Note: Descriptions are shown in the official language in which they were submitted.
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CXCR4 AGONIST TREATMENT OF HEMATOPOIETIC CELLS
FIELD OF THE INVENTION
In one aspect, the invention relates to therapeutic uses of chemokine receptor
agonists, including peptide agonists of CXC chemokine receptor 4 (CXCR4) for
use
in the treatment of hematopoietic cells in vitro and in vivo.
BACKGROUND OF THE INVENTION
Hematopoiesis consists of developmental cascades in which the
hematopoietic stem cells generate lineage-committed cells and repeat the
process of
self-renewal. Hematopoietic stem cells are typically cells that have dual
capacity for
self-renewal and multilineage differentiation.
Cytokines are soluble proteins secreted by a variety of cells including
monocytes or lymphocytes that regulate immune responses. Chemokines are a
superfamily of chemoattractant proteins that may be classified into four
groups,
characterized by the nature of cysteine residues that are involved in
disulfide bond
formation. Chemokines regulate a variety of biological responses and they
promote
the recruitment of multiple lineages of leukocytes and lymphocytes to a body
organ
tissue. Chemokines may be classified into two families according to the
relative
position of the first two cysteine residues in the protein. In CC chemokines,
which
include beta chemokine the first two cysteines are adjacent to each other. In
CXC
chemokines, which include alpha chemokine, the first two cysteines are
separated
by one amino acid residue. Two minor subgroups contain only one of the two
cysteines (C) or have three amino acids between the cysteines (CX3C). In
humans,
the genes of the CXC chemokines are clustered on chromosome 4 (with the
exception of SDF-1 gene, which has been localized to chromosome 10) and those
of
the CC chemokines on chromosome 17.
The molecular targets for chemokines are cell surface receptors. One such
receptor is CXC chemokine receptor 4 (CXCR4), which is a G-protein coupled
seven
transmembrane protein, and was previously called LESTR (Loetscher, M., Geiser,
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T., O'Reilly, T., Zwahlen, R., Baggionlini, M., and Moser, B., (1994) J. Biol.
Chem,
269, 232-237), HUMSTR (Federsppiel, B., Duncan, A.M.V., Delaney, A.,
Schappert,
K., Clark-Lewis, I., and Jirik, F.R. (1993) Genomics 16, 707-712) and Fusin
(Feng,
Y., Broeder, C.C., Kennedy, P.E., and Berger, E.A. (1996) Science 272, 872-
877).
CXCR4 is widely expressed on cells of hemopoietic origin, and is a major co-
receptor with CD4+ for human immunodeficiency virus 1 (HIV-1 ) ( Feng, Y.,
Broeder,
C.C., Kennedy, P.E., and Berger, E.A. (1996) HIV-1 entry cofactor: Functional
cDNA
cloning of a seven-transmembrane G-protein-coupled receptor, Science 272, 872-
877).
Chemokines are thought to mediate their effect by binding to seven-
transmembrane G protein-coupled receptors, and to attract leukocyte subsets to
sites of inflammation (Baglionini et al. (1998) Nature 392: 565-568). Many of
the
chemokines have been shown to be constitutively expressed in lymphoid tissues,
indicating that they may have a homeostatic function in regulating lymphocyte
trafficking between and within lymphoid organs (Kim and Broxmeyer (1999) J.
Leuk.
Biol. 56: 6-15).
Stromal cell derived factor one (SDF-1 ) is a member of the CXC family of
chemokines that has been found to be constitutively secreted from the bone
marrow
stroma (Tashiro, (1993) Science 261, 600-602). The human and mouse SDF-1
predicted protein sequences are approximately 92% identical. Stromal cell
derived
factor-1a (SDF-1a) and stromal cell derived factor-1~3 (SDF-1(3) are closely
related
(together referred to herein as SDF-1 ). The native amino acid sequences of
SDF-1 a
and SDF-1 (3 are known, as are the genomic sequences encoding these proteins
(see
U.S. Patent No. 5,563,048 issued 8 October 1996, and U.S. Patent No. 5,756,084
issued 26 May 1998). Identification of genomic clones has shown that the alpha
and
beta isoforms are a consequence of alternative splicing of a single gene. The
alpha
form is derived from exons 1-3 while the beta form contains an additional
sequence
from exon 4. The entire human gene is approximately 10 Kb. SDF-1 was initially
characterized as a pre-B cell-stimulating factor and as a highly efficient
chemotactic
factor for T cells and monocytes (Bieul et al. (1996) J. Exp. Med. 184:1101-
1110).
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Biological effects of SDF-1 may be mediated by the chemokine receptor
CXCR4 (also known as fusin or LESTR), which is expressed on mononuclear
leukocytes including hematopoietic stem cells. SDF-1 is thought to be the
natural
ligand for CXCR4, and CXCR4 is thought to be the natural receptor for SDF-1
(Nagasawza et al. (1997) Proc. Natl. Acad. Sci. USA 93:726-732). Genetic
elimination of SDF-1 is associated with parinatal lethality, including
abnormalities in
cardiac development, B-cell lymphopoiesis, and bone marrow myelopoiesis
(Nagasawa et al. (1996) Nature 382:635-637).
SDF-1 is functionally distinct from other chemokines in that it is reported to
have a fundamental role in the trafficking, export and homing of bone marrow
progenitor cells (Aiuti, A., Webb, I.J., Bleul, C., Springer, T., and Guierrez-
Ramos,
J.C., (1996) J. Exp. Med. 185, 111-120 and Nagasawa, T., Hirota, S.,
Tachibana, K.,
Takakura N., Nishikawa, S.-I., Kitamura, Y., Yoshida, N., Kikutani, H., and
Kishimoto,
T., (1996) Nature 382, 635-638). SDF-1 is also structurally distinct in that
it has only
about 22% amino acid sequence identity with other CXC chemokines (Bleul, C.C.,
Fuhlbrigge, R.C., Casasnovas, J.M., Aiuti, A., and Springer, T.A., (1996) J.
Exp.
Med. 184, 1101-1109). SDF-1 appears to be produced constitutively by several
cell
types, and particularly high levels are found in bone-marrow stromal cells
(Shirozu,
M., Nakano, T., Inazawa, J., Tashiro, K., Tada, H. Shinohara, T., and Honjo,
T.,
(1995) Genomics, 28, 495-500 and Bleul, C.C., Fuhlbrigge, R.C., Casasnovas,
J.M.,
Aiuti, A., and Springer, T.A., (1996) J. Exp. Med. 184, 1101-1109). A basic
physiological role for SDF-1 is implied by the high level of conservation of
the SDF-1
sequence between species. In vitro, SDF-1 stimulates chemotaxis of a wide
range
of cells including monocytes and bone marrow derived progenitor cells (Aiuti,
A.,
Webb, I.J., Bleul, C., Springer, T., and Guierrez-Ramos, J.C., (1996) J. Exp.
Med.
185, 111-120 and Bleul, C.C., Fuhlbrigge, R.C., Casasnovas, J.M., Aiuti, A.,
and
Springer, T.A., (1996) J. Exp. Med. 184, 1101-1109). SDF-1 also stimulates a
high
percentage of resting and activated T-lymphocytes (Bleul, C.C., Fuhlbrigge,
R.C.,
Casasnovas, J.M., Aiuti, A., and Springer, T.A., (1996) J. Exp. Med. 184, 1101-
1109
and Campbell, J.J., Hendrick, J., Zlotnik, A., Siani, M.A., Thompson, D.A.,
and
Butcher, E.C., (1998) Science, 279 381-383).
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A variety of diseases require treatment with agents which are preferentially
cytotoxic to dividing cells. Cancer cells, for example, may be targeted with
cytotoxic
doses of radiation or chemotherapeutic agents. A significant side-effect of
this
approach to cancer therapy is the pathological impact of such treatments on
rapidly
dividing normal cells. These normal cells may for example include hair
follicles,
mucosal cells and the hematopoietic cells, such as primitive bone marrow
progenitor
cells and stem cells. The indiscriminate destruction of hematopoietic stem,
progenitor or precursor cells can lead to a reduction in normal mature blood
cell
counts, such as leukocytes, lymphocytes and red blood cells. A major impact on
mature cell numbers may be seen particularly with neutrophils (neutropaenia)
and
platelets (thrombocytopenia), cells which naturally have relatively short half-
lives. A
decrease in leukocyte count, with concomitant loss of immune system function,
may
increase a patient's risk of opportunistic infection. Neutropaenia resulting
from
chemotherapy may for example occur within two or three days of cytotoxic
treatments, and may leave the patient vulnerable to infection for up to 2
weeks until
the hematopoietic system has recovered sufficiently to regenerate neutrophil
counts.
A reduced leukocyte count (leukopenia) as a result of cancer therapy may
become
sufficiently serious that therapy must be interrupted to allow the white blood
cell
count to rebuild. Interruption of cancer therapy can in turn lead to survival
of cancer
cells, an increase in the incidence of drug resistance in cancer cells, and
ultimately in
cancer relapse. There is accordingly a need for therapeutic agents and
treatments
which facilitate the preservation of hematopoietic progenitor or stem cells in
patients
subject to treatment with cytotoxic agents.
Bone marrow transplantation has been used in the treatment of a variety of
hematological, autoimmune and malignant diseases. In conjunction with bone
marrow transplantation, ex vivo hematopoietic (bone marrow) cell culture may
be
used to expand the population of hematopoietic progenitor or stem cells. It
may be
desirable to purge an ex vivo hematopoietic cell culture of cancer cells with
cytotoxic
treatments, while preserving the viability of the hematopoietic progenitor or
stem
cells. There is accordingly a need for agents and methods, which facilitate
the
preservation of hematopoietic progenitor or stem cells in ex vivo cell
cultures
exposed to cytotoxic agents.
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A number of proteins have been identified as inhibitors of hematopoietic
progenitor cell development, with potential therapeutic usefulness as
inhibitors of
hematopoeitic cell multiplication: macrophage inflammatory protein 1-alpha
(MIP-1-
alpha) and LD78 (see U.S. Patent No. 5,856,301 ); the alpha globin chain of
hemoglobin and beta globin chain of hemoglobin (see U.S. Patent No.
6,022,848);
and, interferon gamma (see U.S. Patent No. 5,807,744).
Permanent marrow recovery after cytotoxic drug and radiation therapy
depends on the survival of hematopoietic stem cells having long term
reconstituting
(LTR) potential. The major dose limiting sequelae consequent to chemotherapy
and/or radiation therapy are neutropenia and thrombocytopenia. Protocols
involving
dose intensification (i.e., to increase the log-kill of the respective tumour
therapy) or
schedule compression will exacerbate the degree and duration of
myelosuppression
associated with the standard chemotherapy and/or radiation therapy. For
instance,
in the adjuvant setting, repeated cycles of doxorubicin-based treatment have
been
shown to produce cumulative and long-lasting damage in the bone marrow
progenitor cell populations (Lorhrman et al. (1978) Br. J. Haematol. 40:369).
The
effects of short-term hematopoietic cell damage resulting from chemotherapy
has
been overcome to some extent by the concurrent use of G-CSF (Neupogen~), used
to accelerate the regeneration of neutrophils (Le Chevalier (1994) Eur. J.
Cancer
30A:410). This approach has been met with limitations also, as it is
accompanied by
progressive thrombocytopenia and cumulative bone marrow damage as reflected by
a reduction in the quality of mobilized progenitor cells over successive
cycles of
treatment. Because of the current interest in chemotherapy dose
intensification as a
means of improving tumour response rates and perhaps patient survival, the
necessity for alternative therapies to either improve or replace current
treatments to
rescue the myeloablative effects of chemotherapy and/or radiation therapy has
escalated, and is currently one of the major rate limiting factors for tumour
therapy
dose escalations.
Transplanted peripheral blood stem cells (PBSC, or autologous PBSC) may
provide a rapid and sustained hematopoietic recovery after the administration
of
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high-dose chemotherapy or radiation therapy in patients with hematological
malignancies and solid tumours. PBSC transplantation has become the preferred
source of stem cells for autologous transplantation because of the shorter
time to
engraftment and the lack of a need for surgical procedure necessary for bone
marrow harvesting (Demirer et al. (1996) Stem Cells 14:106-116; Pettengel et
al.
(1992) Blood 82:2239-2248). Although the mechanism of stem cell release into
the
peripheral blood from the bone marrow is not well understood, agents that
augment
the mobilization of CD34+ cells may prove to be effective in enhancing
autologous
PBSC transplantation. G-CSF and GM-CSF are currently the most commonly used
hematopoietic growth factors for PBSC mobilization, although the mobilized
cellular
profiles can differ significantly from patient to patient. Therefore, other
agents are
required for this clinical application.
It is generally accepted that stem cell transplants for autoimmune disease
should be initiated using autologous or allogenic grafts, where the former
would be
preferable since they may bear less risk of complication (Burt and Taylor
(1999)
Stem Cells 17:366-372). Lymphocyte depletion has also been recommended, where
lymphocyte depletion is a form of purging autoreactive cells from the graft.
In
practice, aggressive lymphocyte depletion of an allograft can prevent
alloreactivity
(i.e., graft-versus-host disease (GVHD)) even without immunosuppressive
prophylaxis. Therefore, a lymphocyte-depleted autograft may prevent recurrence
of
autoreactivity. As a consequence, any concurrent therapy that may enhance the
survival of the CFU-GEMM myeloid stem cells, or BFU-E and CFU-GM
myelomonocytic stem cells may be beneficial in therapies for autoimmune
diseases
where hematopoietic stem cells could be compromised.
Retrovirus-mediated gene transfer into murine hematopoietic stem cells and
reconstitution of syngeneic mice have demonstrated persistence and functioning
of
the transgenes over extended period of time (Kume et al. (1999) 69:227-233).
Terminally differentiated cells are relatively short-lived, except for memory
B and T
lymphocytes, and a large number of blood cells are replaced daily. Therefore,
when
long-term functional correction of blood cells by gene transfer is required,
the target
cells may be hematopoietic stem cells (Kume et al. (1999) 69:227-233).
Compounds
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that can maintain the survival of the progenitor stem cells may therefore
increase the
efficiency of the gene transfer in that a greater population of hematopoietic
stems
cells are available.
A number of proteins have been identified and are currently being utilized
clinically as inhibitors of hematopoietic progenitor cell development and
hematopoietic cell proliferation (multiplication). These include recombinant-
methionyl human G-CSF (Neupogen°, Filgastim; Amgen), GM-CSF
(Leukine°,
Sargramostim; Immunex), erythropoietin (rhEPO, Epogen°; Amgen),
thrombopoietin
(rhTPO; Genentech), interleukin-11 (rhlL-11, Neumega°; American Home
Products),
FIt3 ligand (Mobista; Immunex), multilineage hematopoietic factor (MARstemTM;
Maret Pharm.), myelopoietin (Leridistem; Searle), IL-3, myeloid progenitor
inhibitory
factor-1 (Mirostipen; Human Genome Sciences), stem cell factor (rhSCF,
Stemgen~;
Amgen).
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the effect of Ara-C (350 mg/kg) on White Blood Cell Count
(WBC) in mice in the presence (triangular data points, solid line, designated
Ara-C +
CTCE0021 in the legend) and absence (circular data points, dashed line,
designated
Ara-C in the legend) of a peptide of the invention (designated CTCE0021 and
described in Examples 1 and 3).
Figure 2A shows a concentration-dependant inhibition of '251-SDF-1 binding to
CXCR4 by SDF-1, obtained as described for the data shown in Figure 2A,
indicating
the affinity of SDF-1 for the CXCR4 receptor.
Figure 2B shows the CXCR4 receptor binding of SDF-1 and the SDF-1
peptide agonist analogs. SDF-1 and the indicated analogs (competing ligands,
described in Examples) were added at the concentrations illustrated in the
presence
of 4nM '251-SDF-1. CEM cells were assessed for '251-SDF-1 binding following 2
hr of
incubation. The results are expressed as percentages of the maximal specific
binding that was determined without competing ligand, and are the mean of
three
independent experiments.
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Figure 3 shows the induction of [Ca2+]; mobilization by SDF-1 and SDF-1
receptor analogs (described in Examples). Fura-2,AM loaded THP-1 cells
(1x106/ml)
were stimulated with SDF-1, CTCE0021 or CTCE0022 at the concentrations
indicated. The values represent the mean +/- one S.D. of n=3 experiments.
Figure 4 shows the induction of [Ca2+]; mobilization by SDF-1 and SDF-1
analogs. Fura-2,AM loaded THP-1 cells (1x106/ml) were stimulated with native
SDF-
1 and the SDF-1 peptide analogs at the concentration of native SDF-1
concentration
that gave the maximum [Ca2+]; stimulation (1 uM). The values represent the
mean
+/- one S.D. of n=3 experiments. The designated compounds are as follows: SDF-
1,
SDF-1 (1-14)-(G)4-SDF-1 (55-67)-K20/E24-cyclic amide (CTCE0021 ), SDF-1 (1-14)-
(G)4-SDF-1(55-67)-E24/K28-cyclic amide (CTCE0022), SDF-1 (1-9)2-C9/C9-cysteine
dimer (CTCE9901), SDF-1(1-17) (CTCE9902), SDF-1 (1-8)2-lysine bridge dimer
(CTCE9904) and SDF-1 (1-14)-(G)4-SDF-1 (55-67) amide (CTCE0017).
Figure 5 shows cyclic proliferative activity of primitive normal colony
forming
cells (CFC) in the adherent layer of a standard long term culture (LTC), in
which
circles represent BFU-E cells (burst forming unit-erythroid precursors), and
squares
represent CFU-GM cells (colony forming unit - granulocyte-monocyte common
precursor), illustrating the inhibitory effect of SDF-1 on cellular
proliferation as
measured by the susceptibility of the cells to an agent preferentially
cytotoxic to
proliferating cells.
Figure 6 shows cyclic proliferative activity of primitive normal CFC in the
adherent layer of standard LTC, when treated with SDF-1, SDF-1 (1-14)-(G)4-SDF-
1 (55-67)- K20/D24-cyclic amide (Compound #1 ), SDF-1 (1-9)2 (Compound #3), as
measured by the susceptibility of the cells to an agent preferentially
cytotoxic to
dividing cells.
Figure 7 shows the effect of SDF-1 and SDF-1 analogs (defined in Examples)
on the cycling of human progenitors from fetal liver transplanted NOD/SCID
mice.
The cycling status of mature and primitive colony forming cells (CFU-GM;
colony
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forming unit-granulocyte-monocyte precursor, BFU-E; burst forming unit-
erythroid
precursor) in the suspension of CD34+ cells isolated from the marrow of
transplanted
NOD/SCID mice was determined by assessing the proportion of these progenitors
that were inactivated (killed) by short term (20 min) or overnight (LTC-
IC;long-term
culture initiating cell) exposure of the cells to 20pg/ml of high specific
activity 3H-
thymidine. Values represent the mean +/- the S.D. of data from up to four
experiments with up to four mice per point in each.
Figure 8 shows data indicating that SDF-1 enhances the detectability of CRU
(colony regenerating units) regenerated in NOD/SCID Mice transplanted with
human
fetal liver.
Figure 9 shows the effect of SDF-1 and SDF-1 Agonists (defined in
Examples) on the engraftment of human cells in human fetal liver transplanted
NOD/SCID mice. A comparison of the number of phenotypically defined
hematopoietic cells detected in the long bones (tibias and femurs) of mice
four
weeks after being transplanted with 10' light-density human fetal liver blood
cells and
then administered SDF-1, CTCE0021 or CTCE 0013 (0.5 mg/kg) three times per
week for two weeks before sacrifice. Values represent the mean +/- one S.D. of
results obtained from three to seven individual mice in three experiments.
Figure 10 shows the effect of CTCE0021 (1 mg/kg, defined in the Examples)
on the recovery of leukocytes following myeloablative chemotherapy with Ara-C
(300mg/kg). Mice were treated with Ara-C alone (Ara-C) or in combination with
CTCE0021. The results represent the mean +/- one S.D. of 6 animals/group.
Figure 11 shows the effect of CTCE0021 (defined in Examples) and
Neupogen~ (G-CSF) on the growth of white blood cells in Ara-C treated mice.
C3Hhen mice (female) were treated with 500mg/kg Ara-C for two cycles - on days
0
and 10. During the second cycle of Ara-C dosing, Ara-C treated mice were
injected
with 1 Omg/kg CTCE0021, 1 Omg/kg Neupogen°, alone or together (on days -
1, 0,
and 1 to 3). Control represents animals treated with Ara-C alone. Blood was
collected from the tail vein into heparin-containing tubes at the onset of the
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experiment, and one day before and 1, 7 and 12 days following the second Ara-C
dose. A total white blood cell count was obtained. The results represent the
mean
+/- one S.D. of 6 animals/group.
Figure 12 shows the effect of CTCE0021 and Neupogen~ on the relative
growth of white blood cells in Ara-C treated mice. C3Hhen mice (female) were
treated with 500mg/kg Ara-C for two cycles - on days 0 and 10. During the
second
cycle of Ara-C dosing, Ara-C treated mice were injected with 10mg/kg CTCE0021
(defined in Examples), 10mg/kg Neupogen°, alone or together (on days -
1, 0, and 1
to 3). Control represents animals treated with Ara-C alone. Blood was
collected
from the tail vein into heparin-containing tubes at the onset of the
experiment, and
one day before 7 and 12 days following the second Ara-C dose. A total white
blood
cell count was obtained. The increase in leukocytes (white blood cells) was
determined relative to the number of cells counted the day before the second
cycle
Ara-C dose was administered. The results represent the mean +/- one S.D. of 6
animals/group.
Figure 13 shows the sequences of human SDF-1 alpha, SDF-1 Precursor
(PBSF) and SDF-1 beta.
SUMMARY OF THE INVENTION
In accordance with various aspects of the invention, CXCR4 agonists may be
used to treat bone marrow progenitor or stem cells to reduce the
susceptibility of the
cells to cytotoxic agents. CXCR4 agonists may be used to treat bone marrow
progenitor cells or stem cells to reduce the rate of cellular multiplication.
CXCR4
agonists may for example be used in vivo or ex vivo in bone marrow or
peripheral
blood stem cell transplantation procedures to treat bone marrow progenitor or
stem
cells. CXCR4 agonists may be used to treat cancer in a mammal in conjunction
with
one or more cytotoxic agents. Cytotoxic agents may for example include
chemotheraputic agents or radiation. CXCR4 agonists may be used
therapeutically
to regulate bone marrow progenitor or stem cell growth in vivo, ex vivo and in
human
diseases, such as cancer.
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DETAILED DESCRIPTION OF THE INVENTION
In accordance with some aspects of the invention, hematopoietic stem cells
may be affected by CXCR4 agonists via a mechanism of cell growth repression.
Since cytotoxic therapies utilized to kill proliferating cancerous cells, such
as
chemotherapeutic and/or radiation therapy, target proliferating cells, the
CXCR4
agonists in accordance with various aspects of this invention may be utilized
to
reduce cytotoxin mediated destruction of hematopoietic cells, such as
primitive bone
marrow and peripheral blood progenitor and stem cells, and thereby to enhance
recovery of mature blood cell counts, such as leukocytes, lymphocytes and red
blood
cells, following cytotoxin treatments. In various aspects of the invention,
CXCR4
agonists may be given to the patient prior to, during or both prior to and
during
cytotoxin treatments, such as myeloablative chemotherapy and/or radiation
therapy,
in order to suppress the growth of the progenitor cells, and thus rescue them
from
destruction by the therapeutic regimen that the patient is being treated with,
for
example to treat a cancer. Therefore, cancers susceptible to treatment with
CXCR4
agonists in accordance with various aspects of the invention may include both
primary and metastatic tumors, and solid tumors, including carcinomas of
breast,
colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas,
liver,
gallbladder and bile ducts, small intestine, urinary tract (including kidney,
bladder
and urothelium), female genital tract, (including cervix, uterus, and ovaries
as well as
choriocarcinoma and gestational trophoblastic disease), male genital tract
(including
prostate, seminal vesicles, testes and germ cell tumors), endocrine glands
(including
the thyroid, adrenal, and pituitary glands), and skin, as well as hemangiomas,
melanomas, sarcomas (including those arising from bone and soft tissues as
well as
Kaposi's sarcoma) and tumors of the brain, nerves, eyes, and meninges
(including
astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas,
neuroblastomas,
Schwannomas, and meningiomas). In some aspects of the invention, CXCR4
agonists may also be useful in treating tumors, such as solid tumors, arising
from
hematopoietic malignancies such as leukemias (i.e. chloromas, plasmacytomas
and
the plaques and tumors of mycosis fungoides and cutaneous T-cell
lymphoma/leukemia) as well as in the treatment of lymphomas (both Hodgkin's
and
non-Hodgkin's lymphomas). In addition, CXCR4 agonists may be useful in the
prevention of metastases from the tumors described above either when used
alone
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or in combination with cytotoxic agents such as radiotherapy or
chemotherapeutic
agents.
In alternative aspects of the invention, CXCR4 agonists may be used to enrich
populations of CD34+ progenitor cells. Such cells may for example be enriched
by
CXCR4 agonists in bone marrow (BM) and peripheral blood (PB) stem cell
transplantation procedures. Such procedures may be used to treat a variety of
diseases (see for example Ball, E.D., Lister, J., and Law, P. Hematopoietic
Stem Cell
Therapy, Chruchill Livingston (of Harcourt Inc.), New York (2000)). CXCR4
agonists
may accordingly be used in such hematopoietic stem cell transplantation (HSCT)
protocols for the purposed of treating diseases, such as the following
diseases that
may be treated with CXCR4 agonists:
Aplastic Anemia;
Acute Lymphoblastic Anemia.;
Acute Myelogenous Leukemia;
Myelodysplasia;
Multiple Myeloma;
Chronic Lymphocytic Leukemia;
Congenital Immunodeficiencies (such as Autoimmune Lymphoproliferative disease,
Wiscott-Aldrich Syndrome, X-linked Lymphoproliferative disease, Chronic
Granulamatous disease, Kostmann Neutropenia, Leukocyte Adhesion
Deficiency);
Metabolic Diseases (for instance those which have been HSCT indicated such as
Hurler Syndrome (MPS I/II), Sly Syndrome (MPS VII), Chilhood onset cerebral
X-adrenoleukodystrophy, Globard cell Leukodystrophy),
In alternative embodiments, CXCR4 agonists may be used to treat a variety of
hematopoietic cells, and such cells may be isolated or may form only part of a
treated cell population in vivo or in vitro. Cells amenable to treatment with
CXCR4
agonists may for example include cells in the hematopoietic lineage, beginning
with
pluripotent stem cells, such as bone marrow stem or progenitor cells, lymphoid
stem
or progenitor cells, myeloid stem cells, CFU-GEMM cells (colony-forming-unit
granulocyte, erythroid, macrophage, megakaryocye), B stem cells, T stem cells,
DC
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stem cells, pre-B cells, prothymocytes, BFU-E cells (burst-forming unit -
erythroid),
BFU-MK cells (burst-forming unit - megakaryocytes), CFU-GM cells (colony-
formng
unit - granulocyte-macrophage), CFU-bas cells (colony-forming unit -
basophil),
CFU-Mast cells (colony forming unit - mast cell), CFU-G cells (colony forming
unit
granulocyte), CFU-M/DC cells (colony forming unit monocyte/dendritic cell),
CFU-Eo
cells (colony forming unit eosinophil), CFU-E cells (colony forming unit
erythroid),
CFU-MK cells (colony forming unit megakaryocyte), myeloblasts, monoblasts, B-
lymphoblasts, T-lymphoblasts, proerythroblasts, neutrophillic myelocytes,
promonocytes, or other hematopoietic cells that differentiate to give rise to
mature
cells such as macrophages, myeloid related dendritic cells, mast cells, plasma
cells,
erythrocytes, platelets, neutrophils, monocytes, eosinophils, basophils, B-
cells, T-
cells or lymphoid related dendritic cells.
In some embodiments, the present invention is concerned with polypeptides
having the amino acid sequences shown in SEQ ID. NO.'s 1, 2 or 3 (Figure 13).
In
some embodiments, a pegylation moiety may be provided at any position on the
sequence. The polypeptides of the present invention may include polypeptides
in
which part of the amino acid sequence is omitted, or polypeptides that consist
of
sequences containing additional or replaced amino acids, or spliced forms of
the
sequences, yet induce activation of the CXCR4. In some embodiments,
polypeptides of the invention may be at least 70%, 80% 90% or 95% identical to
the
polypeptides of Seq. ID. No.'s 1, 2 or 3, when optimally aligned, over a
region of at
least 10, 15, 20, 30, 40, 50, 60 or 80 or more, contiguous amino acids. In
alternative
embodiments, SDF-1 polypeptides of the invention may consist of amino acid
sequences that are less than 70% identical to portions of SEQ ID No.'s 1, 2or
3,
where a polypeptide demonstrates CXCR4 agonist activity, such as activity that
is
comparable (such as within 0.01-, 0.1-, 1.0-, 10-, or 100-fold) to that
obtained with
the SDF-1 polypeptides of Seq. ID. No.'s 1, 2 or 3.
In one aspect of the invention, a putative SDF-1 polypeptide having some
similarity to SEQ ID No.'s 1, 2 or 3 may be assessed for CXCR4 agonist
activity.
Putative SDF-1 polypeptides of the invention may for example be assayed for
CXCR4 receptor binding as determined by receptor binding assays, such as
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radiolabeled ligand receptor competition assays. Activation of CXCR4 by an SDF-
1
polypeptide may be determined through assaying activation of the receptor
through
standard biochemical techniques including intracellular calcium mobilization,
cellular
chemotaxis, chemiluminescence, degranulation assays, measurement of NADPH-
dependent formation of reactive oxygen species, activation of secondary
messenger
enzymes such as G proteins, phospholipase C (PLC), protein kinase C (PKC), or
of
Src and Src family kinases (i.e., Fyn). In some embodiments, CXCR4 agonist
activity, CXCR4 receptor binding or CXCR4 receptor activation of a putative
CXCR4
agonist of the invention may be at least 0.01-, 0.1-, 1.0-, 10-, or 100-fold
of the
corresponding parameter of a polypeptides of Seq. ID. No.'s 1, 2 or 3.
In alternative embodiments, a variety of small SDF-1 peptide analogues may
be used as CXCR4 agonists. One such peptide is a dimer of amino acids 1-9, in
which the amino acid chains are joined by a disulphide bond between each of
the
cysteines at position 9 in each sequence (designated SDF-1 (1-9)2 or KPVSLSYRC-
CRYSLSVPK). An alternative peptide is a dimer of amino acids 1-8, KPVSLSYR-X-
RYSLSVPK, in which the amino acid chains are joined by a linking moiety X
between
each of the arginines at position 8 in each sequence (designated SDF-1(1-8)2).
CXCR4 agonist peptides may for example be selected from the group consisting
of
peptides having the following sequences:
KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKW
IQEYLEKALN; KPVSLSYRCPCRFFESH; KPVSLSYRC; KPVSLSYRC-
CRYSLSVPK; KPVSLSYRC-X-CRYSLSVPK; and, KPVSLSYR-X-RYSLSVPK. In
the foregoing peptides X may be lysine with both the a (alpha) and ~ (epsilon)
amino
groups of the lysine being associated with covalent (amide) bond formation and
the
lysyl carboxyl group being protected. The last two compounds in the forgoing
list
may, for example, be represented as follows, showing the peptide sequences in
the
standard amino-to-carboxyl orientation:
KPVSLSYR KPVSLSYRC
X X
KPVSLSYR KPVSLSYRC
3J
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In various alternative embodiments, such SDF-1 peptide analogs, along with SDF-
1
polypeptides, are included amongst CXCR4 agonists of the invention.
In some embodiments, the CXCR4 agonists for use in the invention may be
substantially purified peptide fragments, modified peptide fragments,
analogues or
pharmacologically acceptable salts of either SDF-1a or SDF-1(3. SDF-1 derived
peptide agonists of CXCR4 may be identified by known biological assays and a
variety of techniques such as the aforementioned or as discussed in Crump et
al.,
1997, The EMBO Journal 16(23) 6996-7007; and Heveker et al., 1998, Current
Biology 8(7): 369-376; each of which are incorporated herein by reference.
Such
SDF-1 derived peptides may include homologs of native SDF-1, such as naturally
occurring isoforms or genetic variants, or polypeptides having substantial
sequence
similarity to SDF-1, such as 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%
sequence identity to at least a portion of the native SDF-1 sequence, the
portion of
native SDF-1 being any contiguous sequence of 10, 15, 20, 30, 40, 50 or more
amino acids, provided the peptides have CXCR4 agonist activity. In some
embodiments, chemically similar amino acids may be substituted for amino acids
in
the native SDF-1 sequence (to provide conservative amino acid substitutions).
In
some embodiments, peptides having an N-terminal LSY sequence motif within 10,
or
7, amino acids of the N-terminus, and/or an N-terminal RFFESH (SEQ ID N0:5)
sequence motif within 20 amino acids of the N-terminus may be used provided
they
have CXCR4 agonistic activity. One family of such peptide agonist candidates
has
an LSY motif at amino acids 5-7. Alternative peptides further include the
RFFESH
(SEQ ID NO: 5) motif at amino acids 12-17. In alternative embodiments, the LSY
motif is located at positions 3-5 of a peptide. The invention also provides
peptide
dimers having two amino acid sequences, which may each have the foregoing
sequence elements, attached by a disulfide bridge within 20, or preferably
within 10,
amino acids of the N terminus, linking cysteine residues or a-aminobutric acid
residues.
The invention further provides pharmaceutical compositions containing
CXCR4 agonists. In one embodiment, such compositions include a CXCR4 agonist
compound in a therapeutically or prophylactically effective amount sufficient
to alter
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bone marrow and/or peripheral progenitor or stem cell growth, maturation
and/or
mobilization, and a pharmaceutically acceptable carrier. In another
embodiment, the
composition includes a CXCR4 agonist compound in a therapeutically or
prophylactically effective amount sufficient to inhibit a cytotoxic effect of
a cytotoxic
agent, such as cytotoxic agents used in chemotherapy or radiation treatment of
cancer, and a pharmaceutically acceptable carrier.
An "effective amount" of a compound of the invention includes a
therapeutically effective amount or a prophylatically effective amount. A
"therapeutically effective amount" refers to an amount effective, at dosages
and for
periods of time necessary, to achieve the desired therapeutic result, such as
reduction of bone marrow progenitor or stem cell multiplication, or reduction
or
inhibition of a cytotoxic effect of a cytotoxic agent. A therapeutically
effective amount
of CXCR4 agonist may vary according to factors such as the disease state, age,
sex,
and weight of the individual, and the ability of the CXCR4 agonist to elicit a
desired
response in the individual. Dosage regimens may be adjusted to provide the
optimum therapeutic response. A therapeutically effective amount is also one
in
which any toxic or detrimental effects of the CXCR4 agonist are outweighed by
the
therapeutically beneficial effects.
A "prophylactically effective amount" refers to an amount effective, at
dosages
and for periods of time necessary, to achieve the desired prophylactic result,
such as
preventing or inhibiting a cytotoxic effect of a cytotoxic agent. Typically, a
prophylactic dose is used in subjects prior to or at an earlier stage of
disease, so that
a prophylactically effective amount may be less than a therapeutically
effective
amount.
In particular embodiments, a preferred range for therapeutically or
prophylactically effective amounts of CXCR4 agonists may be 0.1 nM-0.1 M, 0.1
nM-
0.05M, 0.05 nM-15pM or 0.01 nM-10 M. It is to be noted that dosage values may
vary with the severity of the condition to be alleviated. For any particular
subject,
specific dosage regimens may be adjusted over time according to the individual
need and the professional judgement of the person administering or supervising
the
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administration of the compositions. Dosage ranges set forth herein are
exemplary
only and do not limit the dosage ranges that may be selected by medical
practitioners.
The amount of active compound in the composition may vary according to
factors such as the disease state, age, sex, and weight of the individual.
Dosage
regimens may be adjusted to provide the optimum therapeutic response. For
example, a single bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or increased
as
indicated by the exigencies of the therapeutic situation. It may be
advantageous to
formulate parenteral compositions in dosage unit form for ease of
administration and
uniformity of dosage. "Dosage unit form" as used herein refers to physically
discrete
units suited as unitary dosages for subjects to be treated; each unit
containing a
predetermined quantity of active compound calculated to produce the desired
therapeutic effect in association with the required pharmaceutical carrier.
The
specification for the dosage unit forms of the invention are dictated by and
directly
dependent on (a) the unique characteristics of the active compound and the
particular therapeutic effect to be achieved, and (b) the limitations inherent
in the art
of compounding such an active compound for the treatment of sensitivity in
individuals.
As used herein "pharmaceutically acceptable carrier'° or "exipient"
includes
any and all solvents, dispersion media, coatings, antibacterial and antifungal
agents,
isotonic and absorption delaying agents, and the like that are physiologically
compatible. In one embodiment, the carrier is suitable for parenteral
administration.
Alternatively, the carrier can be suitable for intravenous, intraperitoneal,
intramuscular, sublingual or oral administration. Pharmaceutically acceptable
carriers
include sterile aqueous solutions or dispersions and sterile powders for the
extemporaneous preparation of sterile injectable solutions or dispersion. The
use of
such media and agents for pharmaceutically active substances is well known in
the
art. Except insofar as any conventional media or agent is incompatible with
the
active compound, use thereof in the pharmaceutical compositions of the
invention is
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contemplated. Supplementary active compounds can also be incorporated into the
compositions.
In some embodiments, CXCR4 agonists may be formulated in pharmaceutical
compositions with additional active ingredients, or administered in methods of
treatment in conjunction with treatment with one or more additional
medications,
such as a medicament selected from the following: recombinant-methionyl human
G-
CSF (Neupogen~, Filgastim; Amgen), GM-CSF (Leukine", Sargramostim; Immunex),
erythropoietin (rhEPO, Epogen°; Amgen), thrombopoietin (rhTPO;
Genentech),
interleukin-11 (rhlL-11, Neumega~; American Home Products), FIt3 ligand
(Mobista;
Immunex), multilineage hematopoietic factor (MARstemTM; Maret Pharm.),
myelopoietin (Leridistem; Searle), IL-3, myeloid progenitor inhibitory factor-
1
(Mirostipen; Human Genome Sciences), and stem cell factor (rhSCF,
Stemgen°;
Amgen).
Therapeutic compositions typically must be sterile and stable under the
conditions of manufacture and storage. The composition can be formulated as a
solution, microemulsion, liposome, or other ordered structure suitable to high
drug
concentration. The carrier can be a solvent or dispersion medium containing,
for
example, water, ethanol, polyol (for example, glycerol, propylene glycol, and
liquid
polyethylene glycol, and the like), and suitable mixtures thereof. The proper
fluidity
can be maintained, for example, by the use of a coating such as lecithin, by
the
maintenance of the required particle size in the case of dispersion and by the
use of
surfactants. In many cases, it will be preferable to include isotonic agents,
for
example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride
in the
composition. Prolonged absorption of the injectable compositions can be
brought
about by including in the composition an agent which delays absorption, for
example,
monostearate salts and gelatin. Moreover, the CXCR4 agonists may be
administered in a time release formulation, for example in a composition which
includes a slow release polymer. The active compounds can be prepared with
carriers that will protect the compound against rapid release, such as a
controlled
release formulation, including implants and microencapsulated delivery
systems.
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl
acetate,
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polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid
and
polylactic, polyglycolic copolymers (PLG). Many methods for the preparation of
such
formulations are patented or generally known to those skilled in the art.
Sterile injectable solutions can be prepared by incorporating the active
compound in the required amount in an appropriate solvent with one or a
combination of ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle that contains a basic dispersion medium and
the
required other ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the preferred
methods of
preparation are vacuum drying and freeze-drying which yields a powder of the
active
ingredient plus any additional desired ingredient from a previously sterile-
filtered
solution thereof. In accordance with an alternative aspect of the invention, a
CXCR4
agonist may be formulated with one or more additional compounds that enhance
the
solubility of the CXCR4 agonist.
CXCR4 antagonist compounds of the invention may include SDF-1
derivatives, such as C-terminal hydroxymethyl derivatives, O-modified
derivatives
(e.g., C-terminal hydroxymethyl benzyl ether), N-terminally modified
derivatives
including substituted amides such as alkylamides and hydrazides and compounds
in
which a C-terminal phenylalanine residue is replaced with a phenethylamide
analogue (e.g., Ser-Ile-phenethylamide as an analogue of the tripeptide Ser-
Ile-Phe).
Within a CXCR4 agonist of the invention, a peptidic structure (such as an
SDF-1 derived peptide) maybe coupled directly or indirectly to at least one
modifying
group. The term "modifying group" is intended to include structures that are
directly
attached to the peptidic structure (e.g., by covalent bonding or covalent
coupling), as
well as those that are indirectly attached to the peptidic structure (e.g., by
a stable
non-covalent bond association or by covalent coupling to additional amino acid
residues, or mimetics, analogues or derivatives thereof, which may flank the
SDF-1
core peptidic structure). For example, the modifying group can be coupled to
the
amino-terminus or carboxy-terminus of an SDF-1 peptidic structure, or to a
peptidic
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or peptidomimetic region flanking the core domain. Alternatively, the
modifying group
can be coupled to a side chain of at least one amino acid residue of a SDF-1
peptidic
structure, or to a peptidic or peptido-mimetic region flanking the core domain
(e.g.,
through the epsilon amino group of a lysyl residue(s), through the carboxyl
group of
an aspartic acid residues) or a glutamic acid residue(s), through a hydroxy
group of
a tyrosyl residue(s), a serine residues) or a threonine residues) or other
suitable
reactive group on an amino acid side chain). Modifying groups covalently
coupled to
the peptidic structure can be attached by means and using methods well known
in
the art for linking chemical structures, including, for example, amide,
alkylamino,
sulphide, carbamate or urea bonds.
In some embodiments, the modifying group may comprise a cyclic,
heterocyclic or polycyclic group. The term "cyclic group", as used herein,
includes
cyclic saturated or unsaturated (i.e., aromatic) group having from 3 to 10, 4
to 8, or 5
to 7 carbon atoms. Exemplary cyclic groups include cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, and cyclooctyl. Cyclic groups may be unsubstituted or
substituted at one or more ring positions. A cyclic group may for example be
substituted with halogens, alkyls, cycloalkyls, alkenyls, alkynyls, aryls,
heterocycles,
hydroxyls, aminos, nitros, thiols amines, imines, amides, phosphonates,
phosphines,
carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls, sulfonates,
selenoethers,
ketones, aldehydes, esters, -CF3, -CN.
The term "heterocyclic group" includes cyclic saturated, unsaturated and
aromatic groups having from 3 to 10, 4 to 8, or 5 to 7 carbon atoms, wherein
the ring
structure includes about one or more heteroatoms. Heterocyclic groups include
pyrrolidine, oxolane, thiolane, imidazole, oxazole, piperidine, piperazine,
morpholine.
The heterocyclic ring may be substituted at one or more positions with such
substituents as, for example, halogens, alkyls, cycloalkyls, alkenyls,
alkynyls, aryls,
other heterocycles, hydroxyl, amino, nitro, thiol, amines, imines, amides,
phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers, thioethers,
sulfonyls,
selenoethers, ketones, aldehydes, esters, -CF3, -CN. Heterocycles may also be
bridged or fused to other cyclic groups as described below.
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The term "polycyclic group" as used herein is intended to refer to two or more
saturated, unsaturated or aromatic cyclic rings in which two or more carbons
are
common to two adjoining rings, so that the rings are "fused rings". Rings that
are
joined through non-adjacent atoms are termed "bridged" rings. Each of the
rings of
the polycyclic group may be substituted with such substituents as described
above,
as for example, halogens, alkyls, cycloalkyls, alkenyls, alkynyls, hydroxyl,
amino,
nitro, thiol, amines, imines, amides, phosphonates, phosphines, carbonyls,
carboxyls, silyls, ethers, thioethers, sulfonyls, selenoethers, ketones,
aldehydes,
esters, -CF3, or -CN.
The term "alkyl" refers to the radical of saturated aliphatic groups,
including
straight chain alkyl groups, branched-chain alkyl groups, cycloalkyl
(alicyclic) groups,
alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.
In some
embodiments, a straight chain or branched chain alkyl has 20 or fewer carbon
atoms
in its backbone (C1-C2o for straight chain, C3-C2o for branched chain), or 10
or fewer
carbon atoms. In some embodiments, cycloalkyls may have from 4-10 carbon atoms
in their ring structure, such as 5, 6 or 7 carbon rings. Unless the number of
carbons
is otherwise specified, "lower alkyl" as used herein means an alkyl group, as
defined
above, having from one to ten carbon atoms in its backbone structure.
Likewise,
"lower alkenyl" and "lower alkynyl" have chain lengths of ten or less carbons.
The term "alkyl" (or "lower alkyl") as used throughout the specification and
claims is intended to include both "unsubstituted alkyls" and "substituted
alkyls", the
latter of which refers to alkyl moieties having substituents replacing a
hydrogen on
one or more carbons of the hydrocarbon backbone. Such substituents can
include,
for example, halogen, hydroxyl, carbonyl (such as carboxyl, ketones (including
alkylcarbonyl and arylcarbonyl groups), and esters (including alkyloxycarbonyl
and
aryloxycarbonyl groups), thiocarbonyl, acyloxy, alkoxyl, phosphoryl,
phosphonate,
phosphinate, amino, acylamino, amido, amidine, imino, cyano, nitro, azido,
sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido,
heterocyclyl, aralkyl,
or an aromatic or heteroaromatic moiety. The moieties substituted on the
hydrocarbon chain can themselves be substituted, if appropriate. For instance,
the
substituents of a substituted alkyl may include substituted and unsubstituted
forms of
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aminos, azidos, iminos, amidos, phosphoryls (including phosphonates and
phosphinates), sulfonyls (including sulfates, sulfonamidos, sulfamoyls and
sulfonates), and silyl groups, as well as ethers, alkylthios, carbonyls
(including
ketones, aldehydes, carboxylates, and esters), -CF3, -CN and the like.
Exemplary
substituted alkyls are described below. Cycloalkyls can be further substituted
with
alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted
alkyls, -CF3, -
CN, and the like.
The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups
analogous in length and possible substitution to the alkyls described above,
but that
contain at least one double or triple bond respectively.
The term "aralkyl", as used herein, refers to an alkyl or alkylenyl group
substituted with at least one aryl group. Exemplary aralkyls include benzyl
(i.e.,
phenylmethyl), 2-naphthylethyl, 2-(2-pyridyl)propyl, 5-dibenzosuberyl, and the
like.
The term "alkylcarbonyl", as used herein, refers to -C(O)-alkyl. Similarly,
the
term "arylcarbonyl" refers to -C(O)-aryl. The term "alkyloxycarbonyl", as used
herein,
refers to the group -C(O)-O-alkyl, and the term "aryloxycarbonyl" refers to -
C(O)-O-
aryl. The term "acyloxy" refers to -O-C(O)-R~, in which R~ is alkyl, alkenyl,
alkynyl,
aryl, aralkyl or heterocyclyl.
The term "amino", as used herein, refers to -N(Ra)(Ra), in which Ra and Rp are
each independently hydrogen, alkyl, alkyenyl, alkynyl, aralkyl, aryl, or in
which Ra
and Ra together with the nitrogen atom to which they are attached form a ring
having
4-8 atoms. Thus, the term "amino", as used herein, includes unsubstituted,
monosubstituted (e.g., monoalkylamino or monoarylamino), and disubstituted
(e.g.,
dialkylamino or alkylarylamino) amino groups. The term "amido" refers to -C(O)
N(R8)(R9), in which R$ and R9 are as defined above. The term "acylamino"
refers to
N(R'$)C(O)-R~, in which R~ is as defined above and R'$ is alkyl.
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As used herein, the term "nitro" means -N02; the term "halogen" designates -
F, -CI, -Br or -I; the term "sulfhydryl" means -SH; and the term "hydroxyl"
means -
OH.
The term "aryl" as used herein includes 5-. 6- and 7-membered aromatic
groups that may include from zero to four heteroatoms in the ring, for
example,
phenyl, pyrrolyl, furyl, thiophenyl, imidazolyl, oxazole, thiazolyl,
triazolyl, pyrazolyl,
pyridyl, pyrazinyl, pyridazinyl and pyrimidinyl, and the like. Those aryl
groups having
heteroatoms in the ring structure may also be referred to as "aryl
heterocycles" or
"heteroaromatics". The aromatic ring can be substituted at one or more ring
positions
with such substituents as described above, as for example, halogen, azide,
alkyl,
aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl,
imino, amido,
phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,
sulfonyl,
sulfonamido, ketone, aldehyde, ester, a heterocyclyl, an aromatic or
heteroaromatic
moiety, -CF3, -CN, or the like. Aryl groups can also be part of a polycyclic
group. For
example, aryl groups include fused aromatic moieties such as naphthyl,
anthracenyl,
quinolyl, indolyl, and the like.
Modifying groups may include groups comprising biotinyl structures,
fluorescein-containing groups, a diethylene-triaminepentaacetyl group, a (O)-
menthoxyacetyl group, a N-acetylneuraminyl group, a cholyl structure or an
iminiobiotinyl group. A CXCR4 agonist compound may be modified at its carboxy
terminus with a cholyl group according to methods known in the art (see e.g.,
Wess,
G. et al. (1993) Tetrahedron Letters, 34:817-822; Wess, G. et al. (1992)
Tetrahedron
Letters 33:195-198; and Kramer, W. et al. (1992) J. Biol. Chem. 267:18598-
18604).
Cholyl derivatives and analogues may also be used as modifying groups. For
example, a preferred cholyl derivative is Aic (3-(O-aminoethyl-iso)-cholyl),
which has
a free amino group that can be used to further modify the CXCR4 agonist
compound. A modifying group may be a "biotinyl structure", which includes
biotinyl
groups and analogues and derivatives thereof (such as a 2-iminobiotinyl
group). In
another embodiment, the modifying group may comprise a "fluorescein-containing
group", such as a group derived from reacting an SDF-1 derived peptidic
structure
with 5-(and 6-)-carboxyfluorescein, succinimidyl ester or fluorescein
isothiocyanate.
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In various other embodiments, the modifying groups) may comprise an N-
acetylneuraminyl group, a trans-4-cotininecarboxyl group, a 2-imino-1-
imidazolidineacetyl group, an (S)-(-)-indoline-2-carboxyl group, a (-)-
menthoxyacetyl
group, a 2-norbornaneacetyl group, a y-oxo-5-acenaphthenebutyryl, a (-)-2-oxo-
4-
thiazolidinecarboxyl group, a tetrahydro-3-furoyl group, a 2-iminobiotinyl
group, a
diethylenetriaminepentaacetyl group, a 4-morpholinecarbonyl group, a 2-
thiopheneacetyl group or a 2-thiophenesulfonyl group.
A CXCR4 agonist compound of the invention may be further modified to alter
the specific properties of the compound while retaining the desired
functionality of
the compound. For example, in one embodiment, the compound may be modified to
alter a pharmacokinetic property of the compound, such as in vivo stability,
bioavailability or half-life. The compound may be modified to label the
compound
with a detectable substance. The compound may be modified to couple the
compound to an additional therapeutic moiety. To further chemically modify the
compound, such as to alter its pharmacokinetic properties, reactive groups can
be
derivatized. For example, when the modifying group is attached to the amino-
terminal end of the SDF-1 core domain, the carboxy-terminal end of the
compound
may be further modified. Potential C-terminal modifications include those that
reduce
the ability of the compound to act as a substrate for carboxypeptidases.
Examples of
C-terminal modifiers include an amide group, an ethylamide group and various
non-
natural amino acids, such as D-amino acids and (3-alanine. Alternatively, when
the
modifying group is attached to the carboxy-terminal end of the aggregation
core
domain, the amino-terminal end of the compound may be further modified, for
example, to reduce the ability of the compound to act as a substrate for
aminopeptidases.
A CXCR4 agonist compound can be further modified to label the compound
by reacting the compound with a detectable substance. Suitable detectable
substances include various enzymes, prosthetic groups, fluorescent materials,
luminescent materials and radioactive materials. Examples of suitable enzymes
include horseradish peroxidase, alkaline phosphatase, -galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group complexes include
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streptavidin/biotin and avidin/biotin; examples of suitable fluorescent
materials
include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an
example of a
luminescent material includes luminol; and examples of suitable radioactive
material
include'4C, '231, '241, 1251 1311, 99m-~-c, 35S or 3H. A CXCR4 agonist
compound may be
radioactively labeled with '4C, either by incorporation of'4C into the
modifying group
or one or more amino acid structures in the CXCR4 agonist compound. Labeled
CXCR4 agonist compounds may be used to assess the in vivo pharmacokinetics of
the compounds, as well as to detect disease progression or propensity of a
subject
to develop a disease, for example for diagnostic purposes. Tissue distribution
CXCR4 receptors can be detected using a labeled CXCR4 agonist compound either
in vivo or in an in vitro sample derived from a subject. For use as an in vivo
diagnostic agent, a CXCR4 antagonist compound of the invention may be labeled
with radioactive technetium or iodine. A modifying group can be chosen that
provides a site at which a chelation group for the label can be introduced,
such as
the Aic derivative of cholic acid, which has a free amino group. For example,
a
phenylalanine residue within the SDF-1 sequence (such as aminoacid residue 13)
may be substituted with radioactive iodotyrosyl. Any of the various isotopes
of
radioactive iodine may be incorporated to create a diagnostic agent. '231
(half-
life=13.2 hours) may be used for whole body scintigraphy, '241 (half life=4
days) may
be used for positron emission tomography (PET), '251 (half life=60 days) may
be
used for metabolic turnover studies and'3'I (half life=8 days) may be used for
whole
body counting and delayed low resolution imaging studies.
In an alternative chemical modification, a CXCR4 agonist compound of the
invention may be prepared in a "prodrug" form, wherein the compound itself
does not
act as a CXCR4 agonist, but rather is capable of being transformed, upon
metabolism in vivo, into a CXCR4 agonist compound as defined herein. For
example, in this type of compound, the modifying group can be present in a
prodrug
form that is capable of being converted upon metabolism into the form of an
active
CXCR4 agonist. Such a prodrug form of a modifying group is referred to herein
as a
"secondary modifying group." A variety of strategies are known in the art for
preparing peptide prodrugs that limit metabolism in order to optimise delivery
of the
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active form of the peptide-based drug (see e.g., Moss, J. (1995) in Peptide-
Based
Drug Design: Controlling Transport and Metabolism, Taylor, M. D. and Amidon,
G. L.
(eds), Chapter 18.
CXCR4 agonist compounds of the invention may be prepared by standard
techniques known in the art. A peptide or polypeptide component of a CXCR4
agonist may be composed, at least in part, of a peptide synthesized using
standard
techniques (such as those described in Bodansky, M. Principles of Peptide
Synthesis, Springer Verlag, Berlin (1993); Grant, G. A. (ed.). Synthetic
Peptides: A
User's Guide, W. H. Freeman and Company, New York (1992); or Clark-Lewis, I.,
Dewald, B., Loetscher, M., Moser, B., and Baggiolini, M., (1994) J. Biol.
Chem., 269,
16075-16081). Automated peptide synthesizers are commercially available (e.g.,
Advanced ChemTech Model 396; Milligen/Biosearch 9600). Peptides and
polypeptides may be assayed for CXCR4 agonist activity in accordance with
standard methods. Peptides and polypeptides may be purified by HPLC and
analyzed by mass spectrometry. Peptides and polypeptides may be dimerized via
a
disulfide bridge formed by gentle oxidation of the cysteines using 10% DMSO in
water. Following HPLC purification dimer formation may be verified, by mass
spectrometry. One or more modifying groups may be attached to a SDF-1 derived
peptidic component by standard methods, for example using methods for reaction
through an amino group (e.g., the alpha-amino group at the amino-terminus of a
peptide), a carboxyl group (e.g., at the carboxy terminus of a peptide), a
hydroxyl
group (e.g., on a tyrosine, serine or threonine residue) or other suitable
reactive
group on an amino acid side chain (see e.g., Greene, T. W. and Wuts, P. G. M.
Protective Groups in Organic Synthesis, John Wiley and Sons, Inc., New York
(1991)).
In another aspect of the invention, CXCR4 agonist peptides may be prepared
according to standard recombinant DNA techniques using a nucleic acid molecule
encoding the peptide. A nucleotide sequence encoding the peptide or
polypeptide
may be determined using the genetic code and an oligonucleotide molecule
having
this nucleotide sequence may be synthesized by standard DNA synthesis methods
(e.g., using an automated DNA synthesizer). Alternatively, a DNA molecule
encoding
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a peptide compound may be derived from the natural precursor protein gene or
cDNA (e.g., using the polymerise chain reaction (PCR) and/or restriction
enzyme
digestion) according to standard molecular biology techniques.
The invention also provides an isolated nucleic acid molecule comprising a
nucleotide sequence encoding a peptide of the invention. In some embodiments,
the
peptide may comprise an amino acid sequence having at least one amino acid
deletion compared to native SDF-1. The term "nucleic acid molecule" is
intended to
include DNA molecules and RNA molecules and may be single-stranded or double-
stranded. In alternative embodiments, the isolated nucleic acid encodes a
peptide
wherein one or more amino acids are deleted from the N-terminus, C-terminus
and/or an internal site of SDF-1.
To facilitate expression of a peptide compound in a host cell by standard
recombinant DNA techniques, the isolated nucleic acid encoding the peptide may
be
incorporated into a recombinant expression vector. Accordingly, the invention
also
provides recombinant expression vectors comprising the nucleic acid molecules
of
the invention. As used herein, the term "vector" refers to a nucleic acid
molecule
capable of transporting another nucleic acid to which it has been operatively
linked.
Vectors may include circular double stranded DNA plasmids and/or viral
vectors.
Certain vectors are capable of autologous replication in a host cell into
which they
are introduced (such as bacterial vectors having a bacterial origin of
replication and
episomal mammalian vectors). Other vectors (such as non-episomal mammalian
vectors) may be integrated into the genome of a host cell upon introduction
into the
host cell, and thereby may be replicated along with the host genome. Certain
vectors
may be capable of directing the expression of genes to which they are
operatively
linked. Such vectors are referred to herein as "recombinant expression
vectors" or
"expression vectors".
In recombinant expression vectors of the invention, the nucleotide sequence
encoding a peptide may be operatively linked to one or more regulatory
sequences,
selected on the basis of the host cells to be used for expression. The terms
"operatively linked" or "operably" linked mean that the sequences encoding the
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peptide are linked to the regulatory sequences) in a manner that allows for
expression of the peptide compound. The term "regulatory sequence" includes
promoters, enhancers, polyadenylation signals and other expression control
elements. Such regulatory sequences are described, for example, in Goeddel;
Gene
Expression Technology: Methods in Enzymology 185, Academic Press, San Diego,
Calif. (1990) (incorporated herein be reference). Regulatory sequences include
those
that direct constitutive expression of a nucleotide sequence in many types of
host
cell, those that direct expression of the nucleotide sequence only in certain
host cells
(such as tissue-specific regulatory sequences) and those that direct
expression in a
regulatable manner (such as only in the presence of an inducing agent). The
design
of the expression vector may depend on such factors as the choice of the host
cell to
be transformed and the level of expression of peptide compound desired.
The recombinant expression vectors of the invention may be designed for
expression of peptide compounds in prokaryotic or eukaryotic cells. For
example,
peptide compounds may be expressed in bacterial cells such as E. coli, insect
cells
(using baculovirus expression vectors) yeast cells or mammalian cells.
Suitable host
cells are discussed further in Goeddel, Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the
recombinant expression vector may be transcribed and translated in vitro, for
example using T7 promoter regulatory sequences and T7 polymerase. Examples of
vectors for expression in yeast S. cerivisae include pYepSec1 (Baldari et al.,
(1987)
EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943),
pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYES2 (Invitrogen
Corporation, San Diego, Calif.). Baculovirus vectors available for expression
of
proteins or peptides in cultured insect cells (e.g., Sf 9 cells) include the
pAc series
(Smith et al., (1983) Mol. Cell. Biol. 3:2156-2165) and the pVL series
(Lucklow, V. A.,
and Summers, M. D., (1989) Virology 170:31-39). Examples of mammalian
expression vectors include pCDM8 (Seed, B., (1987) Nature 329:840) and pMT2PC
(Kaufman et al. (1987), EMBO J. 6:187-195). When used in mammalian cells, the
expression vector's control functions are often provided by viral regulatory
elements.
For example, commonly used promoters are derived from polyoma, Adenovirus 2,
cytomegalovirus and Simian Virus 40.
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In addition to regulatory control sequences, recombinant expression vectors
may contain additional nucleotide sequences, such as a selectable marker gene
to
identify host cells that have incorporated the vector. Selectable marker genes
are
well known in the art. To facilitate secretion of the peptide compound from a
host
cell, in particular mammalian host cells, the recombinant expression vector
preferably encodes a signal sequence operatively linked to sequences encoding
the
amino-terminus of the peptide compound, such that upon expression, the peptide
compound is synthesized with the signal sequence fused to its amino terminus.
This
signal sequence directs the peptide compound into the secretory pathway of the
cell
and is then cleaved, allowing for release of the mature peptide compound
(i.e., the
peptide compound without the signal sequence) from the host cell. Use of a
signal
sequence to facilitate secretion of proteins or peptides from mammalian host
cells is
well known in the art.
A recombinant expression vector comprising a nucleic acid encoding a
peptide compound may be introduced into a host cell to produce the peptide
compound in the host cell. Accordingly, the invention also provides host cells
containing the recombinant expression vectors of the invention. The terms
"host cell"
and "recombinant host cell" are used interchangeably herein. Such terms refer
not
only to the particular subject cell but to the progeny or potential progeny of
such a
cell. Because certain modifications may occur in succeeding generations due to
either mutation or environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the scope of the
term as used
herein. A host cell may be any prokaryotic or eukaryotic cell. For example, a
peptide
compound may be expressed in bacterial cells such as E. coli, insect cells,
yeast or
mammalian cells. The peptide compound may be expressed in vivo in a subject to
the subject by gene therapy (discussed further below).
Vector DNA can be introduced into prokaryotic or eukaryotic cells via
conventional transformation, transfection or infection techniques. The terms
"transformation", "transfection" or "infection" refer to techniques for
introducing
foreign nucleic acid into a host cell, including calcium phosphate or calcium
chloride
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co-precipitation, DEAE-dextran-mediated transfection, lipofection,
electroporation,
microinjection and viral-mediated infection. Suitable methods for
transforming,
transfecting or infecting host cells can for example be found in Sambrook et
al.
(Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor
Laboratory press (1989)), and other laboratory manuals. Methods for
introducing
DNA into mammalian cells in vivo are also known, and may be used to deliver
the
vector DNA of the invention to a subject for gene therapy.
For stable transfection of mammalian cells, it is known that, depending upon
the expression vector and transfection technique used, only a small fraction
of cells
may integrate the foreign DNA into their genome. In order to identify and
select these
integrants, a gene that encodes a selectable marker (such as resistance to
antibiotics) may be introduced into the host cells along with the gene of
interest.
Preferred selectable markers include those that confer resistance to drugs,
such as
6418, hygromycin and methotrexate. Nucleic acids encoding a selectable marker
may be introduced into a host cell on the same vector as that encoding the
peptide
compound or may be introduced on a separate vector. Cells stably transfected
with
the introduced nucleic acid may be identified by drug selection (cells that
have
incorporated the selectable marker gene will survive, while the other cells
die).
A nucleic acid of the invention may be delivered to cells in vivo using
methods
such as direct injection of DNA, receptor-mediated DNA uptake or viral-
mediated
infection. Direct injection has been used to introduce naked DNA into cells in
vivo
(see e.g., Acsadi et al. (1991) Nature 332:815-818; Wolff et al. (1990)
Science
247:1465-1468). A delivery apparatus (e.g., a "gene gun") for injecting DNA
into cells
in vivo may be used. Such an apparatus may be commercially available (e.g.,
from
BioRad). Naked DNA may also be introduced into cells by complexing the DNA to
a
cation, such as polylysine, which is coupled to a ligand for a cell-surface
receptor
(see for example Wu, G. and Wu, C. H. (1988) J. Biol. Chem. 263:14621; Wilson
e1
al. (1992) J. Biol. Chem. 267:963-967; and U.S. Pat. No. 5,166,320). Binding
of the
DNA-ligand complex to the receptor may facilitate uptake of the DNA by
receptor-
mediated endocytosis. A DNA-ligand complex linked to adenovirus capsids that
disrupt endosomes, thereby releasing material into the cytoplasm, may be used
to
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avoid degradation of the complex by intracellular lysosomes (see for example
Curiel
et al. (1991) Proc. Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993)
Proc. Natl.
Acad. Sci. USA 90:2122-2126).
Defective retroviruses are well characterized for use in gene transfer for
gene
therapy purposes (for reviews see Miller, A. D. (1990) Blood 76:271, Kume et
al.
(1999) International. J. Hematol. 69:227-233). Protocols for producing
recombinant
retroviruses and for infecting cells in vitro or in vivo with such viruses can
be found in
Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene
Publishing Associates, (1989), Sections 9.10-9.14 and other standard
laboratory
manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM
which
are well known to those skilled in the art. Examples of suitable packaging
virus lines
include .p~i.Crip, .p~i.Cre, .p~i.2 and .p~i.Am. Retroviruses have been used
to
introduce a variety of genes into many different cell types, including
epithelial cells,
endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in
vitro
and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398;
Danos
and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al.
(1988)
Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl.
Acad.
Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-
8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury
et al.
(1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad.
Sci.
USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al.
(1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J.
Immunol.
150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT
Application
WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and
PCT Application WO 92/07573). In various embodiments, a genome of a retrovirus
that encodes and expresses a polypeptide compound of the invention, may be
utilized for the propagation and/or survival of cells, such as hematopoietic
progenitor
stem cells, for the purposes of maintaining and/or growing cells for the
clinical
purposes of blood transfusion or engraftment, host conditioning or
applications
relevant to chemotherapy, radiation therapy or myeloablative therapy.
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For use as a gene therapy vector, the genome of an adenovirus may be
manipulated so that it encodes and expresses a peptide compound of the
invention,
but is inactivated in terms of its ability to replicate in a normal lytic
viral life cycle. See
for example Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991)
~ Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155. Suitable
adenoviral vectors derived from the adenovirus strain Ad type 5 d1324 or other
strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those
skilled in the
art. Recombinant adenoviruses are advantageous in that they do not require
dividing
cells to be effective gene delivery vehicles and can be used to infect a wide
variety of
cell types, including airway epithelium (Rosenfeld et al. (1992) cited supra),
endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad. Sci. USA 89:6482-
6486), hepatocytes (Herz and Gerard (1993) Proc. Natl. Acad. Sci. USA 90:2812-
2816) and muscle cells (Quantin e1 al. (1992) Proc. Natl. Acad. Sci. USA
89:2581-
2584). In various embodiments, a genome of an adenovirus that encodes and
expresses a polypeptide compound of the invention, may be utilized for the
propagation and/or survival of cells, such as hematopoietic progenitor stem
cells,
stromal cells, or mesenchymal cells, for the purposes of maintaining and/or
growing
cells for the clinical purposes of blood transfusion or engraftment, host
conditioning
or applications relevant to chemotherapy, radiation therapy or myeloablative
therapy.
In some embodiments, adeno-associated virus (AAV) may be used as a gene
therapy vector for delivery of DNA for gene therapy purposes. AAV is a
naturally
occurring defective virus that requires another virus, such as an adenovirus
or a
herpes virus, as a helper virus for efficient replication and a productive
life cycle
(Muzyczka et al. Curr. Topics in Micro. and Immunol. (1992) 158:97-129). AAV
may
be used to integrate DNA into non-dividing cells (see for example Flotte et
al. (1992)
Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol.
63:3822-
3828; and McLaughlin et al. (1989) J. Virol. 62:1963-1973). An AAV vector such
as
that described in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 may be
used to
introduce DNA into cells (see for example Hermonat et al. (1984) Proc. Natl.
Acad.
Sci. USA 81:6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081;
Wondisford et al. (1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J.
Virol.
51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790). In some
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embodiments, a genome of an AAV that encodes and expresses a polypeptide
compound of the invention, may be utilized for the propagation and/or survival
of
cells, such as hematopoietic progenitor stem cells, stromal cells or
mesenchymal
cells, for the purposes of maintaining and/or growing cells for the clinical
purposes of
blood transfusion or engraftment, host conditioning or applications relevant
to
chemotherapy, radiation therapy or myeloablative therapy.
General methods for gene therapy are known in the art. See for example,
U.S. Pat. No. 5,399,346 by Anderson et al. A biocompatible capsule for
delivering
genetic material is described in PCT Publication WO 95/05452 by Baetge et al.
Methods for grafting genetically modified cells to treat central nervous
system
disorders are described in U.S. Pat. No. 5,082,670 and in PCT Publications WO
90/06757 and WO 93/10234, all by Gage et al. Methods of gene transfer into
hematopoietic cells have also previously been reported (see Clapp, D. W., et
al.,
Blood 78: 1132-1139 (1991); Anderson, Science 288:627-9 (2000); and ,
Cavazzana-Calvo et al., Science 288:669-72 (2000), all of which are
incorporated
herein by reference).
Cancers susceptible to treatment with CXCR4 agonists in accordance with
various aspects of the invention may include both primary and metastatic
tumors,
such as solid tumors, including carcinomas of the breast, colon, rectum,
oropharynx,
hypopharynx, esophagus, stomach, pancreas, liver, gall bladder and bile ducts,
small intestine, urinary tract (including kidney, bladder, and urothelium),
female
genital tract (including cervix, uterus, and ovaries as well as
choriocarcinoma and
gestational trophoblast disease), mate genital tract (including prostate,
seminal
vesicles, testes, and germ cell tumors), endocrine glands (including the
thyroid,
adrenal and pituitary glands), and skin, as well as hemangiomas, melanomas,
sarcomas (including those arising from bone and soft tissues as well as
Kaposi's
sarcoma) and tumors of the brain, nerves, eyes, and meninges (including
astrocytomas, gliomas, retinoblastomas, neuromas, neuroblastomas,
Schwannomas, and meningiomas). In some aspects of the invention, CXCR4
agonists may also serve in treating solid tumors arising from hematopoietic
malignancies such as leukemias (i.e., chloromas, plasmacytomas and the plaques
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and tumors of mycosis fungoides and cutaneous T-cell lymphoma/leukemia) as
well
as in the treatment of lymphoma (both Hodgkin's and non-Hodgkin's lymphomas).
In
addition, CDCX4 agonists may be therapeutic in the prevention of metastasis
from
the tumors described above either when used alone or in combination with
cytotoxic
agents such as radiotherapy or chemotherapeutic agents.
In alternative aspects of the invention, CXCR4 agonists such as SDF-1
polypeptides may target CD34+ cells to mediate release of CD34+ cells to the
peripheral blood. In these aspects of the invention, CXCR4 agonists such as
SDF-1
may enhance circulating CD34+ cell proliferation and hematopoietic stem or
progenitor cell survival or levels, which may for example be useful in stem
cell
transplantation.
In various aspects of the invention, CXCR4 agonists may be used in reducing
the rate of hematopoietic cell multiplication. Method of the invention may
comprise
administration of an effective amount of CXCR4 agonists to cells selected from
the
group consisting of hematopoietic stem cells and hematopoietic progenitor
cells,
stromal cells or mesenchymal cells. In alternative embodiments, a
therapeutically
effective amount of the CXCR4 agonist may be administered to a patient in need
of
such treatment. Patients in need of such treatments may include, for example:
patients having cancer, patients having an autoimmune disease, patients
requiring
functional gene transfer into hematopoietic stems cells, stromal cells or
mesenchymal cells (such as for the dysfunction of any tissue or organ into
which a
stem cell may differentiate), patients requiring lymphocyte depletion,
patients
requiring depletion of a blood cancer in the form of purging autoreactive or
cancerous cells using autologous or allgenic grafts, or patients requiring
autologous
peripheral blood stem cell transplantation. A patient in need of treatment in
accordance with the invention may also be receiving cytotoxic treatments such
as
chemotherapy or radiation therapy. In some embodiments, CXCR4 agonists may be
used in treatment to purge an ex vivo hematopoietic stem cell culture of
cancer cells
with cytotoxic treatment, while preserving the viability of the hematopoietic
progenitor
or stem cells.
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In alternative embodiments, CXCR4 agonists may be used in accordance with
the invention to treat hematopoietic cells, in patients in need of such
treatment, for
example:
i) In hematopoietic recovery and bone marrow regeneration following
irradiation;
ii) To ameliorate the myelosuppression associated with dose intensive
chemotherapy;
iii) In maintenance of high quality mobilized progenitor cells for harvesting
and
peripheral blood stem cells transplantation;
iv) To enhance hematopoietic recovery after autologous stem cell
transplantation;
v) In immunotherapy of cancer and infectious disease;
vi) In solid organ regeneration (Silberstein and Toy, 2001, JAMA Vol 285,
577-580);
vii) In stem cell gene therapy and retro-virus gene transfer into
hematopoietic
progenitor cells (Hacein-Bey, 2001, Hum. Gene Ther. Vol 12, 291-301; Kaji and
Leiden, 2001, JAMA Vol 285, 545-550), stromal cells, or mesenchymal cells;
viii) In bone development, bone repair, and skeletal regeneration therapy.
Although various embodiments of the invention are disclosed herein, many
adaptations and modifications may be made within the scope of the invention in
accordance with the common general knowledge of those skilled in this art.
Such
modifications include the substitution of known equivalents for any aspect of
the
invention in order to achieve the same result in substantially the same way.
Numeric
ranges are inclusive of the numbers defining the range. In the claims, the
word
"comprising" is used as an open-ended term, substantially equivalent to the
phrase
"including, but not limited to".
EXAMPLES
The following examples illustrate, but do not limit, the present invention.
Example 1
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Peptides of the invention may be synthesized chemically using the Fmoc/tBu
strategy on a continuos flow peptide synthesizer, as for example has been
carried
out using the following protocols:
A) Reagents (solvents, support, chemicals)
Main Solvent: N,N-Dimethylformamide (DMF): certified ACS spectroanalyzed
from Fisher (D131-4) M.W = 73.10. The DMF is treated with activated molecular
sieves, type 4A (from BDH: B54005) for at least two weeks then tested with
FDNB
(2,4-Dinitrofluorobenzene from Eastman).
Procedure: Mix equal volumes of FDNB solution (1 mg/ml in 95% EtOH) and
DMF; Let stand 30 minutes; read the absorbance at 381 nm over a FDNB blank
(0.5m1 FDNB + 0.5m1 95% EtOH). If the absorbance ~ 0.2, the DMF is suitable to
be
used for the synthesis.
Debloking Agent: 20% Piperidine (from Aldrich Chemical company, catalog
No: 10,409-4) in DMF containing 0.5 % triton X100 v/v ( from Sigma , catalg
No: T-
9284 ).
Activating Agents: 2-(H-benzotriazol-lyl) 1,1,3,3-tetramethyluronium
tetrafluoroborate (TBTU: M.W.=321.09. from Quantum Richilieu, catalog No:
80139)/
Hydroxybenzotriazole (HOBt M.W.=135.1 from Quantum Richilieu, catalog No.:
80166-100) respectively, 0.52 M in DMF and 4-Methylmorpholine (NMM ;
M.W.=101.15, d=0.926 from Aldrich, catalog No.: M5,655-7): 0.9 M in DMF or in
the
case of sensitive amino acids to racemization like Cys, we use 2,4,6-
Collidine, 99%
M.W.=121.18,d=0.917, from Aldrich, catalog No: 14,238-7 ): 0.78M in DMF/DCM,
1/1
vlv.
Support: TentaGel R RAM (90 Nm), RinK-type Fmoc (from Peptides
International, catalog No.: RTS -9995-PI): 0.21 mmol/g, 0.5g for 0.1 mmol of
peptide.
Fmoc-L-amino derivative, side-chains protected with: Boc; tBu; Trt groups:
with 4 fold excess (from Peptides International, Bachem, Novabiochem, Chem-
Impex Inc). GIu24 and Lys24 are Allyl-protected (from Millipore/Perseptive
Biosystems).
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B) Initial Amino Loading and Peptide Synthesis Procedure
The first amino acid Asn31 and the remaining residues are doble coupled
at 450°C automatically with 4-fold excess in each coupling. The
synthesis is
interrupted after residue Leu19. The peptide-bound support is removed from the
synthesizer column and placed in a react-vial containing a small magnetic bar
for
gentle stirring.
C) Removal of The Allyl Groups
A solution of tetrakis(triphenylphosphine)Palladium(0) Pd(PPh3)4 (from
Sigma-Aldrich, catalog No: 21,666-6); M.W.=1155.58 x 0.1 mmol peptide x 3 fold
=
347mg dissolved in 5% Acetic Acid; 2.5% NMM in CHC13 to 0.14 M, under argon.
The solution is added to the support-bound peptide previously removed from the
coulmn in a reactvial containing a small mangnetic bar for gentle stirring.
The mixture
is flushed with argon, sealed and stirred at room temperature for 6 hours. The
support-bound peptide is trasferred to a filter funnel, washed with 30 ml of a
solution
made of 0.5% Sodium Diethyldithiocarbonate/ in DMF the DCM; DCM/DMF (1 : 1)
and DMF. A positive Kaiser test indicate the deprotection of the amino side
chaine of
the Lys20.
D) Lactam Formation:
Activating agent: 7-Azabenztriazol-1-yloxytris (pyrrolindino) phosphonium-
hexafluorophosphate (PyAOP: M.W.=521.7 from PerSeptive Biosystems GmbH,
catalog No: GEN076531 ) , 1.4-fold: 0.105mmol x 1.4 x 521.7 = 76.6mg and NMM
1.5-fold: 0.105 x 1.4 x 1.5 = 0.23 mmol v = 0.23/0.9 M NMM solution = 263 NI)
The cyclisation may be carried out in an amino acid vial at room
temperature overnight (~16 hours) with gentle agitation. The completion of
cyclization may be indicated by a negative kaiser test. The support-bound
peptide
may be poured into the column, washed with DMF and the synthesis continues to
completion, with a cyclic amide bridge thereby introduced into the peptide.
E) Final Product Removal From The Support:
The support-bound peptide is removed from the synthesizer in to a
medium filter funnel, washed with DCM to replace the non-volatile DMF and
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thoroughly dried under high vacuum fro at least two hours, or preferably,
overnight.
Cleavage Mixture (reagent K):
TFA/Phenol/Water/Thio-Anisol/EDT (82/5/5/5/2.5) ; 7.5m1
Support: 0.5g resin-peptide.
TFA 6.15m1 ( Biograde from Halocarbon
)
Phenol 0.375m1 ( Aldrich )
Water 0.375m1 ( MiIIQ )
Thio-Anisol 0.375m1 (Aldrich )
EDT 0.187m1 ( Aldrich )
Total 7.5m1
The cleavage may be performed at room temperature for 4 hours with
gentle agitation on a rocker.
F) Precipitatation of The Peptide
The cleaved peptide solution is filtered through a filter funnel in a 50 ml
round bottom flask. The support is rinsed twice with 4 ml TFA. The TFA
solution
is concentrated on a rotavap and added drop wise into a cold diethyl ether
previously treated with activated neutral aluminum oxide to make it free of
peroxide. Approximately 10-fold excess of ether are used. The beads are stored
until the yield is determined a peptide characterized. The precipitate is
collected
at room temperature in screw capped 50 ml polypropylene vial by centrifugation
at 2K rpm, using a top bench centrifuge (4 minutes run time). The pellet is
washed 3x with cold ether, centrifuged and dried with a flow of argon. The
precipitate is dissolved in 20 % acetonitrile 0.1 % TFA and lyophilized.
G) Crude Product Characterization:
The product is characterized by analytical HPLC.
Experimental conditions: Column: Vydac 218TP54: C18 reversed-phase 5Nm,
4.6 mmIDx150mmL.
Eluants: 0.1 % TFA/Hz0 (solvant A); 0.1 % TFA/acetonitrile (solvent B)
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Elution Conditions: 20-50% B (40 min ); 60-90% B (5 min); 90-20% B (5 min);
20% B (10 min). At 1.0 ml/min and A214 nm = 0.5 absorbance unit full scale.
H) Sample Preparation:
An aliquot of the product is weighed and dissolved in 20% acetonitrile 0.1 %
TFA
at a cocentration of 2 mg/ml. The solution is microfuged and 20N1 is applied
onto
the column. The main peak or the major peaks are collected, SpeedVac dried
and molecular weight determined by mass spectrometry.
In accordance with various aspects of the invention, a wide variety of peptide
sequences may be prepared, for which the following nomenclature may be used.
The portions of the peptide corresponding to a chemokine sequence, such as an
SDF-1 sequence may be identified by specifying the corresponding portion of
the
chemokine, wherein for example a reference to an SDF-1 sequence refers to a
sequence having substantial identity to a portion of the sequence of SEQ ID
No: 1.
For example, the nomenclature SDF-1(1-14) connotes the first fourteen amino
acids
of the N-terminal sequence of SDF-1 of SEQ ID No: 1. In some embodiments, N-
terminal and C-terminal portions of an SDF-1 sequence may be linked by various
amino acids, or other linking moieties, denoted by a formula (L)n, wherein "L"
is a
linking moiety which may for example be an amino acid and n is zero or an
integer.
The carboxy terminal of the peptide may be modified to be an amide rather than
a
carboxylic acid. In some embodiments, polypeptides of the invention may be of
the
following formula:
SDF-1 (1-X)-(L)n-SDF-1 (Y-Z)
wherein:
X is an integer from 5 to 20;
L is a linking moiety having at least one carbon atom, such as a substituted
or
unsubstituted alkyl moiety, or an amino acid;
n is an integer from 1 to 50
Y is an integer from 50 to 60
Z is an integer from 60 to 67
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In some embodiments, (CHZ)n may for example be used as a linker (L)
between N- and C-terminal, where n is an integer and may for example be less
than
20, 30, 40, 50 or 100.
Exemplary embodiments of linear polypeptide sequences are as follows:
SDF-1 (1-14)-(G)3-SDF-1 (55-67) acid:
HZNKPVSLSYRCPCRFFGGGLKWIQEYLEKALNCOOH
SDF-1 (1-14)-(G)4-SDF-1 (55-67) acid:
HZNKPVSLSYRCPCRFFGGGGLKWIQEYLEKALNCOOH
SDF-1 (1-14)-(G)3-SDF-1 (55-67) amide:
H2NKPVSLSYRCPCRFFGGGLKWIQEYLEKALNCONH2
SDF-1 (1-14)-(G)4-SDF-1 (55-67) amide:
H2NKPVSLSYRCPCRFFGGGGLKWIQEYLEKALNCONH2
SDF-1 (1-17)-(G)3-SDF-1 (55-67) acid:
H2NKPVSLSYRCPCRFFESHGGGLKWIQEYLEKALNCOOH
SDF-1 (1-17)-(G)4-SDF-1 (55-67) acid:
H2NKPVSLSYRCPCRFFESHGGGGLKWIQEYLEKALNCOOH
SDF-1 (1-17)-(G)3-SDF-1 (55-67) amide:
H2NKPVSLSYRCPCRFFESHGGGLKWIQEYLEKALNCONH2
SDF-1(1-17)-(G)3-SDF-1(55-67) amide:
H2NKPVSLSYRCPCRFFESHGGGGLKWIQEYLEKALNCONH2
In alternative embodiments, peptides of the invention may be cyclized, for
example glutamate (E) and lysine (K) residues may be joined by side chain
cyclization using a lactam formation procedure, as indicated in the following
sequences by double underlining of linked residues. Lactams may for example be
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formed between glutamic acid (E) at amino acid residue 24 and lysine (K) at
either
position 20 or 28 in the polypeptide (which does not correspond necessarily
with the
numbering of that residue in the native sequence). In further alternatives, a
lysine (K)
may be substituted by leucine (L), ornithine (O) or other hydrophobic
residues, such
~ as isoleucine (I), norleucine (Nle), methionine (M), valine (V), alanine
(A), tryptophan
(W) or Phenylalanine (F). Similarly, glutamate (E) may for example be
substituted
with aspartate (D), denoted by nomenclature such as (E24 -> D) indicating a
substitution at position 24 in the peptide wherein aspartate replaces
glutamate.
SDF-1(1-14)-(G)4-SDF-1(55-67)-E24/K28-cyclic acid
HZNKPVSLSYRCPCRFFGGGGLKWIQEYLEKALNCOOH
SDF-1 (1-14)-(G)4-SDF-1 (55-67)-K20/E24-cyclic acid
H2NKPVSLSYRCPCRFFGGGGLKWIQEYLEKALNCOOH
SDF-1(1-14)-(G)4-SDF-1(55-67)-E24/K28-cyclic amide
H2NKPVSLSYRCPCRFFGGGGLKWIQEYLEKALNCONH2
SDF-1(1-14)-(G)4-SDF-1(55-67) K20/E24-cyclic amide
H2NKPVSLSYRCPCRFFGGGGLKWIQEYLEKALNCONH2
In alternative embodiments of the peptides of the invention, underlined spacer
monomers (such as the illustrated glycine G's) may for be used in variable
numbers,
such as 2, 3 or 4 glycines.
In alternative embodiments, internal cyclization of peptides of the invention
may be in alternative positions, or between substituted amino acids. The
nature of
the cyclic linkage may also be varied. For example, the linkage may be
shortened
by replacing the relevant glutamate (E) with an aspartate (D) residue, and/or
replacing the lysine (K) with an ornithine (O) residue. Cyclization is for
example
possible between Aspartic acid 24 (D24) and Lysine 20 or 28 (K20 or K28), as
illustrated in some of the exemplified polypeptides.
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SDF-1 (1-14)-(G)4-SDF-1 (55-67)-K20/D24-cyclic acid
H2NKPVSLSYRCPCRFFGGGGLKWIQDYLEKALNCOOH
SDF-1 (1-14)-(G)4-SDF-1 (55-67)-K20/D24-cyclic amide
HZNKPVSLSYRCPCRFFGGGGLKWIQDYLEKALNCONHZ
Disulphide or sulphide bridging may be used to produce alternative
embodiments of the polypeptides of the invention, in which cysteine residues
may for
example be involved in bridge formation, as indicated in the following
sequences by
~ double underlined residues.
SDF-1(1-14)-(G)4-SDF-1(55-67)-C9/C11-cyclic acid
H2NKPVSLSYRCPCRFFGGGGLKWIQEYLEKALNCOOH
SDF-1 (1-14)-(G)4-SDF-1 (55-67)-C9/C11-cyclic amide
H2NKPVSLSYRCPCRFFGGGGLKWIQEYLEKALNCONH2
In one aspect, polypeptide compounds of the invention may provide a CXCR4
agonist comprising a peptide having: (a) an N-terminal sequence homologous to
a
chemokine, such as an SDF-1 N-terminal sequence; (b) a C-terminal sequence
homologous to a chemokine, such as an SDF-1 C-terminal sequence; (c) a linking
moiety joining the N-terminal sequence to the C-terminal sequence, such as a
polypeptide linker; and, (d) an internal cyclic bridge formed between portions
of the
polypeptide, such as an amide linking a carboxylic acid side chain on a first
amino
acid residue and an amine side chain on a second amino acid residue. In some
embodiments, the C-terminal sequence may comprise the internal cyclic bridge.
As shown above, exemplary embodiments of polypeptides of the invention
have been synthesized, having N-terminal SDF-1 residues (1-14) or (1-17),
linked to
C-terminal SDF-1 residues (55-67) by a three or four-glycine linker. In some
embodiments, peptides are cyclized between glutamic acid (at 24 position) and
lysine (at 20 or 28 position). Lactamization may be affected by removing the
allylic
group from both side chains of lysine and glutamic acid using the palladium
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technique and then effecting internal amide bond formation between the
corresponding lysine and glutamic acid. Selected members of this family of
polypeptides showed high affinity in a CXCR4 receptor binding assay (CEM
cells)
and in activating [Ca2+] mobilization (THP-1 cells). Further embodiments of
polypeptides are listed below:
SDF-1 (1-14)-(G)4-SDF-1 (55-67)-K20/D24-(E24 -> D)-cyclic acid or amide
SDF-1 (1-14)-(G)4-SDF-1 (55-67)-K28/D24-(E24 -> D)-cyclic acid or amide
Cyclization may also take place between ornithine (O) and glutamic acid (E)
SDF-1 (1-14)-(G)4-SDF-1 (55-67)-020/E24-(K20 ->O)-cyclic acid or amide
SDF-1 (1-14)-(G)4-SDF-1 (55-67)-028/E24-(K28 ->O)-cyclic acid or amide
Cyclization may also take place between ornithine (O) and aspartic acid (D):
SDF-1 (1-14)-(G)4-SDF-1 (55-67)-020/D24-(K20 -> O & E24 -> D)-cyclic acid or
amide
SDF-1 (1-14)-(G)4-SDF-1 (55-67)-028/D24-(K28 -> O & E24 -> D)-cyclic acid or
amide
In some embodiments, proline (P) at position 6th may be replaced with serine
(S). In
some embodiments, lysine (K) and glutamic acid (E) may be replaced by
ornithine
(O) and aspartic acid (D), respectively. Similarly, substitutions may be made
in the
LSYR region, replacing lucine (L), serine (S), tyrosine (Y) or arginine (R) by
proline
(P) or other similarly shaped moiety. Alternatively, proline may be
substituted with
P*:
Where P* _
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X
with X= Ar, Ar-OH, alkyl and more
~COOH
N
H
A wide variety of amino acid substitutions may be made in polypeptide
sequences,
such as K to E, K to D, O to E, O to D. Moieties other than naturally
occurring amino
acids may also be substituted, such as Btd:
Where Btd* _
X
or ~ or
N N
H N ~ HzN Hz
COOH O COOH O .OOH
X= Alkyl, Ar, Ar-OH and more
Similarly, polypeptides may be prepared using sequences from chemokines other
than SDF-1. Such as residues 36-50, 10-50 or 55-70 of MIP-1a:
SDF-1(1-14)-(G)4-MIP-1a(36-50)-acid or amide
HZN-KPVSLSYRCPCRFFGGGGSKPGVIFLTKRSRQV-CONH2
SDF-1(1-14)-(G)4-MIP-1a(11-50)- acid or amide
HZN-
KPVSLSYRCPCRFFGGGGCCFSYTSROIPQNFIADYFETSSQCSKPGVIFLTKRSR
OV-CONHz
SDF-1(1-14)-(G)4-MIP-1a(56-70)-acid or amide
H2N-KPVSLSYRCPCRFFGGGGEEWVQKYVDDLELSA-CONH2
In various Figures, compounds are identified by abbreviations, as follows:
Structure of CTCE9901:
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SDF-1 (1-9)Z-C9/C9-cysteine dimer
HZNKPVSLSYRCCOOH
I
H2NKPVSLSYRCCOOH
Structure of CTCE9902:
SDF-1 (1-17)
H2NKPVSLSYRCPCRFFESHCOOH
Structure of CTCE9904:
SDF-1 (1-8)2-lysine bridge dimer
H2NKPVSLSYR
I
K-CONHZ
H2NKPVSLSYR
Structure of CTCE0013:
SDF-1(1-14)-(G)4-SDF-1(55-67) acid
H2NKPVSLSYRCPCRFFGGGGLKWIQEYLEKALNCOOH
Structure of CTCE0017:
SDF-1 (1-14)-(G)4-SDF-1 (55-67) amide
H2NKPVSLSYRCPCRFFGGGGLKWIQEYLEKALNCONH2
Structure of CTCE0022:
SDF-1(1-14)-(G)4-SDF-1(55-67)-E24/K28-cyclic amide
HZNKPVSLSYRCPCRFFGGGGLKWIQEYLEKALNCONHz
Structure of CTCE0021:
SDF-1 (1-14)-(G)4-SDF-1 (55-67)-K20/E24-cyclic amide
H2NKPVSLSYRCPCRFFGGGGLKWIQEYLEKALNCONHz
Example 2
Tables 1 and 2 show the effect of CXCR4 agoinists on bone marrow
progenitor cells, particularly primitive erythroide cells and primitive
granulocytes,
compared to mature granulocytes. To obtain the data in Tables 1 and 2, cells
were
pre-incubated with each of the compounds or saline alone ('no drug' as
control). The
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cells were then exposed to high dose H3-thymidine, a cytotoxic agent. Rapidly
dividing cells accumulate proportionally more of the cytotoxic radioactive
thymidine
and as a result are preferentially killed. The relative proportion of cells
killed by the
thymidine treatment compared to the control is indicative of the relative
effectiveness
of the compounds in reducing cellular multiplication, i.e. decreasing the rate
of cell
cycle progression. A higher (or unchanged) proportion of killed cells compared
to the
control is indicative that a compound does not reduce cellular multiplication
of the
given cell type.
Table 1:
Effect of CXCR4 Agonists on Bone Marrow Progenitor Cells Exposed to H3-
Thymidine.
CELL KILLED
No drug (control) SDF-1 SDF-1(1-9)2
Primitive
Erythroide 71 2 9
Primitive
Granulocyte 46 1 1
Mature
Granulocyte 39 45 42
In Table 1, SDF-1 polypeptide (KPVSL SYRCP CRFFE SHVAR ANVKH
LKILN TPNCA LQIVA RLKNN NRQVC IDPKL KWIQE YLEKA LN) is used at 100
ng/ml on a human bone marrow cell culture. SDF-1(1-9)2 (KPVSLSYRC-X-
CRYSLSVPK) is used at 50 ug/ml on a human bone marrow cell culture.
Table 2 further demonstrates that SDF-1 (1-14)-(G)4-SDF-1 (55-67)-amide and
SDF-1(1-14)-(G)4-SDF-1(55-67)-K20/E24-cyclic amide are both able to inhibit
cell
cycling in human positive erythroid and primitive granulopoietic cells, but
not in
mature granulopoietic cells, in the assay as described above in this Example.
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Table 2
CELL KILLED
No drug (control) Compound A Compound B
Primitive
Erythroide 47 +/- 4 5 +/- 3 -7 +/- 6
Primitive
Granulocyte 42 +/- 3 1 +/- 6 -11 +/- 7
Mature
Granulocyte 48 +/- 3 39 +/- 5 44 +/- 6
Where: Compound A is SDF-1 (1-14)-(G)4-SDF-1 (55-67)-amide;
Compounds B is SDF-1(1-14)-(G)4-SDF-1(55-67)-K20/E24-cyclic amide
Example 3
The present example demonstrates the therapeutic effectiveness of CXCR4
agonists in an animal model, showing protection of hematopoietic cells from
cytotoxic treatments with CXCR4 agonists. In these animal studies, normal mice
were treated with the cytotoxic chemotherapeutic agent arabinose-cytosine (Ara-
C),
which are known to deleteriously affect cells with high rates of DNA synthesis
(reflecting rapid cell cycling).
As shown in the graph of Figure 1, in mice given a single dose of Arabinose
Cytosine (Ara-C) at 350 mg/kg at day zero intravenously, white blood cell
count
(WBC) decreases (due to the cytotoxic action of Ara-C). In contrast, in mice
given
the peptide SDF-1 (1-14)-(G)4-SDF-1 (55-67)-K20/E24-cyclic amide (designated
CTC
in the graph legend) in combination with Ara-C, the extent of white blood cell
count
decrease is significantly ameliorated. In the graph, circular data points
correspond to
the white blood cell count in animals that received Ara-C but did not receive
the
peptide, and triangular data points are for animals that received Ara-C and
SDF-1 (1-
14)-(G)4-SDF-1 (55-67)-K20/E24-cyclic amide. The data clearly demonstrated the
protective action of the peptide of the invention against the cyctotoxic
action of Ara-
C.
Example 4
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The efficacy of SDF-1 and SDF-1 peptide analogs as CXCR4 agonists was
demonstrated through CXCR4 receptor binding assays. A competitive dose
response for binding to the SDF-1 receptor by native SDF-1 and the CXCR4
agonists against '251-SDF-1 is shown in Figures 2A and 2 B respectively. A
concentration-dependent inhibition of '251-SDF-1 is illustrated in Figure 2A,
indicating
the affinity of SDF-1 for the receptor. A Scartchard plot is illustrated, and
the Kp was
determined to be 26nM. SDF-1 and the indicated analogs (competing ligands)
were
added at the concentrations illustrated in the presence of 4nM '251-SDF-1. CEM
cells
were assessed for '251-SDF-1 binding following 2 hr of incubation. The results
are
expressed as percentages of the maximal specific binding that was determined
without competing ligand, and are the mean of three independent experiments.
The
inhibition of '251-SDF-1 by SDF-1 and the SDF-1 analogs is indicative of CXCR4
receptor binding. The compounds illustrated in the figure are as follows: SDF-
1(1-
14)-(G)4-SDF-1 (55-67)-K20/E24-cyclic amide (CTCE0021 ), SDF-1 (1-14)-(G)4-SDF-
1(55-67)-E24/K28-cyclic amide (CTCE0022), SDF-1 (1-9)2-C9/C9-cysteine dimer
(CTCE9901), SDF-1(1-17) (CTCE9902), SDF-1 (1-8)2-lysine bridge dimer
(CTCE9904) and SDF-1(1-14)-(G)4-SDF-1(55-67) amide (CTCE0017).
Example 5
This example illustrates the efficacy of SDF-1 and SDF-1 peptide analogs in
mediating intracellular calcium mobilization ([Ca2+];). To illustrate that the
binding of
SDF-1 and SDF-1 peptide analogs results in the agonistic induction of the
CXCR4
receptor, [Ca2+]; mobilization assays were conducted, the results of which are
shown
in Figure 3. To obtain the data shown in Figure 3, furs-2,AM loaded THP-1
cells
(1x106/ml) were stimulated with SDF-1, SDF-1(1-14)-(G)4-SDF-1(55-67) K20/E24-
cyclic amide or SDF-1(1-14)-(G)4-SDF-1(55-67)-E24/K28-cyclic amide at the
concentrations indicated (the values represent the mean +/- one S.D. of n=3
experiments). As shown by the data in Figure 3, incubation of THP-1 cells with
SDF-
1, SDF-1 (1-14)-(G)4-SDF-1 (55-67) K20/E24-cyclic amide or SDF-1 (1-14)-(G)4-
SDF-
1 (55-67)-E24/K28-cyclic amide resulted in the receptor-mediated induction of
[Ca2+].
mobilization. The ECSO values (the concentration of ligand necessary to
effectively
induce 50% of the full [Ca2+]; mobilization potential) for SDF-1 (1-14)-(G)4-
SDF-1 (55-
67) acid, SDF-1 (1-14)-(G)4-SDF-1 (55-67)-K20/E24-cyclic amide or SDF-1 (1-14)-
(G)4-SDF-1 (55-67)-E24/K28-cyclic amide and native SDF-1 is shown in Table 3:
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Table 3
Com ound I _ECSO (nM)
SDF-1 26.56
SDF-1 (1-14)-(G)4-SDF-1 (55-67)-106.25
E24/K28-c clic amide
SDF-1(1-14)-(G)4-SDF-1(55-67)-147.94
K20/E24-c clic amide
I SDF-1(1-14)-(G)4-SDF-1(55-67)188.30
acid
The comparative ability of SDF-1, SDF-1 (1-14)-(G)4-SDF-1 (55-67)-K20/E24-
cyclic amide (CTCE0021), SDF-1(1-14)-(G)4-SDF-1(55-67)-E24/K28-cyclic amide
(CTCE0022), SDF-1 (1-9)z-C9/C9-cysteine dimer (CTCE9901), SDF-1(1-17)
(CTCE9902), SDF-1 (1-8)2-lysine bridge dimer (CTCE9904) and SDF-1(1-14)-(G)4-
SDF-1 (55-67) amide (CTCE0017) to induce [Ca2+]; mobilization at the ligand
concentration that the native SDF-1 gave maximal [Ca2+]; mobilization (1~M,
refer to
Figure 3) is illustrated in Figure 4. Fura-2,AM loaded THP-1 cells (1x106/ml)
were
stimulated with native SDF-1 and the SDF-1 peptide agonist analogs at the
concentration of native SDF-1 that gave the maximum [Ca2+]; stimulation (1 pM)
(the
values represent the mean +/- one S.D. of n=3 experiments).
Example 6
Primitive high proliferative potential colony forming cells (HHP-CFC) in an
adherent layer in culture are usually in a quiescent state. This long term
culture
(LTC) is established seven to ten days after initiation of the LTC. The cells
may be
stimulated to proliferate by the addition of fresh medium. Both BFU-E (burst
forming
unit - erythroid precursor) cells and CFU-GM (colony forming unit -
granulocyte-
monocyte common precursor) cells of LTC may be maintained in a quiescent state
by the mesenchymally derived stromal cells in an adherent layer, but can be
reversibly stimulated into the cycle by the addition of fresh media. The
ability of
CXCR4 agonists such as SDF-1 and SDF-1 polypeptides to overcome this
activation
may be determined by adding it to the LTC during the medium change. Rapidly
dividing cells will accumulate proportionally more of a cytotoxic agent, such
as
radioactive thymidine, and as a result are preferentially killed.
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The results depicted in Table 4 illustrate the ability of SDF-1, and SDF-1 (1-
14)-(G)4-SDF-1 (55-67)-K20/E24-cyclic amide (CTCE0021 ) and SDF-1 (1-14)-(G)4-
SDF-1 (55-67) acid (CTCE0013) to repress the proliferation of clonogenic
erythroid
and granulopoitic progenitors (which differentiate into erythrocytes,
platelets,
monocytes/macrophages and neutrophils) in an in vitro LTC-IC (long-term
culture-
initiating cells) assay.
Table 4. Effect of SDF-1 and SDF-1 agonists on the cycling of primitive
progenitors in the adherent layer of human LTC.
Kill after 3H-Thymidine
Treatment Dose Primitive BFU-E Primitive CFU-GM
None 48 +/- 4 44 +/- 3
CTCE0013 1 ~g/ml 24 +/- 6 22 +/- 7
10pg/ml 0+/-2 0+/-0
SDF-1 1 pg/ml 4 +/- 3 5 +/- 4
CTCE0021 1 ~g/ml 2 +/- 4 0 +/- 3
To obtain the results set out in Table 4, clonogenic erythroid (BFU-E) and
granulopoietic (CFU-GM) progenitors were assayed in methylcellulose cultures.
Adherent cells were treated with fresh medium alone (as control) or with the
indicated CXCR4 agonist (10 ~g/ml SDF-1, CTCE0021 or CTCE0013). Dishes were
harvested three days later and 3H-thymidine suicide assays performed on the
progenitor cells in the adherent layer to determine the proportion of cells
killed as a
result of accumulation of cytotoxic 3H-thymidine, where the difference between
the
cells in the control and the nuber of cells remaining represent the cells
killed.
Figure 5 illustrates that feeding cultures SDF-1 in conjunction with media
changes results in significantly reduced cell mortality of hematopoietic cells
when the
cells are challenged with an agent that is preferentially cytotoxic to
dividing cells, in
which circles represent BFU-E cells (burst forming unit-erythroid precursors),
and
squares represent CFU-GM cells (colony forming unit-granulocyte-monocyte
common precursor). Figure 6 shows that a similar concentration dependent
effect
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may be obtained with SDF-1 (1-14)-(G)4-SDF-1 (55-67)- K20/D24-cyclic amide
(Compound #1 ) and SDF-1 (1-9)2 (Compound #3). Together, Figures 5 and 6
illustrate that the SDF-1 polypeptide and SDF-1 peptide analogs repress the
cyclic
activation of the BFU-E and CFU-GM progenitor stem cells in the adherent layer
of
LTC.
Example 7
Figures 7 and 9 show the efficacy of CXCR4 agonists such as SDF-1 and
SDF-1 analogues in repressing the proliferation of human progenitor cells in
an in
vivo engraftment model.
In Figure 7, the cycling status of mature and primitive colony forming cells
(CFU-GM; colony forming unit-granulocyte-monocyte precursor, BFU-E; burst
forming unit-erythroid precursor; LTC-IC, long-term culture initiating cell)
in the
suspension of CD34+ cells isolated from the marrow of transplanted NOD/SCID
mice
was determined by assessing the proportion of these progenitors that were
inactivated (killed) by short term (20 min) or overnight (16 hour) exposure of
the cells
to 20~g/ml of high specific activity 3H-thymidine (values represent the mean
+/- the
S.D. of data from up to four experiments with up to four mice per point in
each).
Significant in the results described in Figure 4 is the observation that the
analogs
SDF-1 (1-14)-(G)4-SDF-1 (55-67)-K20/E24-cyclic amide (CTCE0021 ) and SDF-1 (1
14)-(G)4-SDF-1 (55-67) acid (CTCE013) are as effective as native SDF-1 at
inhibiting
the proliferation of "primitive" human progenitor cells, as measured by the
reduction
of cells killed by exposure to high specific activity 3H-thymidine (which only
affects
proliferating cells).
Example 8
SDF-1 enhances the delectability of colony regenerating units (CRU)
regenerated in NOD/SCID mice transplanted with human fetal liver cells (Figure
8).
Three to four NOD/SCID mice per group were sublethally irradiated and injected
with
human cells, in this case 10' light density fetal liver cells, and the mice
then
maintained for an interval of 2.5-3 weeks. As indicated, each group was then
given
2 daily injections of either 1 Opg of SDF-1, or an equivalent volume of
control
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medium, and all mice were then sacrificed one day after the second injection.
The
bone marrow cells from each group were then pooled, and an aliquot removed for
FACS analysis and overnight 3H-thymidine suicide assays to measure the cycling
activity of the human CFC and LTC-IC (long term culture initiating culture)
present.
~ The remainder of the cells were injected into groups of 3-6 secondary
recipients.
These animals were then sacrificed 6 to 8 weeks later and their bone marrow
removed and analysed for the presence of human cells.
This example describes a secondary engraftment. When the bone marrow of
the secondary recipients was evaluated, a considerable difference was observed
in
the level of human cells present in recipients of cells from the different
groups of
primary mice. As shown in Figure 8, for SDF-1-injected mice a far greater
number of
all types of human cells assessed was found in the marrow of the secondary
recipients that had received marrow from primary mice treated with either SDF-
1 by
comparison to recipients of cells from media injected control primary mice.
Example 9
This example illustrates the effect of CXCR4 agonists such as SDF-1 and
SDF-1 polypeptide analogs on the engraftment of human cells in human fetal
liver
transplanted NOD/SCID mice (Figure 9). As shown in this figure, there was a
lack of
short-term effect of CXCR4 agonists on the frequency of different human cells
present in NOD/SCID mice. In these experiments, 6 to 8 weeks post-transplanted
mice were injected two times, one day apart with the test compound (SDF-1, SDF-
1 (1-14)-(G)4-SDF-1 (55-67)-K20/E24-cyclic amide (CTCE0021 ) or SDF-1 (1-14)-
(G)4-
SDF-1(55-67) acid (CTCE013)) and sacrificed one day later. The frequency of
the
phenotypically defined human hematopoietic cells detected in the long bones
(tibias
and femurs) of mice was determined. Administration of 0.5mg/kg of SDF-1 had no
significant effect on the number of CD45/71, CD19/20, or CD34 cells, nor on
the
CFC or LTC-IC. In addition, none of the human cell types were detectably
affected
by this schedule of CXCR4 agonist administration. This data, coupled with that
of
Figures 7 and 8, indicates that SDF-1, SDF-1 analogs and other CXCR4 agonists
may effectively augment secondary engraftment of human progenitor cells.
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Example 10
This example illustrates the effect of an SDF-1 polypeptide analog CTCE0021
(10mg/kg, identified as Compound #1 in Figure 12) on the recovery of
leukocytes
following myeloablative chemotherapy with Ara-C (300mg/kg). In the experiment
described in the example, C3Hhen mice (female) were treated with 500mg/kg Ara-
C
for two cycles - on days 0 and 10. During the second cycle of Ara-C dosing,
Ara-C
treated mice were injected with 10mg/kg CTCE0021 each day. A control was
conducted with animals treated with Ara-C alone. Blood was collected from the
tail
vein into heparin-containing tubes at the onset of the experiment, and one day
before every day following the second Ara-C dose. A total leukocyte count was
determined. As shown in the graph of Figure 10, the CXCR4 agonist CTCE0021
acted to inhibit the cytotoxic effects of Ara-C and to sustain a higher level
of
leukocytes, illustrating the reversal of myelosuppressive effects of a
chemotherapeutic regimen in vivo.
Example 11:
This example illustrates the effect of an SDF-1 polypeptide analog SDF-1(1-
14)-(G)4-SDF-1 (55-67)-K20/E24-cyclic amide (CTCE0021, 1 mg/kg) on the
recovery
of leukocytes following myeloablative chemotherapy with Ara-C (500mg/kg)
compared to G-CSF (Neupogen~) (Figure 11 ). C3Hhen mice (female) were treated
with 500mg/kg Ara-C for two cycles - on days 0 and 10. During the second cycle
of
Ara-C dosing, Ara-C treated mice were injected with 10mg/kg CTCE0021, 10mg/kg
Neupogen°, alone or together (on days -1, 0, and 1 to 3), with controls
receiving no
drug. Blood was collected from the tail vein into heparin-containing tubes at
the
onset of the experiment, and one day before and 1, 7 and 12 days following the
second Ara-C dose. A total white blood cell count was obtained. The results in
this
example indicates that not only does treatment with CTCE0021 enhance the
recovery of white blood cells following myeloablative chemotherapy with Ara-C,
co-
treatment with the SDF-1 polypeptide analog and G-CSF (Neupogen°)
resulted in a
greater recovery compared the animals treated with G-CSF alone during the
early
treatment phase. Furthermore, the recovery following treatment with the SDF-1
polypeptide analog was sustained compared to the G-CSF treated animals.
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Figure 12 depicts the results of an experiment conducted under identical
conditions, but the growth (increase in leukocyte count) relative to the
number of
cells counted in animals treated with Ara-C alone is illustrated. By twelve
days
following Ara-C administration, an approximately 7.5-fold increase in
leukocytes was
observed in mice treated with CTCE0021 relative to animals treated with Ara-C
alone, compared to 180% obtained in animals treated with Neupogen~.
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