##STR00159## MS m/z 429.1 (M + 1) 1.1 199
##STR00160## MS m/z 399.2 (M + 1) 1.6 200 ##STR00161## MS m/z 423.2
(M + 1) 0.001 201 ##STR00162## 202 ##STR00163## 203
##STR00164##
[0161] In specific embodiments, any salt of an aryl hydrocarbon
receptor antagonist, disclosed in U.S. Patent Application No.
2010/0183564 or known in the art, can be used in the methods
described herein. An aryl hydrocarbon receptor antagonist or a salt
thereof for use in the methods described herein can be formulated
in DMSO or some other suitable carrier, as described in U.S. Patent
Application No. 2010/0183564 (see e.g., page 10, [0086],
specifically incorporated by reference herein in its entirety) or
known in the art.
[0162] In one specific embodiment, an aryl hydrocarbon receptor
antagonist is not alpha-naphthoflavone or
3'-methoxy-4'-nitroflavone.
[0163] In some embodiments, an aryl hydrocarbon receptor antagonist
is an organic compound, for example,
2-methyl-2H-pyrazole-3-carboxylic acid
(2-methyl-4-o-tolylazophenyl)amide (CH223191), alpha napthoflavone,
resveratrol (Nutr. Metab. Cardiovasc. Dis., 2003 April;
13(2):104-13), 3'-methoxy-4'-nitroflavone (Biochem. Pharmacol.,
2007 May 15; 73(10):1622-34, Epub 2007 Jan. 30), or
6-methyl-1,3,8-trichlorodibenzofuran (Cancer Res., 2004, Apr. 15;
64(8):2889-97) (see pages 11-12 of U.S. Patent Publication No.
2010/0183564).
[0164] In some embodiments, an aryl hydrocarbon receptor antagonist
is a compound that decreases aryl hydrocarbon receptor activity to
at least 10%, 20%, 30%, 50%, 60%, 70%, 80% or at least 90% the
transcriptional activity of aryl hydrocarbon receptor as observed
under activated conditions. Any assay known in the art can be used
to measure aryl hydrocarbon receptor inhibitory activity, e.g., the
dioxin-induced aryl hydrocarbon receptor dependent luciferase
reporter gene assay as described at page 12 and in the Examples of
U.S. Patent Publication No. 2010/0183564. In one embodiment, an
aryl hydrocarbon receptor antagonist is a compound that has an EC50
of less than 10 .mu.M, preferably less than 5 .mu.M (e.g., as
measured in the dioxin-induced aryl hydrocarbon receptor dependent
luciferase reporter gene assay).
[0165] In one embodiment, the downstream effector of an aryl
hydrocarbon receptor pathway is one or more of: Cyp1B1, Cyp1A1,
AHRR, .beta.-catenin, STATS, STAT1, HES-1, c-Myc, C/EBP, PU.1, p21,
P27, pRb, deoxynucleotidyl transferase, CXCR4, and CXCL12 (SDF-1).
In other embodiments, the downstream effector of aryl hydrocarbon
receptor pathway is one or more of: genes coding for phase I
xenobiotic-metabolizing enzymes (e.g., cytochromes P450 CYP1A1,
CYP1A2, CYP1B1 and CYP2S1), or genes coding for the phase II
enzymes (e.g., UDP-glucuronosyltransferase UGT1A6,
NAD(P)H-dependent quinone oxidoreductase-1 (NQO1), the aldehyde
dehydrogenase ALDH3A1, and several glutathione-5-transferase).
[0166] In the embodiments wherein an aryl receptor antagonist is an
antisense oligonucleotide capable of down-regulating the expression
of aryl hydrocarbon receptor, the design of such oligonucleotides
must enable specific binding of the target mRNA within cells in a
way which inhibits translation, thus, inhibiting aryl hydrocarbon
receptor protein expression. As described at page 12 of U. S.
Patent Publication No. 2010/0183564, sequence suitable for use in
design and synthesis of such antisense oligonucleotides which
specifically bind to aryl hydrocarbon receptor mRNA, genomic DNA
and/or its promoter or other control sequences are available, and
algorithms for identifying sequences with the highest predicted
binding affinity are also known.
[0167] In the embodiments wherein an aryl receptor antagonist is an
siRNA molecule capable of downregulating the expression of aryl
hydrocarbon receptor, synthesis of RNAi molecules can be affected
as described at page 12, [0101] of U.S. Patent Publication No.
2010/0183564. Examples of siRNA molecules which are capable of
down-regulating the expression of aryl hydrocarbon receptor are:
AHR 111S,5' GCG GCA TAG AGA CCG ACT TAA TTT CAA GAG AAT TAA GTC GGT
CTC TAT GCC GCT TTT TTG G 3' (SEQ ID NO:1); AHR 111AS, 5' CGC GCC
AAA AAA GCG GCA TAG AGA CCG ACT TAA TTC TCT TGA AAT TAA GTC GGT CTC
TAT GCC GC 3' (SEQ ID NO:2); AHR 242S,5' GGC TTC TTT GAT GTT GCA
TTA ATT CAA GAG ATT AAT GCA ACA TCA AAG AAG CCT TTT TTG G 3' (SEQ
ID NO:3); AHR 242AS, 5' CGC GCC AAA AAA GGC TTC TTT GAT GTT GCA TTA
ATC TCT TGA ATT AAT GCA ACA TCA AAG AAG CC 3' (SEQ ID NO:4).
[0168] In some embodiments, also contemplated herein is the use of
pharmaceutically acceptable acid salts and derivatives of the
compounds of Formula I, i.e., salts and derivatives that retain the
biological effectiveness and properties as described.
Pharmaceutically acceptable salts can be formed, for example, with
inorganic acids such as hydrochloric acid, hydrobromic acid,
sulfuric acid, nitric acid, phosphoric acid and the like, and
organic acids such as acetic acid, propionic acid, glycolic acid,
pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic
acid, fumaric acid, tartaric acid, citric acid, benzoic acid,
cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic
acid, p-toluenesulfonic acid, salicylic acid, and the like.
[0169] In specific embodiments, the chemical stability of a
composition comprising a compound of Formula I, or a
pharmaceutically acceptable salt or ester thereof, can be enhanced
by any method known in the art, e.g., by addition of an alkanoic
acid ester of a polyethoxylated sorbitol (a polysorbate) in an
amount effective to enhance the chemical stability of the
compound.
[0170] The dose of an aryl hydrocarbon receptor antagonist for use
in the methods described herein can be estimated using one or more
of cell culture assays described at pages 67-69 of U.S. Patent
Publication No. 2010/0183564.
[0171] An aryl hydrocarbon receptor antagonist can be made using
any of the methods known in the art. For example, U.S. Patent
Publication No. 2010/0183564 describes processes for making the
compounds of Formula I (see, e.g., pages 16-27, which are
specifically incorporated by reference herein in their
entireties).
[0172] Exposing HSPC to an aryl hydrocarbon receptor antagonist can
be done prior to, concurrently with, or following exposure of the
cells to a Notch agonist. In one embodiment, HSPC are exposed to
both a Notch agonist and an aryl receptor antagonist for the entire
period of ex vivo expansion of HSPC. In some embodiments, HSPC are
exposed to both a Notch agonist and an aryl receptor antagonist for
more than 80%, 85%, 90%, 95%, 98%, or 99% of the period of ex vivo
expansion of HSPC. In another embodiment, HSPC are exposed to a
Notch agonist and/or an aryl receptor antagonist for less than the
entire period of ex vivo expansion of HSPC. In yet another
embodiment, HSPC are exposed to a Notch agonist for the entire
period of ex vivo expansion of HSPC, but are exposed to an aryl
receptor antagonist for less than the entire period of ex vivo
expansion (e.g., for less than 100%, 99%, 98%, 95%, 90%, 85%, 80%,
75%, 70%, 60%, or 50% of the ex vivo expansion period).
[0173] 2.4 Growth Factors/Cytokines
[0174] In a preferred embodiment of the present invention, HSPC are
expanded by culturing the cells in the presence of an agonist of
Notch function, an aryl hydrocarbon receptor antagonist, discussed
supra, and one of more growth factors or cytokines for a given
period of time. In some embodiments, HSPC are cultured in the
presence of two or more growth factors. In yet another embodiment,
HSPC are cultured in the presence of three or more growth factors,
four or more growth factors, or five or more growth factors. When
expansion of HSPC without differentiation is to be achieved, HSPC
are cultured in the presence of growth factors that support growth
but not differentiation. The growth factor can be any type of
molecule, such as a protein or a chemical compound, that promotes
cellular proliferation and/or survival.
[0175] The description of growth factors provided herein is at
least in part found in sec. 5.2 of U.S. Pat. No. 7,399,633, and at
least in part found in U.S. Patent Publication No. U.S.
2010/0183564 (e.g., pages 13-14).
[0176] Exposing HSPC to one or more growth factors can be done
prior to, concurrently with, or following exposure of the cells to
a Notch agonist and/or an aryl hydrocarbon receptor antagonist. In
some embodiments, HSPC are exposed to one or more growth factors
for at least a portion of the time or the minimal culture time,
most preferably the majority or all of the time, that HSPC are
exposed to a Notch agonist and/or an aryl hydrocarbon receptor
antagonist. The minimal culture time is the amount of time at which
the cell would die or stop proliferating in the absence of the
Notch agonist, the aryl hydrocarbon receptor antagonist and the
growth factors (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6
weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13
weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks,
20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, or 25 weeks). In
specific embodiments, the minimal culture time is from 3 to 4
weeks.
[0177] In specific exemplary embodiments, the growth factors
present in the expansion medium include one or more of the
following growth factors: stem cell factor (SCF), also known as the
c-kit ligand or mast cell growth factor, Flt-3 ligand (Flt-3L),
interleukin-6 (IL-6), interleukin-3 (IL-3), interleukin-7 (IL-7),
interleukin-11 (IL-11), thrombopoietin (TPO),
granulocyte-macrophage colony stimulating factor (GM-CSF),
granulocyte colony stimulating factor (G-CSF), angiopoietin-like
proteins (Angptls) (Angptl2, Angptl3, Angptl5, Angptl7, and Mfap4),
insulin growth factor-2 (IFG-2), and fibroblast growth factor-1
(FGF-1).
[0178] In some embodiments, the growth factors present in the
expansion medium include one or more of the following growth
factors: IL-1, IL-3, IL-6, IL-11, G-CSF, GM-CSF, SCF, FIT3-L, TPO,
erythropoietin and analogs thereof (wherein the analogs include any
structural variants of the growth factors having the biological
activity of the naturally occurring growth factor and cytokine
receptor agonists, e.g., agonist antibody against the TPO receptor
such as VB22B sc(Fv)2 described in WO 2007/145227) (see page 13 of
U.S. Patent Publication No. 2010/0183564). In one embodiment, SCF,
Flt3-L and TPO are used in the expansion methods provided herein.
In another embodiment, IL-6, SCF, Flt3-L and TPO are used in the
expansion methods provided herein. In some embodiments, one or more
growth factors (e.g., TPO) are used in a serum-free medium.
[0179] The amount of SCF, Flt-3L, IL-6, or TPO can be in the range
of 5-1000 ng/ml, more preferably about 25-250 ng/ml or about 25-100
ng/ml, most preferably about 50-100 ng/ml. In certain specific
embodiments, the amount of SCF, Flt-3L, IL-6, or TPO is 25, 30, 50,
100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,
425 or 450 ng/ml. The amount of IL-3, IL-11, G-CSF, or GM-CSF can
be in the range of 2-100 ng/ml, more preferably about 5-50 ng/ml,
more preferably about 7.5-25 ng/ml or about 5-15 ng/ml, most
preferably about 10-15 ng/ml. In certain specific embodiments, the
amount of II-3, IL-11, G-CSF, or GM-CSF is 5, 6, 7, 8, 9, 10, 12.5,
or 15 ng/ml. In one embodiment, one or more growth factors are
added to HSPC in serum free medium.
[0180] The amount or concentration of growth factors suitable for
expanding HSPC of the present invention will depend on the activity
of the growth factor preparation, and the species correspondence
between the growth factors and HSPC, etc. Generally, when the
growth factor(s) and HSPC are of the same species, the total amount
of growth factor in the culture medium ranges from 1 ng/ml to 5
.mu.g/ml, more preferably from 5 ng/ml to 1 .mu.g/ml, and most
preferably from about 5 ng/ml to 250 ng/ml. In one embodiment, HSPC
are expanded by exposing HSPC to a Notch agonist, an aryl
hydrocarbon receptor antagonist, and 50 ng/ml or 100 ng/ml of SCF.
In another embodiment, HSPC are expanded by exposing the HSPC to a
Notch agonist, an aryl hydrocarbon receptor antagonist, and 50
ng/ml or 100 ng/ml of each of Flt-3L, IL-6, TPO and SCF. In yet
another embodiment, HSPC are expanded by exposing the HSPC to a
Notch agonist, an aryl hydrocarbon receptor antagonist, 50 ng/ml or
100 ng/ml of each of Flt-3L, IL-6, TPO and SCF, and 10 ng/ml of
IL-11 or IL-3.
[0181] In some embodiments, the amount or concentration of growth
factors suitable for expanding HSPC of the present invention is the
amount or concentration effective to promote proliferation of HSPC
but substantially no differentiation of HSPC.
[0182] In a preferred embodiment for expanding HSPC, the cells are
cultured in a tissue culture dish onto which an extracellular
matrix protein is bound. In a preferred mode of the embodiment, the
extracellular matrix protein is fibronectin (FN), or a fragment
thereof. Such a fragment can be but is not limited to CH-296 (Dao
et al., 1998, Blood 92(12):4612-21) or RetroNectin.RTM. (a
recombinant human fibronectin fragment) (Clontech Laboratories,
Inc., Madison, Wis.). In certain embodiments to the foregoing
culture conditions, fibronectin is excluded from the tissue culture
dishes or is replaced by another extracellular matrix protein. See
also U.S. Pat. No. 7,399,633 to Bernstein et al. for additional
exemplary culture conditions for HSPC expansion.
[0183] In a specific embodiment for expanding HSPC of the present
invention, the cells are cultured on a plastic tissue culture dish
containing immobilized Delta ligand, e.g., the extracellular domain
of Delta, and fibronectin in the presence of an aryl hydrocarbon
receptor antagonist, and about 25 ng/ml or about 100 ng/ml (or any
range in between these values), and preferably about 50 ng/ml, of
each of SCF and TPO. In another specific embodiment for expanding
HSPC of the present invention, the cells are cultured on a plastic
tissue culture dish containing immobilized Delta ligand and
fibronectin in the presence of an aryl hydrocarbon receptor
antagonist, and about 25 ng/ml or about 100 ng/ml (or any range in
between these values), and preferably about 50 ng/ml of each of SCF
and Flt-3L. In another specific embodiment for expanding HSPC, the
cells are cultured on a plastic tissue culture dish containing
immobilized Delta ligand and fibronectin in the presence of an aryl
hydrocarbon receptor antagonist, and about 25 ng/ml or about 100
ng/ml (or any range in between these values), and preferably about
50 ng/ml, of each of SCF, Flt-3L and TPO. In another specific
embodiment for expanding HSPC, the cells are cultured on a plastic
tissue culture dish containing immobilized Delta ligand and
fibronectin in the presence of an aryl hydrocarbon receptor
antagonist, and about 25 ng/ml or about 100 ng/ml (or any range in
between these values), and preferably about 50 ng/ml, of each of
SCF, Flt-3L, TPO and IL-6. In some of these embodiments, the HSPC
are cultured further in the presence of about 5 to 15 ng/ml, and
preferably about 10 ng/ml of IL-3. While in other embodiments, the
HSPC are cultured further in the presence of about 5 to 15 ng/ml,
and preferably about 10 ng/ml, GM-CSF. In some embodiments, the one
or more growth factors used in compositions and methods described
herein is not GM-SCF or IL-7. In some alternative embodiments,
fibronectin is excluded from the tissue culture dishes or is
replaced by another extracellular matrix protein.
[0184] Where differentiation of HSPC is desired, HSPC (e.g.,
enriched HSPC or expanded HSPC) can be exposed to one or more
growth factors that promote differentiation. The growth factors and
cell culture conditions that promote differentiation are known in
the art (see, e.g., U.S. Pat. No. 7,399,633 at Section 5.2 and
Section 5.5, the disclosures of which are specifically incorporated
by reference herein in their entireties). For example, SCF can be
used in combination with GM-SCF or IL-7 to differentiate HSPC
(e.g., expanded HSPC) into myeloid stem/progenitor cells or
lymphoid stem/progenitor cells, respectively. In specific
embodiments, HSPC can be differentiated into a lymphoid
stem/progenitor cell by exposing HSPC to about 100 ng/ml of each of
SCF and IL-7. In other embodiments, HSPC can be differentiated into
a myeloid stem/progenitor cell by exposing HSPC to about 100 ng/ml
of each of SCF and GM-SCF. In some embodiments, a retinoic acid
receptor (RAR) agonist, or preferably all trans retinoic acid
(ATRA) is used to promote the differentiation of HSPC (e.g.,
expanded HSPC). In certain embodiments, HSPC (e.g., expanded HSPC)
are differentiated before engraftment/in vivo repopulation (i.e.,
before administration of Expanded HSPC to the patient).
[0185] The growth factors utilized by the methods of the invention
can be obtained commercially, produced by recombinant expression,
or chemically synthesized. For example, Flt-3L (human), IGF-1
(human), IL-6 (human and mouse), IL-11 (human), SCF (human), TPO
(human and murine) can be purchased from Sigma (St. Louis, Mo.).
IL-6 (human and murine), IL-7 (human and murine), and SCF (human)
can be purchased from Life Technologies, Inc. (Rockville, Md.).
[0186] In other embodiments, the growth factors are produced by
recombinant expression or by chemical peptide synthesis (e.g. by a
peptide synthesizer). Methods that can be used for recombinantly
expressing the growth factors are described in, e.g., sec. 5.3 of
U.S. Pat. No. 7,399,633, which is specifically incorporated herein
by reference in its entirety. Growth factor nucleic acid and
peptide sequences are generally available from GenBank.
[0187] Preferably, but not necessarily, the growth factor(s) used
to expand HSPC in the presence of a Notch agonist and an aryl
hydrocarbon receptor antagonist by the methods of the invention is
derived from the same species as HSPC.
[0188] 2.5 Hematopoietic Stem/Progenitor Cells
[0189] U.S. Pat. No. 7,399,633 describes hematopoietic
stem/progenitor cells (HSPC) that can be used in the methods
described herein (see sections 5.4 and 5.4.1, specifically
incorporated by reference herein in their entireties). U.S. Pat.
No. 7,399,633 also describes hematopoietic cell markers (see
section 5.4.1.1, specifically incorporated by reference herein in
its entirety). The above-identified sections of U.S. Pat. No.
7,399,633 also describe isolation, separation and enrichment of
HSPC (and, thus, specifically incorporated herein to supply such
disclosures). Contemplated herein are compositions and methods for
isolation, separation and enrichment of HSPC in accordance with the
teachings of U.S. Pat. No. 7,399,633 and/or using other methods
known in the art. Further, U.S. Patent Publication No. 2010/0183564
describes hematopoietic stem/progenitor cells (HSPC) that can be
used in the methods described herein and their isolation,
separation, enrichment and expansion (see section "Utility" and
"Methods for Expanding Hematopoietic Cells" at pages 10-13,
specifically incorporated by reference herein in their entireties).
Compositions and methods that can be used for isolation, separation
and enrichment of HSPC are also described hereinbelow.
[0190] Sources of HSPC include but are not limited to: umbilical
cord blood, placental blood, peripheral blood (e.g., mobilized
peripheral blood), bone marrow (e.g., from femurs, hips, ribs,
sternum and other bones), embryonic cells (including embryonic stem
cells), aortal-gonadal-mesonephros derived cells, lymph, liver
(e.g., fetal liver), thymus, and spleen. Sources of HSPC further
include fetal blood, neonatal blood (from an infant in the first 28
days after birth), blood from an infant under 12 months of age,
blood from a toddler between 1 year and 3 years of age, blood form
a child between 3 and 18 years of age, and adult blood (i.e.,
derived from a subject who is older than 18 years of age).
[0191] HSPC can be collected from any species, including without
limitation, any vertebrate, preferably any mammal (such as a human,
a primate, a mouse, a rat, a rabbit, a guinea pig, a dog, a cat, a
horse, a cow, a pig, a sheep, a goat, etc.). In a preferred
embodiment, HSPC are collected from one or more humans. In one
embodiment, HSPC are obtained from a tissue of a patient to whom
they are to be administered after expansion (and, optionally,
differentiation). Collection of cord blood is described in further
detail in the section below. Methods that can be used for
collection of HSPC from bone marrow, peripheral blood and/or other
sources can be any of the methods known in the art.
[0192] HSPC from bone marrow can be obtained, e.g., directly from
bone marrow from the posterior iliac crest by needle aspiration
(see, e.g., Kodo et al., 1984, J. Clin Invest. 73:1377-1384), or
from the blood following pre-treatment with cytokines (such as
G-CSF) that induce cells to be released from the bone marrow
compartment.
[0193] HSPC from peripheral blood can be collected from the blood
through a syringe or catheter inserted into a patient's vein. For
example, the peripheral blood can be collected using an apheresis
machine. Blood flows from the vein through the catheter into an
apheresis machine, which separates the stem cells from the rest of
the blood and then returns the blood to the patient's body.
Apheresis can be performed for several days (e.g., 1 to 5 days)
until enough stem cells have been collected.
[0194] Peripheral blood is preferably mobilized prior to its
collection. Peripheral blood can be mobilized by any method known
in the art. Peripheral blood can be mobilized by treating the
subject from whom HSPC are to be collected with any agent(s),
described herein or known in the art, that increase the number of
HSPC circulating in the peripheral blood of a subject. For example,
in some embodiments, peripheral blood is mobilized by treating the
subject from whom HSPC are to be collected with one or more
cytokines or growth factors (e.g., G-CSF, kit ligand (KL), IL-1,
IL-7, IL-8, IL-11, Flt3 ligand, SCF, thrombopoietin, or GM-CSF
(such as sargramostim)). Different types of G-CSF that can be used
in the methods for mobilization of peripheral blood include,
without limitation, filgrastim and longer acting
G-CSF-pegfilgrastim. In certain embodiments, peripheral blood is
mobilized by treating the subject from whom HSPC are to be
collected with one or more chemokines (e.g., macrophage
inflammatory protein-la (MIP1.alpha./CCL3)), chemokine receptor
ligands (e.g., chemokine receptor 2 ligands GRO.beta. and
GRO.beta..DELTA.4), chemokine receptor analogs (e.g., stromal cell
derived factor-la (SDF-la) peptide analogs such as CTCE-0021 and
CTCE-0214, or SDF-la analog such as Met-SDF-113), or chemokine
receptor antagonists (e.g., chemokine (C-X-C motif) receptor 4
(CXCR4) antagonists such as AMD3100). In some embodiments,
peripheral blood is mobilized by treating the subject from whom
HSPC are to be collected with one or more anti-integrin signaling
agents (e.g., function blocking anti-very late antigen 4 (VLA-4)
antibody, or anti-vascular cell adhesion molecule 1 (VCAM-1)). In
other embodiments, peripheral blood is mobilized by treating the
subject from whom HSPC are to be collected with one or more
cytotoxic drugs such as cyclophosphamid, etoposide or paclitaxel.
In particular, peripheral blood can be mobilized by administering
to a subject one or more of the agents listed above for a certain
period of time. For example, the subject can be treated with one or
more agents (e.g., G-CSF) via injection (e.g., subcutaneous,
intravenous or intraperitoneal), once daily or twice daily, for 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days prior to
collection of HSPC. In specific embodiments, HSPC are collected
within 1, 2, 3, 4, 5, 6, 7, 8, 12, 14, 16, 18, 20 or 24 hours after
the last dose of an agent used for mobilization of peripheral
blood. In some embodiments, peripheral blood is mobilized by
treating the subject from whom HSPC are to be collected with two or
more different types of agents described above or known in the art,
such as a growth factor (e.g., G-CSF) and a chemokine receptor
antagonist (e.g., CXCR4 receptor antagonist such as AMD3100), or a
growth factor (e.g., G-CSF or KL) and an anti-integrin agent (e.g.,
function blocking VLA-4 antibody). In particular embodiments,
different types of mobilizing agents are administered concurrently
or sequentially. Methods of mobilization of peripheral blood are
known in the art (see, e.g., Craddock et al., 1997, Blood
90(12):4779-4788; Jin et al., 2008, Journal of Translational
Medicine 6:39; Pelus, 2008, Curr. Opin. Hematol. 15(4):285-292;
Papayannopoulou et al., 1998, Blood 91(7):2231-2239; Tricot et al.,
2008, Haematologica 93(11):1739-1742; Weaver et al., 2001, Bone
Marrow Transplantation 27(2):S23-S29).
[0195] In one aspect, HSPC used in the methods described herein can
be collected from a single human. In another aspect, HSPC used in
the methods described herein can be collected from two or more
humans. In some aspects, HSPC used in the methods described herein
are collected from a single human at birth or not more than two
humans at birth. In one embodiment, one or more HSPC samples (from
one, two or more humans) can be pooled prior to enriching for HSPC,
prior to expansion of HSPC, and/or prior to engraftment of the
expanded HSPC. In another embodiment, individual HSPC samples (from
one, two or more humans) can be pooled after enriching for HSPC,
and/or after expansion of such HSPC. In specific embodiments, the
number of HSPC samples that are pooled is 2, 3, 4, 5, 6, 7, 8, 9,
10, 15, 20, 25, 30, 35, or 40, or at least any of the foregoing
numbers, or no more than 2, 3, 4, 5, 10, 15 or 20 HSPC samples,
respectively. In some embodiments, the HSPC samples are pooled
without regard to the HLA type of the HSPC. In some embodiments,
the HSPC samples (e.g., one or more HSPC samples from one, two or
more humans) are for administration to a patient without regard to
the HLA type of the HSPC or without HLA matching. In a specific
embodiment, the Expanded HSPC are administered to a patient without
regard to the HLA type of the HSPC or without HLA matching. In
certain embodiments, the samples in the pool are derived from HSPC
from individuals of the same race, e.g., African-American,
Caucasian, Asian, Hispanic, Native-American, Australian Aboriginal,
Inuit, Pacific Islander, or derived from HSPC from individuals of
the same ethnicity, e.g., Irish, Italian, Indian, Japanese,
Chinese, Russian, etc.
[0196] 2.6 Collecting Umbilical Cord Blood or Placental Blood
[0197] Sources of HSPC include human umbilical cord blood and/or
human placental blood. Such blood can be obtained by any method
known in the art. The use of cord or placental blood as a source of
CB Stem Cells provides numerous advantages, including that the cord
and placental blood can be obtained easily and without trauma to
the donor. See, e.g., U.S. Pat. No. 5,004,681 for a discussion of
collecting cord and placental blood at the birth of a human. In one
embodiment, cord blood collection is performed by the method
disclosed in U.S. Pat. No. 7,147,626 to Goodman et al.
[0198] Collections should be made under sterile conditions.
Immediately upon collection, cord or placental blood should be
mixed with an anticoagulant. Such an anticoagulant can be any known
in the art, including but not limited to CPD
(citrate-phosphate-dextrose), ACD (acid citrate-dextrose),
Alsever's solution (Alsever et al., 1941, N. Y. St. J. Med.
41:126), De Gowin's Solution (De Gowin, et al., 1940, J. Am. Med.
Ass. 114:850), Edglugate-Mg (Smith, et al., 1959, J. Thorac.
Cardiovasc. Surg. 38:573), Rous-Turner Solution (Rous and Turner,
1916, J. Exp. Med. 23:219), other glucose mixtures, heparin, ethyl
biscoumacetate, etc. See, generally, Hurn, 1968, Storage of Blood,
Academic Press, New York, pp. 26-160). In one embodiment, ACD can
be used.
[0199] The cord blood can preferably be obtained by direct drainage
from the cord and/or by needle aspiration from the delivered
placenta at the root and at distended veins. See, generally, U.S.
Pat. No. 5,004,681. Preferably, the collected human cord blood
and/or placental blood is free of contamination.
[0200] In certain embodiments, HSPC are obtained from the fetal
blood from the fetal circulation at the placental root with the use
of needle guided ultrasound, by placentocentisis, or by fetoscopy
as described in sec. 5.4.5 of U.S. Pat. No. 7,399,633. In specific
embodiments, HSPC are obtained from Wharton's jelly as described in
sec. 5.4.5 of U.S. Pat. No. 7,399,633.
[0201] In certain embodiments, the following tests on the collected
blood sample can be performed either routinely, or where clinically
indicated:
[0202] (i) Bacterial culture: To ensure the absence of microbial
contamination, established assays can be performed, such as routine
hospital cultures for bacteria under aerobic and anaerobic
conditions.
[0203] (ii) Diagnostic screening for pathogenic microorganisms: To
ensure the absence of specific pathogenic microorganisms, various
diagnostic tests can be employed. Diagnostic screening for any of
the numerous pathogens transmissible through blood can be done by
standard procedures. As one example, the collected blood sample (or
a maternal blood sample) can be subjected to diagnostic screening
for the presence of Human Immunodeficiency Virus-1 or 2 (HIV-1 or
HIV-2). Any of numerous assay systems can be used, based on the
detection of virions, viral-encoded proteins, HIV-specific nucleic
acids, antibodies to HIV proteins, etc. The collected blood can
also be tested for other infectious diseases, including but not
limited to human T-Cell lymphotropic virus I and II (HTLV-I and
HTLV-II), Hepatitis B, Hepatitis C, Cytomegalovirus, Syphilis, West
Nile Virus.
[0204] Preferably, prior to collection of the cord blood, maternal
health history is determined in order to identify risks that the
cord blood cells might pose in transmitting genetic or infectious
diseases, such as cancer, leukemia (e.g., acute myeloid leukemia),
immune disorders, neurological disorders, hepatitis or AIDS. The
collected cord blood samples can undergo testing for one or more of
cell viability, HLA typing, ABO/Rh typing, CD34+ cell count, and
total nucleated cell count.
[0205] In one aspect, umbilical cord blood or placental blood for
use in the methods described herein can be collected from a single
human at birth. In another aspect, umbilical cord blood or
placental blood for use in the methods described herein can be
collected from two or more humans. In one embodiment, one or more
umbilical cord blood and/or placental blood samples can be pooled
prior to enriching for CB HSPC, prior to expansion of such samples,
and/or prior to engraftment of expanded samples of umbilical cord
blood and/or placental blood. In another embodiment, individual
umbilical cord blood and/or placental blood samples can be pooled
after enriching for HSPC, and/or after expansion of such cells. In
specific embodiments, the number of umbilical cord blood and/or
placental blood samples, or CB Stem Cell samples, that are pooled
is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40, or at
least any of the foregoing numbers, or no more than 3, 5, 10, 20 or
25, umbilical cord blood and/or placental blood samples, or CB Stem
Cell samples, respectively. In one embodiment, the umbilical cord
blood and/or placental blood samples or CB Stem Cell samples are
pooled without regard to the HLA type of the hematopoietic
stem/progenitor cells. In some embodiments, the umbilical cord
blood and/or placental blood samples or CB Stem Cell samples (e.g.,
one or more samples from one, two or more humans) are for
administration to a patient without regard to the HLA type of the
HSPC or without HLA matching. In a specific embodiment, the
Expanded HSPC, obtained from umbilical cord blood or placental
blood using the methods described herein, are administered to a
patient without regard to the HLA type of the HSPC or without HLA
matching. In certain embodiments, the samples in the pool are
derived from the umbilical cord blood and/or placental blood of
individuals of the same race, e.g., African-American, Caucasian,
Asian, Hispanic, Native-American, Australian Aboriginal, Inuit,
Pacific Islander, or derived from umbilical cord blood and/or
placental blood of individuals of the same ethnicity, e.g., Irish,
Italian, Indian, Japanese, Chinese, Russian, etc.
[0206] 2.7 Enrichment of HSPC
[0207] Once HSPC are isolated or collected, the blood is processed
to produce an enriched hematopoietic stem and progenitor cell
population. Enriched HSPC produced from umbilical cord blood or
placental blood form a population of CB Stem Cells.
[0208] The hematopoietic stem/progenitor cells can be positive for
a specific marker expressed in increased levels on the
hematopoietic stem/progenitor cells relative to other types of
hematopoietic cells. For example, such markers can be, but are not
limited to, CD34, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117,
CD133, CD166, HLA DR, or a combination thereof. The hematopoietic
stem/progenitor cells also can be negative for a specific marker
relative to other types of hematopoietic cells. For example, such
markers can be, but are not limited to, Lin, CD38, or a combination
thereof. Preferably, the hematopoietic stem/progenitor cells are
CD34+ cells. Preferably, the Enriched HSPC are enriched in CD34+
stem/progenitor cells (and, thus, T cell depleted). Enrichment thus
refers to a process wherein the percentage of hematopoietic
stem/progenitor cells in the sample is increased (relative to the
percentage in the sample before the enrichment procedure).
Purification is one example of enrichment. In certain embodiments,
the increase in the number of CD34+ cells (or other suitable
antigen-positive cells) as a percentage of cells in the enriched
sample, relative to the sample prior to the enrichment procedure,
is at least 25-, 50-, 75-, 100-, 150-, 200-, 250-, 300-, 350-fold,
and preferably is 100-200 fold. In a preferred embodiment, the
CD34+ cells are enriched using a monoclonal antibody to CD34, which
antibody is conjugated to a magnetic bead, and a magnetic cell
separation device to separate out the CD34+ cells. In some
embodiments, using anti-CD34 antibodies, HSPC are enriched from
1-2% of a normal bone marrow cell population to approximately
50-80% of the population, as described in sec. 5.4.1.1 of U.S. Pat.
No. 7,399,633.
[0209] In certain embodiment, prior to processing for enrichment,
the collected HSPC sample (derived, e.g., from peripheral blood,
bone marrow, umbilical cord blood, or placental blood) is fresh and
has not been previously cryopreserved. In one embodiment, prior to
processing for enrichment, the collected cord and/or placental
blood is fresh and has not been previously cryopreserved. In other
embodiments, prior to processing for enrichment, the collected HSPC
sample has been cryopreserved and thawed.
[0210] Any technique known in the art for cell separation/selection
can be used to carry out the enrichment for hematopoietic
stem/progenitor cells. For example, methods which rely on
differential expression of cell surface markers can be used. For
example, cells expressing the cell surface marker CD34 can be
positively selected using a monoclonal antibody to CD34, such that
cells expressing CD34 are retained, and cells not expressing CD34
are not retained. Moreover, the separation techniques employed
should maximize the viability of the cell to be selected. The
particular technique employed will depend upon efficiency of
separation, cytotoxicity of the methodology, ease and speed of
performance, and necessity for sophisticated equipment and/or
technical skill.
[0211] Procedures for separation may include magnetic separation,
using antibody-coated magnetic beads; fluorescence activated cell
sorting (FACS); affinity chromatography; cytotoxic agents joined to
a monoclonal antibody or used in conjunction with a monoclonal
antibody, e.g., complement and cytotoxins; and "panning" with
antibody attached to a solid matrix, e.g., plate, or other
convenient technique. Techniques providing accurate
separation/selection include fluorescence activated cell sorters,
which can have varying degrees of sophistication, e.g., a plurality
of color channels, low angle and obtuse light scattering detecting
channels, impedance channels, etc.
[0212] The antibodies may be conjugated with markers, such as
magnetic beads, which allow for direct separation, biotin, which
can be removed with avidin or streptavidin bound to a support,
fluorochromes, which can be used with a fluorescence activated cell
sorter, or the like, to allow for ease of separation of the
particular cell type. Any technique may be employed which is not
unduly detrimental to the viability of the remaining cells.
[0213] In one embodiment, the enrichment of HSPC is affected by
contacting an HSPC sample with a solid substrate (e.g., beads,
flask, magnetic particles) to which antibodies are bound, and by
removing any unbound cells, wherein the Enriched HSPC can be found
either in the cells bound to the solid substrate or in the unbound
cells depending on the antibodies used.
[0214] In one embodiment of the present invention, an HSPC sample
(e.g., a fresh cord blood unit) is processed to select for, i.e.,
enrich for, CD34+ cells using anti-CD34 antibodies directly or
indirectly conjugated to magnetic particles in connection with a
magnetic cell separator, for example, the CliniMACS.RTM. Cell
Separation System (Miltenyi Biotec, Bergisch Gladbach, Germany),
which employs nano-sized super-paramagnetic particles composed of
iron oxide and dextran coupled to specific monoclonal antibodies.
The CliniMACS.RTM. Cell Separator is a closed sterile system,
outfitted with a single-use disposable tubing set. The disposable
set can be used for and discarded after processing a single unit of
collected cord and/or placental blood to enrich for CD34+ cells.
Similarly, CD133+ cells can be enriched using anti-CD133
antibodies. In a specific embodiment, CD34+CD90+ cells are enriched
for. Similarly, cells expressing CD43, CD45RO, CD45RA, CD59, CD90,
CD109, CD117, CD166, HLA DR, or a combination of the foregoing, can
be enriched for using antibodies against the antigen.
[0215] In one embodiment, HSPC express CD34 (CD34+) and lack CD38
expression (CD38-). In some embodiments, HSPC are selected and/or
enriched for CD34+CD38- cells. In specific embodiments, HSPC are
CD34+ and CD33-, CD38-, HLA DR- and/or Thy-1lo. In some
embodiments, HSPC are selected and/or enriched for CD34+ and CD33-,
CD38-, HLA DR- and/or Thy-1lo cells. In particular embodiments,
human HSPC are CD45Ra-, CD19- and/or c-kit+. In some embodiments,
HSPC are selected and/or enriched for CD45Ra-, CD19- and/or c-kit+
cells. In one embodiment, HSPC express vascular endothelial growth
factor receptor 2 (VEGFR2). In some embodiments, HSPC are selected
and/or enriched for VEGFR2, which can be used as a marker for
HSPC.
[0216] HSPC can also be enriched as described in sec. 5.4.1.1 of
U.S. Pat. No. 7,399,633. In particular, human HSPC can be enriched
by incubating a sample with antibodies that recognize one or more
of glycophorin A, CD3, CD24, CD16, CD14, CD45Ra, CD36, CD56, CD2,
CD19, CD20, CD66a and CD66b, and separating the antibody-bound
cells from non-antibody bound cells. In some of these embodiments,
the non-antibody bound cell population would be enriched for HSPC.
In some embodiments My10 and HLA-DR are used to obtain enriched
HSPC. In some embodiments, T lymphocyte depletion is used to enrich
for HSPC, e.g., by pretreating cells with a monoclonal antibody
that recognizes a T cell antigen plus complement. In one
embodiment, glycophorin A antibody is used to select for or against
erythrocytes. In other embodiments, antibodies against CD14, CD16,
CD66a and CD66b are used to select for or against monocytes. In
other embodiments, antibodies against CD24, CD3, CD19, CD20, CD56,
CD2 are used to select for or against B and T lymphocytes and NK
cells. In yet another embodiment, antibodies against CD45RA and
CD36 are used to select for or against T-cells, B-cells,
granulocytes, platelets, monocytes, differentiated erythroid
precursors, and some committed mature progenitors. T-cell markers
for use in the subject invention include CD7, CD5, TCD-2, and
either CD4 or CD8. CD7 and terminal deoxyribonucleotidyl
transferase (Tdt), which are markers of pre-T progenitor cells.
Markers of pre-B progenitor cells can be MHC class II antigens.
CD21 is a marker of mature B cells. In specific embodiments,
antibodies which can be used for enrichment of HSPC include My-10
and 3C5 (which recognize CD34), or RFB-1 (which recognizes CD99 and
identifies populations of BFU-E cells). Other antibodies against
the above-mentioned hematopoietic antigens are disclosed in U.S.
Pat. No. 5,877,299.
[0217] The above-mentioned antibodies can be used alone or in
combination with procedures such as "panning" (Broxmeyer et al.,
1984, J. Clin. Invest. 73:939-953) or fluorescence activated
cell-sorting (FACS) (Williams et al., 1985, J. Immunol. 135:1004;
Lu et al., 1986, Blood 68(1):126-133) to isolate the cells
containing surface determinants recognized by these antibodies, as
described in sec. 5.4.1.1 of U.S. Pat. No. 7,399,633. HSPC can also
be separated and/or enriched using selective agglutination using a
lectin such as soybean (Reisner et al., 1980, Proc. Natl. Acad.
Sci. U.S.A. 77:1164).
[0218] In particular embodiments, HSPC separated and/or enriched as
described herein still contain accessory or helper cells
(non-stem/progenitor cells that influence the growth of
stem/progenitor cells). In other embodiments, HSPC separated and/or
enriched as described herein do not contain accessory or helper
cells.
[0219] Optionally, prior to enrichment for HSPC, the red blood
cells and white blood cells of the HSPC sample can be separated.
Once the separation of the red blood cells and the white blood
cells has taken place, the red blood cell fraction can be
discarded, and the white blood cell fraction can be processed in
the magnetic cell separator as above. Separation of the white and
red blood cell fractions can be performed by any method known in
the art, including centrifugation techniques. Other separation
methods that can be used include the use of commercially available
products FICOLL.TM. or FICOLL-PAQUE.TM. or PERCOLL.TM. (GE
Healthcare, Piscataway, N.J.). FICOLL-PAQUE.TM. is normally placed
at the bottom of a conical tube, and the whole blood is layered
above. After being centrifuged, the following layers will be
visible in the conical tube, from top to bottom: plasma and other
constituents, a layer of mono-nuclear cells called buffy coat
containing the peripheral blood mononuclear cells (white blood
cells), FICOLL-PAQUE.TM., and erythrocytes and granulocytes, which
should be present in pellet form. This separation technique allows
easy harvest of the peripheral blood mononuclear cells.
[0220] Optionally, prior to CD34.sup.+ cell selection, an aliquot
of the HSPC sample (e.g., a fresh cord blood unit) can be checked
for total nucleated cell count and/or CD34.sup.+ content. In a
specific embodiment, after the CD34.sup.+ cell selection, both
CD34.sup.+ and CD34- cell fractions are recovered. Optionally, DNA
can be extracted from a sample of the CD34- cell fraction for
initial HLA typing and future chimerism studies. The CD34.sup.+
enriched stem cell fraction can be subsequently processed prior to
expansion, for example, the HSPC can be suspended in an appropriate
cell culture medium for transport or storage. In a specific
embodiment, the cell culture medium consists of STEMSPAN.TM. Serum
Free Expansion Medium (StemCell Technologies, Vancouver, British
Columbia) supplemented with 10 ng/ml recombinant human
Interleukin-3 (rhlL-3), 50 ng/ml recombinant human Interleukin-6
(rhlL-6), 50 ng/ml recombinant human Thrombopoietin (rhTPO), 50
ng/ml recombinant human Flt-3 Ligand (rhFlt-3L), 50 ng/ml and
recombinant human stem cell factor (rhSCF).
In a specific embodiment, the HSPC (e.g., from umbilical cord blood
and/or placental blood) sample are red cell depleted, and the
number of CD34.sup.+ cells in the red cell depleted fraction is
calculated. Preferably, the HSPC (e.g. umbilical cord blood and/or
placental blood) samples containing more than 3.5 million
CD34.sup.+ cells are enriched by the enrichment methods described
above.
[0221] 2.8 Methods of HSPC Expansion
[0222] After HSPC have been isolated according to the enrichment
methods described above or other methods known in the art, the
Enriched HSPC can be expanded in order to increase the number of
hematopoietic stem/progenitor cells, e.g., CD34+ cells. In less
preferred embodiments, the methods described herein can be applied
to HSPC without prior enrichment, or prior to enrichment.
[0223] In some embodiments, HSPC that are subjected to expansion
using the methods described herein are fresh, i.e., they have not
been previously cryopreserved and thawed. In other embodiments,
HSPC that are subjected to expansion using the methods described
herein have been cryopreserved and thawed. The HSPC can be derived,
e.g., from peripheral blood (such as mobilized peripheral blood),
bone marrow, umbilical cord blood, or placental blood.
In certain embodiments, described herein are methods for expansion
of HSPC (e.g., the Enriched HSPC) using a composition comprising a
Notch agonist and an aryl hydrocarbon receptor antagonist. In one
embodiment, a Notch agonist as described herein is immobilized
(e.g., immobilized on a solid phase surface to which the cells are
exposed during cell culturing), while the aryl hydrocarbon receptor
antagonist is present in the cell culture medium. In specific
embodiments, the Notch agonist (e.g., an extracellular domain of a
Notch ligand) is fused to a fusion partner before immobilization.
The fusion partners can be, but are not limited to, an Fc domain of
IgG or tags that contain antigenic determinants such as a myc tag.
The fusion partner can be any protein or peptide preferably of at
least six amino acids in length.
[0224] The solid phase surface on which a Notch agonist is
immobilized can be any surface known in the art, e.g., the inside
surface of a cell culture dish, flask, or container, or the surface
of a bead, etc. The immobilization of a Notch agonist on the solid
surface can be by any method known in the art, and can be covalent
or noncovalent, by adsorption or cross-linking, etc. In a specific
embodiment, an antibody to the fusion partner of an extracellular
domain of a Notch ligand (e.g., a Delta or a Serrate protein, or a
Notch-binding portion thereof) can be bound (e.g., covalently) to
the solid phase surface, and then immunospecifically bound to the
fusion partner. In one embodiment, the solid phase surface (e.g.,
an inside surface of a cell culture dish, flask, or container, or
the surface of a bead) is pre-coated with an antibody to a fusion
partner protein (e.g., an anti-myc where the fusion partner is a
myc tag, or an anti-IgG Fc domain antibody where the fusion partner
is an Fc domain of an IgG) before addition of an extracellular
domain of a Notch ligand fused to the fusion partner.
[0225] Preferably, the Notch agonist (e.g., an extracellular domain
of a Notch ligand) is immobilized on the inside surface of a cell
culture dish, flask or another container. In specific embodiments,
Delta.sup.ext-IgG (e.g., Delta1.sup.ext-IgG) or
Delta.sup.ext-myc(e.g., Delta1.sup.ext-myc) is immobilized on the
inside surface of a cell culture dish, flask or another container.
In some embodiments, to present Delta.sup.ext-IgG (e.g.,
Delta1.sup.ext-IgG) or Delta.sup.ext-myc (e.g., Delta1.sup.ext-myc)
in immobilized form, Delta.sup.ext-IgG or Delta.sup.ext-myc is
attached to the surface of the cell culture dish by binding to an
anti-myc tag antibody (e.g., 9E10), or anti-human IgG Fc domain
antibody, respectively, that had previously been adsorbed to the
surface of the cell culture dish.
[0226] In a specific embodiment, a Notch agonist (e.g., an
extracellular domain of a Notch ligand) is immobilized on beads
(e.g., Sepharose beads, agarose beads, or another type of bead
known in the art). A Notch agonist can be attached to the beads
utilizing any methodology known in the art including, but not
limited to, cross-linking, binding via antibody, or sticking. For
example, an extracellular domain of a Notch ligand can be fused to
a myc tag and bound to Sepharose beads crosslinked to an anti-myc
tag antibody (e.g., 9E10) (see, e.g., methodology described in
Varnum Finney et al., 1998, Blood 91(11):4084-4091).
[0227] In certain embodiments, a Notch agonist is any one of the
compounds described in Section 6.2 above. In some embodiments, a
Notch agonist is a Notch-interacting domain of a Delta (e.g.,
Delta-1, Delta-3 or Delta-4), a Jagged (e.g., Jagged-1 or
Jagged-2), or a Serrate protein. In some embodiments, a Notch
agonist comprises an extracellular domain of a Delta protein or a
Serrate (e.g., Jagged) protein. In preferred embodiments, a Notch
agonist comprises a human or rodent Delta protein or a human or
rodent Jagged protein (e.g., an extracellular domain of a human
Delta protein or a human Jagged protein). Any ligand immobilization
technique known in the art can be used in the methods of the
invention to immobilize the Notch agonist. In specific embodiments,
a Notch agonist (e.g., an extracellular domain of a Notch ligand)
is fused to a fusion partner protein. Any fusion partner protein
known in the art can be used in the methods, kits and compositions
of the invention. For example, a tag (with an antigenic
determinant) or an intracellular domain of a receptor can be used
as the fusion partner protein. Fusion partner proteins include, but
are not limited to, an Fc domain of an IgG, a myc tag, and a his
tag. In one embodiment, the Notch agonist is the extracellular
domain of a Delta protein or a Serrate (e.g., Jagged) protein fused
to the Fc domain of human IgG (e.g., Delta1.sup.ext-IgG). In
another embodiment, the Notch agonist is the extracellular domain
of a Delta protein or a Serrate (e.g., Jagged) protein fused to a
myc epitope tag (e.g., Delta1.sup.ext-myc). Preferably, a Notch
agonist (e.g., Delta1.sup.ext-IgG) is immobilized on the surface of
the tissue culture dish during HSPC expansion. In specific
embodiments, a Notch agonist (e.g., Delta1.sup.ext-IgG) is
immobilized on beads (e.g., Sepharose beads, agarose beads, or
other types of beads known in the art).
[0228] In preferred embodiments, an aryl hydrocarbon receptor
antagonist is any one of the compounds described in Section 6.3
above. In one embodiment, an aryl hydrocarbon receptor antagonist
is SR1.
[0229] In some embodiments, described herein are methods for
expansion of HSPC (e.g., the Enriched HSPC) using a composition
comprising a Notch agonist, an aryl hydrocarbon receptor
antagonist, and one or more growth factors. Growth factors that can
be used in the methods for expansion of HSPC (e.g., the Enriched
HSPC) are described in Section 6.4 above. In certain embodiments,
the one or more growth factors can be selected from the following
human growth factors: stem cell factor, Flt-3-ligand,
thrombopoietin, interleukin-6, and interleukin-3. In some
embodiments, HSPC (e.g., the Enriched HSPC) are expanded in the
presence of two or more, or three or more growth factors. In one
embodiment, the following four human growth factors are present
during HSPC expansion: stem cell factor, Flt-3-ligand,
thrombopoietin and interleukin-6. In one embodiment, the following
five human growth factors are present during HSPC expansion: stem
cell factor, Flt-3-ligand, thrombopoietin, interleukin-6 and
interleukin-3. In specific embodiments, described herein are
methods for expansion of HSPC (e.g., the Enriched HSPC) using a
composition comprising a Notch agonist, an aryl hydrocarbon
receptor antagonist, one or more growth factors, and an immobilized
fibronectin or a fragment thereof. In one embodiment, an
immobilized fibronectin or a fragment thereof is CH-296 or
RetroNectin.RTM. (a recombinant human fibronectin fragment).
[0230] Preferably, HSPC (e.g., the Enriched HSPC) are cultured
under cell growth conditions (e.g., promoting mitosis) such that
the HSPC grow and divide (proliferate) to obtain a population of
Expanded HSPC. In some embodiments, HSPC (e.g., the Enriched HSPC)
used for ex vivo expansion are derived from a single human (e.g.,
CB Stem Cells derived from a single human at birth). In another
embodiment, HSPC (e.g., the Enriched HSPC) used for ex vivo
expansion are derived from two or more humans and pooled prior to
the expansion methods described herein. In some embodiments, the
Expanded HSPC are pooled after the expansion methods described
herein. In one embodiment, individual populations of CB Stem Cells
each derived from the umbilical cord blood and/or placental blood
of a single human at birth can be pooled prior to or after the
expansion technique. In another embodiment, the sample that is
expanded is not a pool of samples but is obtained from a single
individual at birth.
[0231] In one embodiment, the HSPC sample that is expanded as
described herein is derived from only one cord blood unit. In one
embodiment, the HSPC sample that is expanded as described herein is
derived from one or two cord blood units. In one embodiment, the
HSPC sample that is expanded as described herein is derived from
mobilized peripheral blood from only one patient (e.g., human).
[0232] In one specific embodiment where the HSPC sample essentially
consists of CD34+ enriched cells from one or two cord blood units,
described herein are methods for expansion wherein HSPC are
cultured from about 3 days to about 90 days, e.g., between 7 and 2
days, and/or until the fold expansion or the characteristic cell
populations described herein are obtained. In one embodiment,
described herein are methods for expansion wherein HSPC are
cultured not more than 21 days, 16 days, 14 days or 7 days.
[0233] In particular embodiments, the HSPC sample that is expanded
as described herein contains at least 50% CD34+ cells or more than
90% of CD34+ cells. In one embodiment, the HSPC sample that is
expanded as described herein contains between 10.sup.5 and 10.sup.9
nucleated cells. In specific embodiments, the sample that is
expanded as described herein is derived from mobilized peripheral
blood (e.g., human) which have been enriched in CD34.sup.+
cells.
[0234] In specific embodiments, wherein the HSPC sample that is
expanded as described herein is derived from not more than one or
two cord blood units, the Expanded HSPC contain a total amount of
cells of at least 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8 or
10.sup.9 cells, with between 20-100%, e.g., between 40-80%, of
total cells being CD34+ cells. In one embodiment, the Expanded HSPC
contain a total amount of cells between 0.1-40%, e.g., between
0.1-10%, of total cells being CD34+Thy1+ and 20-80% of cells being
CD34+CD45RA+. In some embodiments, the Expanded HSPC contain
between 10-95% of cells being CD38+ and between 5-70% of cells
being CD133+.
[0235] In one embodiment, described herein are methods for
expansion wherein HSPC are cultured for a period of time sufficient
to reach an absolute number of CD34+ cells of at least 10.sup.5,
10.sup.6, 10.sup.7, 10.sup.8 or 10.sup.9 cells. In another
embodiment, described herein are methods for expansion wherein HSPC
are cultured for a period of time sufficient to achieve a 10 to
50000 fold expansion of CD34+ cells, e.g., between 100 and 10000
fold expansion.
[0236] Preferably, the technique used for expansion is one that has
been shown to (i) result in an increase in the number of
hematopoietic stem/progenitor cells, e.g., CD34+ cells, in the
expanded sample relative to the unexpanded HSPC sample, or (ii)
results in an increased number of SCID repopulating cells in the
expanded sample determined by limiting-dilution analysis as shown
by enhanced engraftment in NOD/SCID mice infused with the expanded
sample, relative to that seen with the unexpanded sample, where the
unexpanded sample and expanded sample are from different aliquots
of the same sample, wherein the expanded sample but not the
unexpanded sample is subjected to the expansion technique.
[0237] In certain embodiments, the technique results in (or more
than) a 50-, 75-, 100-, 150-, 200-, 250-, 300-, 350-, 400-, 450-,
500-, 1000-, 2000-, 3000-, 4000-, 5000-fold increase in the number
of hematopoietic stem/progenitor cells in the expanded sample,
relative to the unexpanded sample. The hematopoietic
stem/progenitor cells can be positive for one or more of CD34,
CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD133, CD166, and
HLA DR and/or negative for Lin and/or CD38. In a specific
embodiment, the enhanced engraftment can be detected by detecting
an increased percentage of human CD45.sup.+ cells in the bone
marrow of mice infused with an aliquot of the expanded sample
relative to mice infused with an aliquot of the unexpanded sample
at, e.g., 10 days, 3 weeks or 9 weeks post-infusion (see Delaney et
al., 2010, Nature Med. 16(2): 232-236). In some embodiments, the
technique results in (or more than) a 50-, 75-, 100-, 150-, 200-,
250-, 300-, 350-, 400-, 450-, 500-, 1000-, 2000-, 3000-, 4000-,
5000-fold increase in the number of CD34+ hematopoietic
stem/progenitor cells in the expanded sample, relative to the
unexpanded sample.
[0238] Such expansion techniques include, but are not limited to
those described in U.S. Pat. No. 7,399,633; Delaney et al., 2010,
Nature Med. 16(2): 232-236; Zhang et al., 2008, Blood
111:3415-3423; and Himburg et al., 2010, Nature Medicine
doi:10.1038/nm.2119 (advanced online publication), as well as those
described below.
[0239] In one embodiment of the invention, HSPC (e.g., the Enriched
HSPC) are cultured with a Notch agonist, an aryl hydrocarbon
receptor antagonist, and growth factors, and are exposed to cell
growth conditions (e.g., promoting mitosis) such that the HSPC
proliferate to obtain an Expanded HSPC population according to the
present invention. In certain embodiments of the invention, HSPC
(e.g., the Enriched HSPC) are cultured with an amount of an agonist
of Notch function and an amount of an aryl hydrocarbon receptor
antagonist, where the amounts of both of these agents together are
effective to expand HSPC. In particular, in certain embodiments of
the invention, HSPC (e.g., the Enriched HSPC) are cultured with an
amount of an agonist of Notch function and an amount of an aryl
hydrocarbon receptor antagonist, where the amounts of both of these
agents together are effective to expand HSPC, and are exposed to
cell growth conditions (e.g., promoting mitosis) such that the HSPC
proliferate to obtain an Expanded HSPC population according to the
present invention (optionally, HSPC are cultured in the presence of
one or more growth factors). In one embodiment of the invention,
HSPC (e.g., the Enriched HSPC) are cultured with an amount of an
agonist of Notch function effective to inhibit differentiation, and
an amount of an aryl hydrocarbon receptor antagonist effective to
promote cell proliferation or block cell differentiation, and are
exposed to cell growth conditions (e.g., promoting mitosis) such
that the HSPC proliferate to obtain an Expanded HSPC population
according to the present invention. In another embodiment, HSPC
(e.g., the Enriched HSPC) are cultured with an amount of an agonist
of Notch function effective to inhibit differentiation, an amount
of an aryl hydrocarbon receptor antagonist effective to promote
cell proliferation or block cell differentiation, and in the
presence of one or more growth factors, and are exposed to cell
growth conditions (e.g., promoting mitosis) such that the HSPC
proliferate to obtain an Expanded HSPC population according to the
present invention. The Expanded HSPC population so obtained can be
frozen and stored for later use, for example, to provide
hematopoietic function to an immunodeficient human patient.
Optionally, the Notch pathway agonist and/or an aryl hydrocarbon
receptor antagonist is/are inactivated or removed from the Expanded
HSPC population prior to transplantation into the patient (e.g., by
separation, dilution).
[0240] In one embodiment, one or more agents used in the expansion
methods described herein, in addition to the Notch agonist and the
aryl hydrocarbon receptor antagonist, are: an agonist antibody
against the TPO receptor (e.g., VB22B sc(Fv)2 as described inWO
2007/145227), SCF, IL-6, Flt-3 ligand, TPO or aTPO mimetic (e.g.,
such as described in WO/2007/022269; WO/2007/009120;
WO/2004/054515; WO/2003/103686; WO/2002/085343; WO/2002/049413;
WO/2001/089457; WO/2001/039773; WO/2001/034585; WO/2001/021180;
WO/2001/021180; WO/2001/017349; WO/2000/066112; WO/2000/035446;
WO/2000/028987; WO/2008/028645), granulocyte colony stimulating
factor (G-CSF), granulocyte macrophage colony stimulating factor
(GM-CSF), a prostaglandin or a prostaglandin receptor agonist
(e.g., prostaglandin E2 receptor-1 (EP-1) agonist, prostaglandin E2
receptor-2 (EP-2) agonist, prostaglandin E2 receptor-3 (EP-3)
agonist and prostaglandin E2 receptor-4 (EP-4) agonists, as
described in WO/2008/073748), tetraethylenepentamine (TEPA), and/or
a WNT agonist (see U.S. Patent Publication No. 2010/0183564). In
particular embodiments, HSPC are cultured in the presence of
mesenchymal stem cells (MSCs).
[0241] In specific embodiments, HSPC (e.g., the Enriched HSPC) are
cultured for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, or 30 days or more; or,
preferably, the HSPC are cultured for at least 10 days or at least
16 days (in the presence of the combination of a Notch agonist and
an aryl hydrocarbon receptor antagonist, and, optionally, one or
more growth factors). In other embodiments, HSPC (e.g., the
Enriched HSPC) are cultured for 1 week, 2 weeks, 3 weeks, 4 weeks,
5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks or 10 weeks; or,
preferably, the HSPC are cultured for at least 3 or 4 weeks (in the
presence of the combination of a Notch agonist and an aryl
hydrocarbon receptor antagonist, and, optionally, one or more
growth factors). In yet other embodiments, HSPC (e.g., the Enriched
HSPC) are cultured for less than 4 weeks (in the presence of the
combination of a Notch agonist and an aryl hydrocarbon receptor
antagonist, and, optionally, one or more growth factors). In yet
other embodiments, HSPC (e.g., the Enriched HSPC) are cultured for
more than 10 weeks, e.g., 12, 15, 18, 20 or 25 weeks (in the
presence of the combination of a Notch agonist and an aryl
hydrocarbon receptor antagonist, and, optionally, one or more
growth factors).
[0242] Exemplary culture conditions for expanding HSPC (e.g., the
Enriched HSPC) comprise, as set forth in Section 7 infra, culturing
the HSPC for about 16 days or about 16-21 days in the presence of
fibronectin fragments and the extracellular domain of a Delta
protein fused to the Fc domain of human IgG (e.g.,
Delta1.sup.ext-IgG) in a serum free medium supplemented with the
following four human growth factors: stem cell factor, Flt-3
receptor ligand, thrombopoietin and interleukin-6, or in the
presence of the following five human growth factors: stem cell
factor, Flt-3 receptor ligand, thrombopoietin, interleukin-6 and
interleukin-3. In some embodiments, the cell culture dishes are
coated overnight at 4.degree. C. (or for a minimum of 2 hours at
37.degree. C.) with 0.2 to 10 .mu.g/ml Delta1.sup.ext-IgG (e.g.,
0.5, 1, 1.25, 1.5, 2. 2.5, 5, 7.5, or 10 .mu.g/ml
Delta1.sup.ext-IgG) and 5 .mu.g/ml RetroNectin.RTM. (a recombinant
human fibronectin fragment) in phosphate buffered saline, before
adding HSPC (e.g., the Enriched HSPC) and an aryl hydrocarbon
receptor antagonist.
[0243] In certain embodiments, HSPC (e.g., the Enriched HSPC) are
expanded in the presence of a Notch agonist, and in particular
Delta.sup.ext-IgG (e.g., Delta1.sup.ext-IgG), at a concentration
that is equal to or more than 0.5, 1, 1.25, 1.5, 2, 2.5, 5, 7.5 or
10 .mu.g/ml, e.g., in a fluid applied to a solid phase surface. In
some embodiments, HSPC (e.g., the Enriched HSPC) are expanded in
the presence of a Notch agonist, and in particular
Delta.sup.ext-IgG (e.g., Delta1.sup.ext-IgG), at a concentration
that is below 25, 20, 17.5, 15, 12.5, 10, 9, 8, 7.5, 7, 6, 5, 4, 3,
2.5, 2, 1.5 or 1.25 .mu.g/ml, e.g., in a fluid applied to a solid
phase surface. In yet other embodiments, HSPC (e.g., the Enriched
HSPC) are expanded in the presence of a Notch agonist, and in
particular Delta1.sup.ext-IgG, at a concentration between 0.5 and
10 .mu.g/ml, between 1 and 15 .mu.g/ml, between 1.25 and 15
.mu.g/ml, between 1.5 and 15 .mu.g/ml, between 1 and 10 .mu.g/ml,
between 1.25 and 10 .mu.g/ml, between 1.5 and 10 .mu.g/ml, between
2 and 10 .mu.g/ml, between 1 and 7.5 .mu.g/ml, between 1.25 and 7.5
.mu.g/ml, between 1.5 and 7.5 .mu.g/ml, between 2 and 7.5 .mu.g/ml,
between 1 and 5 .mu.g/ml, between 1.25 and 5 .mu.g/ml, between 1.5
and 6 .mu.g/ml, between 2 and 6 .mu.g/ml, between 2.5 and 6
.mu.g/ml, or between 2.5 and 5 .mu.g/ml, e.g., in a fluid applied
to a solid phase surface. In a specific embodiment, a Notch
agonist, and in particular Delta.sup.ext-IgG (e.g.,
Delta1.sup.ext-IgG), is used for HSPC expansion at a concentration
of 0.5, 1, 1.25, 1.5, 2. 2.5, 5 or 7.5 .mu.g/ml, e.g., in a fluid
applied to a solid phase surface.
[0244] In certain embodiments, an aryl hydrocarbon receptor
antagonist (e.g., SR1) is added to the medium in which HSPC (e.g.,
the Enriched HSPC) are cultured. In some embodiments, an aryl
hydrocarbon receptor antagonist is added to the medium in which
HSPC (e.g., the Enriched HSPC) are cultured during all of the
feedings of the cells. In these embodiments, an aryl hydrocarbon
receptor antagonist is present in the HSPC cell culture at all
times during the HSPC expansion. In yet other embodiments, an aryl
hydrocarbon receptor antagonist is not present in the HSPC cell
culture at all times during the HSPC expansion. In a specific
embodiment, an aryl hydrocarbon receptor antagonist compound is
made fresh before addition of such compound to HSPC (e.g., the
Enriched HSPC) or the medium in which HSPC (e.g., the Enriched
HSPC) are cultured. In certain embodiment, an aryl hydrocarbon
compound (e.g., SR1) is added to the culture medium so as to be
present at a concentration between about 100 nM and 1500 nM, 100 nM
and 1000 nM, 250 nM and 1500 nM, 250 nM and 1000 nM, 500 nM and
1500 nM, 500 nM and 1000 nM, 600 nM and 1000 nM, 600 nM and 900 nM,
700 nM and 900 nM, or 700 nM and 800 nM. In certain embodiments, an
aryl hydrocarbon compound (e.g., SR1) is added to the culture
medium so as to be present at a concentration of 500 nM, 550 nM,
600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM or
1000 nM. In some embodiments, an aryl hydrocarbon compound (e.g.,
SR1) is added to the culture medium so as to be present at a
concentration of no more than 1000 nM. In specific embodiments, an
aryl hydrocarbon compound (e.g., SR1) is added to the culture
medium so as to be present at a concentration in the range of 200
nM to 1000 nM. In particular embodiments, an aryl hydrocarbon
receptor antagonist is added to the culture medium in which HSPC
are expanded at a concentration between 1 pM and 100 .mu.M, between
10 pM and 10 .mu.M, or between 100 pM and 1 .mu.M. In some
embodiments, an aryl hydrocarbon receptor antagonist is formulated
in DMSO or another suitable carrier (e.g., the DMSO formulation can
contain 0.3 mg/ml of the aryl hydrocarbon receptor antagonist in
60% DMSO/40% water solution) for use in the expansion technique
provided herein.
[0245] In certain embodiments, the foregoing growth factors are
present in the culture condition for expanding HSPC (e.g., the
Enriched HSPC) at the following concentrations: 25-300 ng/ml stem
cell factor, 25-300 ng/ml Flt-3 receptor ligand, 25-100 ng/ml
thrombopoietin, 25-100 ng/ml interleukin-6 and 10 ng/ml
interleukin-3. In more specific embodiments, 50, 100 or 200 ng/ml
stem cell factor, 50, 100 or 200 ng/ml of Flt-3 receptor ligand, 50
or 100 ng/ml thrombopoietin, 50 or 100 ng/ml interleukin-6 and
about 10 ng/ml interleukin-3 are used.
[0246] Other exemplary culture condition for expanding HSPC (e.g.,
the Enriched HSPC) are set forth in Zhang et al., 2008, Blood
111:3415-3423. In a specific embodiment, HSPC (e.g., the Enriched
HSPC) can be cultured in serum free medium supplemented with
heparin, stem cell factor, thrombopoietin, insulin-like growth
factor-2 (IGF-2), fibroblast growth factor-1 (FGF-1), and Angptl3
or Angptl5. In a specific embodiment, the medium is supplemented
with 10 .mu.g/ml heparin, 10 ng/ml stem cell factor, 20 ng/ml
thrombopoietin, 20 ng/ml IGF-2, and 10 ng/ml FGF-1, and 100 ng/ml
Angptl3 or Angptl5 and the cells are cultured for 19-23 days. In
another specific embodiment, the HSPC can be expanded by culturing
the HSPC in serum free medium supplemented with 10 .mu.g/ml
heparin, 10 ng/ml stem cell factor, 20 ng/ml thrombopoietin, 10
ng/ml FGF-1, and 100 ng/ml Angptl5 for 11-19 days. In another
specific embodiment, HSPC (e.g., the Enriched HSPC) can be expanded
by culturing the HSPC in serum free medium supplemented with 50
ng/ml stem cell factor, 10 ng/ml thrombopoietin, 50 ng/ml Flt-3
receptor ligand, and 100 ng/ml insulin-like growth factor binding
protein-2 (IGFBP2) or 500 ng/ml Angptl5 for 10 days. In yet another
embodiment, HSPC (e.g., the Enriched HSPC) can be expanded by
culturing HSPC (e.g., the Enriched HSPC) in serum free medium
supplemented with 10 .mu.g/ml heparin, 10 ng/ml stem cell factor,
20 ng/ml thrombopoietin, 10 ng/ml FGF-1, 500 ng/ml Angptl5, and 500
ng/ml IGFBP2 for 11 days. See Zhang et al., 2008, Blood
111:3415-3423.
[0247] Another exemplary culture condition for expanding HSPC
(e.g., the Enriched HSPC) is set forth in Himburg et al., 2010,
Nature Medicine doi:10.1038/nm.2119 (advanced online publication).
In a specific embodiment, the HSPC can be cultured in liquid
suspension culture supplemented with thrombopoietin, stem cell
factor, Flt-3 receptor ligand, and pleiotrophin. In a specific
embodiment, the liquid suspension culture is supplemented with 20
ng/ml thrombopoietin, 125 ng/ml stem cell factor, 50 ng/ml Flt-3
receptor ligand, and 10, 100, 500, or 1000 ng/ml pleiotrophin and
the HSPC are cultured for 7 days.
[0248] In specific embodiments, HSPC (e.g., the Enriched HSPC) are
expanded in a basal medium, which can be supplemented with one or
more growth factors described herein. A basal medium can comprise
amino acids, carbon sources, vitamins, serum proteins (e.g.
albumin), inorganic salts, divalent cations, buffers or any other
element suitable for use in expansion of HSPC as described at page
13 of U.S. Patent Publication No. 2010/0183564. Examples of such
basal medium appropriate include, without limitation, StemSpan.RTM.
SFEM--Serum-Free Expansion Medium (StemCell Technologies,
Vancouver, Canada), StemSpan.RTM. H3000--Defined Medium (StemCell
Technologies, Vancouver, Canada), CellGro.RTM. SCGM (CellGenix,
Freiburg Germany), and StemPro.RTM.-34 SFM (Invitrogen).
[0249] It is further contemplated herein that HSPC (e.g., the
Enriched HSPC) are expanded in the presence of a composition
comprising a Notch agonist and an aryl hydrocarbon receptor
antagonist, and further comprising any additional component
disclosed in U.S. Pat. No. 7,399,633 and U.S. Patent Publication
No. 2010/0183564, which are incorporated by reference herein in
their entireties.
[0250] In a preferred embodiment of the invention, after expansion
of the HSPC, the total number of cells, viable CD34.sup.+, and/or
viable CD34.sup.+CD90.sup.+, cells are determined to measure the
potency of the sample to provide hematopoietic function. Numerous
clinical studies have shown that the total nucleated cell dose and
the CD34.sup.+ cell dose in stem cell grafts are highly correlated
with neutrophil and platelet engraftment as well as the incidence
of graft failure and early transplant-related complications
(primarily lethal infections) following stem cell transplantation.
Further, CD34.sup.+CD90.sup.+ cells have been shown to represent a
subpopulation of CD34.sup.+ cells capable of generating long-term
engraftment. For example, at day 5-16 post culture initiation
during expansion, a sample can be taken for determination of the
total viable nucleated cell count. In addition, the total number of
CD34.sup.+ cells and/or CD34.sup.+CD90.sup.+ cells can be
determined by multi-parameter flow cytometry, as well as the
percentage of such cells in the sample. Preferably, cultures that
have not resulted in at least a 10-fold increase in the absolute
number of CD34.sup.+ cells at this time are discontinued.
Similarly, prior to cryopreservation or after thawing, an aliquot
of the Expanded HSPC sample can be taken for determination of total
nucleated cells and percentage of viable CD34.sup.+ cells and/or
viable CD34.sup.+CD90.sup.+ cells in order to calculate the total
viable CD34.sup.+ and/or CD34.sup.+CD90.sup.+ cell number in the
Expanded HSPC sample. In a preferred embodiment, those Expanded
HSPC samples containing less than 75 million CD34.sup.+ viable
cells can be discarded.
[0251] In some specific embodiments, the Expanded HSPC contain at
least 10.sup.5 cells, at least 10.sup.6 cells, at least 10.sup.7
cells, at least 10.sup.8 cells or at least 10.sup.9 cells, wherein
between 20% to 100% of total cells are CD34+, e.g., between 40% to
80%, of total cells are CD34+. In particular embodiments, the
Expanded HSPC have at least 10%, 20%, 30%, 40% or 50% or more cells
relative to the number of cells prior to expansion or relative to
the number of cells in the control cell population not subjected to
the expansion technique. In particular embodiments, the Expanded
HSPC have at least 10%, 20%, 30%, 40% or 50% or more CD34+ cells
relative to the number of CD34+ cells prior to expansion or
relative to the number of cells in the control cell population not
subjected to the expansion technique. Differentiation properties of
the CD34+ cells can be assessed by analyzing the colony forming
units (CFU) as described in U.S. Patent Publication No.
2010/0183564.
[0252] In a specific embodiment, total viable CD34+, CD34+CD90+
cells (or other antigen-positive) cell numbers can be considered
the potency assay for release of the final product for therapeutic
use. Viability can be determined by any method known in the art,
for example, by trypan blue exclusion or 7-AAD exclusion.
Preferably, the total nucleated cell count (TNC) and other data are
used to calculate the potency of the product. The percentage of
viable CD34+ cells and/or viable CD34+CD90+ cells can be assessed
by flow cytometry and use of a stain that is excluded by viable
cells. The percentage of viable CD34+ cells=the number of CD34+
cells that exclude 7-AAD (or other appropriate stain) in an aliquot
of the sample divided by the TNC (both viable and non-viable) of
the aliquot. Viable CD34+ cells in the sample can be calculated as
follows: Viable CD34+ cells=TNC of sample x % viable CD34+ cells in
the sample. The proportional increase during enrichment or
expansion in viable CD34+ cells can be calculated as follows: Total
Viable CD34+ cells Post-culture/Total Viable CD34+ cells
Pre-culture. As will be apparent, antigens other than or in
addition to CD34 can be used.
[0253] In certain embodiments, after expansion of the HSPC in the
presence of an amount of a Notch agonist and an amount of an aryl
hydrocarbon receptor antagonist (e.g., after culturing HSPC (e.g.,
the Enriched HSPC) for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20 or more days, or 1, 2, 3, 4, 5, 6, 7, or 8 or more
weeks), the percentage of CD34.sup.+ and/or CD34.sup.+CD90.sup.+
cells increases relative to the percentage of CD34.sup.+ and/or
CD34.sup.+CD90.sup.+ cells cultured in the presence of the same
amount of a Notch agonist alone and/or the same amount of an aryl
hydrocarbon receptor antagonist alone. In other embodiments, after
expansion of the HSPC in the presence of an amount of a Notch
agonist and an amount of an aryl hydrocarbon receptor antagonist
(e.g., after culturing HSPC (e.g., the Enriched HSPC) for 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days, or
1, 2, 3, 4, 5, 6, 7, 8 or more weeks), the percentage of CD34.sup.+
and/or CD34.sup.+CD90.sup.+ cells increases relative to the
percentage of CD34.sup.+ and/or CD34.sup.+CD90.sup.+ cells before
the expansion (at day 0), or relative to the percentage of
CD34.sup.+ and/or CD34.sup.+CD90.sup.+ cells in the HSPC expanded
under conditions lacking either a Notch agonist and/or without an
aryl hydrocarbon receptor antagonist.
[0254] In some embodiments, after expansion of the HSPC in the
presence of an amount of a Notch agonist and an amount of an aryl
hydrocarbon receptor antagonist (e.g., after culturing HSPC (e.g.,
the Enriched HSPC) for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20 or more days, or 1, 2, 3, 4, 5, 6, 7, 8, or more
weeks), the percentage of CD34-CD14+ cells decreases relative to
the percentage of CD34-CD14+ cells cultured in the presence of the
same amount of a Notch agonist alone and/or the same amount of an
aryl hydrocarbon receptor antagonist alone. In other embodiments,
after expansion of the HSPC in the presence of an amount of a Notch
agonist and an amount of an aryl hydrocarbon receptor antagonist
(e.g., after culturing HSPC (e.g., the Enriched HSPC) for 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days, or
1, 2, 3, 4, 5, 6, 7, 8 or more weeks), the percentage of CD34-CD14+
cells decreases relative to the percentage of CD34-CD14+ cells in
the HSPC expanded under conditions lacking either a Notch agonist
and/or without an aryl hydrocarbon receptor antagonist.
[0255] The Expanded HSPC can be used without further purification
or selection, or can be subject to further purification or
selection. Once the Expanded HSPC are obtained, the Expanded HSPC
may then be washed to remove the aryl hydrocarbon receptor
antagonist (and, optionally, one or more other agents used during
the expansion procedure). Upon washing, the Expanded HSPC can be
resuspended in an appropriate cell suspension medium for short term
use or in a long-term storage medium, for example, a medium
suitable for cryopreservation.
[0256] The cell sample containing isolated HSPC, the Enriched HSPC
and/or the Expanded HSPC can also contain supporting cells as
described, e.g., at pages 10-11 of U.S. Patent Publication No.
2010/0183564. Supporting cells can be cells that are naturally
found in the vicinity of HSPC. Supporting cells secrete or express
on their cell surface the factors necessary for the maintenance,
growth or differentiation of HPSC. Supporting cells include, but
are not limited to, lymphoreticular stromal cells. Lymphoreticular
stromal cells include, but are not limited to, all cell types
present in a lymphoid tissue which are not lymphocytes or
lymphocyte precursors or progenitors (e.g., osteoblasts, epithelial
cells, endothelial cells, mesothelial cells, dendritic cells,
splenocytes and macrophages). Lymphoreticular stromal cells may
also include fibroblasts which have been genetically altered to
secrete or express on their cell surface the factors necessary for
the maintenance, growth or differentiation of HSPC. Lymphoreticular
stromal cells can be derived from the lymphoid tissue (e.g., bone
marrow, peripheral blood (e.g., mobilized peripheral blood),
umbilical cord blood, placental blood, fetal liver, embryonic cells
(e.g., embryonic stem cells), aortal-gonadal-mesonephros derived
cells, or lymphoid soft tissue such as thymus, spleen, liver, lymph
node, skin, tonsil, adenoids and Peyer's patch). Lymphoreticular
stromal cells can be autologous (self) or non-autologous (non-self,
e.g., heterologous, allogeneic, syngeneic or xenogeneic) with
respect to HSPC. Lymphoid tissue (e.g., lymphoreticular stroma
cells) can be obtained from a subject (e.g., human) at any time
after such tissue has developed to a stage at which it can support
the maintenance, growth or differentiation of HSPC. HSPC can be
cultured with supporting cells as described in U.S. Patent
Publication No. 2010/0183564 (see, e.g., page 11). In an
alternative embodiment, the cell sample containing isolated HSPC,
the Enriched HSPC and/or the Expanded HSPC does not contain
supporting cells.
[0257] 2.9 Cryopreservation and Thawing
[0258] 2.9.1 Cryopreservation
[0259] Once the isolated HSPC, the Enriched HSPC or the Expanded
HSPC are obtained, such isolated HSPC, Enriched HSPC or Expanded
HSPC can be cryopreserved in accordance with the methods described
below or known in the art.
[0260] In one embodiment, an Expanded HSPC population can be
divided and frozen in one or more bags (or units). In another
embodiment, two or more Expanded HSPC populations can be pooled,
divided into separate aliquots, and each aliquot is frozen. In a
preferred embodiment, a maximum of approximately 4 billion
nucleated cells is frozen in a single bag. In a preferred
embodiment, the Expanded HSPC are fresh, i.e., they have not been
previously frozen prior to expansion or cryopreservation. The terms
"frozen/freezing" and "cryopreserved/cryopreserving" are used
interchangeably in the present application. Cryopreservation can be
by any method in known in the art that freezes cells in viable
form. The freezing of cells is ordinarily destructive. On cooling,
water within the cell freezes. Injury then occurs by osmotic
effects on the cell membrane, cell dehydration, solute
concentration, and ice crystal formation. As ice forms outside the
cell, available water is removed from solution and withdrawn from
the cell, causing osmotic dehydration and raised solute
concentration which eventually destroy the cell. For a discussion,
see Mazur, P., 1977, Cryobiology 14:251-272.
[0261] These injurious effects can be circumvented by (a) use of a
cryoprotective agent, (b) control of the freezing rate, and (c)
storage at a temperature sufficiently low to minimize degradative
reactions.
[0262] Cryoprotective agents which can be used include but are not
limited to dimethyl sulfoxide (DMSO) (Lovelock and Bishop, 1959,
Nature 183:1394-1395; Ashwood-Smith, 1961, Nature 190:1204-1205),
glycerol, polyvinylpyrrolidine (Rinfret, 1960, Ann. N.Y. Acad. Sci.
85:576), polyethylene glycol (Sloviter and Ravdin, 1962, Nature
196:548), albumin, dextran, sucrose, ethylene glycol, i-erythritol,
D-ribitol, D-mannitol (Rowe et al., 1962, Fed. Proc. 21:157),
D-sorbitol, i-inositol, D-lactose, choline chloride (Bender et al.,
1960, J. Appl. Physiol. 15:520), amino acids (Phan The Tran and
Bender, 1960, Exp. Cell Res. 20:651), methanol, acetamide, glycerol
monoacetate (Lovelock, 1954, Biochem. J. 56:265), and inorganic
salts (Phan The Tran and Bender, 1960, Proc. Soc. Exp. Biol. Med.
104:388; Phan The Tran and Bender, 1961, in Radiobiology,
Proceedings of the Third Australian Conference on Radiobiology,
Ilbery ed., Butterworth, London, p. 59). In a preferred embodiment,
DMSO is used, a liquid which is nontoxic to cells in low
concentration. Being a small molecule, DMSO freely permeates the
cell and protects intracellular organelles by combining with water
to modify its freezability and prevent damage from ice formation.
Addition of plasma (e.g., to a concentration of 20-25%) can augment
the protective effect of DMSO. After addition of DMSO, cells should
be kept at 0.degree. C. until freezing, since DMSO concentrations
of about 1% are toxic at temperatures above 4.degree. C.
[0263] A controlled slow cooling rate can be critical. Different
cryoprotective agents (Rapatz et al., 1968, Cryobiology 5(1):18-25)
and different cell types have different optimal cooling rates (see
e.g., Rowe and Rinfret, 1962, Blood 20:636; Rowe, 1966, Cryobiology
3(1):12-18; Lewis, et al., 1967, Transfusion 7(1):17-32; and Mazur,
1970, Science 168:939-949 for effects of cooling velocity on
survival of marrow-stem cells and on their transplantation
potential). The heat of fusion phase where water turns to ice
should be minimal. The cooling procedure can be carried out by use
of, e.g., a programmable freezing device or a methanol bath
procedure.
[0264] Programmable freezing apparatuses allow determination of
optimal cooling rates and facilitate standard reproducible cooling.
Programmable controlled-rate freezers such as Cryomed or Planar
permit tuning of the freezing regimen to the desired cooling rate
curve. For example, for marrow cells in 10% DMSO and 20% plasma,
the optimal rate is 1.degree. to 3.degree. C./minute from 0.degree.
C. to -80.degree. C. In a preferred embodiment, this cooling rate
can be used for CB cells. The container holding the cells must be
stable at cryogenic temperatures and allow for rapid heat transfer
for effective control of both freezing and thawing. Sealed plastic
vials (e.g., Nunc, Wheaton cryules) or glass ampules can be used
for multiple small amounts (1-2 ml), while larger volumes (100-200
ml) can be frozen in polyolefin bags (e.g., Delmed) held between
metal plates for better heat transfer during cooling. Bags of bone
marrow cells have been successfully frozen by placing them in
-80.degree. C. freezers which, fortuitously, gives a cooling rate
of approximately 3.degree. C./minute).
[0265] In an alternative embodiment, the methanol bath method of
cooling can be used. The methanol bath method is well-suited to
routine cryopreservation of multiple small items on a large scale.
The method does not require manual control of the freezing rate nor
a recorder to monitor the rate. In a preferred embodiment,
DMSO-treated cells are pre-cooled on ice and transferred to a tray
containing chilled methanol which is placed, in turn, in a
mechanical refrigerator (e.g., Harris or Revco) at -80.degree. C.
Thermocouple measurements of the methanol bath and the samples
indicate the desired cooling rate of 1.degree. to 3.degree.
C./minute. After at least two hours, the specimens have reached a
temperature of -80.degree. C. and can be placed directly into
liquid nitrogen (-196.degree. C.) for permanent storage.
[0266] After thorough freezing, the Expanded HSPC can be rapidly
transferred to a long-term cryogenic storage vessel. In a preferred
embodiment, samples can be cryogenically stored in liquid nitrogen
(-196.degree. C.) or its vapor (-165.degree. C.). Such storage is
greatly facilitated by the availability of highly efficient liquid
nitrogen refrigerators, which resemble large Thermos containers
with an extremely low vacuum and internal super insulation, such
that heat leakage and nitrogen losses are kept to an absolute
minimum.
[0267] Suitable racking systems are commercially available and can
be used for cataloguing, storage, and retrieval of individual
specimens.
[0268] Considerations and procedures for the manipulation,
cryopreservation, and long-term storage of the hematopoietic stem
cells, particularly from bone marrow or peripheral blood (e.g.,
mobilized peripheral blood), which are also largely applicable to
the Expanded HSPC can be found, for example, in the following
references, incorporated by reference herein: Gorin, 1986, Clinics
In Haematology 15(1):19-48; Bone-Marrow Conservation, Culture and
Transplantation, Proceedings of a Panel, Moscow, Jul. 22-26, 1968,
International Atomic Energy Agency, Vienna, pp. 107-186.
[0269] Other methods of cryopreservation of viable cells, or
modifications thereof, are available and envisioned for use (e.g.,
cold metal-mirror techniques; Livesey and Linner, 1987, Nature
327:255; Linner et al., 1986, J. Histochem. Cytochem.
34(9):1123-1135; see also U.S. Pat. No. 4,199,022 by Senkan et al.,
U.S. Pat. No. 3,753,357 by Schwartz, U.S. Pat. No. 4,559,298 by
Fahy).
[0270] In other embodiments, isolated HSPC, the Enriched HSPC or
the Expanded HSPC are preserved by freeze-drying (see Simione,
1992, J. Parenter. Sci. Technol. 46(6):226-32).
[0271] 2.9.2 Thawing
[0272] Following cryopreservation, frozen isolated HSPC, frozen
Enriched HSPC or frozen Expanded HSPC can be thawed in accordance
with the methods described below or known in the art.
[0273] Frozen cells are preferably thawed quickly (e.g., in a water
bath maintained at 37.degree.-41.degree. C.) and chilled
immediately upon thawing. In a specific embodiment, the vial
containing the frozen cells can be immersed up to its neck in a
warm water bath; gentle rotation will ensure mixing of the cell
suspension as it thaws and increase heat transfer from the warm
water to the internal ice mass. As soon as the ice has completely
melted, the vial can be immediately placed in ice.
[0274] In an embodiment of the invention, the Expanded HSPC sample
as thawed, or a portion thereof, can be infused for providing
hematopoietic function in a human patient in need thereof. Several
procedures, relating to processing of the thawed cells are
available, and can be employed if deemed desirable.
[0275] It may be desirable to treat the cells in order to prevent
cellular clumping upon thawing. To prevent clumping, various
procedures can be used, including but not limited to, the addition
before and/or after freezing of DNase (Spitzer et al., 1980, Cancer
45:3075-3085), low molecular weight dextran and citrate,
hydroxyethyl starch (Stiff et al., 1983, Cryobiology 20:17-24),
etc.
[0276] The cryoprotective agent, if toxic in humans, should be
removed prior to therapeutic use of the thawed Expanded HSPC. In an
embodiment employing DMSO as the cryopreservative, it is preferable
to omit this step in order to avoid cell loss, since DMSO has no
serious toxicity. However, where removal of the cryoprotective
agent is desired, the removal is preferably accomplished upon
thawing.
[0277] One way in which to remove the cryoprotective agent is by
dilution to an insignificant concentration. This can be
accomplished by addition of medium, followed by, if necessary, one
or more cycles of centrifugation to pellet cells, removal of the
supernatant, and resuspension of the cells. For example,
intracellular DMSO in the thawed cells can be reduced to a level
(less than 1%) that will not adversely affect the recovered cells.
This is preferably done slowly to minimize potentially damaging
osmotic gradients that occur during DMSO removal.
[0278] After removal of the cryoprotective agent, cell count (e.g.,
by use of a hemocytometer) and viability testing (e.g., by trypan
blue exclusion; Kuchler, 1977, Biochemical Methods in Cell Culture
and Virology, Dowden, Hutchinson & Ross, Stroudsburg, Pa., pp.
18-19; 1964, Methods in Medical Research, Eisen et al., eds., Vol.
10, Year Book Medical Publishers, Inc., Chicago, pp. 39-47) can be
done to confirm cell survival. The percentage of viable antigen
(e.g., CD34) positive cells in a sample can be determined by
calculating the number of antigen positive cells that exclude 7-AAD
(or other suitable dye excluded by viable cells) in an aliquot of
the sample, divided by the total number of nucleated cells (TNC)
(both viable and non-viable) in the aliquot of the sample. The
number of viable antigen positive cells in the sample can be then
determined by multiplying the percentage of viable antigen positive
cells by TNC of the sample. Prior to cryopreservation and/or after
thawing, the total number of nucleated cells, or in a specific
embodiment, the total number of CD34.sup.+ or CD133.sup.+ cells can
be determined. For example, total nucleated cell count can be
performed by using a hemocytometer and exclusion of trypan blue
dye. Specimens that are of high cellularity can be diluted to a
concentration range appropriate for manual counting. Final cell
counts for products are corrected for any dilution factors. Total
nucleated cell count=viable nucleated cells per mL.times.volume of
product in mL. The number of CD34.sup.+ or CD133.sup.+ positive
cells in the sample can be determined, e.g., by the use of flow
cytometry using anti-CD34 or anti-CD133 monoclonal antibodies
conjugated to a fluorochrome.
[0279] Optionally, the Expanded HSPC sample can undergo HLA typing
either prior to cryopreservation and/or after cryopreservation and
thawing. HLA typing can be performed using serological methods with
antibodies specific for identified HLA antigens, or using DNA-based
methods for detecting polymorphisms in the HLA antigen-encoding
genes for typing HLA alleles. In a specific embodiment, HLA typing
can be performed at intermediate resolution using a sequence
specific oligonucleotide probe method for HLA-A and HLA-B or at
high resolution using a sequence based typing method (allele
typing) for HLA-DRB1.
[0280] In certain embodiments, the identity and purity of the
starting HSPC, the enriched HSPC, and the Expanded HSPC prior to
cryopreservation, or the Expanded HSPC after thawing can be
subjected to multi-parameter flow cytometric immunophenotyping,
which provides the percentage of viable antigen positive cells
present in a sample. Each sample can be tested for one or more of
the following cell phenotypes using a panel of monoclonal
antibodies directly conjugated to fluorochromes: [0281] 1.
CD34.sup.+ HPC [0282] 2. T cells (CD3.sup.+, including both
CD4.sup.+ and CD8.sup.+ subsets) [0283] 3. B cells (CD19.sup.+ or
CD20.sup.+) [0284] 4. NK cells (CD56.sup.+) [0285] 5. Monocytes
(CD14.sup.+) [0286] 6. Myelomonocytes (CD15.sup.+) [0287] 7.
Megakaryocytes (CD41.sup.+) [0288] 8. Dendritic Cells (lineage
negative/HLA-DRbright and CD123bright, or lineage
negative/HLA-DRbright and CD11cbright).
[0289] 2.10 Genetically Engineered HSPC
[0290] In a preferred embodiment, the Expanded HSPC administered to
the patient are non-recombinant. However, in a different
embodiment, the isolated HSPC, the Enriched HSPC prior to expansion
or the Expanded HSPC can be genetically engineered to produce gene
products beneficial upon transplantation of the genetically
engineered cells to a subject. Such gene products include but are
not limited to anti-inflammatory factors, e.g., anti-TNF,
anti-IL-1, anti-IL-2, etc. In some embodiments, HSPC can be
genetically engineered to "knock out" expression of MHC. The HSPC
can be genetically engineered for use in gene therapy to adjust the
level of gene activity in a subject to assist or improve the
results of transplantation or to treat a disease caused by, for
example, a deficiency in the recombinant gene. The HSPC are made
recombinant by the introduction of a recombinant nucleic acid into
the isolated HSPC, the Enriched HSPC or into the Expanded HSPC. The
description of genetically engineered HSPC provided herein is
largely found in sec. 5.1 of U.S. Pat. No. 7,399,633.
[0291] In its broadest sense, gene therapy refers to therapy
performed by the administration of a nucleic acid to a subject. The
nucleic acid, either directly or indirectly via its encoded
protein, mediates a therapeutic effect in the subject. The present
invention provides methods of gene therapy wherein a nucleic acid
encoding a protein of therapeutic value (preferably to humans) is
introduced into the HSPC, before or after expansion, such that the
nucleic acid is expressible by the HSPC and/or their progeny,
followed by administration of the recombinant Expanded HSPC to a
subject.
[0292] The recombinant HSPC of the present invention can be used in
any of the methods for gene therapy available in the art. Thus, the
nucleic acid introduced into the cells may encode any desired
protein, e.g., a protein missing or dysfunctional in a disease or
disorder. The descriptions below are meant to be illustrative of
such methods. It will be readily understood by those of skill in
the art that the methods illustrated represent only a sample of all
available methods of gene therapy.
[0293] For general reviews of the methods of gene therapy, see
Gardlik et al., 2005, Med. Sci. Monit. 11:RA110-121; Lundstrom,
1999, J. Recept. Signal Transduct. Res. 19:673-686; Robbins and
Ghivizzani, 1998, Pharmacol. Ther. 80:35-47; Pelegrin et al., 1998,
Hum. Gene Ther. 9:2165-2175; Harvey and Caskey, 1998, Curr. Opin.
Chem. Biol. 2:512-518; Guntaka and Swamynathan, 1998, Indian J.
Exp. Biol. 36:539-535; Desnick and Schuchman, 1998, Acta Paediatr.
Jpn. 40:191-203; Vos, 1998, Curr. Opin. Genet. Dev. 8:351-359;
Tarahovsky and Ivanitsky, 1998, Biochemistry (Mosc) 63:607-618;
Morishita et al., 1998, Circ. Res. 2:1023-1028; Vile et al., 1998,
Mol. Med. Today 4:84-92; Branch and Klotman, 1998, Exp. Nephrol.
6:78-83; Ascenzioni et al., 1997, Cancer Lett. 118:135-142; Chan
and Glazer, 1997, J. Mol. Med. 75:267-282. Methods commonly known
in the art of recombinant DNA technology which can be used are
described in Ausubel et al. (eds.), 1993, Current Protocols in
Molecular Biology, John Wiley & Sons, NY; and Kriegler, 1990,
Gene Transfer and Expression, A Laboratory Manual, Stockton Press,
NY.
[0294] In an embodiment in which recombinant HSPC are used in gene
therapy, a gene whose expression is desired in a subject is
introduced into the HSPC such that it is expressible by the cells
and/or their progeny, and the recombinant cells are then
administered in vivo for therapeutic effect.
[0295] Recombinant Expanded HSPC can be used in any appropriate
method of gene therapy, as would be recognized by those in the art
upon considering this disclosure. The resulting action of
recombinant cell populations administered to a subject can, for
example, lead to the activation or inhibition of a pre-selected
gene in the subject, thus leading to improvement of the diseased
condition afflicting the subject.
[0296] In this embodiment, the desired gene is introduced into the
HSPC or its progeny prior to administration in vivo of the
resulting recombinant cell. Such introduction can be carried out by
any method known in the art, including but not limited to
transfection, electroporation, microinjection, lipofection, calcium
phosphate mediated transfection, infection with a viral or
bacteriophage vector containing the gene sequences, cell fusion,
chromosome-mediated gene transfer, microcell-mediated gene
transfer, spheroplast fusion, etc. Numerous techniques are known in
the art for the introduction of foreign genes into cells (see e.g.,
Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et al.,
1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther.
29:69-92) and may be used in accordance with the present invention,
provided that the necessary developmental and physiological
functions of the recipient cells are not disrupted. The technique
should provide for the stable transfer of the gene to the cell, so
that the gene is expressible by the cell and preferably heritable
and expressible by its cell progeny. Usually, the method of
transfer includes the transfer of a selectable marker to the cells.
The cells are then placed under selection to isolate those cells
that have taken up and are expressing the transferred gene. Those
cells are then delivered to a subject.
[0297] Retroviral vectors (see Miller et al., 1993, Meth. Enzymol.
217:581-599) can be used in gene therapy. In such embodiments, the
gene to be used in gene therapy is cloned into the retroviral
vector for its delivery into HSPC. In particular embodiments, a
retroviral vector for use in gene therapy contains all of the
cis-acting sequences necessary for the packaging and integration of
the viral genome, i.e., (a) a long terminal repeat (LTR), or
portions thereof, at each end of the vector; (b) primer binding
sites for negative and positive strand DNA synthesis; and (c) a
packaging signal, necessary for the incorporation of genomic RNA
into virions.
[0298] More detail about retroviral vectors can be found in Boesen
et al., 1994, Biotherapy 6:291-302, Clowes et al., 1994, J. Clin.
Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons
and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and
Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114.
[0299] Adenoviruses are also of use in gene therapy. See Kozarsky
and Wilson, 1993, Current Opinion in Genetics and Development
3:499-503, Rosenfeld et al., 1991, Science 252:431-434; Rosenfeld
et al., 1992, Cell 68:143-155; and Mastrangeli et al., 1993, J.
Clin. Invest. 91:225-234.
[0300] It has been proposed that adeno-associated virus (AAV) be
used in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol.
Med. 204:289-300). It has also been proposed that alphaviruses be
used in gene therapy (Lundstrom, 1999, J. Recept. Signal Transduct.
Res. 19:673-686).
[0301] Other methods of gene delivery in gene therapy include the
use of mammalian artificial chromosomes (Vos, 1998, Curr. Op.
Genet. Dev. 8:351-359); liposomes (Tarahovsky and Ivanitsky, 1998,
Biochemistry (Mosc) 63:607-618); ribozymes (Branch and Klotman,
1998, Exp. Nephrol. 6:78-83); and triplex DNA (Chan and Glazer,
1997, J. Mol. Med. 75:267-282).
[0302] A desired gene can be introduced intracellularly and
incorporated within HSPC DNA for expression, by homologous
recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci.
USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).
[0303] In a specific embodiment, isolated HSPC or the Enriched HSPC
or the Expanded HSPC are genetically engineered to express a gene
that is deficient in the patient to whom such HSPC are to be
administered.
[0304] In a specific embodiment, the desired gene recombinantly
expressed in the HSPC or their progeny after expansion to be
introduced for purposes of gene therapy comprises an inducible
promoter operably linked to the coding region, such that expression
of the recombinant gene is controllable by controlling the presence
or absence of the appropriate inducer of transcription.
[0305] 2.11 Therapeutic Methods
[0306] The ideal therapeutic product for treatment of chemotherapy
or radiation induced pancytopenia is one that, when infused, would
give rise to rapid hematopoietic reconstitution, especially of
granulocytes, and also facilitate autologous recovery of
hematopoiesis.
[0307] The Expanded HSPC populations, whether recombinantly
expressing a desired gene or not, can be administered into a human
patient in need thereof for hematopoietic function for the
treatment of disease or injury or for gene therapy by any method
known in the art which is appropriate for the Expanded HSPC and the
transplant site. Preferably, the Expanded HSPC are transplanted
(infused) intravenously. In one embodiment, the Expanded HSPC
differentiate into cells of the myeloid lineage in the patient. In
another embodiment, the Expanded HSPC differentiate into cells of
the lymphoid lineage in the patient.
[0308] In one embodiment, the transplantation of the Expanded HSPC
is autologous. In such embodiments, before expansion, the HSPC are
isolated from tissues of a subject to whom the Expanded HSPC are to
be administered. In other embodiments, the transplantation of the
Expanded HSPC is non-autologous. In some of these embodiments, the
transplantation of the Expanded HSPC is allogeneic. For
non-autologous transplantation, the recipient can be given an
immunosuppressive drug to reduce the risk of rejection of the
transplanted cells. In some embodiments, the transplantation of the
Expanded HSPC is syngeneic.
[0309] In specific embodiments, HSPC are isolated from a subject
for expansion prior to the subject's exposure to chemotherapy, and
the Expanded HSPC obtained using the methods described herein from
the isolated HSPC of the subject are administered to the subject
following exposure to chemotherapy.
[0310] In specific embodiments, the Expanded HSPC are not
administered to the patient within 12 hours of administration of a
myeloid progenitor cell population as defined in International
Patent Publication Nos. WO 2006/047569 A2 and/or WO 2007/095594 A2.
In other specific embodiments, the Expanded HSPC are not
administered to the patient within 18 or 24 or 36 or 48 or 72 or 96
hours or within 7, 10, 14, 21, 30 days of administration of such a
myeloid progenitor cell population to the patient.
[0311] In a specific embodiment, the Expanded HSPC sample that is
administered to the patient is not a pooled sample, i.e., it is
derived from one individual (e.g., the umbilical cord blood and/or
placental blood of one individual). In other embodiments, the
Expanded HSPC sample that is administered to the patient is a
pooled sample, i.e., it is derived from two or more
individuals.
[0312] In some embodiments, the Expanded HSPC sample that is
administered to the patient has been cryopreserved and thawed prior
to administration. In other embodiments, the Expanded HSPC sample
that is administered to the patient is fresh, i.e., it has not been
cryopreserved prior to administration.
[0313] In a specific embodiment, the methods of the invention
described herein further comprise administering one or more
umbilical cord blood/placental blood samples (hereinafter called
"Grafts" or "cord blood transplants"). Such Grafts are umbilical
cord blood and/or placental blood samples from humans that are
whole blood samples, except that red blood cells have been removed
from the whole blood samples, but which samples have not been
further fractionated and have not been expanded. In a specific
embodiment, the Grafts have been cryopreserved and are thawed prior
to administration. The Grafts can be administered concurrently
with, sequentially with respect to, before, or after the Expanded
HSPC sample is administered to the patient. In a specific
embodiment, the Expanded HSPC sample that is administered to the
patient is administered within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days
of administering the one or more Grafts. In a specific embodiment,
the Expanded HSPC sample is administered before administering the
one or more Grafts. In another specific embodiment, the Expanded
HSPC sample is administered after administering the one or more
Grafts. In a specific embodiment, the Expanded HSPC sample is
administered 1 to 24 hours, 2 to 12 hours, 3 to 8 hours, or 3 to 5
hours before or after administering the one or more Grafts. In
other specific embodiments, the Expanded HSPC sample is
administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24
hours before or after administering the one or more Grafts. In a
preferred embodiment, the Expanded HSPC sample is administered
about 4 hours after administering the one or more Grafts. In a
specific embodiment, a single Graft is administered that is derived
from the cord and/or placental blood of a single human individual.
In a specific embodiment, two Grafts are administered, each derived
from the cord and/or placental blood of a different human
individual. In another specific embodiment, a single Graft is
administered that is a combination of cord and/or placental blood
derived from two or more different human individuals. In the
foregoing embodiments, the Graft is intended to provide long-term
engraftment.
[0314] In certain embodiments, the Expanded HSPC are intended to
provide short-term engraftment. Short-term engraftment usually
refers to engraftment that lasts for up to a few days to few weeks,
preferably 4 weeks, post-transplantation of the Expanded HSPC. In
some embodiments, the Expanded HSPC are effective to provide
engraftment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days; or 1, 2, 3, 4 weeks
after administration of the Expanded HSPC to a patient (e.g., a
human patient). In other embodiments, the Expanded HSPC are
intended to provide long-term engraftment. Long-term engraftment
usually refers to engraftment that is present months to years
post-transplantation of the Expanded HSPC. In some embodiments, the
Expanded HSPC are effective to provide engraftment when assayed at
8, 9, 10 weeks; 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months (or more
than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months); or 1, 2, 3, 4, 5
years (or more than 1, 2, 3, 4, 5 years) after administration of
the Expanded HSPC to a patient. In some embodiments, the Expanded
HSPC are intended to provide both short-term and long-term
engraftment. In certain embodiments, the Expanded HSPC provide
short-term and/or long-term engraftment in a patient, preferably, a
human.
[0315] In some embodiments, the Expanded HSPC are effective to
provide engraftment when assayed at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10
days (or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days); 1, 2, 3, 4,
5, 6, 7, 8, 9, 10 weeks (or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10
weeks); 1; 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months (or more than
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months); or 1, 2, 3, 4, 5
years (or more than 1, 2, 3, 4, 5 years) after administration of
the Expanded HSPC to a patient (e.g., a human patient). In other
embodiments, the Expanded HSPC are effective to provide engraftment
when assayed within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days (or less
than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days); 1, 2, 3, 4, 5, 6, 7, 8,
9, 10 weeks (or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks); or
1; 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months (or less than 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12 months) after administration of the
Expanded HSPC to a patient (e.g., a human patient). In specific
embodiments, the Expanded HSPC are effective to provide engraftment
when assayed within 10 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 6
weeks, or 13 weeks after administration of the Expanded HSPC to a
patient (e.g., a human patient).
[0316] The HSPC expanded using the methods described herein have
been shown to provide short-term and/or long-term engraftment when
infused into sublethally irradiated immunodeficient mice (e.g.,
NOD-SCID mice). The HSPC expanded using the methods described
herein have been shown to provide short-term and/or long-term
engraftment that is superior to the short-term and/or long-term
engraftment obtained using cells expanded with a Notch agonist
alone (i.e., without an aryl hydrocarbon receptor antagonist) or
with an aryl hydrocarbon receptor antagonist (i.e., without a Notch
agonist), e.g., in an animal model (NOD-SCID mice).
[0317] The inventors of the present invention observed that
expansion of HSPC cells under conditions described herein, in the
presence of a Notch agonist (e.g., Delta1.sup.Ext-IgG) and an aryl
hydrocarbon receptor antagonist (e.g., SR1), increases generation
of repopulating cells capable of early myeloid repopulation in NSG
mice as compared to a Notch agonist alone and an aryl hydrocarbon
receptor antagonist alone, suggesting this combination may further
enhance generation of short-term repopulating cells. This could
have clear clinical benefit if these expanded cells were able to
further reduce time to neutrophil engraftment in transplant
recipients. Thus, the Expanded HSPC can be used for short-term in
vivo repopulation/engraftment.
[0318] In addition, while significant generation of HSPC capable of
sustained long-term repopulation in immunodeficient mice or humans
remained elusive, the inventors of the present invention observed
that expansion of HSPC cells under conditions described herein, in
the presence of a Notch agonist (e.g., Delta1.sup.Ext-IgG) and an
aryl hydrocarbon receptor antagonist (e.g., SR1), increased
generation of cells capable of long-term repopulation in NSG mice.
Thus, the Expanded HSPC can be used for long-term in vivo
repopulation/engraftment. Furthermore, the inventors found that
expansion of HSPC cells under conditions described herein, in the
presence of a Notch agonist (e.g., Delta1.sup.Ext-IgG) and an aryl
hydrocarbon receptor antagonist (e.g., SR1), increased in vivo
generation of cells with multi-lineage potential that may give rise
to cells of, e.g., myeloid, lymphoid and progenitor lineage. In
addition, the inventors found that expansion of HSPC cells under
conditions described herein, in the presence of a Notch agonist
(e.g., Delta1.sup.Ext-IgG) and an aryl hydrocarbon receptor
antagonist (e.g., SR1), are capable of long-term maintenance of
progenitor cells upon in vivo repopulation. In certain embodiments,
the described effects of the Expanded HSPC are superior to the
effects observed with HSPC expanded in the presence of a Notch
agonist alone, an aryl hydrocarbon receptor antagonist alone, or
the effects observed with non-manipulated HSPC.
[0319] Suitable methods of administration of the Expanded HSPC are
encompassed by the present invention. The Expanded HSPC populations
can be administered by any convenient route, for example by
infusion or bolus injection, and may be administered together with
other biologically active agents. Administration can be systemic or
local.
[0320] The titer of Expanded HSPC administered which will be
effective in the treatment of a particular disorder or condition
will depend on the nature of the disorder or condition, and can be
determined by standard clinical techniques. In addition, in vitro
and in vivo assays may optionally be employed to help identify
optimal dosage ranges. The precise dose to be employed in the
formulation will also depend on the route of administration, and
the seriousness of the disease or disorder, and should be decided
according to the judgment of the practitioner and each subject's
circumstances. In specific embodiments, suitable dosages of
Expanded HSPC for administration are generally about at least
5.times.10.sup.6, 10.sup.7, 5.times.10.sup.7, 75.times.10.sup.6,
10.sup.7, 5.times.10.sup.7, 108, 5.times.10.sup.8,
1.times.10.sup.9, 5.times.10.sup.9, 1.times.10.sup.10,
5.times.10.sup.10, 1.times.10.sup.11, 5.times.10.sup.11 or
10.sup.12 CD34.sup.+ cells per kilogram patient weight, and most
preferably about 10.sup.7 to about 10.sup.12 CD34.sup.+ cells per
kilogram patient weight, and can be administered to a patient once,
twice, three or more times with intervals as often as needed. In a
specific embodiment, a single Expanded HSPC sample provides one or
more doses for a single patient. In one specific embodiment, a
single Expanded HSPC sample provides four doses for a single
patient.
[0321] In certain embodiments, the patient is a human patient,
preferably an immunodeficient human patient.
[0322] In a specific embodiment, the Expanded HSPC population
administered to a human patient in need thereof can be a pool of at
least two individual Expanded HSPC samples, each sample derived
from a single human (e.g., the umbilical cord blood and/or
placental blood of a single human). For example, an aliquot of a
frozen, thawed, expanded sample that is a pool of samples (i.e., a
pooled sample) can be administered. In one embodiment, the
individual samples in the pool are all derived from HSPC (e.g.,
umbilical cord blood and/or placental blood) of individuals of the
same race, e.g., African-American, Caucasian, Asian, Hispanic,
Native-American, Australian Aboriginal, Inuit, Pacific Islander, or
are all derived from HSPC (e.g., umbilical cord blood and/or
placental blood) of individuals of the same ethnicity, e.g., Irish,
Italian, Indian, Japanese, Chinese, Russian, etc. In an alternative
embodiment, the administered sample is not a pool of samples.
[0323] 2.12 Pharmaceutical Compositions
[0324] The invention provides methods of treatment by
administration to a patient of a pharmaceutical (therapeutic)
composition comprising a therapeutically effective amount of
recombinant or non-recombinant Expanded HSPC produced by the
methods of the present invention as described herein above.
[0325] The present invention provides pharmaceutical compositions.
Such compositions comprise a therapeutically effective amount of
the Expanded HSPC, and a pharmaceutically acceptable carrier or
excipient. Such a carrier can be but is not limited to saline,
buffered saline, dextrose, water, glycerol, ethanol, and
combinations thereof. The carrier and composition preferably are
sterile. Suitable pharmaceutical carriers are described in
Remington: The Science and Practice of Pharmacy, 21st Edition,
David B. Troy, ed., Lippicott Williams & Wilkins (2005), which
is incorporated by reference herein in its entirety, and
specifically for the material related to pharmaceutical carriers
and compositions. The pharmaceutical compositions described herein
can be formulated in any manner known in the art.
[0326] The formulations (including, e.g., carriers, excipients and
medium) and modes of administration of pharmaceutical compositions
described at pages 14-15 of U.S. Patent Publication No.
2010/0183564, can also be used in the methods described herein. For
example, as described at page 14 of U.S. Patent Publication No.
2010/0183564, in some embodiments, the carrier or excipient is
selected to minimize degradation of the active ingredient and/or to
minimize adverse side effects on the cells or in the patient.
[0327] The formulation should suit the mode of administration.
Expanded HSPC can be resuspended in a pharmaceutically acceptable
medium suitable for administration to a mammalian host. In
preferred embodiments, the pharmaceutical composition is acceptable
for therapeutic use in humans. The composition, if desired, can
also contain pH buffering agents.
[0328] The pharmaceutical compositions described herein can be
administered via any route known to one skilled in the art to be
effective. In a preferred embodiment, the composition is formulated
in accordance with routine procedures as a pharmaceutical
composition adapted for intravenous administration to a patient
(e.g., a human). Typically, compositions for intravenous
administration are solutions in sterile isotonic aqueous buffer.
Where necessary, the composition may also include a solubilizing
agent and a local anesthetic such as lidocaine to ease pain at the
site of the injection. In one embodiment, a pharmaceutically
acceptable carrier for infusion of a composition comprising the
Expanded HSPC into a patient comprises buffered saline with 5% HSA
or unsupplemented basal medium or any medium known in the art or
described herein.
[0329] As described at page 14 of U.S. Patent Publication No.
2010/0183564, the number of the Expanded HSPC to be administered to
a patient depends on such factors as sex, age, weight, the types of
disease or disorder, stage of the disorder, the percentage of the
desired cells in the cell population and the amount of cells needed
to produce a therapeutic benefit. In one embodiment, the
composition is administered by intravenous infusion and comprises
at least 10.sup.4 cells/kg, from 10.sup.5 to 5.times.10.sup.7
cells/kg or more cells. In one embodiment, the composition
comprises 10.sup.6 to 10.sup.8 cells/ml. In a specific embodiment,
all of the infused cells are derived from HSPC isolated from a
single human at birth.
[0330] As described at page 15 of U.S. Patent Publication No.
2010/0183564, the compositions can be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. In particular embodiments, the compositions can be
formulated for local administration, e.g., by injection into the
bone marrow of a bone (e.g., long bone).
[0331] In specific embodiments, the compositions described herein
are formulated for administration to a patient with one or more
additional therapeutic active ingredients.
[0332] 2.13 Therapeutic Uses of the Expanded HSPC
[0333] The Expanded HSPC of the present invention can be used to
provide hematopoietic function to a patient in need thereof,
preferably a human patient. In other embodiments, the patient is a
cow, a pig, a horse, a dog, a cat, or any other animal, preferably
a mammal.
[0334] The Expanded HSPC that are administered to a patient in need
thereof can be derived from the umbilical cord blood, placental
blood, peripheral blood (e.g., mobilized peripheral blood), bone
marrow or other sources of HSPC. In one embodiment, the Expanded
HSPC are derived from the umbilical cord blood and/or placental
blood, such as the umbilical cord blood and/or placental blood of a
single human at birth, or the umbilical cord blood and/or placental
blood of more than 1 human at birth (pool of samples), as described
above. In another embodiment, the Expanded HSPC are derived from
the peripheral blood (e.g., mobilized peripheral blood).
[0335] The description of therapeutic uses of the Expanded HSPC
provided herein is largely found in sec. 5.6.1 of U.S. Pat. No.
7,399,633.
[0336] In one embodiment, administration of Expanded HSPC of the
invention is for the treatment of immunodeficiency. In a preferred
embodiment, administration of Expanded HSPC of the invention is for
the treatment of pancytopenia or for the treatment of neutropenia.
The immunodeficiency in the patient, for example, pancytopenia or
neutropenia, can be the result of an intensive chemotherapy
regimen, myeloablative regimen for hematopoietic cell
transplantation (HCT), or exposure to acute ionizing radiation.
Exemplary chemotherapeutics that can cause prolonged pancytopenia
or prolonged neutropenia include, but are not limited to alkylating
agents such as cisplatin, carboplatin, and oxaliplatin,
mechlorethamine, cyclophosphamide, chlorambucil, and ifosfamide.
Other chemotherapeutic agents that can cause prolonged pancytopenia
or prolonged neutropenia include azathioprine, mercaptopurine,
vinca alkaloids, e.g., vincristine, vinblastine, vinorelbine,
vindesine, and taxanes. In particular, a chemotherapy regimen that
can cause prolonged pancytopenia or prolonged neutropenia is the
administration of clofarabine and Ara-C.
[0337] In one embodiment, the patient is in an acquired or induced
aplastic state.
[0338] The immunodeficiency in the patient also can be caused by
exposure to acute ionizing radiation following a nuclear attack,
e.g., detonation of a "dirty" bomb in a densely populated area, or
by exposure to ionizing radiation due to radiation leakage at a
nuclear power plant, or exposure to a source of ionizing radiation,
raw uranium ore.
[0339] Transplantation of Expanded HSPC of the invention can be
used in the treatment or prevention of hematopoietic disorders and
diseases. In one embodiment, the Expanded HSPC are administered to
a patient with a hematopoietic deficiency. In one embodiment, the
Expanded HSPC are used to treat or prevent a hematopoietic disorder
or disease characterized by a failure or dysfunction of normal
blood cell production and cell maturation. In another embodiment,
the Expanded HSPC are used to treat or prevent a hematopoietic
disorder or disease resulting from a hematopoietic malignancy. In
yet another embodiment, the Expanded HSPC are used to treat or
prevent a hematopoietic disorder or disease resulting from
immunosuppression, particularly immunosuppression in subjects with
malignant, solid tumors. In yet another embodiment, the Expanded
HSPC are used to treat or prevent an autoimmune disease affecting
the hematopoietic system. In yet another embodiment, the Expanded
HSPC are used to treat or prevent a genetic or congenital
hematopoietic disorder or disease.
[0340] Examples of particular hematopoietic diseases and disorders
which can be treated by the Expanded HSPC of the invention include
but are not limited to those listed in Table 2, infra.
TABLE-US-00002 TABLE 2 DISEASES OR DISORDERS WHICH CAN BE TREATED
BY ADMINISTERING EXPANDED HSPC OF THE INVENTION I. Diseases
Resulting from a Failure or Dysfunction of Normal Blood Cell
Production and Maturation hyperproliferative stem cell disorders
aplastic anemia pancytopenia agranulocytosis thrombocytopenia red
cell aplasia Blackfan-Diamond syndrome due to drugs, radiation, or
infection Idiopathic II. Hematopoietic malignancies acute
lymphoblastic (lymphocytic) leukemia chronic lymphocytic leukemia
acute myelogenous leukemia chronic myelogenous leukemia acute
malignant myelosclerosis multiple myeloma polycythemia vera
agnogenic myelometaplasia Waldenstrom's macroglobulinemia Hodgkin's
lymphoma non-Hodgkin's lymphoma III. Immunosuppression in patients
with malignant, solid tumors malignant melanoma carcinoma of the
stomach ovarian carcinoma breast carcinoma small cell lung
carcinoma retinoblastoma testicular carcinoma glioblastoma
rhabdomyosarcoma neuroblastoma Ewing's sarcoma lymphoma IV
Autoimmune diseases rheumatoid arthritis diabetes type I chronic
hepatitis multiple sclerosis systemic lupus erythematosus V.
Genetic (congenital) disorders anemias familial aplastic Fanconi's
syndrome (Fanconi anemia) Bloom's syndrome pure red cell aplasia
(PRCA) dyskeratosis congenital Blackfan-Diamond syndrome congenital
dyserythropoietic syndromes I-IV Chwachmann-Diamond syndrome
dihydrofolate reductase deficiencies formamino transferase
deficiency Lesch-Nyhan syndrome congenital spherocytosis congenital
elliptocytosis congenital stomatocytosis congenital Rh null disease
paroxysmal nocturnal hemoglobinuria G6PD (glucose-6-phosphate
dehydrogenase) variants 1, 2, 3 pyruvate kinase deficiency
congenital erythropoietin sensitivity deficiency sickle cell
disease and trait (Sickle cell anemia) thalassemia alpha, beta,
gamma met-hemoglobinemia congenital disorders of immunity severe
combined immunodeficiency disease (SCID) bare lymphocyte syndrome
ionophore-responsive combined immunodeficiency combined
immunodeficiency with a capping abnormality nucleoside
phosphorylase deficiency granulocyte actin deficiency infantile
agranulocytosis Gaucher's disease adenosine deaminase deficiency
Kostmann's syndrome reticular dysgenesis congenital leukocyte
dysfunction syndromes VI. Others osteopetrosis myelosclerosis
acquired hemolytic anemias acquired immunodeficiencies infectious
disorders causing primary or secondary immunodeficiencies bacterial
infections (e.g., Brucellosis, Listerosis, tuberculosis, leprosy)
parasitic infections (e.g., malaria, Leishmaniasis) fungal
infections disorders involving disproportions in lymphoid cell sets
and impaired immune functions due to aging phagocyte disorders
Kostmann's agranulocytosis chronic granulomatous disease
Chediak-Higachi syndrome neutrophil actin deficiency neutrophil
membrane GP-180 deficiency metabolic storage diseases
mucopolysaccharidoses mucolipidoses miscellaneous disorders
involving immune mechanisms Wiskott-Aldrich Syndrome
.alpha.1-antitrypsin deficiency
[0341] In one embodiment, the Expanded HSPC are administered to a
patient with a hematopoietic deficiency. Hematopoietic deficiencies
whose treatment with the Expanded HSPC of the invention is
encompassed by the methods of the invention include but are not
limited to decreased levels of either myeloid, erythroid, lymphoid,
or megakaryocyte cells of the hematopoietic system or combinations
thereof, including those listed in Table 2. In one embodiment, the
Expanded HSPC are administered prenatally to a fetus diagnosed with
a hematopoietic deficiency.
[0342] Among conditions susceptible to treatment with the Expanded
HSPC of the present invention is leukopenia, a reduction in the
number of circulating leukocytes (white cells) in the peripheral
blood. Leukopenia may be induced by exposure to certain viruses or
to radiation. It is often a side effect of various forms of cancer
therapy, e.g., exposure to chemotherapeutic drugs, radiation and of
infection or hemorrhage.
[0343] Expanded HSPC also can be used in the treatment or
prevention of neutropenia and, for example, in the treatment of
such conditions as aplastic anemia, cyclic neutropenia, idiopathic
neutropenia, Chediak-Higashi syndrome, systemic lupus erythematosus
(SLE), leukemia, myelodysplastic syndrome, myelofibrosis,
thrombocytopenia. Severe thrombocytopenia may result from genetic
defects such as Fanconi's Anemia, Wiscott-Aldrich, or May-Hegglin
syndromes and from chemotherapy and/or radiation therapy or cancer.
Acquired thrombocytopenia may result from auto- or allo-antibodies
as in Immune Thrombocytopenia Purpura, Systemic Lupus
Erythromatosis, hemolytic anemia, or fetal maternal
incompatibility. In addition, splenomegaly, disseminated
intravascular coagulation, thrombotic thrombocytopenic purpura,
infection or prosthetic heart valves may result in
thrombocytopenia. Thrombocytopenia may also result from marrow
invasion by carcinoma, lymphoma, leukemia or fibrosis.
[0344] Many drugs may cause bone marrow suppression or
hematopoietic deficiencies. Examples of such drugs are AZT, DDI,
alkylating agents and anti-metabolites used in chemotherapy,
antibiotics such as chloramphenicol, penicillin, gancyclovir,
daunomycin and sulfa drugs, phenothiazones, tranquilizers such as
meprobamate, analgesics such as aminopyrine and dipyrone,
anticonvulsants such as phenytoin or carbamazepine, antithyroids
such as propylthiouracil and methimazole and diuretics.
Transplantation of the Expanded HSPC can be used in preventing or
treating the bone marrow suppression or hematopoietic deficiencies
which often occur in subjects treated with these drugs.
[0345] Hematopoietic deficiencies may also occur as a result of
viral, microbial or parasitic infections and as a result of
treatment for renal disease or renal failure, e.g., dialysis.
Transplantation of the Expanded HSPC populations may be useful in
treating such hematopoietic deficiency.
[0346] Various immunodeficiencies, e.g., in T and/or B lymphocytes,
or immune disorders, e.g., rheumatoid arthritis, may also be
beneficially affected by treatment with the Expanded HSPC.
Immunodeficiencies may be the result of viral infections (including
but not limited to HIVI, HIVII, HTLVI, HTLVII, HTLVIII), severe
exposure to radiation, cancer therapy or the result of other
medical treatment.
[0347] In specific embodiments, the Expanded HSPC are used for the
treatment of multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's
disease, neuroblastoma, germ cell tumors, autoimmune disorder
(e.g., Systemic lupus erythematosus (SLE) or systemic sclerosis),
amyloidosis, acute myeloid leukemia, acute lymphoblastic leukemia,
chronic myeloid leukemia, chronic lymphocytic leukemia,
myeloproliferative disorder, myelodysplastic syndrome, aplastic
anemia, pure red cell aplasia, paroxysmal nocturnal hemoglobinuria,
Fanconi anemia, Thalassemia major, Sickle cell anemia, Severe
combined immunodeficiency (SCID), Wiskott-Aldrich syndrome,
Hemophagocytic lymphohistiocytosis (HLH), or inborn errors of
metabolism (e.g., mucopolysaccharidosis, Gaucher disease,
metachromatic leukodystrophies or adrenoleukodystrophies). In some
embodiments, the Expanded HSPC are used for the treatment of an
inherited immunodeficient disease, an autoimmune disease and/or a
hematopoietic disorder.
[0348] In one embodiment, the Expanded HSPC are for replenishment
of hematopoietic cells in a patient who has undergone chemotherapy
or radiation treatment. In a specific embodiment, the Expanded HSPC
are administered to a patient that has undergone chemotherapy or
radiation treatment. In a specific embodiment, the Expanded HSPC
are administered to a patient who has HIV (e.g., for replenishment
of hematopoietic cells in a patient who has HIV).
[0349] In certain embodiments, the Expanded HSPC are administered
into the appropriate region of a patient's body, for example, by
injection into the patient's bone marrow.
[0350] In specific embodiments, a Notch agonist is inactivated or
removed prior to administration of the Expanded HSPC to a patient.
In other specific embodiments, an aryl hydrocarbon antagonist is
inactivated or removed prior to administration of the Expanded HSPC
to a patient.
[0351] In some embodiments, the patient to whom the Expanded HSPC
are administered is a bone marrow donor, at risk of depleted bone
marrow, or at risk for depleted or limited blood cell levels. In
one embodiment, the patient to whom the Expanded HSPC is
administered is a bone marrow donor prior to harvesting of the bone
marrow. In one embodiment, the patient to whom the Expanded HSPC is
administered is a bone marrow donor after harvesting of the bone
marrow. In one embodiment, the patient to whom the Expanded HSPC is
administered is a recipient of a bone marrow transplant. In one
embodiment, the patient to whom the Expanded HSPC is administered
is elderly, has been exposed or is to be exposed to an immune
depleting or myeloablative treatment (e.g., chemotherapy,
radiation), has a decreased blood cell level, or is at risk of
developing a decreased blood cell level as compared to a control
blood cell level. In one embodiment, the patient has anemia or is
at risk for developing anemia. In one embodiment, the patient has
blood loss due to, e.g., trauma, or is at risk for blood loss. The
Expanded HSPC can be administered to a patient, e.g., before, at
the same time, or after chemotherapy, radiation therapy or a bone
marrow transplant. In specific embodiments, the patient has
depleted bone marrow related to, e.g., congenital, genetic or
acquired syndrome characterized by bone marrow loss or depleted
bone marrow. In one embodiment, the patient is in need of
hematopoiesis. In one embodiment, HSPC are isolated from peripheral
blood of a patient that will undergo an immune depleting procedure
(e.g., chemotherapy, radiation, or bone marrow extraction from
donor), the HSPC are expanded as described herein, and after the
treatment the Expanded HSPC are administered to the patient.
[0352] 2.14 Kits
[0353] The invention also provides a pharmaceutical pack or kit
comprising one or more containers. In a preferred embodiment, a kit
of the invention comprises, in one or more containers, a Notch
agonist (such as purified Notch agonist) and an aryl hydrocarbon
receptor antagonist. In a specific embodiment, the Notch agonist is
Delta1.sup.Ext-IgG, and the aryl hydrocarbon receptor antagonist is
SR1. In one embodiment, a Notch agonist and an aryl hydrocarbon
antagonist are stored in two separate containers of the kit. In
certain embodiments, each of the ingredients of the kit listed
herein is provided in a separate container. In other embodiments,
two or more of the ingredients of the kit listed herein are
provided in a same container.
[0354] The kit may additionally comprise one or more purified
growth factors, for example, one or more growth factors that
promote proliferation but not differentiation of HSPC. Such one or
more growth factors may be stored in a container separate from the
container comprising a Notch agonist and/or in a container separate
from the container comprising an aryl hydrocarbon receptor
antagonist. In some embodiments, the kit may further comprise, in a
separate container, one or more purified growth factors that
promote the differentiation of HSPC.
[0355] In certain embodiments, cell culture medium is also provided
in the kit. In other embodiments, the solid phase on which
Delta1.sup.Ext-IgG can be coated is also provided in the kit (for
example, such a kit may contain one or more tissue culture dishes
coated with Delta1.sup.Ext-IgG). In certain embodiments, the kit
also comprises fibronectin (e.g., an immobilized fibronectin) or a
fragment thereof (e.g., CH-296). In certain embodiments,
fibronectin or a fragment thereof are provided in a separate
container. In some embodiments, fibronectin or a fragment thereof
is provided in the same container as a Notch agonist. In a
particular embodiment, fibronectin or a fragment thereof is
provided in the same container as a Notch agonist, wherein both
fibronectin or a fragment thereof and the Notch agonist are coated
on a solid phase.
[0356] The kit may further comprise one or more containers filled
with isolated HSPC or the Enriched HSPC. The Notch agonist, the
aryl hydrocarbon antagonist and the one or more growth factors
provided in the described kit are together effective to expand the
Enriched HSPC exposed to these ingredients of the kit in culture.
In certain embodiment, a kit comprises one or more containers
filled with the Enriched HSPC or with the Expanded HSPC produced by
the methods of the invention and/or reagents to prepare said cells,
or with reagents for the genetic manipulation of the cells.
[0357] The kit may additionally comprise a solution or a buffer (in
a separate container, or in the same container as the aryl
hydrocarbon receptor antagonist and/or the Notch agonist).
[0358] In some embodiments, the kit comprises a container with one
or more antibodies (e.g., anti-CD34, anti-CD133, anti-CD38,
anti-CD45R, anti-Thy 1 antibodies, or any other antibodies to
markers/antigens described herein or known in the art).
[0359] In specific embodiments, the kit comprises a
pharmaceutically acceptable carrier or a stabilizer (in a separate
container, or in the same container as an aryl hydrocarbon receptor
antagonist and/or a Notch agonist). Examples of physiologically
acceptable carriers include buffers such as phosphate, citrate, and
other organic acids; antioxidants including ascorbic acid; low
molecular weight (less than about 10 residues) polypeptide;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, arginine or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA; sugar
alcohols such as mannitol or sorbitol; salt-forming counterions
such as sodium; and/or nonionic surfactants such as TWEEN.TM.
(Uniqema, United Kingdom), polyethylene glycol (PEG), and
PLURONICS.TM. (BASF, Germany) (U.S. Patent Publication No.
2010/0183564).
[0360] In some embodiments, an aryl hydrocarbon antagonist in the
kit is formulated as a suspension, solution or emulsion in oily or
aqueous vehicle that, optionally, contains a suspending, a
stabilizing, a dispersing agent, and/or a preservative. The
stabilizing agent can be sodium bisulfate, sodium sulfite, ascorbic
acid, citric acid or its salt, and/or sodium
ethylenediaminetetraacetic acid (EDTA). The preservative can be
benzalkonium chloride, methyl- or propyl-paraben, or chlorobutanol.
In some embodiments, an aryl hydrocarbon antagonist in the kit is
formulated in a suitable carrier, e.g., in water, suitable oil,
saline, aqueous dextrose (glucose), related sugar solutions or
glycol (e.g., propylene glycol or polyethylene glycol). In specific
embodiments, an aryl hydrocarbon antagonist in the kit is
formulated in a powder form (for reconstitution with a suitable
vehicle, e.g., sterile pyrogen-free water, before use).
[0361] Optionally associated with such container(s) can be a notice
in the form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration.
EXAMPLES
[0362] The data presented herein show advantageous properties of a
combination of an agonist of Notch function, e.g.,
Delta1.sup.ext-IgG, and an aryl hydrocarbon receptor antagonist,
e.g., SR1, for ex vivo expansion of HSPC. The data show that HSPC
expanded using such combination of agents maintain immature
progenitor cells and display superior in vivo engraftment
properties relative to the engraftment properties of HSPC expanded
using a Notch agonist alone or an aryl hydrocarbon receptor
antagonist alone. The inventors found that expansion of HSPC using
a combination of a Notch agonist alone or an aryl hydrocarbon
receptor antagonist leads to superior transient myeloid and
progenitor engraftment and generation of cells with multi-lineage
potential capable of long-term repopulation. Data also show that
HSPC that display improved engraftment properties when expanded
using such combination of agents include cord blood hematopoietic
stem/progenitor cells and, surprisingly, peripheral blood stem
cells (in particular, mobilized peripheral blood stem
cells--mPBSC). While previous attempts to expand mPBSC ex vivo
generated modest expansion of progenitor cells with no difference
in engraftment, the data presented herein shows that the described
combination of agents leads to enhanced expansion and engraftment
of mPBSC.
[0363] Materials and Methods Utilized for Examples 1-8
[0364] Cell processing. Human cord blood samples for research were
obtained from normal deliveries under Swedish Medical Center
Institutional Review Board (Seattle) approval and after consent was
obtained. The units were incubated in ammonium chloride red blood
cell lysis buffer (consisting of 16.6 g NH4Cl, 2 g NaHCO3 and 74.4
mg EDTA per 2L of water) and resuspended in PBS with 2% human AB
serum. Cells were incubated with CD34 Microbeads from Miltenyi
Biotec and purified with an Automacs (Miltenyi Biotec). Cells were
then frozen and thawed at time of use with PBS+1% FBS. Pools of 2
or more cord blood units were used for all experiments. Peripheral
blood stem cells (PBSC) were obtained from a single donor and
cryopreserved after CD34+ cell selection.
[0365] Cell culture. Cells were cultured for 16 days in
non-tissue-cultured-treated tissue culture flasks (25, 75, and
175-cm.sup.2). Flasks were precoated with Delta1.sup.ext-IgG
(ligand preparation described in Delaney C. et al., 2005, Blood
106(8):2693-2699; and Varnum-Finney et al., 2000, J. Cell Science
113: 4313-4318), or human control IgG at 2.5 .mu.g/ml and
retronectin 5 .mu.g/ml. Delta1.sup.ext-IgG titrations were
performed with concentrations of 0.5, 1.25 (for PBSC), 2.5, or 5
.mu.g/ml. Flasks were incubated at 4.degree. C. overnight or
37.degree. C. for 2 hours and then washed with PBS and blocked with
PBS 2% BSA for 30 minutes at 37.degree. C. Cells were cultured in
StemSpan serum-free expansion medium in the presence of 4 growth
factors (IL6 (50 ng/ml), thrombopoietin (50 ng/ml), Flt-3 ligand
(50 ng/ml), stem cell factor (50 ng/ml)) or 5 growth factors (4
growth factors plus IL3 (10 ng/ml)). SR-1 was made fresh and added
to cells with all feedings at a concentration of 750 nM. Cultures
in 25-cm.sup.2 flasks were initiated with between 7.times.10.sup.4
and 1.3.times.10.sup.5 CD34+ cells. Cultures in 75-cm.sup.2 flasks
were initiated with 3.times.10.sup.5 CD34+ cells. Cells were
expanded to larger flasks when they exceeded cell density
>1.times.10.sup.6 cells/ml. Expansions typically occurred on day
7 and 10 of culture. Fresh media with cytokines was added very 3-4
days including the day prior to transplantation.
[0366] Flow cytometric analysis. Immunofluorescence analysis was
performed as described (see Ohishi et al., 2002, J Clin Invest.
110(8): 1165-1174), with FITC-labeled antibodies against human CD3,
CD14, CD15, CD33, CD34, CD90, phycoerythryin (PE)-labeled
antibodies against human CD19, CD56, CD90, CD133, Glycophorin A,
CXCR4, PERCP-labeled antibodies against human CD14, CD34,
APC-labeled antibodies against human CD45, CD90 and PECy7 labeled
antibody against mouse CD45.1.
[0367] In vivo repopulation studies. Sublethally irradiated
NOD-SCID IL-2R.gamma.-null mice (obtained from an established
breeding colony at the Fred Hutchinson Cancer Research Center and
approved for use by the Fred Hutchinson Cancer Research Center
Institutional Animal Care and Use Committee) were infused with the
progeny 10,000 starting CD34+ cells per mouse via tail vein for
cord blood experiments and the progeny of 20,000 for PBSC
experiments. Repopulating ability of infused cells was assessed at
2-3 weeks post-transplant by bone marrow aspiration and at 13 weeks
by bone marrow harvest.
[0368] Statistical analyses. Data are presented graphically with
means. Significance of differences between groups was determined
used non-parametric two-tailed t-tests (GraphPad software).
Example 1: Delta1.sup.ext-IgG and SR-1 in Combination Expand CB
HSPC Ex Vivo
[0369] This example shows that the combination of a Notch agonist
and an aryl hydrocarbon receptor antagonist, specifically the
combination of Delta1.sup.Ext-IgG and SR1, is effective to expand
CD34+CB HSPC ex vivo.
[0370] Both Delta1.sup.Ext-IgG and SR1 have been previously shown
to generate CD34+CB HSPC ex vivo as compared to cytokine-containing
control (see Boitano et al., 2010, Science 329(5997): 1345-1348;
Ohishi et al., 2002, J Clin Invest. 110(8): 1165-1174; Delaney et
al., 2005, Blood 106(9): 2693-2699).
[0371] To determine whether Delta1.sup.Ext-IgG and SR1 in
combination were successfully able to expand CD34+CB HSPC ex vivo,
CD34+ cell generation, an HSPC enriched population routinely used
in clinical application, was assessed at multiple time points in
culture as compared to IgG control. CD34+CB progenitors were
isolated by bead selection and Automacs and then cultured for 21
days in StemSpan serum-free expansion media supplemented with
cytokines (TPO, SCF, IL-6, and Flt3-ligand at 50 ng/ml). This
cytokine combination has previously been shown to optimize CD34+
HSPC expansion in the presence of SR1 (see Boitano et al., 2010,
Science 329(5997): 1345-1348). Cells were cultured in the presence
of SR1 (750 nM) and Delta1.sup.Ext-IgG (0.5 or 2.5 .mu.g/ml) or an
IgG control. The engineered Notch ligand, Delta1.sup.Ext-IgG,
contains the extracellular domain of Delta1 fused to the Fc portion
of human IgG1 and is immobilized on the plastic surface of tissue
culture flasks at varying densities. Immobilization of the Fc
portion of human IgG1 served as a control construct.
7.0.times.10.sup.3 CD34+ selected cells were seeded per flask from
a pool of 2 CB units. At 7, 14, 18, and 21 days in culture
Delta1.sup.Ext-IgG and SR1 combination groups had greater CD34-fold
expansion than IgG control (FIG. 1). This effect was most
pronounced at later time points with 425 and 161-fold CD34
expansion in the combination groups and only 38 fold in the IgG
groups. Delta1.sup.Ext-IgG and SR1 alone groups (data not shown)
also demonstrated enhanced CD34 expansion compared to IgG control
consistent with previous reports.
Example 2: Delta1.sup.ext-IgG and SR-1 in Combination Maintain CB
HSPC with More Immature Phenotype Ex Vivo
[0372] This example shows that expansion of HSPC in the presence of
the combination of a Notch agonist and an aryl hydrocarbon receptor
antagonist, specifically Delta1.sup.Ext-IgG and SR1, leads to an
increase in HSPC-enriched cell population (CD34+ cells), an
increase in cells capable of generating long-term engraftment
(CD34+CD90+ cells), and a decrease in differentiated myeloid cells
that have lost multi-potent repopulation capacity (CD34-CD14+
cells).
[0373] The phenotype of cells expanded ex vivo in the presence of
Delta1.sup.Ext-IgG, SR1, or the combination was evaluated to
determine how the combination of these agents affected generation
of CD34+ and CD34+CD90+CB HSPC. The CD34+ cell population has
previously been shown to be enriched for HSPC based on the presence
of both long-term culture initiating cells (LTC-IC) and cells
capable of sustained long-term in vivo repopulation (see Srour et
al., 1991, Blood Cells 17(2): 287-295; Berenson et al., 1988, J
Clin Invest. 81(3): 951-955). CD34+CD90+ cells represent a
subpopulation of CD34+ cells capable of generating long-term
engraftment when transplanted in isolation (see Baum et al., 1992,
Proc Natl Acad Sci. 89: 2804-2808; Craig et al., 1993, J Exp Med.
177: 1331-1342; Majeti et al., 2007, Cell Stem Cell 1(6): 635-645).
Thus, these two phenotypes were used to determine how the culture
conditions affected generation of CB HSPC.
[0374] Cells were cultured in serum-free StemSpan with four
cytokines (TPO, SCF, IL-6, Flt-3 ligand at 50 ng/ml) for 16 days in
the presence of Delta1.sup.Ext-IgG 2.5 .mu.g/ml, SR1 (with IgG), or
the combination (with two Delta1.sup.Ext-IgG densities). Cell
surface analysis by FACS of cell populations generated after 16
days in culture revealed possibly greater percentage of CD34+ and
CD34+CD90+ cells in both combination groups compared to either
approach alone (FIGS. 2A-2B). While statistical comparisons between
the combined groups and Delta1.sup.Ext-IgG or SR1 alone did not
achieve significance, this trend was present for 6 independent
experiments. Delta1.sup.Ext-IgG and SR1 in combination additionally
result in a lower percentage of CD34-CD14+ cells, differentiated
myeloid cells that have lost multi-potent repopulation capacity, as
compared to Delta1.sup.Ext-IgG (not significant) or SR1
(significant) alone, suggesting maintenance of a more immature
cellular phenotype (FIG. 2C).
Example 3: Maintenance of CD34+CD90+ Cells Ex Vivo Correlates with
In Vivo Engraftment
[0375] This example shows that maintenance of the CD34+CD90+ cell
phenotype correlates with improved in vivo repopulating ability of
CB HSPC expanded in the presence of a combination of a Notch
agonist and an aryl hydrocarbon receptor antagonist
(Delta1.sup.Ext-IgG and SR1).
[0376] Cells were cultured as described above and transplanted into
sublethally irradiated immunodeficient NSG mice. Total human
engraftment was defined as percent of human CD45+ murine CD45.1-
cells based on bone marrow aspirate at 2 weeks post-transplant.
Total myeloid engraftment was defined as percent human CD45+CD33+
murine CD45.1- cells on bone marrow aspirate at the same time
point. Then, mean engraftment for six mice per group was compared
to the percent CD34+CD90+ cells infused at time of transplant
(FIGS. 3A, 3B). Total human engraftment correlated significantly
with percent CD34+CD90+ cells infused at time of transplant
(R=0.8117, p-value=0.0079, FIG. 3A). Percent CD34+CD90+ cells also
may predict early myeloid engraftment at this time point, although
the correlation did not achieve significance (R=0.4136,
p-value=0.2685, FIG. 3B). As indicated in FIG. 3, those mice
receiving cells cultured in the presence of both Delta1.sup.Ext-IgG
and SR1 maintain the highest percentage of this immature CB HSPC
population and have on average higher engraftment than those
cultured with either approach alone suggesting the CD34+CD90+
phenotype may represent or contain cells responsible for in vivo
repopulation consistent with previous reports. Thus, culture with
Delta1.sup.Ext-IgG and SR1 in combination results in generation of
HSPC with a more immature cellular phenotype, and this phenotype
correlates with enhanced in vivo repopulating ability.
Example 4: SR-1 Enhances Expansion of CB HSPC; Delta1.sup.Ext-IgG
Blocks Differentiation
[0377] This example shows that blockade of differentiation of
progenitor cells via Notch signaling (using Notch agonist
Delta1.sup.Ext-IgG) and enhancement of cellular expansion via an
AhR antagonist (SR1) may be responsible for enhanced generation of
CB HSPC when Delta1.sup.Ext-IgG and SR1 are used in
combination.
[0378] Cells were cultured as described in previous sections in the
presence of SR1 (with IgG), IgG, or increasing densities of
Delta1.sup.Ext-IgG. Cells were counted at various time points in
culture to determine expansion of total nucleated cells (TNC). TNC
expansion was defined as cell count at given time in culture
divided by starting TNC. SR1 enhanced total nucleated cell (TNC)
expansion over IgG control and all Delta1.sup.Ext-IgG conditions
(FIG. 4A). Addition of SR1 to Delta1.sup.Ext-IgG increased TNC
generation over Delta1.sup.Ext-IgG alone (data not shown). While
apoptosis and cell cycle studies are pending, each condition
demonstrated comparable overall cell viability at each time point
(data not shown) suggesting this expansion was due to enhanced
cellular proliferation.
[0379] Cultured cells were analyzed after 16 days in culture for
cell surface markers of immature progenitor and differentiated cell
populations. Induction of Notch signaling through increasing
densities of Delta1.sup.Ext-IgG decreased TNC generation in a
stepwise manner (FIG. 4B). This was due primarily to decreased
generation of mature cells of the myeloid lineage (CD34-CD14+) with
increased induction of Notch signaling (FIG. 4C) suggesting
Delta1.sup.Ext-IgG may be blocking or delaying differentiation of
these cells in culture. The decreased number of myeloid cells
generated with increasing densities of Delta1.sup.Ext-IgG was the
result of both greater TNC generation and percentage of myeloid
cells generated (FIG. 2C) suggesting a qualitative difference in
cells generated in the presence of Notch signaling. This is
consistent with previous data demonstrating Delta1.sup.Ext-IgG
inhibits generation of CD14+ cells by inhibiting differentiation
from CD34+ cells (see Delaney et al., 2005, Blood 106(9):
2693-2699).
[0380] Generation of immature progenitor and precursor cells,
however, was relatively spared by increasing Delta1.sup.Ext-IgG
signaling where total numbers of immature HSPC (CD34+CD90+) and
lymphoid progenitors (CD34+CD7+) were more equivalent between
conditions despite large differences in TNC (FIGS. 4D, 4E). In
addition, greater numbers and percentages of these immature
progenitor cells were generated in the presence of SR1 and
Delta1.sup.Ext-IgG as compared to Delta1.sup.Ext-IgG alone (data
not shown).
Example 5: Higher Densities of Delta1.sup.ext-IgG in Combination
with SR-1 Enhance In Vivo Engraftment Despite Greater In Vitro
Expansion with Lower Delta1.sup.ext-IgG Densities
[0381] This example demonstrates optimal Delta1.sup.Ext-IgG
densities for maximal generation of cells with in vivo repopulating
ability. In particular, it shows that higher densities of
Delta1.sup.ext-IgG in combination with SR-1 enhance in vivo
engraftment despite greater in vitro expansion with lower
Delta1.sup.ext-IgG densities.
[0382] It was noted that generation of CD34+ progenitor cells was
increased in the presence of SR1 and the lowest Delta1.sup.Ext-IgG
density (0.5 .mu.g/ml). Thus, this combination was selected for
initial transplant experiments (data not shown), which showed some
in vivo repopulation. Next, CB HSPC were expanded in the presence
of SR1, Delta1.sup.Ext-IgG, or the combination using varying
concentrations of the immobilized Delta1.sup.Ext-gG. Greatest
expansion of CD34+CB HSPC was noted with SR1 and the lowest
Delta1.sup.Ext-IgG ligand density (FIG. 5A). The two groups with
higher Delta1.sup.Ext-IgG densities (2.5 and 5 .mu.g/ml)
demonstrated inferior ex vivo CD34+ cell expansion to SR1.
[0383] The progeny of 1.times.10.sup.4 CD34+ cells were then
transplanted into 10 sublethally irradiated NSG mice per condition
and engraftment was assessed by bone marrow aspirate at 2 weeks
post-transplant. Total human engraftment was defined as percent of
human CD45+ murine CD45.1- cells based on bone marrow aspirate at 2
weeks post-transplant. Total myeloid engraftment was defined as
percent human CD45+CD33+ murine CD45.1- cells on bone marrow
aspirate at the same time point. Significant enhancement of
repopulating cell activity was seen for total human engraftment and
total myeloid engraftment with higher densities of
Delta1.sup.Ext-IgG as compared to the lowest density (FIGS. 5B,
5C). Total human engraftment and total myeloid engraftment for
cells cultured with Delta1.sup.Ext-IgG 2.5 .mu.g/ml and SR1 were
both 3.7-fold higher (p-values 0.0144, 0.0099) than the lower
density combination. Total human engraftment and total myeloid
engraftment for cells cultured with Delta1.sup.Ext-IgG 5 .mu.g/ml
and SR1 were 3 and 3.7-fold higher (p-values 0.0284, 0.0165) than
the lower density combination. Furthermore, addition of SR1 to
higher densities of Delta1.sup.Ext-IgG significantly enhanced
repopulating cell activity as compared to Delta1.sup.Ext-IgG 2.5
.mu.g/ml control. For example, total human engraftment was 3-fold
higher for with Delta1.sup.Ext-IgG 2.5 .mu.g/ml and SR1 compared to
Delta1.sup.Ext-IgG 2.5 .mu.g/ml alone (p-value 0.0458). Other
comparisons with Delta1.sup.Ext-IgG 2.5 .mu.g/ml control also
reached significance. In this individual experiment, in vivo
repopulating cell activity was not significantly enhanced with
higher Delta1.sup.Ext-IgG densities and SR1 as compared to SR1
alone, although comparisons trended towards significance. For
example total human engraftment with Delta1.sup.Ext-IgG 2.5
.mu.g/ml and SR1 compared to SR1 alone was 2-fold higher (p-value
0.0832) and total myeloid engraftment was 2.3-fold higher (p-value
0.0732). Thus, despite increased generation of CD34.sup.+ CB HSPC
in vitro, the lowest dose of Delta1.sup.Ext-IgG ligand with SR1
resulted in inferior generation of cell with in vivo repopulating
activity in comparison to the combination of SR1 and higher doses
of Delta1.sup.Ext-IgG ligand. Thus, the combination of SR1 and
higher doses of Delta1.sup.Ext-IgG ligand demonstrates the most
robust in vivo repopulating activity.
Example 6: Delta1.sup.ext-IgG and SR-1 in Combination Enhance
Generation of Short-Term Repopulating Cells and Maintain CB HSPC In
Vivo
[0384] This example demonstrates that the combination of a Notch
agonist and an aryl hydrocarbon antagonist, specifically the
combination of Delta1.sup.ext-IgG and SR1, enhances total human
engraftment, enhances engraftment of myeloid cells (CD45+CD33+)
that are capable of early short-term repopulation, and enhances
engraftment of immature progenitor cells (CD45+CD34+).
[0385] The in vivo repopulating cell potential of cells expanded
using optimized density of Delta1.sup.Ext-IgG and SR1 was
investigated. Because delayed neutrophil recovery remains a major
clinical challenge in cord blood transplantation resulting in
increased morbidity and mortality from infectious complications, it
was investigated whether Delta1.sup.Ext-IgG and SR1 in combination
improved early total human (human CD45.sup.+ murine CD45.1.sup.-),
myeloid (human CD45.sup.+CD33.sup.+ murine CD45.1.sup.-), and
progenitor cell (human CD45.sup.+CD34.sup.+ murine CD45.1.sup.-)
engraftment in immunodeficient NSG mice.
[0386] Transplants into NSG mice were performed as described above,
with the cultured progeny of 1.times.10.sup.4 CD34+ cells. Cells
were cultured in the presence of SR1, Delta1.sup.Ext-IgG 2.5
.mu.g/ml, or SR1 and Delta1.sup.Ext-IgG (2.5 or 5 .mu.g/ml) for 16
days. In four combined experiments, there was a trend towards
improved total human engraftment in the combination groups compared
to either control (FIG. 6A). This reached significance when
compared to Delta1.sup.Ext-IgG 2.5 .mu.g/ml (p-values 0.0056,
0.0026 for comparison with SR1 and Delta1.sup.Ext-IgG 2.5 and 5
.mu.g/ml respectively) and approached significance compared to SR1
alone (p-values 0.1624, 0.1699 for comparison with SR1 and
Delta1.sup.Ext-IgG 2.5 and 5 .mu.g/ml respectively).
[0387] Both combination groups (SR1 and Delta1.sup.Ext-IgG 2.5 and
5 .mu.g/ml) demonstrated significantly enhanced total myeloid
engraftment compared to SR1 and Delta1.sup.Ext-IgG 2.5 .mu.g/ml
controls, an important measure of the early repopulating potential
of these expanded cells (FIG. 6B). SR1 and Delta1.sup.Ext-IgG at
2.5 or 5 .mu.g/ml resulted in approximately 2-fold greater
generation of early myeloid repopulating cells as compared to
either alone (all comparison achieve significance, p-values listed
in FIG. 6B). Notably, the combination of SR1 and Delta1.sup.Ext-IgG
also resulted in significantly enhanced engraftment of immature
progenitor cells (FIG. 6C) suggesting enhanced generation of
short-term repopulating cells did not result in the exhaustion of
more immature HSPC. SR1 and Delta1.sup.Ext-IgG 2.5 and 5 .mu.g/ml
resulted in approximately 2.5-fold greater engraftment of CD34+
HSPC compared to SR1 and almost 4-fold greater engraftment than
Delta1.sup.Ext-IgG 2.5 alone (all p-values achieved significance,
listed in FIG. 6C).
Example 7: Delta1.sup.Ext-IgG and SR1 Combination Results in
Generation of Cells with Multi-Lineage Potential Capable of
Long-Term Repopulation
[0388] This example demonstrates that HSPC expanded in the presence
of both a Notch agonist (Delta1.sup.Ext-IgG) and an aryl
hydrocarbon antagonist (SR1) are capable of long-term in vivo
repopulation, long-term generation of cells of various lineage
(myeloid, lymphoid, progenitor), and maintenance of long-term
repopulating progenitor cells.
[0389] To determine how cells cultured in the presence of both SR1
and Delta1.sup.Ext-IgG contributed to engraftment over time, total
human (human CD45.sup.+ murine CD45.1.sup.-), progenitor cell
(human CD45.sup.+CD34.sup.+ murine CD45.1.sup.-), myeloid (human
CD45.sup.+CD33.sup.+ murine CD45.1.sup.-), and lymphoid (human
CD45.sup.+CD19.sup.+ murine CD45.1.sup.-), engraftment was assessed
at 2, 8, and 13 weeks post-transplant (FIGS. 7A-7D). These results
were compared to IgG, SR1, and Delta1.sup.Ext-IgG 2.5 .mu.g/ml
controls.
[0390] Cells were cultured and transplanted as described above with
the progeny of 1.times.10.sup.4 CD34+ cells transplanted per mouse.
Total human, progenitor cell, myeloid, and lymphoid assessments
were performed on bone marrow samples. Bone marrow aspirates were
performed in the mice at 2 and 8 weeks post-transplant, and mice
were sacrificed for bone marrow harvest at 13 weeks.
Delta1.sup.Ext-IgG and SR1 in combination maintained total human
engraftment long-term with almost no decrease in total human
engraftment in the Delta1.sup.Ext-IgG 5 .mu.g/ml and SR1 13 weeks
after transplant. This was in clear contrast to the control groups
(IgG, SR1, and Delta1.sup.Ext-IgG alone) and lowest combination
group where there was a clear reduction in overall long-term
repopulation (as evidenced by engraftment at 8 and 13 weeks).
Furthermore, engraftment was seen in the myeloid, lymphoid, and
progenitor cells compartments suggesting the presence of cells with
multi-lineage, long-term repopulating potential.
[0391] To determine whether generation of HSPC with long-term
repopulating potential was enhanced when cultured with
Delta1.sup.ext-IgG and SR-1 in combination, long-term engraftment
at 13 weeks post-transplant was compared with either condition
alone (FIGS. 8A, 8B). While not all comparisons were significant,
there was a trend towards enhanced total human and progenitor cell
engraftment in the combination groups, particularly with the
highest Delta1.sup.Ext-IgG density (5 .mu.g/ml) (p-values listed in
FIGS. 8A, 8B).
Example 8: Delta1.sup.ext-IgG and SR1 in Combination Expand Adult
HSPC Capable of Enhanced In Vivo Repopulation
[0392] This example shows that, unlike Delta1.sup.ext-IgG alone or
SR1 alone, the combination of a Notch agonist and an aryl
hydrocarbon antagonist is capable of successful expansion and
engraftment of adult HSPC derived from peripheral blood.
[0393] To test whether the combination of Delta1.sup.ext-IgG and
SR1 had similar effects on adult HSPC as cord blood-derived HSPC,
mobilized peripheral blood stem cells (mPBSC) were cultured in the
presence of Delta1.sup.ext-IgG, SR1 or the combination for 16 days
and transplanted into immunodeficient mice. Previous attempts to
expand mPBSC have met limited success including experiments using
Delta alone (unpublished). In vitro, expansion with SR1 alone or
lower dose Delta1.sup.ext-IgG (1.25 .mu.g/ml) resulted in greatest
TNC and CD34.sup.+ cell expansion; however, addition of
Delta1.sup.ext-IgG (1.25 or 5 .mu.g/ml) resulted in greater
maintenance of the immature progenitor CD34.sup.+CD90.sup.+ cell
population (at least 2-fold higher in the presence of
Delta1.sup.ext-IgG 5 .mu.g/ml and SR1 as compared to SR1 alone for
4 or 5 growth factor conditions). When the progeny of 20,000
CD34.sup.+ cells were transplanted into immunodeficient mice, the
combination groups (cultured with 4 or 5 growth factors) had
significantly greater total human and early myeloid engraftment at
3 weeks compared to SR1 alone or 20,000 non-manipulated mPBSC from
the same donor (FIGS. 9A, 9B). Engraftment was comparable to that
achieved with 250,000 starting cells.
Example 9: Enhanced Generation of Cord Blood HSPC by Expansion
Using a Combination of Delta1.sup.ext-IgG and SR1
[0394] Delayed myeloid engraftment is a known risk factor in
patients undergoing cord blood transplantation (CBT) as a
consequence of inadequate cell doses provided by the stem cell
graft. Furthermore, this delay in engraftment contributes to an
increased risk of early transplant related mortality, primarily
from infectious complications, as compared to conventional
allogeneic stem cell donor transplants.
[0395] Ex vivo culture of CB HSPC in the presence of StemRegenin1
(SR1), an aryl hydrocarbon receptor antagonist, and growth factors
(SCF, Flt3-ligand, IL3, and TPO) previously was shown to result in
significant expansion of CD34+ HSPC with enhanced in vivo
repopulating capability (Boitano et al., 2010, Science
329(5997):1345-1348). Ex vivo expansion and enhanced in vivo
repopulation with CD34.sup.+ CB HSPC cultured in the presence of
the immobilized Notch ligand Delta1.sup.Ext-IgG and growth factors
(SCF, Flt3-ligand, IL6, IL3, TPO) has also been previously
demonstrated (Delaney et al, 2010, Nat Med. 16(2):232-236; Delaney
et al., 2005, Blood 106(8):2693-2699; Ohishi et al., 2002, J Clin
Invest. 110(8):1165-1174). In a phase I trial, patients undergoing
cord blood transplantation with cells cultured in the presence of
the immobilized Notch ligand Delta1.sup.Ext-IgG experienced a
significant reduction (50%) in time to neutrophil engraftment as
compared to a concurrent cohort receiving a double cord blood
transplantation of non-manipulated units (Delaney et al., 2010).
However, there appears to be a dose dependent effect on reducing
time to neutrophil engraftment with greater numbers of CD34+ cells
infused, and thus, there is a need for methods that further enhance
expansion of HSPC capable of rapid marrow repopulation until
long-term engraftment occurs.
[0396] The data presented in this example demonstrate that whereas
both a Notch agonist (specifically, Delta1.sup.Ext-IgG) alone and
an aryl hydrocarbon receptor antagonist (specifically, SR1) alone,
enhance generation of CD34+ cells, including NOD/SCID repopulating
cells (SRC), the addition of a Notch agonist to an aryl hydrocarbon
receptor antagonist (specifically, the addition of
Delta1.sup.Ext-IgG to SR1) for use in expansion of cord blood HSPC
further enhances the generation of rapidly repopulating myeloid
cells, but not the generation of absolute numbers of CD34+ cells.
The analysis of developing cells within the cultures presented in
this example suggests that enhanced generation of rapidly
repopulating cells using the combination of a Notch agonist and an
aryl hydrocarbon receptor antagonist (specifically, the combination
of Delta1.sup.Ext-IgG and SR1) resulted from the delayed
differentiation of cells due to Delta1.sup.Ext-IgG.
[0397] Further, the data presented in this example demonstrate that
expanding HSPC using both a Notch agonist (specifically,
Delta1.sup.Ext-IgG) and an aryl hydrocarbon receptor antagonist
(specifically, SR1) enhances generation of early myeloid and
progenitor repopulating cells in vivo relative to HSPC expanded
using a Notch agonist alone and/or an aryl hydrocarbon receptor
antagonist alone (despite generation of fewer absolute total number
of CD34.sup.+ cells relative to HSPC expanded using an aryl
hydrocarbon receptor antagonist alone). Furthermore, the data
presented in this example demonstrate that expanding HSPC using
both a Notch agonist (specifically, Delta1.sup.Ext-IgG) and an aryl
hydrocarbon receptor antagonist (specifically, SR1) enhances
long-term multiple lineage engraftment and total human and
B-lymphoid engraftment in vivo relative to HSPC expanded using a
Notch agonist alone and/or an aryl hydrocarbon receptor antagonist
alone.
[0398] Materials and Methods
[0399] Human cord blood (CB) samples for research were obtained
from normal deliveries under Swedish Medical Center Institutional
Review Board (Seattle) approval and after consent was obtained. CB
samples were red blood cell depleted (Delaney et al., 2010, Nat Med
16(2):232-236), CD34 enriched using CD34+ immunomagnetic particles
(Miltenyi Biotec), purified with Automacs (Miltenyi Biotec), and
cryopreserved. Cultures were performed using thawed and pooled cord
blood units with Delta1.sup.Ext-IgG or IgG (2.5 .mu.g/ml) (Delaney
et al., 2010) in StemSpan serum-free expansion medium in the
presence of four growth factors that have been previously used for
SR1-induced HSPC expansion (IL6 (50 ng/ml), thrombopoietin (50
ng/ml), Flt-3 ligand (50 ng/ml), stem cell factor (50 ng/ml)) with
SR-1 (750 nM) added fresh to cells with all feedings. Cultures were
initiated with 7.times.10.sup.4 to 1.3.times.10.sup.5 CD34+ cells
for 25-cm.sup.2 flasks and with 3.times.10.sup.5 CD34+ cells for
75-cm.sup.2 flasks. Immunofluorescence analysis was performed as
described (Ohishi et al., 2002, J Clin Invest. 110(8):1165-1174)
with labeled antibodies: FITC (human CD14, CD33, and Lineage); PE
(human CD7, CD14, CD15, CD19, CD90, and CD123); PERCP (human CD34);
APC (human CD10, CD45, IL3Ra); PECy7 (mouse CD45.1, human CD34);
Alexa Fluor 700 (CD38); APC-eFluor 780 (CD45RA). Transplants were
performed in sublethally irradiated (275 rad) NOD-SCID
IL-2R.gamma.-null mice (NSG). In vivo engraftment was assessed by
immunofluorescence analysis of human cell subsets (human CD45.sup.+
murine CD45.1-). On average, the total number of cells infused was
4.25.times.10.sup.6/mouse for Delta1.sup.ext-IgG alone,
1.88.times.10.sup.7/mouse for SR1 alone, and
4.76.times.10.sup.6/mouse for the combination. Statistical analyses
were performed as indicated. For limiting dilution analysis, mice
were injected with the cultured progeny of the number of CD34.sup.+
cells indicated and bone marrow aspirates performed at 2 weeks. Two
engraftment cut-offs were chosen based on percent engraftment
observed in non-limiting transplantation studies. The frequency of
SRCs was determined by the method of maximum likelihood with L-CALC
software (StemCell Technologies) from the proportions of engrafted
recipients (FIGS. 9A, 9B).
RESULTS AND CONCLUSIONS
1. Immobilized Delta1.sup.Ext-IgG Delays Differentiation of CB HSPC
Cultured with SR1 and Delta1.sup.Ext-IgG
[0400] The data presented herein show that a Notch agonist
(specifically, immobilized Delta1.sup.ExtIgG) delays
differentiation of HSPC (specifically, cord blood HSPC) cultured
using the combination of a Notch agonist and an aryl receptor
antagonist (specifically, the combination of Delta1.sup.Ext-IgG and
SR1).
[0401] SR1 significantly enhanced the ex vivo generation of total
nucleated cells (TNC) as compared to immobilized Delta1.sup.Ext-IgG
(2.5 .mu.g/ml) or SR1 (750 nM) and Delta1.sup.Ext-IgG (5 .mu.g/ml)
combined (p<0.001, p=0.04, FIG. 10A). Dose titration of
Delta1.sup.Ext-IgG with standard dose SR1 trended towards enhanced
generation of the highly immature Lin-CD34+CD38-CD45RA-CD90+ subset
(Majeti et al., 2007, Cell Stem Cell 1(6): 635-645) with increasing
concentration of Delta1.sup.ext-IgG (p=0.07) while significantly
decreasing TNC and total CD34+ cell generation (p=0.02, p=0.04,
FIG. 10A). Similar numbers of common myeloid progenitors (CMP) were
generated between cultures containing SR1 or SR1 and
Delta1.sup.Ext-IgG combined; however, there was a trend towards
maintenance of greater percentages of these cells with increasing
Delta1.sup.Ext-IgG doses (p=0.18, FIG. 10B) (Manz et al., 2002,
Proc Natl Acad Sci USA. 99(18):11872-11877). Together, these data
suggest impeded differentiation of early myeloid precursors due to
Delta1.sup.Ext-IgG. Consistent with this notion were the
significantly fewer CD14/15.sup.+ mature myeloid cells generated
with increasing Delta1.sup.Ext-IgG dose (p=0.005), presumably due
to inhibition of precursor differentiation towards mature myeloid
progeny (FIG. 10C) (Manz et al., 2002). No differences were
observed for granulocyte-monocyte progenitors ("GMP," FIG. 10C) and
megakaryocyte-erythrocyte progenitors ("MEP," FIG. 10D); however,
there was a suggestion of decreased cell numbers generated with
increased Delta1.sup.Ext-IgG doses in these cell populations.
Megakaryocyte generation was similar across all conditions despite
decreased generation of MEP with increasing doses of
Delta1.sup.Ext-IgG. Delta1.sup.Ext-IgG containing cultures had a
greater percentage of CD41.sup.+CD14.sup.- cells consistent with
the role of Notch in megakaryocyte differentiation (FIG. 10D).
There was no difference in generation of erythroid precursors
(CD235a.sup.+CD71+, data not shown). Taken together, these data
suggest that decreased generation of more mature myeloid cell
populations together with greater maintenance of the immature
Lin-CD34+CD38-CD45RA-CD90+ and CMP cell populations in the presence
of Delta1.sup.Ext-IgG results from inhibition of differentiation
and potentially enhanced self-renewal of the least mature
precursors.
2. Expansion of HSPC in the Presence of the Combination of
Delta1.sup.Ext-IgG and SR1 Enhances Generation of Early Progenitor
and Myeloid Repopulating Cells In Vivo
[0402] The data presented herein show that expansion of HSPC
(specifically, cord blood HSPC) in the presence of the combination
of a Notch agonist and an aryl hydrocarbon receptor antagonist
(specifically, the Delta1.sup.Ext-IgG and SR1) enhances generation
of early progenitor and myeloid repopulating cells in vivo, and
enhances long-term total and B-lymphocyte engraftment.
[0403] In vivo repopulating capability of HSPC generated from
culture with Delta1.sup.Ext-IgG, SR1, or the combination of
Delta1.sup.Ext-IgG and SR1, was assessed. Using Delta1.sup.Ext-IgG
at 5 .mu.g/ml, concentration found to be optimal in preliminary
studies (data not shown), significantly enhanced rapid myeloid
reconstitution (percent CD45.sup.+CD33.sup.+) was observed using
cells cultured in the presence of the combination of
Delta1.sup.Ext-IgG and SR1 in comparison with cells cultured in the
presence of either factor alone (FIG. 11A). Moreover, a
significant, nearly 3-fold, increase in CD33.sup.+ NOD/SCID
repopulating cells (SRC) frequency was found in mice that received
cells cultured in the presence of the combination of
Delta1.sup.Ext-IgG and SR1 as compared to mice that received cells
cultured in the presence of either factor alone, based on robust,
greater than 5% marrow repopulation (FIG. 11A, and Table 3). At a
more limiting level of marrow repopulation (1%), a significant
3-fold difference was also found between cells cultured in the
presence of SR1 alone and the cells cultured in the presence of the
combination of Delta1.sup.Ext-IgG and SR1. However, there was a
non-significant 1%-fold difference between cells cultured in the
presence of Delta1.sup.Ext-IgG vs. the combination of
Delta1.sup.Ext-IgG and SR1. This suggests that the difference in
SRC frequency between cells cultured in the presence of SR1 and
Delta1.sup.Ext-IgG at a limiting level of marrow repopulation may
reflect generation of cells with more robust repopulating
capability when cultured in Delta1.sup.Ext-IgG, perhaps due to
retention of properties of less mature repopulating cells.
TABLE-US-00003 TABLE 3 CD33.sup.+ SRC frequency determined by
limiting dilution analysis Engraftment with .gtoreq.1% CD33.sup.+
cells Engraftment with .gtoreq.5% CD33.sup.+ cells
Delta1.sup.Ext-IgG Delta1.sup.Ext-IgG Delta1.sup.Ext-IgG
Delta1.sup.Ext-IgG No. CD34.sup.+ 2.5 .mu.g/ml IgG + SR1 5 .mu.g/ml
+ SR1 2.5 .mu.g/ml IgG + SR1 5 .mu.g/ml + SR1 cells/mouse No. mice
engrafted No. mice engrafted 1000 2/9 3/9 4/9 0/9 1/9 2/9 4000 9/9
5/9 9/9 3/9 1/9 6/9 20000 7/7 7/7 7/7 6/7 7/7 7/7 SRC frequency
1/1719* 1/3868** 1/1276 .sup. 1/11314.sup.# 1/10063.sup.## 1/3652
per starting cell *p = 0.47, **p = 0.02; .sup.#p = 0.02, .sup.##p =
0.04, reference group Delta1.sup.Ext-IgG 5 .mu.g/ml + SR1
[0404] Further, progenitor cell repopulation (percent
CD45.sup.+CD34.sup.+) was significantly enhanced with cell cultured
in the presence of the combination of Delta1.sup.Ext-IgG and SR1
(FIG. 11B). A 2- to 3-fold increase in CD34.sup.+ SRC frequency was
suggested by limiting dilution analysis at 5% marrow repopulation,
with lesser differences detected at 1% repopulation (FIG. 11B, and
Table 4).
TABLE-US-00004 TABLE 4 CD34.sup.+ SRC frequency determined by
limiting dilution analysis Engraftment with .gtoreq.0.5% CD34.sup.+
cells Engraftment with .gtoreq.1% CD34.sup.+ cells
Delta1.sup.Ext-IgG Delta1.sup.Ext-IgG Delta1.sup.Ext-IgG
Delta1.sup.Ext-IgG No. CD34.sup.+ 2.5 .mu.g/ml IgG + SR1 5 .mu.g/ml
+ SR1 2.5 .mu.g/ml IgG + SR1 5 .mu.g/ml + SR1 cells/mouse No. mice
engrafted No. mice engrafted 1000 0/9 1/9 1/9 0/9 1/9 0/9 4000 1/9
2/9 6/9 0/9 1/9 3/9 20000 7/7 7/7 7/7 6/7 6/7 7/7 SRC frequency
1/11739* 1/8495** 1/4200 .sup. 1/19121.sup.# 1/13514.sup.## 1/8273
per starting cell *p = 0.04, **p = 0.15; .sup.#p = 0.11, .sup.##p =
0.33, reference group Delta1.sup.Ext-IgG 5 .mu.g/ml + SR1
[0405] Analysis of progressively maturing precursor populations
contributing to engraftment revealed that enhanced myeloid and
progenitor repopulation was due to enhanced engraftment of the
CD45.sup.+CD34+33.sup.- and CD45.sup.+CD34.sup.+33.sup.+ cell
populations but not the most mature CD45.sup.+CD34.sup.-CD33 cell
subset, consistent with the above-presented in vitro data that
addition of Delta1.sup.Ext-IgG to SR1 resulted in generation of
more immature progenitor cells (FIG. 11C). Monocytic/granulocytic
cell engraftment (percent CD45.sup.+ that are CD14.sup.+ and/or
CD15.sup.+) was also enhanced at this early time point by cells
cultured in the presence of the combination of SR1 and
Delta1.sup.Ext-IgG compared to either agent alone (FIG. 11D). In
contrast, rapid repopulation by B-lymphoid cells (percent
CD45.sup.+CD33.sup.-CD19.sup.+) was significantly enhanced by cells
cultured in the presence of SR1 alone in comparison with cells
cultured in the presence of Delta1.sup.Ext-IgG alone or cultured in
the presence of the combination of Delta1.sup.Ext-IgG and SR1 (FIG.
11E). This may be a result of Notch1-mediated inhibition of B-cell
differentiation combined with greater generation of immature
B-cells in the presence of aryl hydrocarbon receptor (AhR)
inhibition as previously demonstrated in AhR receptor-null mice
(Lehar et al., 2005, Blood 105(4):1440-1447; Jaleco et al., 2001, J
Exp Med. 194(7):991-1002; Singh et al., 2011, Stem Cells Dev.
20(5):769-784). Minimal megakaryocyte (CD14.sup.-CD41.sup.+),
erythroid (Glycophorin A.sup.+), and T-cell (CD3.sup.+) engraftment
was observed at this early time point preventing in vivo comparison
of these cell populations.
[0406] Cells cultured in the presence of the combination of SR1 and
Delta1.sup.Ext-IgG were found to be capable of longer-term,
multi-lineage engraftment (FIG. 11F). Evaluation at 12-14 weeks
post-infusion revealed overall enhanced human engraftment in the
combination group (FIG. 11F); however this was primarily due to
enhanced B-lymphoid engraftment (FIG. 11F) as total marrow myeloid
(CD33+ and CD14/15+), progenitor (CD34+), T-lymphoid (CD3+), and
erythroid (Glycophorin A+) cell engraftment was similar between the
groups (not shown). It is not clear whether these lymphoid cells
were newly derived or represented longer lived cells developed
during earlier time points. Low-level human engraftment was
observed in all groups upon secondary transplantation demonstrating
stable, long-term engraftment of the expanded cells (average
CD45+0.22% in Delta1.sup.Ext-IgG, 0.11% in SR1, and 0.18% in the
combination); however, these differences were not statistically
significant suggesting these cells have similar long-term
engraftment potential.
3. Conclusions
[0407] The data presented herein show that culturing of cord blood
hematopoietic stem/progenitor cells in the presence of both a Notch
agonist and an aryl hydrocarbon receptor antagonist, specifically
the combination of Delta1.sup.Ext-IgG and SR1, enhances in vitro
generation of a highly immature progenitor cell population
(Lin-CD34+CD38-CD45RA-CD90+) and decreases generation of mature
myeloid populations (CD14.sup.+ and CD15.sup.+ cells). Importantly,
greater expansion of clinically relevant, rapidly repopulating
cells occurred in the presence of the combination of a Notch
agonist and an aryl hydrocarbon receptor antagonist in comparison
with either agent alone. In particular, the data presented herein
show that combining Delta1.sup.Ext-IgG and SR1 achieved enhanced
generation of cells capable of rapidly repopulating bone marrow
with early myeloid and progenitor cells. Unexpectedly, this
occurred despite decreased generation of CD34.sup.+ cells in
culture when Delta1.sup.Ext-IgG was added to SR1, indicating that
generation of particular subsets of CD34.sup.+ cells, rather than
the absolute number of CD34.sup.+ cells, may be more predictive of
in vivo reconstitution. The data presented herein also suggest that
a Notch agonist (specifically, Delta1.sup.ext-IgG) enhances an aryl
hydrocarbon receptor antagonist (specifically, SR1) induced
generation of less mature precursors by delaying their
differentiation, thereby enabling generation of greater numbers of
clinically relevant repopulating cells.
[0408] The ability to significantly enhance early myeloid
reconstitution has clear clinical relevance for reducing duration
of neutropenia and early infectious complications in recipients of
stem cell transplants or other intensive chemotherapy. Therefore,
the methods presented in this example can be used for generation of
increased numbers of HSPC that may be capable of achieving
clinically significant reduction in time to myeloid engraftment in
neutropenic patients. In particular, the inventors expect that use
of the combination of a Notch agonist (e.g., Delta1.sup.ext-IgG)
and an aryl hydrocarbon receptor antagonist (e.g., SR1), such as
described herein, can be used to produce an economically feasible,
non-HLA matched, expanded cell product where greater numbers of
repopulating cells may be required to overcome greater HLA
disparity.
REFERENCES
[0409] 1. Delaney C, Heimfeld S, Brashem-Stein C, Voorhies H,
Manger R L, Bernstein I D. Notch-mediated expansion of human cord
blood progenitor cells capable of rapid myeloid reconstitution. Nat
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et al. Aryl hydrocarbon receptor antagonists promote the expansion
of human hematopoietic stem cells. Science. 2010;
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al. Pre-clinical development of cord blood-derived progenitor cell
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chelator tetraethylenepentamine. Cytotherapy. 2004; 6(4):344-355.
[0412] 4. de Lima M, McMannis J, Gee A, et al. Transplantation of
ex vivo expanded cord blood cells using the copper chelator
tetraethylenepentamine: a phase I/II clinical trial. Bone marrow
transplant. 2008; 41(9):771-778. [0413] 5. Peled T, Shoham H,
Aschengrau D, et al. Nicotinamide, a SIRT1 inhibitor, inhibits
differentiation and facilitates expansion of hematopoietic
progenitor cells with enhanced bone marrow homing and engraftment.
Exp Hematol. 2012; 40(4):342-355 e341. [0414] 6. McNiece I,
Harrington J, Turney J, Kellner J, Shpall E J. Ex vivo expansion of
cord blood mononuclear cells on mesenchymal stem cells.
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J, et al. Mesenchymal stem cell based cord blood expansion leads to
rapid engraftment of platelets and neutrophils [abstract]. Blood.
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I D. Ex vivo expansion of human hematopoietic stem and progenitor
cells. Blood. 2011; 117(23):6083-6090. [0417] 9. Delaney C,
Varnum-Finney B, Aoyama K, Brashem-Stein C, Bernstein I D.
Dose-dependent effects of the Notch ligand Delta1 on ex vivo
differentiation and in vivo marrow repopulating ability of cord
blood cells. Blood. 2005; 106(8):2693-2699. [0418] 10. Ohishi K,
Varnum-Finney B, Bernstein I D. Delta-1 enhances marrow and thymus
repopulating ability of human CD34(+)CD38(-) cord blood cells. J
Clin Invest. 2002; 110(8):1165-1174. [0419] 11. Majeti R, Park C Y,
Weissman I L. Identification of a hierarchy of multipotent
progenitors in human cord blood. Cell Stem Cell. 2007; 1(6):
635-645. [0420] 12. Manz M G, Miyamoto T, Akashi K, Weissman I L.
Prospective isolation of human clonogenic common myeloid
progenitors. Proc Natl Acad Sci USA. 2002; 99(18):11872-11877.
[0421] 13. Lehar S M, Dooley J, Farr A G, Bevan M J. Notch ligands
Delta 1 and Jagged1 transmit distinct signals to T-cell precursors.
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Hooijberg E, et al. Differential effects of Notch ligands Delta-1
and Jagged-1 in human lymphoid differentiation. J Exp Med. 2001;
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[0424] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and accompanying figures. Such modifications
are intended to fall within the scope of the appended claims.
[0425] Various publications, including patents, patent application
publications, and scientific literature, are cited herein, the
disclosures of which are incorporated by reference in their
entireties for all purposes.
Sequence CWU 1
1
4161DNAArtificial SequencesiRNA molecule capable of down-regulating
the expression of aryl hydrocarbon receptor 1gcggcataga gaccgactta
atttcaagag aattaagtcg gtctctatgc cgcttttttg 60g 61265DNAArtificial
SequencesiRNA molecule capable of down-regulating the expression of
aryl hydrocarbon receptor 2cgcgccaaaa aagcggcata gagaccgact
taattctctt gaaattaagt cggtctctat 60gccgc 65361DNAArtificial
SequencesiRNA molecule capable of down-regulating the expression of
aryl hydrocarbon receptor 3ggcttctttg atgttgcatt aattcaagag
attaatgcaa catcaaagaa gccttttttg 60g 61465DNAArtificial
SequencesiRNA molecule capable of down-regulating the expression of
aryl hydrocarbon receptor 4cgcgccaaaa aaggcttctt tgatgttgca
ttaatctctt gaattaatgc aacatcaaag 60aagcc 65
* * * * *