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AU2018378804B2 - Dosing regimens for the mobilization of hematopoietic stem and progenitor cells - Google Patents
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AU2018378804B2 - Dosing regimens for the mobilization of hematopoietic stem and progenitor cells - Google Patents

Dosing regimens for the mobilization of hematopoietic stem and progenitor cells

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AU2018378804B2
AU2018378804B2 AU2018378804A AU2018378804A AU2018378804B2 AU 2018378804 B2 AU2018378804 B2 AU 2018378804B2 AU 2018378804 A AU2018378804 A AU 2018378804A AU 2018378804 A AU2018378804 A AU 2018378804A AU 2018378804 B2 AU2018378804 B2 AU 2018378804B2
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cells
fold
per day
donor
cd45ra
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AU2018378804A1 (en
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Anthony Boitano
Michael P. Cooke
Patrick C. Falahee
Kevin A. Goncalves
Dwight Morrow
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Ensoma Inc
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Ensoma Inc
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Description

WO wo 2019/113375 PCT/US2018/064335
DOSING REGIMENS FOR THE MOBILIZATION OF HEMATOPOIETIC STEM AND PROGENITOR CELLS
Cross Reference to Related Applications
[0001] This application claims the benefit of and priority to U.S. Application No.
15/834,017, filed on December 6, 2017, U.S. Provisional Patent Application No. 62/596,056,
filed on December 7, 2017, U.S. Application No. 16/101,676, filed on August 13, 2018, U.S.
Provisional Patent Application No. 62/753,656, filed on October 31, 2018, and U.S.
Provisional Patent Application No. 62/773,954, filed on November 30, 2018, the disclosure
of each of which is hereby incorporated by reference in its entirety.
Field of the Invention
[0002] The invention relates to the mobilization of hematopoietic stem and progenitor cells
from a donor, such as a human donor, and to the treatment of patients suffering from various
pathologies, such as blood diseases, metabolic disorders, cancers, and autoimmune diseases,
among others.
Background of the Invention
[0003] Despite advances in the medicinal arts, there remains a demand for treating
pathologies of the hematopoietic system, such as diseases of a particular blood cell, metabolic
disorders, cancers, and autoimmune conditions, among others. While hematopoietic stem
cells have significant therapeutic potential, a limitation that has hindered their use in the
clinic has been the difficulty associated with releasing hematopoietic stem cells from the
bone marrow into the peripheral blood of a donor, from which the hematopoietic stem cells
may be isolated for infusion into a patient.
[0004] A further limitation is that up to 80% of mobilized peripheral blood (mPB) allogeneic
recipients will experience graft-versus-host disease (GVHD). Despite the higher levels of
CD3+ T cells in mPB grafts compared to bone marrow transplants, the level of acute GVHD
observed following transplant of HLA-matched mPB is comparable to HLA-matched bone
marrow. One explanation is that G-CSF mobilized grafts contain myeloid-derived suppressor
cells (MDSCs) possessing potent immunosuppressive properties capable of inhibiting T cell
proliferation in vitro. The percentage of MDSCs is variable in grafts mobilized with G-CSF,
and clinical data suggest that patients transplanted with mPB grafts that contain higher
WO wo 2019/113375 PCT/US2018/064335
numbers of MDSCs may have better outcomes including lower rates of acute GVHD
(Vendramin et al., (2014) BBMT 20(12):2049-2055) BBMT20(12):2049-2055).
[0005] Accordingly, there is currently a need for compositions and methods for promoting
the mobilization of hematopoietic stem and progenitor cells, and particularly for methods of
identifying populations of mobilized cells that are suitable for therapeutic use. There is also a
need for compositions and methods for promoting the mobilization of hematopoietic stem
and progenitor cells that consistently produce higher numbers of MDSCs than do prior art
methods.
Summary of the Invention
[0006] The present invention provides compositions and methods for mobilizing
hematopoietic stem and progenitor cells in a subject. For example, the subject may be a
hematopoietic stem and progenitor cell donor (i.e., a donor), such as a mammalian donor, and
particularly a human donor. The invention additionally provides compositions and methods
for the treatment of disorders, such as stem cell disorders, in a patient, such as a human
patient. Using the compositions and methods described herein, a C-X-C chemokine receptor
type 2 (CXCR2) agonist, such as Gro-B Gro-ß or a variant thereof, such as a truncated form of Gro-
ß (e.g., Gro-B Gro-ß T), as described herein, optionally in combination with a C-X-C chemokine
receptor type 4 (CXCR4) antagonist, such as 1,1'-[1,4-phenylenebis(methylene)]-bis ,1'-[1,4-phenylenebis(methylene)]-bis-
1,4,8,11-tetra-azacyclotetradecane or a variant thereof, may be administered to a subject in
amounts sufficient to mobilize hematopoietic stem and progenitor cells. Significantly, the
compositions and methods described herein may be used to mobilize hematopoietic stem and
progenitor cells from a stem cell niche within a donor, such as a human donor, into the
circulating peripheral blood of the donor while reducing the mobilization of other cells of the
hematopoietic lineage, such as white blood cells, neutrophils, lymphocytes, and monocytes.
The compositions and methods described herein thus enable the selective mobilization of
hematopoietic stem and progenitor cells in a donor, which may then be isolated from a donor
for therapeutic use.
[0007] In some embodiments, the hematopoietic stem or progenitor cells may be mobilized
from the bone marrow of the donor to the peripheral blood, from which the hematopoietic
stem or progenitor cells may be collected and/or isolated. Upon collection of the mobilized
cells, the withdrawn hematopoietic stem or progenitor cells may then be infused into a
WO wo 2019/113375 PCT/US2018/064335
patient, which may be the donor or another subject, such as a subject that is HLA-matched to
the donor, for the treatment of one or more pathologies of the hematopoietic system. In some
embodiments, the withdrawn hematopoietic stem or progenitor cells are first expanded ex
vivo prior to infusion of these cells, and/or progeny thereof, into the patient. The
compositions and methods described herein provide the important clinical benefit of enabling
the production of populations of cells that are enriched in hematopoietic stem cells relative to
other cell types, such as leukocytes, neutrophils, and monocytes. Thus, the populations of
mobilized hematopoietic stem and progenitor cells produced using the compositions and
methods described herein are particularly suitable for hematopoietic stem cell transplantation
therapy, optionally preceded by ex vivo expansion in order to increase the quantity of
hematopoietic stem and progenitor cells available for infusion into a patient.
[0008] Further, the methods described herein provide the advantage of inhibiting
leukocytosis in the donor. Leukocytosis may lead to adverse effects such as splenic rupture,
renal dysfunction, acute febrile noninfectious pneumonitis (i.e., pulmonary toxicity),
cardiovascular toxicity (e.g., hypercoagulation, heart attack, dyspnea, angina, arrhythmia,
atherosclerotic plaque rupture due to proinflammatory actions related o high neutrophil
counts), neurological disturbances (e.g., blurred vision, headache, and retinal hemorrhage),
and sickle cell crisis. See, e.g., D'Souza et al. (2008) Transfusion Medicine Reviews
22(4):280-290.
[0009] As described herein, hematopoietic stem cells are capable of differentiating into a
multitude of cell types in the hematopoietic lineage and can thus be administered to a patient
in order to populate or repopulate a cell type that is defective or deficient in the patient. The
patient may be one, for example, that is suffering from one or more blood disorders, such as
an autoimmune disease, cancer, hemoglobinopathy, or other hematopoietic pathology, and is
therefore in need of hematopoietic stem cell transplantation. The invention thus provides
methods of treating a variety of hematopoietic conditions, such as sickle cell anemia,
thalassemia, Fanconi anemia, Wiskott-Aldrich syndrome, adenosine deaminase deficiency-
severe combined immunodeficiency, metachromatic leukodystrophy, Diamond-Blackfan
anemia and Schwachman-Diamond syndrome, human immunodeficiency virus infection, and
acquired immune deficiency syndrome, as well as cancers and autoimmune diseases, among
others.
WO wo 2019/113375 PCT/US2018/064335
[0010] In a first aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to produce a population of cells having a ratio of
CD34+ cellsto CD34 cells toleukocytes leukocytesof offrom fromabout about0.0008 0.0008to toabout about0.0021 0.0021in inaasample sampleof ofperipheral peripheral
blood of the donor following administration of the CXCR2 agonist and CXCR4 antagonist.
In some embodiments, the ratio of CD34+ cells to CD34 cells to leukocytes leukocytes in in the the sample sample may may be be about about
0.00080, 0.00081, 0.00082, 0.00083, 0.00084, 0.00085, 0.00086, 0.00087, 0.00088, 0.00089,
0.00090, 0.00091, 0.00092, 0.00093, 0.00094, 0.00095, 0.00096, 0.00097, 0.00098, 0.00099,
0.00100, 0.00101, 0.00102, 0.00103, 0.00104, 0.00105, 0.00106, 0.00107, 0.00108, 0.00109,
0.00110, 0.00111, 0.00112, 0.00113, 0.00114, 0.00115, 0.00116, 0.00117, 0.00118, 0.00119,
0.00120, 0.00121, 0.00122, 0.00123, 0.00124, 0.00125, 0.00126, 0.00127, 0.00128, 0.00129,
0.00130, 0.00131, 0.00132, 0.00133, 0.00134, 0.00135, 0.00136, 0.00137, 0.00138, 0.00139,
0.00140, 0.00141, 0.00142, 0.00143, 0.00144, 0.00145, 0.00146, 0.00147, 0.00148, 0.00149,
0.00150, 0.00151, 0.00152, 0.00153, 0.00154, 0.00155, 0.00156, 0.00157, 0.00158, 0.00159,
0.00160, 0.00161, 0.00162, 0.00163, 0.00164, 0.00165, 0.00166, 0.00167, 0.00168, 0.00169,
0.00170, 0.00171, 0.00172, 0.00173, 0.00174, 0.00175, 0.00176, 0.00178, 0.00179, 0.00180,
0.00181, 0.00182, 0.00183, 0.00184, 0.00185, 0.00186, 0.00187, 0.00188, 0.00189, 0.00190,
0.00191, 0.00192, 0.00193, 0.00194, 0.00195, 0.00196, 0.00197, 0.00198, 0.00199, 0.00200,
0.00201, 0.00202, 0.00203, 0.00204, 0.00205, 0.00206, 0.00207, 0.00208, 0.00209, 0.00210,
0.00211, 0.00212, 0.00213, 0.00214, 0.00215, 0.00216, 0.00217, 0.00218, 0.00219, 0.00220,
0.00221, 0.00222, 0.00223, 0.00224, or 0.00225. In some embodiments, the ratio of CD34+ CD34
cells to leukocytes in the sample is from about 0.0009 to about 0.002, about 0.001 to about
0.0019, about 0.0011 to about 0.0018, about 0.0012 to about 0.0017, about 0.0013 to about
0.0016, or about 0.0014 to about 0.0015. In some embodiments, the ratio of CD34 cells to
leukocytes in the sample is from about 0.0010 to about 0.0018, such as a ratio of
hematopoietic stem cells to leukocytes in the sample of about 0.00100, 0.00101, 0.00102,
0.00103, 0.00104, 0.00105, 0.00106, 0.00107, 0.00108, 0.00109, 0.00110, 0.00111, 0.00112,
0.00113, 0.00114, 0.00115, 0.00116, 0.00117, 0.00118, 0.00119, 0.00120, 0.00121, 0.00122,
0.00123, 0.00124, 0.00125, 0.00126, 0.00127, 0.00128, 0.00129, 0.00130, 0.00131, 0.00132,
0.00133, 0.00134, 0.00135, 0.00136, 0.00137, 0.00138, 0.00139, 0.00140, 0.00141, 0.00142,
0.00143, 0.00144, 0.00145, 0.00146, 0.00147, 0.00148, 0.00149, 0.00150, 0.00151, 0.00152,
0.00153, 0.00154, 0.00155, 0.00156, 0.00157, 0.00158, 0.00159, 0.00160, 0.00161, 0.00162,
WO wo 2019/113375 PCT/US2018/064335
0.00163, 0.00164, 0.00165, 0.00166, 0.00167, 0.00168, 0.00169, 0.00170, 0.00171, 0.00172,
0.00173, 0.00174, 0.00175, 0.00176, 0.00178, 0.00179, or 0.00180. In some embodiments,
the the ratio ratioofofCD34+ CD34cells to to cells leukocytes in the leukocytes in sample is fromisabout the sample from0.0012 aboutto0.0012 about 0.0016, to about 0.0016,
such as a ratio of CD34 cells to leukocytes in the sample of about 0.00120, 0.00121,
0.00122, 0.00123, 0.00124, 0.00125, 0.00126, 0.00127, 0.00128, 0.00129, 0.00130, 0.00131,
0.00132, 0.00133, 0.00134, 0.00135, 0.00136, 0.00137, 0.00138, 0.00139, 0.00140, 0.00141,
0.00142, 0.00143, 0.00144, 0.00145, 0.00146, 0.00147, 0.00148, 0.00149, 0.00150, 0.00151,
0.00152, 0.00153, 0.00154, 0.00155, 0.00156, 0.00157, 0.00158, 0.00159, or 0.00160. In
some embodiments, the ratio of CD34+ cellsto CD34 cells toleukocytes leukocytesin inthe thesample sampleis isabout about0.0014. 0.0014.
[0011] In another aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to enrich the peripheral blood of the donor with
CD34+ cells relative CD34 cells relative to to leukocytes leukocytes by by aa ratio ratio of of from from about about 3.40:1 3.40:1 to to about about 6.90:1 6.90:1 as as assessed assessed
by comparing a sample of peripheral blood of the donor following administration of the
CXCR2 agonist and CXCR4 antagonist to a sample of peripheral blood of the donor prior to
administration of the CXCR2 agonist and CXCR4 antagonist. In some embodiments, the
peripheral blood of the donor may be enriched with CD34+ cellsrelative CD34 cells relativeto toleukocytes leukocytesby byaa
ratio of about 3.40:1, 3.45:1, 3.50:1, 3.55:1, 3.60:1, 3.65:1, 3.70:1, 3.75:1, 3.80:1, 3.85:1,
3.90:1, 3.95:1, 4.00:1, 4.05:1, 4.10:1, 4.15:1, 4.20:1, 4.25:1, 4.30:1, 4.35:1, 4.40:1, 4.45:1,
4.50:1, 4.55:1, 4.60:1, 4.65:1, 4.70:1, 4.75:1, 4.80:1, 4.85:1, 4.90:1, 4.95:1, 5.00:1, 5.05:1,
5.10:1, 5.15:1, 5.20:1, 5.25:1, 5.30:1, 5.35:1, 5.40:1, 5.45:1, 5.50:1, 5.55:1, 5.60:1, 5.65:1,
5.70:1, 5.75:1, 5.80:1, 5.85:1, 5.90:1, 5.95:1, 6.00:1, 6.05:1, 6.10:1, 6.15:1, 6.20:1, 6.25:1,
6.30:1, 6.35:1, 6.40:1, 6.45:1, 6.50:1, 6.55:1, 6.60:1, 6.65:1, 6.70:1, 6.75:1, 6.80:1, 6.85:1, or
6.90:11. In some embodiments, the peripheral blood of the donor is enriched with CD34+
cells relative to leukocytes by a ratio of from about 3.5:1 to about 6.8:1, about 3.6:1 to about
6.7:1, about 3.8:1 to about 6.6:1, about 3.9:1 to about 6.5:1, about 4:1 to about 6.4:1, about
4.1:1 to about 6.3:1, about 4.2:1 to about 6.2:1, about 4.3:1 to about 6.1:1, about 4.4:1 to
about 6:1, about 4.5:1 to about 6:1, about 4.6:1 to about 5.9:1, about 4.7:1 to about 5.8:1, or
about 4.8:1 to about 5.7:1. In some embodiments, the peripheral blood of the donor is
enriched enrichedwith withCD34+ CD34cells relative cells to leukocytes relative by a ratio to leukocytes by a of aboutoffrom ratio about about 4.0:1 from to about about 4.0:1 to about
6.0:1, such as a ratio of about 4.00:1, 4.05:1, 4.10:1, 4.15:1, 4.20:1, 4.25:1, 4.30:1, 4.35:1,
4.40:1, 4.45:1, 4.50:1, 4.55:1, 4.60:1, 4.65:1, 4.70:1, 4.75:1, 4.80:1, 4.85:1, 4.90:1, 4.95:1,
WO wo 2019/113375 PCT/US2018/064335
5.00:1, 5.05:1, 5.10:1, 5.15:1, 5.20:1, 5.25:1, 5.30:1, 5.35:1, 5.40:1, 5.45:1, 5.50:1, 5.55:1,
5.60:1, 5.65:1, 5.70:1, 5.75:1, 5.80:1, 5.85:1, 5.90:1, 5.95:1, or 6.00:1. In some
embodiments, the peripheral blood of the donor is enriched with CD34+ cellsrelative CD34 cells relativeto to
leukocytes by a ratio of about from about 4.5:1 to about 5.5:1, such as a ratio of about 4.50:1,
4.55:1,4.60:1, 4.55:1, 4.60:1,4.65:1, 4.65:1,4.70:1, 4.70:1,4.75:1, 4.75:1,4.80:1, 4.80:1,4.85:1, 4.85:1,4.90:1, 4.90:1,4.95:1, 4.95:1,5.00:1, 5.00:1,5.05:1, 5.05:1,5.10:1, 5.10:1,
5.15:1, 5.20:1, 5.25:1, 5.30:1, 5.35:1, 5.40:1, 5.45:1, or 5.50. In some embodiments, the
peripheral blood of the donor is enriched with CD34+ cells relative CD34 cells relative to to leukocytes leukocytes by by aa ratio ratio of of
about 5.1:1.
[0012] In another aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density
of of CD34+ CD34 cells cells ofofatatleast about least 38,000 about cells/ml, 38,000 such as cells/ml, a density such of CD34+ of as a density cells of cells CD34 from of from
about 38,000 cells/ml to about 100,000 cells/ml, about 40,000 cells/ml to about 90,000
cells/ml, about 50,000 cells/ml to about 80,000 cells/ml, or about 60,000 cells/ml to about
70,000 cells/ml (e.g., about 38,00 cells/ml, 39,000 cells/ml, 40,000 cells/ml, 41,000 cells/ml,
42,000 cells/ml, 43,000 cells/ml, 44,000 cells/ml, 45,000 cells/ml, 46,000 cells/ml, 47,000
cells/ml, 48,000 cells/ml, 49,000 cells/ml, 50,000 cells/ml, 51,000 cells/ml, 52,000 cells/ml,
53,000 cells/ml, 54,000 cells/ml, 55,000 cells/ml, 56,000 cells/ml, 57,000 cells/ml, 58,000
cells/ml, 59,000 cells/ml, 60,000 cells/ml, 61,000 cells/ml, 62,000 cells/ml, 63,000 cells/ml,
64,000 cells/ml, 65,000 cells/ml, 66,000 cells/ml, 67,000 cells/ml, 68,000 cells/ml, 69,000
cells/ml, 70,000 cells/ml, 71,000 cells/ml, 72,000 cells/ml, 73,000 cells/ml, 74,000 cells/ml,
75,000 cells/ml, 76,000 cells/ml, 77,000 cells/ml, 78,000 cells/ml, 79,000 cells/ml, 80,000
cells/ml, 81,000 cells/ml, 82,000 cells/ml, 83,000 cells/ml, 84,000 cells/ml, 85,000 cells/ml,
86,000 cells/ml, 87,000 cells/ml, 88,000 cells/ml, 89,000 cells/ml, 90,000 cells/ml, 91,000
cells/ml, 92,000 cells/ml, 93,000 cells/ml, 94,000 cells/ml, 95,000 cells/ml, 96,000 cells/ml,
97,000 cells/ml, 98,000 cells/ml, 99,000 cells/ml, 100,000 cells/ml, or more), and having a
density densityofofleukocytes of no leukocytes of more than than no more about about 5.3 X 107 5.3 cells/ml, such as a X 10 cells/ml, density such as aofdensity of
leukocytes leukocytesofof about 2.32.3 about X 107 cells/ml X 10 to about cells/ml 5.3 X 5.3 to about 107 cells/ml, about 2.5 X 10 cells/ml, x 1072.5 about cells/ml to x 10 cells/ml to
about about 5.1 5.1X X107 10cells/ml, cells/ml,2.92.9 x 107 cells/ml x 10 to about cells/ml 4.5 107 to about 4.5cells/ml, about 3 X about X 10 cells/ml, 107 cells/ml 3 x 10 cells/ml
to about 4x 4 X10 10cells/ml cells/ml(e.g., (e.g.,5.3 5.3x X107 10 cells/ml, 5.2 x X 107 cells/ml, 5.1 10 cells/ml, 5.1 Xx 10 107 cells/ml, cells/ml, 5 5 x x 10107
cells/ml, 4.9 x 107 cells/ml, 4.8 10 cells/ml, 4.8 xx 10 107 cells/ml, cells/ml, 4.7 4.7 x x 10107 cells/ml, cells/ml, 4.64.6x10 cells/ml, 4.5 X 10 cells/ml, 4.5 xx 10 107
cells/ml, 4.4x 4.4 X107 10 cells/ml, 4.3 X 107 cells/ml4.2 10 cells/ml 4.2Xx10 10cells/ml, cells/ml,4.1 4.1Xx10 107 cells/ml cells/ml 4 4x 107 x 10
WO wo 2019/113375 PCT/US2018/064335
cells/ml, 3.9 x X 107 cells/ml, 3.8 10 cells/ml, 3.8 xx 10 107 cells/ml, cells/ml, 3.7 3.7 x x 10107 cells/ml, cells/ml, 3.63.6x107 cells/ml, x 10 cells/ml, 3.5 3.5 x x 10107
cells/ml, 3.4x10' 3.4 X 10cells/ml, cells/ml,3.3 3.3x X107 10 cells/ml, 3.2x1 3.2 X 107 cells/ml, 3.1 10 cells/ml, 3.1 Xx 10 107 cells/ml, cells/ml, 3 3 X x 10107
cells/ml, 2.9 x X 107 cells/ml, 2.8 10 cells/ml, 2.8 Xx 10 107 cells/ml, cells/ml, 2.7 2.7 X x 10107 cells/ml, cells/ml, 2.62.6xx107 cells/ml, x 10 cells/ml, 2.52.5 X 107 x 10
cells/ml, cells/ml,2.4 2.4x 107 x 10cells/ml, 2.3 2.3 cells/ml, X 107 X cells/ml, or less). 10 cells/ml, In someInembodiments, or less). the methodthe method some embodiments,
includes administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts
sufficient sufficienttotoproduce a population produce of cells a population having having of cells a density of CD34+ of a density cells of cells CD34 from about of from about
38,000 cells/ml to about 100,000 cells/ml, and having a density of leukocytes of from about
2.3 X x 107 cells/ml to 10 cells/ml to about about 5.3 5.3 XX 10 107 cells/ml. cells/ml. InIn some some embodiments, embodiments, the the method method includes includes
administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient
to produce a population of cells having a density of CD34+ cells of CD34 cells of from from about about 40,000 40,000
cells/ml cells/mltotoabout 80,000 about cells/ml, 80,000 and having cells/ml, a density and having of leukocytes a density of from about of leukocytes 2.5 X about of from 107 2.5 X 10
cells/ml to about 107 5 X cells/ml. In some 10 cells/ml. embodiments, In some the the embodiments, method includes method administering includes administering
to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a
population of cells having a density of CD34+ cells of CD34 cells of from from about about 50,000 50,000 cells/ml cells/ml to to about about
90,000 90,000 cells/ml, cells/ml,andand having a density having of leukocytes a density of from of of leukocytes about 3 Xabout from 107 cells/ml 3 X 10 to about 4 to about 4 cells/ml
107 cells/ml. x 10 cells/ml.
[0013] In a further aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to produce a population of cells having a ratio of
CD34+ cells to CD34 cells to neutrophils neutrophils of of from from about about 0.0018 0.0018 to to about about 0.0058 0.0058 in in aa sample sample of of peripheral peripheral
blood of the donor following administration of the CXCR2 agonist and CXCR4 antagonist.
In some embodiments, the ratio of CD34+ cells to CD34 cells to neutrophils neutrophils in in the the sample sample may may be be about about
0.00180, 0.00181, 0.00182, 0.00183, 0.00184, 0.00185, 0.00186, 0.00187, 0.00188, 0.00189,
0.00190, 0.00191, 0.00192, 0.00193, 0.00194, 0.00195, 0.00196, 0.00197, 0.00198, 0.00199,
0.00200, 0.00201, 0.00202, 0.00203, 0.00204, 0.00205, 0.00206, 0.00207, 0.00208, 0.00209,
0.00210, 0.00211, 0.00212, 0.00213, 0.00214, 0.00215, 0.00216, 0.00217, 0.00218, 0.00219,
0.00220, 0.00221, 0.00222, 0.00223, 0.00224, 0.00225, 0.00226, 0.00227, 0.00228, 0.00229,
0.00230, 0.00231, 0.00232, 0.00233, 0.00234, 0.00235, 0.00236, 0.00237, 0.00238, 0.00239,
0.00240, 0.00241, 0.00242, 0.00243, 0.00244, 0.00245, 0.00246, 0.00247, 0.00248, 0.00249,
0.00250, 0.00251, 0.00252, 0.00253, 0.00254, 0.00255, 0.00256, 0.00257, 0.00258, 0.00259,
0.00260, 0.00261, 0.00262, 0.00263, 0.00264, 0.00265, 0.00266, 0.00267, 0.00268, 0.00269,
0.00270, 0.00271, 0.00272, 0.00273, 0.00274, 0.00275, 0.00276, 0.00277, 0.00278, 0.00279,
0.00280, 0.00281, 0.00282, 0.00283, 0.00284, 0.00285, 0.00286, 0.00287, 0.00288, 0.00289,
0.00290, 0.00291, 0.00292, 0.00293, 0.00294, 0.00295, 0.00296, 0.00297, 0.00298, 0.00299,
0.00300, 0.00300, 0.00301, 0.00302, 0.00303, 0.00304, 0.00305, 0.00306, 0.00307, 0.00308,
0.00309, 0.00310, 0.00311, 0.00312, 0.00313, 0.00314, 0.00315, 0.00316, 0.00317, 0.00318,
0.00319, 0.00320, 0.00321, 0.00322, 0.00323, 0.00324, 0.00325, 0.00326, 0.00327, 0.00328,
0.00329, 0.00330, 0.00331, 0.00332, 0.00333, 0.00334, 0.00335, 0.00336, 0.00337, 0.00338,
0.00339, 0.00340, 0.00341, 0.00342, 0.00343, 0.00344, 0.00345, 0.00346, 0.00347, 0.00348,
0.00349, 0.00350, 0.00351, 0.00352, 0.00353, 0.00354, 0.00355, 0.00356, 0.00357, 0.00358,
0.00359, 0.00360, 0.00361, 0.00362, 0.00363, 0.00364, 0.00365, 0.00366, 0.00367, 0.00368,
0.00369, 0.00370, 0.00371, 0.00372, 0.00373, 0.00374, 0.00375, 0.00376, 0.00377, 0.00378,
0.00379, 0.00380, 0.00381, 0.00382, 0.00383, 0.00384, 0.00385, 0.00386, 0.00387, 0.00388,
0.00389, 0.00390, 0.00391, 0.00392, 0.00393, 0.00394, 0.00395, 0.00396, 0.00397, 0.00398,
0.00399, 0.00400, 0.00401, 0.00402, 0.00403, 0.00404, 0.00405, 0.00406, 0.00407, 0.00408,
0.00409, 0.00410, 0.00411, 0.00412, 0.00413, 0.00414, 0.00415, 0.00416, 0.00417, 0.00418,
0.00419, 0.00420, 0.00421, 0.00422, 0.00423, 0.00424, 0.00425, 0.00426, 0.00427, 0.00428,
0.00429, 0.00430, 0.00431, 0.00432, 0.00433, 0.00434, 0.00435, 0.00436, 0.00437, 0.00438,
0.00439, 0.00440, 0.00441, 0.00442, 0.00443, 0.00444, 0.00445, 0.00446, 0.00447, 0.00448,
0.00449, 0.00450, 0.00451, 0.00452, 0.00453, 0.00454, 0.00455, 0.00456, 0.00457, 0.00458,
0.00459, 0.00460, 0.00461, 0.00462, 0.00463, 0.00464, 0.00465, 0.00466, 0.00467, 0.00468,
0.00469, 0.00470, 0.00471, 0.00472, 0.00473, 0.00474, 0.00475, 0.00476, 0.00477, 0.00478,
0.00479, 0.00480, 0.00481, 0.00482, 0.00483, 0.00484, 0.00485, 0.00486, 0.00487, 0.00488,
0.00489, 0.00490, 0.00491, 0.00492, 0.00493, 0.00494, 0.00495, 0.00496, 0.00497, 0.00498,
0.00499, 0.00500, 0.00501, 0.00502, 0.00503, 0.00504, 0.00505, 0.00506, 0.00507, 0.00508,
0.00509, 0.00510, 0.00511, 0.00512, 0.00513, 0.00514, 0.00515, 0.00516, 0.00517, 0.00518,
0.00519, 0.00519, 0.00520, 0.00520, 0.00521, 0.00521, 0.00522, 0.00522, 0.00523, 0.00523, 0.00524, 0.00524, 0.00525, 0.00525, 0.00526, 0.00526, 0.00527, 0.00527, 0.00528, 0.00528,
0.00529, 0.00530, 0.00531, 0.00532, 0.00533, 0.00534, 0.00535, 0.00536, 0.00537, 0.00538,
0.00539, 0.00540, 0.00541, 0.00542, 0.00543, 0.00544, 0.00545, 0.00546, 0.00547, 0.00548,
0.00549, 0.00550, 0.00551, 0.00552, 0.00553, 0.00554, 0.00555, 0.00556, 0.00557, 0.00558,
0.00559, 0.00560, 0.00561, 0.00562, 0.00563, 0.00564, 0.00565, 0.00566, 0.00567, 0.00568,
0.00569, 0.00570, 0.00571, 0.00572, 0.00573, 0.00574, 0.00575, 0.00576, 0.00577, 0.00578,
0.00579, or 0.00580. In some embodiments, the ratio of CD34+ cells to CD34 cells to neutrophils neutrophils in in the the
sample is from about 0.002 to about 0.0056, about 0.0022 to about 0.0054, about 0.0024 to
about 0.0052, about 0.0026 to about 0.005, about 0.0028 to about 0.0048, or about 0.003 to
WO wo 2019/113375 PCT/US2018/064335
about 0.0046. In some embodiments, the ratio of CD34+ cells to CD34 cells to neutrophils neutrophils in in the the sample sample is is
from from about about0.0026 to to 0.0026 about 0.0046, about such as 0.0046, a ratio such as a of CD34+ofcells ratio CD34tocells neutrophils in the to neutrophils in the
sample of about 0.00260, 0.00261, 0.00262, 0.00263, 0.00264, 0.00265, 0.00266, 0.00267,
0.00268, 0.00269, 0.00270, 0.00271, 0.00272, 0.00273, 0.00274, 0.00275, 0.00276, 0.00277,
0.00278, 0.00279, 0.00280, 0.00281, 0.00282, 0.00283, 0.00284, 0.00285, 0.00286, 0.00287,
0.00288, 0.00289, 0.00290, 0.00291, 0.00292, 0.00293, 0.00294, 0.00295, 0.00296, 0.00297,
0.00298, 0.00299, 0.00300, 0.00300, 0.00301, 0.00302, 0.00303, 0.00304, 0.00305, 0.00306,
0.00307, 0.00308, 0.00309, 0.00310, 0.00311, 0.00312, 0.00313, 0.00314, 0.00315, 0.00316,
0.00317, 0.00318, 0.00319, 0.00320, 0.00321, 0.00322, 0.00323, 0.00324, 0.00325, 0.00326,
0.00327, 0.00328, 0.00329, 0.00330, 0.00331, 0.00332, 0.00333, 0.00334, 0.00335, 0.00336,
0.00337, 0.00338, 0.00339, 0.00340, 0.00341, 0.00342, 0.00343, 0.00344, 0.00345, 0.00346,
0.00347, 0.00348, 0.00349, 0.00350, 0.00351, 0.00352, 0.00353, 0.00354, 0.00355, 0.00356,
0.00357, 0.00358, 0.00359, 0.00360, 0.00361, 0.00362, 0.00363, 0.00364, 0.00365, 0.00366,
0.00367, 0.00368, 0.00369, 0.00370, 0.00371, 0.00372, 0.00373, 0.00374, 0.00375, 0.00376,
0.00377, 0.00378, 0.00379, 0.00380, 0.00381, 0.00382, 0.00383, 0.00384, 0.00385, 0.00386,
0.00387, 0.00388, 0.00389, 0.00390, 0.00391, 0.00392, 0.00393, 0.00394, 0.00395, 0.00396,
0.00397, 0.00398, 0.00399, 0.00400, 0.00401, 0.00402, 0.00403, 0.00404, 0.00405, 0.00406,
0.00407, 0.00408, 0.00409, 0.00410, 0.00411, 0.00412, 0.00413, 0.00414, 0.00415, 0.00416,
0.00417, 0.00418, 0.00419, 0.00420, 0.00421, 0.00422, 0.00423, 0.00424, 0.00425, 0.00426,
0.00427, 0.00428, 0.00429, 0.00430, 0.00431, 0.00432, 0.00433, 0.00434, 0.00435, 0.00436,
0.00437, 0.00438, 0.00439, 0.00440, 0.00441, 0.00442, 0.00443, 0.00444, 0.00445, 0.00446,
0.00447, 0.00448, 0.00449, 0.00450, 0.00451, 0.00452, 0.00453, 0.00454, 0.00455, 0.00456,
0.00457, 0.00458, 0.00459, or 0.00460. In some embodiments, the ratio of CD34+ cells to CD34 cells to
neutrophils in the sample is about 0.0036.
[0014] In an additional aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to enrich the peripheral blood of the donor with
CD34+ cells relative CD34 cells relative to to neutrophils neutrophils by by aa ratio ratio of of from from about about 2.1:1 2.1:1 to to about about 8.1:1 8.1:1 as as assessed assessed
by comparing a sample of peripheral blood of the donor following administration of the
CXCR2 agonist and CXCR4 antagonist to a sample of peripheral blood of the donor prior to
administration of the CXCR2 agonist and CXCR4 antagonist. In some embodiments, the
peripheral blood of the donor may be enriched with CD34+ cells relative CD34 cells relative to to neutrophils neutrophils by by aa ratio of about 2.10:1, 2.15:1, 2.20:1, 2.25:1, 2.30:1, 2.35:1, 2.40:1, 2.45:1, 2.50:1, 2.55:1,
2.60:1, 2.65:1, 2.70:1, 2.75:1, 2.80:1, 2.85:1, 2.90:1, 2.95:1, 3.00:1, 3.05:1, 3.10:1, 3.15:1,
3.20:1, 3.25:1, 3.30:1, 3.35:1, 3.40:1, 3.45:1, 3.50:1, 3.55:1, 3.60:1, 3.65:1, 3.70:1, 3.75:1,
3.80:1, 3.85:1, 3.90:1, 3.95:1, 4.00:1, 4.05:1, 4.10:1, 4.15:1, 4.20:1, 4.25:1, 4.30:1, 4.35:1,
4.40:1, 4.45:1, 4.50:1, 4.55:1, 4.60:1, 4.65:1, 4.70:1, 4.75:1, 4.80:1, 4.85:1, 4.90:1, 4.95:1,
5.00:1, 5.05:1, 5.10:1, 5.15:1, 5.20:1, 5.25:1, 5.30:1, 5.35:1, 5.40:1, 5.45:1, 5.50:1, 5.55:1,
5.60:1, 5.65:1, 5.70:1, 5.75:1, 5.80:1, 5.85:1, 5.90:1, 5.95:1, 6.00:1, 6.05:1, 6.10:1, 6.15:1,
6.20:1, 6.25:1, 6.30:1, 6.35:1, 6.40:1, 6.45:1, 6.50:1, 6.55:1, 6.60:1, 6.65:1, 6.70:1, 6.75:1,
6.80:1, 6.85:1, 6.90:1, 6.95:1, 7.00:1, 7.05:1, 7.10:1, 7.15:1, 7.20:1, 7.25:1, 7.30:1, 7.35:1,
7.40:1, 7.45:1, 7.50:1, 7.55:1, 7.60:1, 7.65:1, 7.70:1, 7.75:1, 7.80:1, 7.85:1, 7.90:1, 7.95:1, or
8.00:1. In some embodiments, the peripheral blood of the donor is enriched with CD34+ cells CD34 cells
relative to neutrophils by a ratio of from about 2.5:1 to about 7:1, about 2.6:1 to about 6.9:1,
about 2.7:1 to about 6.8:1, about 2.8:1 to about 6.7:1, about 2.9:1 to about 6.6:1, about 3:1 to
about 6.5:1, about 3.2:1 to about 6.4:1, about 3.3:1 to about 6.3:1, about 3.4:1 to about 6.2:1,
or about 3.5:1 to about 6.1:1 In some embodiments, the peripheral blood of the donor is
enriched enrichedwith withCD34+ CD34cells relative cells to neutrophils relative by a ratio to neutrophils by a of from of ratio about from from aboutfrom about 5.4:1about to 5.4:1 to
about 7.4:1, such as a ratio of about 5.40:1, 5.45:1, 5.50:1, 5.55:1, 5.60:1, 5.65:1, 5.70:1,
5.75:1, 5.80:1, 5.85:1, 5.90:1, 5.95:1, 6.00:1, 6.05:1, 6.10:1, 6.15:1, 6.20:1, 6.25:1, 6.30:1,
6.35:1, 6.40:1, 6.45:1, 6.50:1, 6.55:1, 6.60:1, 6.65:1, 6.70:1, 6.75:1, 6.80:1, 6.85:1, 6.90:1,
6.95:1, 7.00:1, 7.05:1, 7.10:1, 7.15:1, 7.20:1, 7.25:1, 7.30:1, 7.35:1, or 7.40:1. In some
CD34 cells embodiments, the peripheral blood of the donor is enriched with CD34+ cells relative relative to to
neutrophils by a ratio of about 6.4:1.
[0015] In
[0015] Inyet yetanother aspect, another the invention aspect, features the invention a methoda of features mobilizing method a population of mobilizing a of population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to produce a population of cells having a ratio of
CD34+ cells to CD34 cells to lymphocytes lymphocytes of of from from about about 0.0021 0.0021 to to about about 0.0094 0.0094 in in aa sample sample of of peripheral peripheral
blood of the donor following administration of the CXCR2 agonist and CXCR4 antagonist.
CD34 cells In some embodiments, the ratio of CD34+ cells to to lymphocytes lymphocytes in in the the sample sample may may be be about about
0.00210, 0.00211, 0.00212, 0.00213, 0.00214, 0.00215, 0.00216, 0.00217, 0.00218, 0.00219,
0.00220, 0.00221, 0.00222, 0.00223, 0.00224, 0.00225, 0.00226, 0.00227, 0.00228, 0.00229,
0.00230, 0.00231, 0.00232, 0.00233, 0.00234, 0.00235, 0.00236, 0.00237, 0.00238, 0.00239,
0.00240, 0.00241, 0.00242, 0.00243, 0.00244, 0.00245, 0.00246, 0.00247, 0.00248, 0.00249,
0.00250, 0.00251, 0.00252, 0.00253, 0.00254, 0.00255, 0.00256, 0.00257, 0.00258, 0.00259,
0.00260, 0.00261, 0.00262, 0.00263, 0.00264, 0.00265, 0.00266, 0.00267, 0.00268, 0.00269,
0.00270, 0.00270, 0.00271, 0.00271, 0.00272, 0.00272, 0.00273, 0.00273, 0.00274, 0.00274, 0.00275, 0.00275, 0.00276, 0.00276, 0.00277, 0.00277, 0.00278, 0.00278, 0.00279, 0.00279,
0.00280, 0.00281, 0.00282, 0.00283, 0.00284, 0.00285, 0.00286, 0.00287, 0.00288, 0.00289,
0.00290, 0.00291, 0.00292, 0.00293, 0.00294, 0.00295, 0.00296, 0.00297, 0.00298, 0.00299,
0.00300, 0.00300, 0.00301, 0.00302, 0.00303, 0.00304, 0.00305, 0.00306, 0.00307, 0.00308,
0.00309, 0.00310, 0.00311, 0.00312, 0.00313, 0.00314, 0.00315, 0.00316, 0.00317, 0.00318,
0.00319, 0.00320, 0.00321, 0.00322, 0.00323, 0.00324, 0.00325, 0.00326, 0.00327, 0.00328,
0.00329, 0.00330, 0.00331, 0.00332, 0.00333, 0.00334, 0.00335, 0.00336, 0.00337, 0.00338,
0.00339, 0.00340, 0.00341, 0.00342, 0.00343, 0.00344, 0.00345, 0.00346, 0.00347, 0.00348,
0.00349, 0.00350, 0.00351, 0.00352, 0.00353, 0.00354, 0.00355, 0.00356, 0.00357, 0.00358,
0.00359, 0.00360, 0.00361, 0.00362, 0.00363, 0.00364, 0.00365, 0.00366, 0.00367, 0.00368,
0.00369, 0.00370, 0.00371, 0.00372, 0.00373, 0.00374, 0.00375, 0.00376, 0.00377, 0.00378,
0.00379, 0.00379, 0.00380, 0.00380, 0.00381, 0.00381, 0.00382, 0.00382, 0.00383, 0.00383, 0.00384, 0.00384, 0.00385, 0.00385, 0.00386, 0.00386, 0.00387, 0.00387, 0.00388, 0.00388,
0.00389, 0.00390, 0.00391, 0.00392, 0.00393, 0.00394, 0.00395, 0.00396, 0.00397, 0.00398,
0.00399, 0.00400, 0.00401, 0.00402, 0.00403, 0.00404, 0.00405, 0.00406, 0.00407, 0.00408,
0.00409, 0.00410, 0.00411, 0.00412, 0.00413, 0.00414, 0.00415, 0.00416, 0.00417, 0.00418,
0.00419, 0.00420, 0.00421, 0.00422, 0.00423, 0.00424, 0.00425, 0.00426, 0.00427, 0.00428,
0.00429, 0.00430, 0.00431, 0.00432, 0.00433, 0.00434, 0.00435, 0.00436, 0.00437, 0.00438,
0.00439, 0.00440, 0.00441, 0.00442, 0.00443, 0.00444, 0.00445, 0.00446, 0.00447, 0.00448,
0.00449, 0.00450, 0.00451, 0.00452, 0.00453, 0.00454, 0.00455, 0.00456, 0.00457, 0.00458,
0.00459, 0.00460, 0.00461, 0.00462, 0.00463, 0.00464, 0.00465, 0.00466, 0.00467, 0.00468,
0.00469, 0.00470, 0.00471, 0.00472, 0.00473, 0.00474, 0.00475, 0.00476, 0.00477, 0.00478,
0.00479, 0.00480, 0.00481, 0.00482, 0.00483, 0.00484, 0.00485, 0.00486, 0.00487, 0.00488,
0.00489, 0.00490, 0.00491, 0.00492, 0.00493, 0.00494, 0.00495, 0.00496, 0.00497, 0.00498,
0.00499, 0.00500, 0.00501, 0.00502, 0.00503, 0.00504, 0.00505, 0.00506, 0.00507, 0.00508,
0.00509, 0.00509, 0.00510, 0.00510, 0.00511, 0.00511, 0.00512, 0.00512, 0.00513, 0.00513, 0.00514, 0.00514, 0.00515, 0.00515, 0.00516, 0.00516, 0.00517, 0.00517, 0.00518, 0.00518,
0.00519, 0.00520, 0.00521, 0.00522, 0.00523, 0.00524, 0.00525, 0.00526, 0.00527, 0.00528,
0.00529, 0.00530, 0.00531, 0.00532, 0.00533, 0.00534, 0.00535, 0.00536, 0.00537, 0.00538,
0.00539, 0.00540, 0.00541, 0.00542, 0.00543, 0.00544, 0.00545, 0.00546, 0.00547, 0.00548,
0.00549, 0.00550, 0.00551, 0.00552, 0.00553, 0.00554, 0.00555, 0.00556, 0.00557, 0.00558,
0.00559, 0.00560, 0.00561, 0.00562, 0.00563, 0.00564, 0.00565, 0.00566, 0.00567, 0.00568,
0.00569, 0.00570, 0.00571, 0.00572, 0.00573, 0.00574, 0.00575, 0.00576, 0.00577, 0.00578,
0.00579, 0.00580, 0.00581, 0.00582, 0.00583, 0.00584, 0.00585, 0.00586, 0.00587, 0.00588,
0.00589, 0.00590, 0.00591, 0.00592, 0.00593, 0.00594, 0.00595, 0.00596, 0.00597, 0.00598,
0.00599, 0.00600, 0.00601, 0.00602, 0.00603, 0.00604, 0.00605, 0.00606, 0.00607, 0.00608,
0.00609, 0.00610, 0.00611, 0.00612, 0.00613, 0.00614, 0.00615, 0.00616, 0.00617, 0.00618,
0.00619, 0.00620, 0.00621, 0.00622, 0.00623, 0.00624, 0.00625, 0.00626, 0.00627, 0.00628,
0.00629, 0.00630, 0.00631, 0.00632, 0.00633, 0.00634, 0.00635, 0.00636, 0.00637, 0.00638,
0.00639, 0.00640, 0.00641, 0.00642, 0.00643, 0.00644, 0.00645, 0.00646, 0.00647, 0.00648,
0.00649, 0.00650, 0.00651, 0.00652, 0.00653, 0.00654, 0.00655, 0.00656, 0.00657, 0.00658,
0.00659, 0.00660, 0.00661, 0.00662, 0.00663, 0.00664, 0.00665, 0.00666, 0.00667, 0.00668,
0.00669, 0.00670, 0.00671, 0.00672, 0.00673, 0.00674, 0.00675, 0.00676, 0.00677, 0.00678,
0.00679, 0.00680, 0.00681, 0.00682, 0.00683, 0.00684, 0.00685, 0.00686, 0.00687, 0.00688,
0.00689, 0.00689, 0.00690, 0.00690, 0.00691, 0.00691, 0.00692, 0.00692, 0.00693, 0.00693, 0.00694, 0.00694, 0.00695, 0.00695, 0.00696, 0.00696, 0.00697, 0.00697, 0.00698, 0.00698,
0.00699, 0.00700, 0.00701, 0.00702, 0.00703, 0.00704, 0.00705, 0.00706, 0.00707, 0.00708,
0.00709, 0.00709, 0.00710, 0.00710, 0.00711, 0.00711, 0.00712, 0.00712, 0.00713, 0.00713, 0.00714, 0.00714, 0.00715, 0.00715, 0.00716, 0.00716, 0.00717, 0.00717, 0.00718, 0.00718,
0.00719, 0.00720, 0.00721, 0.00722, 0.00723, 0.00724, 0.00725, 0.00726, 0.00727, 0.00728,
0.00729, 0.00730, 0.00731, 0.00732, 0.00733, 0.00734, 0.00735, 0.00736, 0.00737, 0.00738,
0.00739, 0.00740, 0.00741, 0.00742, 0.00743, 0.00744, 0.00745, 0.00746, 0.00747, 0.00748,
0.00749, 0.00750, 0.00751, 0.00752, 0.00753, 0.00754, 0.00755, 0.00756, 0.00757, 0.00758,
0.00759, 0.00760, 0.00761, 0.00762, 0.00763, 0.00764, 0.00765, 0.00766, 0.00767, 0.00768,
0.00769, 0.00770, 0.00771, 0.00772, 0.00773, 0.00774, 0.00775, 0.00776, 0.00777, 0.00778,
0.00779, 0.00780, 0.00781, 0.00782, 0.00783, 0.00784, 0.00785, 0.00786, 0.00787, 0.00788,
0.00789, 0.00790, 0.00791, 0.00792, 0.00793, 0.00794, 0.00795, 0.00796, 0.00797, 0.00798,
0.00799, 0.00800, 0.00801, 0.00802, 0.00803, 0.00804, 0.00805, 0.00806, 0.00807, 0.00808,
0.00809, 0.00810, 0.00811, 0.00812, 0.00813, 0.00814, 0.00815, 0.00816, 0.00817, 0.00818,
0.00819, 0.00819, 0.00820, 0.00820, 0.00821, 0.00821, 0.00822, 0.00822, 0.00823, 0.00823, 0.00824, 0.00824, 0.00825, 0.00825, 0.00826, 0.00826, 0.00827, 0.00827, 0.00828, 0.00828,
0.00829, 0.00830, 0.00831, 0.00832, 0.00833, 0.00834, 0.00835, 0.00836, 0.00837, 0.00838,
0.00839, 0.00840, 0.00841, 0.00842, 0.00843, 0.00844, 0.00845, 0.00846, 0.00847, 0.00848,
0.00849, 0.00850, 0.00851, 0.00852, 0.00853, 0.00854, 0.00855, 0.00856, 0.00857, 0.00858,
0.00859, 0.00860, 0.00861, 0.00862, 0.00863, 0.00864, 0.00865, 0.00866, 0.00867, 0.00868,
0.00869, 0.00870, 0.00871, 0.00872, 0.00873, 0.00874, 0.00875, 0.00876, 0.00877, 0.00878,
0.00879, 0.00880, 0.00881, 0.00882, 0.00883, 0.00884, 0.00885, 0.00886, 0.00887, 0.00888,
0.00889, 0.00890, 0.00891, 0.00892, 0.00893, 0.00894, 0.00895, 0.00896, 0.00897, 0.00898,
0.00899, 0.00900, 0.00901, 0.00902, 0.00903, 0.00904, 0.00905, 0.00906, 0.00907, 0.00908,
WO wo 2019/113375 PCT/US2018/064335 PCT/US2018/064335
0.00909, 0.00910, 0.00911, 0.00912, 0.00913, 0.00914, 0.00915, 0.00916, 0.00917, 0.00918,
0.00919, 0.00920, 0.00921, 0.00922, 0.00923, 0.00924, 0.00925, 0.00926, 0.00927, 0.00928,
0.00929, 0.00930, 0.00931, 0.00932, 0.00933, 0.00934, 0.00935, 0.00936, 0.00937, 0.00938,
0.00939, or 0.00940. In some embodiments, the ratio of CD34+ CD34 cells cells to to lymphocytes lymphocytes in in the the
sample is from about 0.0022 to about 0.0093, about 0.0023 to about 0.0092, about 0.0024 to
about 0.0091, about 0.003 to about 0.0085, about 0.0035 to about 0.0075, or about 0.0045 to
about 0.0065. In some embodiments, the ratio of CD34 cells to lymphocytes in the sample is
from about 0.0025 to about 0.0035, such as a ratio of CD34 cells to lymphocytes in the
sample of about 0.00250, 0.00251, 0.00252, 0.00253, 0.00254, 0.00255, 0.00256, 0.00257,
0.00258, 0.00259, 0.00260, 0.00261, 0.00262, 0.00263, 0.00264, 0.00265, 0.00266, 0.00267,
0.00268, 0.00269, 0.00270, 0.00271, 0.00272, 0.00273, 0.00274, 0.00275, 0.00276, 0.00277,
0.00278, 0.00279, 0.00280, 0.00281, 0.00282, 0.00283, 0.00284, 0.00285, 0.00286, 0.00287,
0.00288, 0.00289, 0.00290, 0.00291, 0.00292, 0.00293, 0.00294, 0.00295, 0.00296, 0.00297,
0.00298, 0.00299, 0.00300, 0.00300, 0.00301, 0.00302, 0.00303, 0.00304, 0.00305, 0.00306,
0.00307, 0.00308, 0.00309, 0.00310, 0.00311, 0.00312, 0.00313, 0.00314, 0.00315, 0.00316,
0.00317, 0.00318, 0.00319, 0.00320, 0.00321, 0.00322, 0.00323, 0.00324, 0.00325, 0.00326,
0.00327, 0.00328, 0.00329, 0.00330, 0.00331, 0.00332, 0.00333, 0.00334, 0.00335, 0.00336,
0.00337, 0.00338, 0.00339, 0.00340, 0.00341, 0.00342, 0.00343, 0.00344, 0.00345, 0.00346,
CD34 cells 0.00347, 0.00348, 0.00349, or 0.00350. In some embodiments, the ratio of CD34+ cells to to
lymphocytes in the sample is about 0.0031.
[0016] In an additional aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to enrich the peripheral blood of the donor with
CD34+ cells relative CD34 cells relativetoto lymphocytes by a by lymphocytes ratio of from a ratio ofabout from 4.8:1 aboutto4.8:1 about to 8.4:1 as assessed about 8.4:1 as assessed
by comparing a sample of peripheral blood of the donor following administration of the
CXCR2 agonist and CXCR4 antagonist to a sample of peripheral blood of the donor prior to
administration of the CXCR2 agonist and CXCR4 antagonist. In some embodiments, the
peripheral blood of the donor may be enriched with CD34+ cellsrelative CD34 cells relativeto tolymphocytes lymphocytesby byaa
ratio of about 4.80:1, 4.85:1, 4.90:1, 4.95:1, 5.00:1, 5.05:1, 5.10:1, 5.15:1, 5.20:1, 5.25:1,
5.30:1, 5.35:1, 5.40:1, 5.45:1, 5.50:1, 5.55:1, 5.60:1, 5.65:1, 5.70:1, 5.75:1, 5.80:1, 5.85:1,
5.90:1, 5.95:1, 6.00:1, 6.05:1, 6.10:1, 6.15:1, 6.20:1, 6.25:1, 6.30:1, 6.35:1, 6.40:1, 6.45:1,
6.50:1, 6.55:1, 6.60:1, 6.65:1, 6.70:1, 6.75:1, 6.80:1, 6.85:1, 6.90:1, 6.95:1, 7.00:1, 7.05:1,
WO wo 2019/113375 PCT/US2018/064335
7.10:1, 7.15:1, 7.20:1, 7.25:1, 7.30:1, 7.35:1, 7.40:1, 7.45:1, 7.50:1, 7.55:1, 7.60:1, 7.65:1,
7.70:1, 7.75:1, 7.80:1, 7.85:1, 7.90:1, 7.95:1, 8.00:1, 8.05:1, 8.10:1, 8.15:1, 8.20:1, 8.25:1,
8.30:1, 8.35:1, or 8.40:1. In some embodiments, the peripheral blood of the donor is enriched
with with CD34+ CD34 cells cellsrelative relativeto to lymphocytes by a by lymphocytes ratio of fromofabout a ratio from5:1 to about about 7:1,about 5:1 to about 7:1, about
5.5:1 to about 6.5:1, or about 5.2:1 to about 5.7:1. In some embodiments, the peripheral
blood of the donor is enriched with CD34+ cells relative CD34 cells relative to to lymphocytes lymphocytes by by aa ratio ratio of of from from
about 5.0:1 to about 6.5:1, such as a ratio of about 5.00:1, 5.05:1, 5.10:1, 5.15:1, 5.20:1,
5.25:1, 5.30:1, 5.35:1, 5.40:1, 5.45:1, 5.50:1, 5.55:1, 5.60:1, 5.65:1, 5.70:1, 5.75:1, 5.80:1,
5.85:1, 5.90:1, 5.95:1, 6.00:1, 6.05:1, 6.10:1, 6.15:1, 6.20:1, 6.25:1, 6.30:1, 6.35:1, 6.40:1,
6.45:1, or 6.50:1. In some embodiments, the peripheral blood of the donor is enriched with
CD34 cells relative to lymphocytes by a ratio of about 5.7:1.
[0017] In another aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density
of CD34 cells of at least about 38,000 cells/ml, such as a density of CD34+ cells of CD34 cells of from from
about 38,000 cells/ml to about 100,000 cells/ml, about 40,000 cells/ml to about 90,000
cells/ml, about 50,000 cells/ml to about 80,000 cells/ml, or about 60,000 cells/ml to about
70,000 cells/ml (e.g., about 38,00 cells/ml, 39,000 cells/ml, 40,000 cells/ml, 41,000 cells/ml,
42,000 cells/ml, 43,000 cells/ml, 44,000 cells/ml, 45,000 cells/ml, 46,000 cells/ml, 47,000
cells/ml, 48,000 cells/ml, 49,000 cells/ml, 50,000 cells/ml, 51,000 cells/ml, 52,000 cells/ml,
53,000 cells/ml, 54,000 cells/ml, 55,000 cells/ml, 56,000 cells/ml, 57,000 cells/ml, 58,000
cells/ml, 59,000 cells/ml, 60,000 cells/ml, 61,000 cells/ml, 62,000 cells/ml, 63,000 cells/ml,
64,000 cells/ml, 65,000 cells/ml, 66,000 cells/ml, 67,000 cells/ml, 68,000 cells/ml, 69,000
cells/ml, 70,000 cells/ml, 71,000 cells/ml, 72,000 cells/ml, 73,000 cells/ml, 74,000 cells/ml,
75,000 cells/ml, 76,000 cells/ml, 77,000 cells/ml, 78,000 cells/ml, 79,000 cells/ml, 80,000
cells/ml, 81,000 cells/ml, 82,000 cells/ml, 83,000 cells/ml, 84,000 cells/ml, 85,000 cells/ml,
86,000 cells/ml, 87,000 cells/ml, 88,000 cells/ml, 89,000 cells/ml, 90,000 cells/ml, 91,000
cells/ml, 92,000 cells/ml, 93,000 cells/ml, 94,000 cells/ml, 95,000 cells/ml, 96,000 cells/ml,
97,000 cells/ml, 98,000 cells/ml, 99,000 cells/ml, 100,000 cells/ml, or more), and having a
density of lymphocytes of no more than about 2.4 x X 107 cells/ml, such 10 cells/ml, such as as aa density density of of
lymphocytes lymphocytesofof about 1 X 1107 about cells/ml X 10 to about cells/ml 2.3 X 2.3 to about 107 cells/ml, about 1.3 x 10 cells/ml, X 1071.3 about cells/ml to x 10 cells/ml to
about 2.1 X 107 cells/ml, or 10 cells/ml, or about about 1.5 1.5 XX 10 107 cells/ml cells/ml toto about about 1.9 1.9 X X 10107 cells/ml cells/ml (e.g., (e.g., about about 2.42.4
WO wo 2019/113375 PCT/US2018/064335
X 107cells/ml, x 10 cells/ml, 2.3 2.3 X 10x cells/ml, 107 cells/ml, 2.2 X o 2.2x07 cells/ml, cells/ml, 2.1 X 10 2.1 x 107 2cells/ml, cells/ml, cells/ml, x 10 cells/ml, 1.9 x 1.9 x
107 cells/ml, 1.8 10 cells/ml, 1.8 xx 10 107 cells/ml, cells/ml, 1.7 1.7 X x 10107 cells/ml, cells/ml, 1.61.6x1 X 10 107 cells/ml, cells/ml, 1.5 1.5x X 10 107 cells/ml cells/ml 1.4 1.4 X
107cells/ml, 10 cells/ml,1.3 1.3Xx10 107 cells/ml, cells/ml, 1.2 1.2 x 107 X 10 cells/ml, cells/ml, 1.11.1 X cells/ml, X 10 107 cells/ml, X 107 1 x 10 cells/ml, cells/ml, or less, or less,
0.9 X x 107 cells/ml, 0.8 10 cells/ml, 0.8 XX 10 107 cells/ml, cells/ml, oror less). less). InIn some some embodiments, embodiments, the the method method includes includes
administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient
to produce a population of cells having a density of CD34+ cells of CD34 cells of from from about about 38,000 38,000
cells/ml to about 100,000 cells/ml, and having a density of lymphocytes of from about 1 x X 107 10
cells/ml to about 2.3 X 107 cells/ml. In 10 cells/ml. In some some embodiments, embodiments, the the method method includes includes
administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient
to produce a population of cells having a density of CD34+ cells of CD34 cells of from from about about 40,000 40,000
cells/ml to about 80,000 cells/ml, and having a density of lymphocytes of from about 1.3 X
107 cells/ml to 10 cells/ml to about about 2.3 2.3 XX 10 107 cells/ml. cells/ml. InIn some some embodiments, embodiments, the the method method includes includes
administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient
to produce a population of cells having a density of CD34+ cells of CD34 cells of from from about about 50,000 50,000
cells/ml to about 90,000 cells/ml, and having a density of lymphocytes of from about 1.5 X x
107 cells/ml to 10 cells/ml toabout about2 X 2 107 x 10cells/ml. cells/ml.
[0018] In another aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to produce a population of cells having a ratio of
CD34+ cells to CD34 cells to monocytes monocytes of of from from about about 0.0071 0.0071 to to about about 0.0174 0.0174 in in aa sample sample of of peripheral peripheral
blood of the donor following administration of the CXCR2 agonist. In some embodiments,
the ratio of CD34+ cells to CD34 cells to monocytes monocytes in in the the sample sample may may be be about about 0.00710, 0.00710, 0.00711, 0.00711, 0.00712, 0.00712,
0.00713, 0.00714, 0.00715, 0.00716, 0.00717, 0.00718, 0.00719, 0.00720, 0.00721, 0.00722,
0.00723, 0.00724, 0.00725, 0.00726, 0.00727, 0.00728, 0.00729, 0.00730, 0.00731, 0.00732,
0.00733, 0.00734, 0.00735, 0.00736, 0.00737, 0.00738, 0.00739, 0.00740, 0.00741, 0.00742,
0.00743, 0.00744, 0.00745, 0.00746, 0.00747, 0.00748, 0.00749, 0.00750, 0.00751, 0.00752,
0.00753, 0.00754, 0.00755, 0.00756, 0.00757, 0.00758, 0.00759, 0.00760, 0.00761, 0.00762,
0.00763, 0.00764, 0.00765, 0.00766, 0.00767, 0.00768, 0.00769, 0.00770, 0.00771, 0.00772,
0.00773, 0.00774, 0.00775, 0.00776, 0.00777, 0.00778, 0.00779, 0.00780, 0.00781, 0.00782,
0.00783, 0.00784, 0.00785, 0.00786, 0.00787, 0.00788, 0.00789, 0.00790, 0.00791, 0.00792,
0.00793, 0.00794, 0.00795, 0.00796, 0.00797, 0.00798, 0.00799, 0.00800, 0.00801, 0.00802,
0.00803, 0.00804, 0.00805, 0.00806, 0.00807, 0.00808, 0.00809, 0.00810, 0.00811, 0.00812,
WO wo 2019/113375 PCT/US2018/064335
0.00813, 0.00814, 0.00815, 0.00816, 0.00817, 0.00818, 0.00819, 0.00820, 0.00821, 0.00822,
0.00823, 0.00824, 0.00825, 0.00826, 0.00827, 0.00828, 0.00829, 0.00830, 0.00831, 0.00832,
0.00833, 0.00834, 0.00835, 0.00836, 0.00837, 0.00838, 0.00839, 0.00840, 0.00841, 0.00842,
0.00843, 0.00844, 0.00845, 0.00846, 0.00847, 0.00848, 0.00849, 0.00850, 0.00851, 0.00852,
0.00853, 0.00854, 0.00855, 0.00856, 0.00857, 0.00858, 0.00859, 0.00860, 0.00861, 0.00862,
0.00863, 0.00864, 0.00865, 0.00866, 0.00867, 0.00868, 0.00869, 0.00870, 0.00871, 0.00872,
0.00873, 0.00874, 0.00875, 0.00876, 0.00877, 0.00878, 0.00879, 0.00880, 0.00881, 0.00882,
0.00883, 0.00884, 0.00885, 0.00886, 0.00887, 0.00888, 0.00889, 0.00890, 0.00891, 0.00892,
0.00893, 0.00894, 0.00895, 0.00896, 0.00897, 0.00898, 0.00899, 0.00900, 0.00901, 0.00902,
0.00903, 0.00904, 0.00905, 0.00906, 0.00907, 0.00908, 0.00909, 0.00910, 0.00911, 0.00912,
0.00913, 0.00914, 0.00915, 0.00916, 0.00917, 0.00918, 0.00919, 0.00920, 0.00921, 0.00922,
0.00923, 0.00924, 0.00925, 0.00926, 0.00927, 0.00928, 0.00929, 0.00930, 0.00931, 0.00932,
0.00933, 0.00934, 0.00935, 0.00936, 0.00937, 0.00938, 0.00939, 0.00940, 0.00941, 0.00942,
0.00943, 0.00944, 0.00945, 0.00946, 0.00947, 0.00948, 0.00949, 0.00950, 0.00951, 0.00952,
0.00953, 0.00954, 0.00955, 0.00956, 0.00957, 0.00958, 0.00959, 0.00960, 0.00961, 0.00962,
0.00963, 0.00964, 0.00965, 0.00966, 0.00967, 0.00968, 0.00969, 0.00970, 0.00971, 0.00972,
0.00973, 0.00974, 0.00975, 0.00976, 0.00977, 0.00978, 0.00979, 0.00980, 0.00981, 0.00982,
0.00983, 0.00984, 0.00985, 0.00986, 0.00987, 0.00988, 0.00989, 0.00990, 0.00991, 0.00992,
0.00993, 0.00994, 0.00995, 0.00996, 0.00997, 0.00998, 0.00999, 0.0100, 0.0101, 0.0103,
0.0104, 0.0105, 0.0106, 0.0107, 0.0108, 0.0109, 0.0110, 0.0111, 0.0112, 0.0113, 0.0114,
0.0115, 0.0116, 0.0117, 0.0118, 0.0119, 0.0120, 0.0121, 0.0122, 0.0123, 0.0124, 0.0125,
0.0126, 0.0127, 0.0128, 0.0129, 0.0130, 0.0131, 0.0132, 0.0133, 0.0134, 0.0135, 0.0136,
0.0137, 0.0138, 0.0139, 0.0140, 0.0141, 0.0142, 0.0143, 0.0144, 0.0145, 0.0146, 0.0147,
0.0148, 0.0149, 0.0150, 0.0151, 0.0152, 0.0153, 0.0154, 0.0155, 0.0156, 0.0157, 0.0158,
0.0159, 0.0160, 0.0161, 0.0162, 0.0163, 0.0164, 0.0165, 0.0166, 0.0167, 0.0168, 0.0169,
0.0170, 0.0171, 0.0172, 0.0173, or 0.0174. In some embodiments, the ratio of CD34+ cells to CD34 cells to
monocytes in the sample is from 0.008 to about 0.016, about 0.009 to about 0.015, about 0.01
to about 0.014, or about 0.011 to about 0.013. In some embodiments, the ratio of CD34+ CD34
cells to monocytes in the sample is from about 0.0100 to about 0.0140, such as a ratio of
CD34+ cells to CD34 cells to monocytes monocytes in in the the sample sample of of about about 0.0100, 0.0100, 0.0101, 0.0101, 0.0103, 0.0103, 0.0104, 0.0104, 0.0105, 0.0105,
0.0106, 0.0107, 0.0108, 0.0109, 0.0110, 0.0111, 0.0112, 0.0113, 0.0114, 0.0115, 0.0116,
0.0117, 0.0118, 0.0119, 0.0120, 0.0121, 0.0122, 0.0123, 0.0124, 0.0125, 0.0126, 0.0127,
0.0128, 0.0129, 0.0130, 0.0131, 0.0132, 0.0133, 0.0134, 0.0135, 0.0136, 0.0137, 0.0138,
WO wo 2019/113375 PCT/US2018/064335
0.0139, or 0.0140. In some embodiments, the ratio of CD34+ cellsto CD34 cells tomonocytes monocytesin inthe the
sample is about 0.0118.
[0019] In an additional aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density
of of CD34+ CD34 cells cellsofofatatleast about least 38,000 about cells/ml, 38,000 such as cells/ml, a density such of CD34+ of as a density cells of cells CD34 from of from
about 38,000 cells/ml to about 100,000 cells/ml, about 40,000 cells/ml to about 90,000
cells/ml, about 50,000 cells/ml to about 80,000 cells/ml, or about 60,000 cells/ml to about
70,000 cells/ml (e.g., about 38,00 cells/ml, 39,000 cells/ml, 40,000 cells/ml, 41,000 cells/ml,
42,000 cells/ml, 43,000 cells/ml, 44,000 cells/ml, 45,000 cells/ml, 46,000 cells/ml, 47,000
cells/ml, 48,000 cells/ml, 49,000 cells/ml, 50,000 cells/ml, 51,000 cells/ml, 52,000 cells/ml,
53,000 cells/ml, 54,000 cells/ml, 55,000 cells/ml, 56,000 cells/ml, 57,000 cells/ml, 58,000
cells/ml, 59,000 cells/ml, 60,000 cells/ml, 61,000 cells/ml, 62,000 cells/ml, 63,000 cells/ml,
64,000 cells/ml, 65,000 cells/ml, 66,000 cells/ml, 67,000 cells/ml, 68,000 cells/ml, 69,000
cells/ml, 70,000 cells/ml, 71,000 cells/ml, 72,000 cells/ml, 73,000 cells/ml, 74,000 cells/ml,
75,000 cells/ml, 76,000 cells/ml, 77,000 cells/ml, 78,000 cells/ml, 79,000 cells/ml, 80,000
cells/ml, 81,000 cells/ml, 82,000 cells/ml, 83,000 cells/ml, 84,000 cells/ml, 85,000 cells/ml,
86,000 cells/ml, 87,000 cells/ml, 88,000 cells/ml, 89,000 cells/ml, 90,000 cells/ml, 91,000
cells/ml, 92,000 cells/ml, 93,000 cells/ml, 94,000 cells/ml, 95,000 cells/ml, 96,000 cells/ml,
97,000 cells/ml, 98,000 cells/ml, 99,000 cells/ml, 100,000 cells/ml, or more), and having a
density of monocytes of no more than about 6 X 106 cells/ml, such 10 cells/ml, such as as aa density density of of monocytes monocytes
of from 3.4x 3.4 x106 10 cells/ml to about 5.9 x 106 cells/ml, about 10 cells/ml, about 3.5 3.5 xx 10 106 cells/ml cells/ml toto about about 5.7 5.7 X X
106 cells/ml, or 10 cells/ml, or about about 44 XX 10 106 cells/ml cells/ml toto about about 5 5 X X 10106 cells/ml cells/ml (e.g., (e.g., 5.95.9 x 106 X 10 cells/ml, cells/ml, 5.8 5.8 X X
106 cells/ml, 10 cells/ml, 5.75.7 x 10x cells/ml, 106 cells/ml, 5.6x10 5.6 X 10 cells/ml, cells/ml, 5.5 5.5 x 10 x 106 cells/ml, cells/ml, 5.4x cells/ml, 5.4 x 10 cells/ml, 5.3 x 5.3 X
106cells/ml, 10 cells/ml,5.2 5.2X x10 106 cells/ml, cells/ml, 5.1 5.1 x 1cells/ml, x 10 106 cells/ml, 5 cells/ml, 5 x 10 x 106 cells/ml, 4.9cells/ml, 4.9 X 10 x 1 106 cells/ml, 4.8 x 4.8 X
106cells/ml, 10 cells/ml,4.7 4.7Xx10 106 cells/ml, cells/ml, 4.6 4.6 x 106 X 10 cells/ml, cells/ml, 4.54.5 x cells/ml, X 10 106 cells/ml, 4.4x 4.4 X 106 cells/ml, 10 cells/ml, 4.3 X 4.3 x
106 cells/ml,4.2 10 cells/ml, 4.2Xx10 106 cells/ml, cells/ml, 4.1 4.1 X x 10106 cells/ml, cells/ml, 4 x410 x 106 cells/ml, cells/ml, 3.9 3.9 X 10xcells/ml, 106 cells/ml, 3.8 x3.8 X
106 cells/ml,3.7 10 cells/ml, 3.7x x 106 cells/ml,3.6 10 cells/ml, 3.6X106 10 cells/ml, 3.5 106 X 10cells/ml, cells/ml,3.4 3.4Xx106 10 cells/ml, or
less). less). In In some some embodiments, embodiments, the the method method includes includes administering administering to to the the donor donor aa CXCR2 CXCR2
agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells
having a density of CD34+ cellsof CD34 cells offrom fromabout about38,000 38,000cells/ml cells/mlto toabout about100,000 100,000cells/ml, cells/ml,and and
having having a adensity densityof of monocytes of from monocytes about about of from 3.4 x 106 3.4 cells/ml to aboutto X 10 cells/ml 6 Xabout 106 cells/ml. 6 X 10 cells/ml. InIn
WO wo 2019/113375 PCT/US2018/064335
some embodiments, the method includes administering to the donor a CXCR2 agonist and a
CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of
CD34+ cells of CD34 cells of from from about about 40,000 40,000 cells/ml cells/ml to to about about 80,000 80,000 cells/ml, cells/ml, and and having having aa density density of of
monocytes of from about 106 4 x cells/ml to about 10 cells/ml 5.5 5.5 to about X 106 cells/ml. x 10 In In cells/ml. some embodiments, some embodiments,
the method includes administering to the donor a CXCR2 agonist and a CXCR4 antagonist in
amounts sufficient to produce a population of cells having a density of CD34+ cells of CD34 cells of from from
about 50,000 cells/ml to about 90,000 cells/ml, and having a density of monocytes of from
about X 4 106 X 10cells/ml cells/mlto toabout about5 5X X106 10 cells/ml.
[0020] In another aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to enrich the peripheral blood of the donor with
CD34+ cells relative CD34 cells relativetoto monocytes by aby monocytes ratio of from a ratio of about from 1.1:1 aboutto1.1:1 about to 2.3:1 as assessed about 2.3:1 asbyassessed by
comparing a sample of peripheral blood of the donor following administration of the CXCR2
agonist and CXCR4 antagonist to a sample of peripheral blood of the donor prior to
administration of the CXCR2 agonist and CXCR4 antagonist. In some embodiments, the
peripheral blood of the donor may be enriched with CD34+ cellsrelative CD34 cells relativeto tomonocytes monocytesby byaa
ratio of about 1.10:1, 1.15:1, 1.20:1, 1.25:1, 1.30:1, 1.35:1, 1.40:1, 1.45:1, 1.50:1, 1.55:1,
1.60:1, 1.65:1, 1.70:1, 1.75:1, 1.80:1, 1.85:1, 1.90:1, 1.95:1, 2.00:1, 2.05:1, 2.10:1, 2.15:1,
2.20:1, 2.25:1, or 2.30:1. In some embodiments, the peripheral blood of the donor is enriched
with with CD34+ CD34 cells cellsrelative relativeto to monocytes by a by monocytes ratio of about a ratio of from aboutabout from1.3:1 to 1.3:1 about about 1.9:1, to about 1.9:1,
such as a ratio of about 1.30:1, 1.35:1, 1.40:1, 1.45:1, 1.50:1, 1.55:1, 1.60:1, 1.65:1, 1.70:1,
1.75:1, 1.80:1, 1.85:1, or 1.90:1.
[0021] In another aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to produce a population of cells having a
frequency of CD34+ CD34 cells cells of of from from about about 0.051% 0.051% to to about about 0.140% 0.140% in in aa sample sample of of peripheral peripheral
blood of the donor following administration of the CXCR2 agonist and CXCR4 antagonist.
In some embodiments, the population of cells may have a frequency of CD34+ cellsof CD34 cells ofabout about
0.051%, 0.052%, 0.053%, 0.054%, 0.055%, 0.056%, 0.057%, 0.058%, 0.059%, 0.060%,
0.061%, 0.062%,0.063%, 0.061%, 0.062%, 0.063%, 0.064%, 0.064%, 0.065%, 0.065%, 0.066%, 0.066%, 0.067%, 0.067%, 0.068%,0.068%, 0.069%, 0.069%, 0.070%, 0.070%,
0.071%, 0.072%, 0.073%, 0.074%, 0.075%, 0.076%, 0.077%, 0.078%, 0.079%, 0.080%,
WO wo 2019/113375 PCT/US2018/064335
0.081%, 0.082%, 0.083%, 0.084%, 0.085%, 0.086%, 0.087%, 0.088%, 0.089%, 0.090%,
0.091%, 0.092%, 0.093%, 0.094%, 0.095%, 0.096%, 0.097%, 0.098%, 0.099%, 0.100%,
0.101%, 0.102%, 0.103%, 0.104%, 0.105%, 0.106%, 0.107%, 0.108%, 0.109%, 0.110%,
0.111%, 0.112%, 0.113%, 0.114%, 0.115%, 0.116%, 0.117%, 0.118%, 0.119%, 0.120%,
0.121%, 0.122%, 0.123%, 0.124%, 0.125%, 0.126%, 0.127%, 0.128%, 0.129%, 0.130%,
0.131%, 0.132%, 0.133%, 0.134%, 0.135%, 0.136%, 0.137%, 0.138%, 0.139%, or 0.140%.
In some embodiments, the population of cells has a frequency of CD34+ cells of CD34 cells of from from about about
0.050% to about 0.120%, about 0.060% to about 0.110%, or about 0.080% to about 0.100%.
In some embodiments, the population of cells has a frequency of CD34+ cells of CD34 cells of from from about about
0.080% to about 0.120%, such as a frequency of hematopoietic stem cells of about 0.080%,
0.081%, 0.082%, 0.083%, 0.084%, 0.085%, 0.086%, 0.087%, 0.088%, 0.089%, 0.090%,
0.091%, 0.092%, 0.093%, 0.094%, 0.095%, 0.096%, 0.097%, 0.098%, 0.099%, 0.100%,
0.101%, 0.102%, 0.103%, 0.104%, 0.105%, 0.106%, 0.107%, 0.108%, 0.109%, 0.110%,
0.111%, 0.112%, 0.113%, 0.114%, 0.115%, 0.116%, 0.117%, 0.118%, 0.119%, or 0.120%.
In some embodiments, the population of cells has a frequency of CD34+ cells of CD34 cells of about about
0.097%.
[0022] In an additional aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to induce an increase in the frequency of CD34+ CD34
cells in the peripheral blood of the donor by at least 3-fold as assessed by comparing a sample
of peripheral blood of the donor following administration of the CXCR2 agonist and CXCR4
antagonist to a sample of peripheral blood of the donor prior to administration of the CXCR2
agonist and CXCR4 antagonist (e.g., by from about 3.4-fold to about 7.1-fold, such as by
about 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold, 4.0-fold, 4.1-fold, 4.2-fold, 4.3-
fold, 4.4-fold, 4.5-fold, 4.6-fold, 4.7-fold, 4.8-fold, 4.9-fold, 5.0-fold, 5.1-fold, 5.2-fold, 5.3-
fold, 5.4-fold, 5.5-fold, 5.6-fold, 5.7-fold, 5.8-fold, 5.9-fold, 6.0-fold, 6.1-fold, 6.2-fold, 6.3-
fold, 6.4-fold, 6.5-fold, 6.6-fold, 6.7-fold, 6.8-fold, 6.9-fold, 7.0-fold, or 7.1-fold. In some
embodiments, the frequency of CD34+ cells in CD34 cells in the the peripheral peripheral blood blood of of the the donor donor is is increased increased
by from about 4-fold to about 7-fold, about 4.5-fold to about 6.5-fold, or about 5-fold to
about 6-fold following administration of the CXCR2 agonist and CXCR4 antagonist. In
some embodiments, the frequency of CD34+ cellsin CD34 cells inthe theperipheral peripheralblood bloodof ofthe thedonor donoris is
increased by from about 4.0-fold to about 6.0-fold following administration of the CXCR2 agonist and CXCR4 antagonist, such as by about 4.0-fold, 4.1-fold, 4.2-fold, 4.3-fold, 4.4- fold, 4.5-fold, 4.6-fold, 4.7-fold, 4.8-fold, 4.9-fold, 5.0-fold, 5.1-fold, 5.2-fold, 5.3-fold, 5.4- fold, 5.5-fold, 5.6-fold, 5.7-fold, 5.8-fold, 5.9-fold, or 6.0-fold. In some embodiments, the frequency frequencyofofCD34+ CD34cells in in cells thethe peripheral blood blood peripheral of theof donor the is increased donor by about 4.8-fold. is increased by about 4.8-fold.
[0023] In a further aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to produce a population of cells having a ratio of
CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells toto leukocytes leukocytes ofof from from about about 0.0003 0.0003 toto about about 0.0016 0.0016 inin a a
sample of peripheral blood of the donor following administration of the CXCR2 agonist and
CXCR4 CXCR4 antagonist. antagonist.In In some embodiments, some the ratio embodiments, of CD34+ the ratio of CD90+ CD34 CD45RA cells to CD90 CD45RA cells to
leukocytes in the sample may be about 0.00030, 0.00031, 0.00032, 0.00033, 0.00034,
0.00035, 0.00036, 0.00037, 0.00038, 0.00039, 0.00040, 0.00041, 0.00042, 0.00043, 0.00044,
0.00045, 0.00046, 0.00047, 0.00048, 0.00049, 0.00050, 0.00051, 0.00052, 0.00053, 0.00054,
0.00055, 0.00056, 0.00057, 0.00058, 0.00059, 0.00060, 0.00061, 0.00062, 0.00063, 0.00064,
0.00065, 0.00066, 0.00067, 0.00068, 0.00069, 0.00070, 0.00071, 0.00072, 0.00073, 0.00074,
0.00075, 0.00076, 0.00077, 0.00078, 0.00079, 0.00080, 0.00081, 0.00082, 0.00083, 0.00084,
0.00085, 0.00086, 0.00087, 0.00088, 0.00089, 0.00090, 0.00091, 0.00092, 0.00093, 0.00094,
0.00095, 0.00096, 0.00097, 0.00098, 0.00099, 0.00100, 0.00101, 0.00102, 0.00103, 0.00104,
0.00105, 0.00106, 0.00107, 0.00108, 0.00109, 0.00110, 0.00111, 0.00112, 0.00113, 0.00114,
0.00115, 0.00116, 0.00117, 0.00118, 0.00119, 0.00120, 0.00121, 0.00122, 0.00123, 0.00124,
0.00125, 0.00126, 0.00127, 0.00128, 0.00129, 0.00130, 0.00131, 0.00132, 0.00133, 0.00134,
0.00135, 0.00136, 0.00137, 0.00138, 0.00139, 0.00140, 0.00141, 0.00142, 0.00143, 0.00144,
0.00145, 0.00146, 0.00147, 0.00148, 0.00149, 0.00150, 0.00151, 0.00152, 0.00153, 0.00154,
0.00155, 0.00156, 0.00157, 0.00158, 0.00159, or 0.00160. In some embodiments, the ratio of
CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells toto leukocytes leukocytes inin the the sample sample isis from from about about 0.0008 0.0008 toto about about
0.0010. In some embodiments, the ratio of CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells toto leukocytes leukocytes inin
the sample is from about 0.0006 to about 0.0012, such as a ratio of hematopoietic stem cells
to leukocytes in the sample of about 0.00060, 0.00061, 0.00062, 0.00063, 0.00064, 0.00065,
0.00066, 0.00067, 0.00068, 0.00069, 0.00070, 0.00071, 0.00072, 0.00073, 0.00074, 0.00075,
0.00076, 0.00077, 0.00078, 0.00079, 0.00080, 0.00081, 0.00082, 0.00083, 0.00084, 0.00085,
0.00086, 0.00087, 0.00088, 0.00089, 0.00090, 0.00091, 0.00092, 0.00093, 0.00094, 0.00095,
0.00096, 0.00097, 0.00098, 0.00099, 0.00100, 0.00101, 0.00102, 0.00103, 0.00104, 0.00105,
20 wo 2019/113375 WO PCT/US2018/064335
0.00106, 0.00107, 0.00108, 0.00109, 0.00110, 0.00111, 0.00112, 0.00113, 0.00114, 0.00115,
0.00116, 0.00117, 0.00118, 0.00119, or 0.00120. In some embodiments, the ratio of CD34+ CD34
CD90+ CD45RAcells CD90 CD45RA cellsto toleukocytes leukocytesin inthe thesample sampleis isabout about0.0009. 0.0009.
[0024] In another aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to enrich the peripheral blood of the donor with
CD34 CD90 CD45RA cells relative to leukocytes by a ratio of from about 5.5:1 to about
26.9:1 as assessed by comparing a sample of peripheral blood of the donor following
administration of the CXCR2 agonist and CXCR4 antagonist to a sample of peripheral blood
of the donor prior to administration of the CXCR2 agonist and CXCR4 antagonist. In some
embodiments, the peripheral blood of the donor may be enriched with CD34+ CD90 CD34 CD90
CD45RA cells relative to leukocytes by a ratio of about 5.50:1, 5.55:1, 5.60:1, 5.65:1, 5.70:1,
5.75:1, 5.80:1, 5.85:1, 5.90:1, 5.95:1, 6.00:1, 6.05:1, 6.10:1, 6.15:1, 6.20:1, 6.25:1, 6.30:1,
6.35:1, 6.40:1, 6.45:1, 6.50:1, 6.55:1, 6.60:1, 6.65:1, 6.70:1, 6.75:1, 6.80:1, 6.85:1, 6.90:1,
6.95:1, 7.00:1, 7.05:1, 7.10:1, 7.15:1, 7.20:1, 7.25:1, 7.30:1, 7.35:1, 7.40:1, 7.45:1, 7.50:1,
7.55:1, 7.60:1, 7.65:1, 7.70:1, 7.75:1, 7.80:1, 7.85:1, 7.90:1, 7.95:1, 8.00:1, 8.05:1, 8.10:1,
8.15:1, 8.20:1, 8.25:1, 8.30:1, 8.35:1, 8.40:1, 8.45:1, 8.50:1, 8.55:1, 8.60:1, 8.65:1, 8.70:1,
8.75:1, 8.80:1, 8.85:1, 8.90:1, 8.95:1, 9.00:1, 9.05:1, 9.10:1, 9.15:1, 9.20:1, 9.25:1, 9.30:1,
9.35:1, 9.40:1, 9.45:1, 9.50:1, 9.55:1, 9.60:1, 9.65:1, 9.70:1, 9.75:1, 9.80:1, 9.85:1, 9.90:1,
9.95:1, 10.00:1, 10.05:1, 10.10:1, 10.15:1, 10.20:1, 10.25:1, 10.30:1, 10.35:1, 10.40:1,
10.45:1, 10.50:1, 10.55:1, 10.60:1, 10.65:1, 10.70:1, 10.75:1, 10.80:1, 10.85:1, 10.90:1,
10.95:1, 11.00:1, 11.05:1, 11.10:1, 11.15:1, 11.20:1, 11.25:1, 11.30:1, 11.35:1, 11.40:1,
11.45:1, 11.50:1, 11.55:1, 11.60:1, 11.65:1, 11.70:1, 11.75:1, 11.80:1, 11.85:1, 11.90:1,
11.95:1, 12.00:1, 12.05:1, 12.10:1, 12.15:1, 12.20:1, 12.25:1, 12.30:1, 12.35:1, 12.40:1,
12.45:1, 12.50:1, 12.55:1, 12.60:1, 12.65:1, 12.70:1, 12.75:1, 12.80:1, 12.85:1, 12.90:1,
12.95:1, 13.00:1, 13.05:1, 13.10:1, 13.15:1, 13.20:1, 13.25:1, 13.30:1, 13.35:1, 13.40:1,
13.45:1, 13.50:1, 13.55:1, 13.60:1, 13.65:1, 13.70:1, 13.75:1, 13.80:1, 13.85:1, 13.90:1,
13.95:1, 14.00:1, 14.05:1, 14.10:1, 14.15:1, 14.20:1, 14.25:1, 14.30:1, 14.35:1, 14.40:1,
14.45:1, 14.50:1, 14.55:1, 14.60:1, 14.65:1, 14.70:1, 14.75:1, 14.80:1, 14.85:1, 14.90:1,
14.95:1, 15.00:1, 15.05:1, 15.10:1, 15.15:1, 15.20:1, 15.25:1, 15.30:1, 15.35:1, 15.40:1,
15.45:1, 15.50:1, 15.55:1, 15.60:1, 15.65:1, 15.70:1, 15.75:1, 15.80:1, 15.85:1, 15.90:1,
15.95:1, 16.00:1, 16.05:1, 16.10:1, 16.15:1, 16.20:1, 16.25:1, 16.30:1, 16.35:1, 16.40:1, wo 2019/113375 WO PCT/US2018/064335
16.45:1, 16.50:1, 16.55:1, 16.60:1, 16.65:1, 16.70:1, 16.75:1, 16.80:1, 16.85:1, 16.90:1,
16.95:1, 17.00:1, 17.05:1, 17.10:1, 17.15:1, 17.20:1, 17.25:1, 17.30:1, 17.35:1, 17.40:1,
17.45:1, 17.50:1, 17.55:1, 17.60:1, 17.65:1, 17.70:1, 17.75:1, 17.80:1, 17.85:1, 17.90:1,
17.95:1, 18.00:1, 18.05:1, 18.10:1, 18.15:1, 18.20:1, 18.25:1, 18.30:1, 18.35:1, 18.40:1,
18.45:1, 18.50:1, 18.55:1, 18.60:1, 18.65:1, 18.70:1, 18.75:1, 18.80:1, 18.85:1, 18.90:1,
18.95:1, 19.00:1, 19.05:1, 19.10:1, 19.15:1, 19.20:1, 19.25:1, 19.30:1, 19.35:1, 19.40:1,
19.45:1, 19.50:1, 19.55:1, 19.60:1, 19.65:1, 19.70:1, 19.75:1, 19.80:1, 19.85:1, 19.90:1,
19.95:1, 20.00:1, 20.05:1, 20.10:1, 20.15:1, 20.20:1, 20.25:1, 20.30:1, 20.35:1, 20.40:1,
20.45:1, 20.50:1, 20.55:1, 20.60:1, 20.65:1, 20.70:1, 20.75:1, 20.80:1, 20.85:1, 20.90:1,
20.95:1, 21.00:1, 21.05:1, 21.10:1, 21.15:1, 21.20:1, 21.25:1, 21.30:1, 21.35:1, 21.40:1,
21.45:1, 21.50:1, 21.55:1, 21.60:1, 21.65:1, 21.70:1, 21.75:1, 21.80:1, 21.85:1, 21.90:1,
21.95:1, 22.00:1, 22.05:1, 22.10:1, 22.15:1, 22.20:1, 22.25:1, 22.30:1, 22.35:1, 22.40:1,
22.45:1, 22.50:1, 22.55:1, 22.60:1, 22.65:1, 22.70:1, 22.75:1, 22.80:1, 22.85:1, 22.90:1,
22.95:1, 23.00, 23.05:1, 23.10:1, 23.15:1, 23.20:1, 23.25:1, 23.30:1, 23.35:1, 23.40:1,
23.45:1, 23.50:1, 23.55:1, 23.60:1, 23.65:1, 23.70:1, 23.75:1, 23.80:1, 23.85:1, 23.90:1,
23.95:1, 24.00:1, 24.05:1, 24.10:1, 24.15:1, 24.20:1, 24.25:1, 24.30:1, 24.35:1, 24.40:1,
24.45:1, 24.50:1, 24.55:1, 24.60:1, 24.65:1, 24.70:1, 24.75:1, 24.80:1, 24.85:1, 24.90:1,
24.95:1, 25.05:1, 25.10:1, 25.15:1, 25.20:1, 25.25:1, 25.30:1, 25.35:1, 25.40:1, 25.45:1,
25.50:1, 25.55:1, 25.60:1, 25.65:1, 25.70:1, 25.75:1, 25.80:1, 25.85:1, 25.90:1, 25.95:1,
26.00:1, 26.05:1, 26.10:1, 26.15:1, 26.20:1, 26.25:1, 26.30:1, 26.35:1, 26.40:1, 26.45:1,
26.50:1, 26.55:1, 26.60:1, 26.65:1, 26.70:1, 26.75:1, 26.80:1, 26.85:1, 26.90:1, or 26.95:1. In
some embodiments, the peripheral blood of the donor is enriched with CD34+ CD90 CD34 CD90
CD45RA cells relative to leukocytes by a ratio of about from about 5.5:1 to about 6.5:1, such
as a ratio of about 5.50:1, 5.55:1, 5.60:1, 5.65:1, 5.70:1, 5.75:1, 5.80:1, 5.85:1, 5.90:1, 5.95:1,
6.00:1, 6.05:1, 6.10:1, 6.15:1, 6.20:1, 6.25:1, 6.30:1, 6.35:1, 6.40:1, 6.45:1, or 6.50:1. In
some embodiments, the peripheral blood of the donor is enriched with CD34+ CD90 CD34 CD90
CD45RA cells relative to leukocytes by a ratio of about 6.0:1.
[0025] In another aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density
of CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells ofof atat least least about about 16,000 16,000 cells/ml, cells/ml, such such asas a a density density ofof from from
about 20,000 cells/ml to about 75,000 cells/ml, about 25,000 cells/ml to about 70,000
WO wo 2019/113375 PCT/US2018/064335
cells/ml, about 30,000 cells/ml to about 65,000 cells/ml, about 35,000 cells/ml to about
60,000 cells/ml, about 40,000 cells/ml to about 55,000 cells/ml, or about 45,000 cells/ml to
about 50,000 cells/ml (e.g., about 16,000 cells/ml, 17,000 cells/ml, 18,000 cells/ml, 19,000
cells/ml, 20,000 cells/ml, 21,000 cells/ml, 22,000 cells/ml, 23,000 cells/ml, 24,000 cells/ml,
25,000 cells/ml, 26,000 cells/ml, 27,000 cells/ml, 28,000 cells/ml, 29,000 cells/ml, 30,000
cells/ml, 31,000 cells/ml, 32,000 cells/ml, 33,000 cells/ml, 34,000 cells/ml, 35,000 cells/ml,
36,000 cells/ml, 37,000 cells/ml, 38,000 cells/ml, 39,000 cells/ml, 40,000 cells/ml, 41,000
cells/ml, 42,000 cells/ml, 43,000 cells/ml, 44,000 cells/ml, 45,000 cells/ml, 46,000 cells/ml,
47,000 cells/ml, 48,000 cells/ml, 49,000 cells/ml, 50,000 cells/ml, 51,000 cells/ml, 52,000
cells/ml, 53,000 cells/ml, 54,000 cells/ml, 55,000 cells/ml, 56,000 cells/ml, 57,000 cells/ml,
58,000 cells/ml, 59,000 cells/ml, 60,000 cells/ml, 61,000 cells/ml, 62,000 cells/ml, 63,000
cells/ml, 64,000 cells/ml, 65,000 cells/ml, 66,000 cells/ml, 67,000 cells/ml, 68,000 cells/ml,
69,000 cells/ml, 70,000 cells/ml, 71,000 cells/ml, 72,000 cells/ml, 73,000 cells/ml, 74,000
cells/ml, 75,000 cells/ml, 76,000 cells/ml, 77,000 cells/ml, or more), and having a density of
leukocytes of no more than about 5.3 107 X 10cells/ml, cells/ml,such suchas asa adensity densityof ofleukocytes leukocytesof ofabout about
2.3 2.3 xx 107 10 cells/ml cells/mltoto about 5.35.3 about x 107 cells/ml, X 10 aboutabout cells/ml, 2.5 x 2.5 107 cells/ml to about x 10 cells/ml to5.1 X 1075.1 x 10 about
cells/ml, cells/ml,2.9 2.9X 107 X 10cells/ml to about cells/ml 4,5 x4.5 to about 107Xcells/ml, about 3 10 cells/ml, X 1073cells/ml about to about to X 10 cells/ml 4 xabout 107 4 x 10
cells/ml (e.g., 5.3 x X 10 cells/ml, 5.2 x X 107 cells/ml, 5.1 10 cells/ml, 5.1 Xx 10 107 cells/ml, cells/ml, 5 5 x x 10107 cells/ml, cells/ml, 4.94.9 X X
107 cells/ml, 4.8 10 cells/ml, 4.8 Xx 10 107 cells/ml, cells/ml, 4.7 4.7 X x 10107 cells/ml, cells/ml, 4.64.6 x 107 X 10 cells/ml, cells/ml, 4.5 4.5x X 10 cells/ml, 4.4 x X
107 cells/ml, 4.3 10 cells/ml, 4.3x X107 10cells/ml cells/ml4.24.2 x 10 X cells/ml, 4.1 x 4.1 10 cells/ml, 107 x cells/ml 4 x 10 4 10 cells/ml cells/ml, 3.9 x 107 3.9 x 10 X 10 cells/ml,
cells/ml, 3.8 x X 107 cells/ml, 3.7 10 cells/ml, 3.7 Xx 10 107 cells/ml, cells/ml, 3.6 3.6 X x 10107 cells/ml, cells/ml, 3.53.5x X 10cells/ml, cells/ml,3.4 3.4x X107 10
cells/ml, 3.3 x X 107 cells/ml, 3.2 10 cells/ml, 3.2 Xx 10 107 cells/ml, cells/ml, 3.1 3.1 X x 10107 cells/ml, cells/ml, 3 X3 10 x 107 cells/ml, cells/ml, 2.9 2.9 x 10x 107
cells/ml, 2.8 x X 107 cells/ml,2.7 10 cells/ml, 2.7Xx10 107 cells/ml, cells/ml, 2.6 2.6 X x 10107 cells/ml, cells/ml, 2.52.5 107 X 10 cells/ml, cells/ml, 2.4 2.4 X x 10107
cells/ml, 2.3 x X 107 cells/ml, or 10 cells/ml, or less). less). In In some some embodiments, embodiments, the the method method includes includes
administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient
to produce a population of cells having a density of CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells ofof from from
about 20,000 cells/ml to about 75,000 cells/ml, and having a density of leukocytes of from
about 2.3 X x 107 cells/ml to 10 cells/ml to about about 5.3 5.3 Xx 10 107 cells/ml. cells/ml. InIn some some embodiments, embodiments, the the method method
includes administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts
sufficient to produce a population of cells having a density of CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells
of from about 30,000 cells/ml to about 60,000 cells/ml, and having a density of leukocytes of
from about 2.5 X 107 cells/mlto 10 cells/ml toabout about55Xx10 107 cells/ml. cells/ml. InIn some some embodiments, embodiments, the the method method
includes administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts
PCT/US2018/064335
sufficient to produce a population of cells having a density of CD34+ CD90 CD45RA CD34 CD90 CD45RA cells cells
of from about 40,000 cells/ml to about 50,000 cells/ml, and having a density of leukocytes of
from from about about3 3X x 10710cells/ml to to cells/ml about 4x 107 about 4 Xcells/ml. 10 cells/ml.
[0026] In a further aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to produce a population of cells having a ratio of
CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells toto neutrophils neutrophils ofof from from about about 0.0007 0.0007 toto about about 0.0043 0.0043 inin a a
sample of peripheral blood of the donor following administration of the CXCR2 agonist and
CXCR4 antagonist. In some embodiments, the ratio of CD34+ CD90 CD45RA CD34 CD90 CD45RA cells cells to to
neutrophils in the sample may be about 0.00070, 0.00071, 0.00072, 0.00073, 0.00074,
0.00075, 0.00076, 0.00077, 0.00078, 0.00079, 0.00080, 0.00081, 0.00082, 0.00083, 0.00084,
0.00085, 0.00086, 0.00087, 0.00088, 0.00089, 0.00090, 0.00091, 0.00092, 0.00093, 0.00094,
0.00095, 0.00096, 0.00097, 0.00098, 0.00099, 0.00100, 0.00101, 0.00102, 0.00103, 0.00104,
0.00105, 0.00106, 0.00107, 0.00108, 0.00109, 0.00110, 0.00111, 0.00112, 0.00113, 0.00114,
0.00115, 0.00116, 0.00117, 0.00118, 0.00119, 0.00120, 0.00121, 0.00122, 0.00123, 0.00124,
0.00125, 0.00126, 0.00127, 0.00128, 0.00129, 0.00130, 0.00131, 0.00132, 0.00133, 0.00134,
0.00135, 0.00136, 0.00137, 0.00138, 0.00139, 0.00140, 0.00141, 0.00142, 0.00143, 0.00144,
0.00145, 0.00146, 0.00147, 0.00148, 0.00149, 0.00150, 0.00151, 0.00152, 0.00153, 0.00154,
0.00155, 0.00156, 0.00157, 0.00158, 0.00159, 0.00160, 0.00161, 0.00162, 0.00163, 0.00164,
0.00165, 0.00166, 0.00167, 0.00168, 0.00169, 0.00170, 0.00171, 0.00172, 0.00173, 0.00174,
0.00175, 0.00176, 0.00177, 0.00178, 0.00179, 0.00180, 0.00181, 0.00182, 0.00183, 0.00184,
0.00185, 0.00186, 0.00187, 0.00188, 0.00189, 0.00190, 0.00191, 0.00192, 0.00193, 0.00194,
0.00195, 0.00196, 0.00197, 0.00198, 0.00199, 0.00200, 0.00201, 0.00202, 0.00203, 0.00204,
0.00205, 0.00206, 0.00207, 0.00208, 0.00209, 0.00210, 0.00211, 0.00212, 0.00213, 0.00214,
0.00215, 0.00216, 0.00217, 0.00218, 0.00219, 0.00220, 0.00221, 0.00222, 0.00223, 0.00224,
0.00225, 0.00226, 0.00227, 0.00228, 0.00229, 0.00230, 0.00231, 0.00232, 0.00233, 0.00234,
0.00235, 0.00236, 0.00237, 0.00238, 0.00239, 0.00240, 0.00241, 0.00242, 0.00243, 0.00244,
0.00245, 0.00246, 0.00247, 0.00248, 0.00249, 0.00250, 0.00251, 0.00252, 0.00253, 0.00254,
0.00255, 0.00256, 0.00257, 0.00258, 0.00259, 0.00260, 0.00261, 0.00262, 0.00263, 0.00264,
0.00265, 0.00266, 0.00267, 0.00268, 0.00269, 0.00270, 0.00271, 0.00272, 0.00273, 0.00274,
0.00275, 0.00276, 0.00277, 0.00278, 0.00279, 0.00280, 0.00281, 0.00282, 0.00283, 0.00284,
0.00285, 0.00286, 0.00287, 0.00288, 0.00289, 0.00290, 0.00291, 0.00292, 0.00293, 0.00294,
0.00295, 0.00296, 0.00297, 0.00298, 0.00299, 0.00300, 0.00300, 0.00301, 0.00302, 0.00303,
0.00304, 0.00305, 0.00306, 0.00307, 0.00308, 0.00309, 0.00310, 0.00311, 0.00312, 0.00313,
0.00314, 0.00315, 0.00316, 0.00317, 0.00318, 0.00319, 0.00320, 0.00321, 0.00322, 0.00323,
0.00324, 0.00325, 0.00326, 0.00327, 0.00328, 0.00329, 0.00330, 0.00331, 0.00332, 0.00333,
0.00334, 0.00335, 0.00336, 0.00337, 0.00338, 0.00339, 0.00340, 0.00341, 0.00342, 0.00343,
0.00344, 0.00345, 0.00346, 0.00347, 0.00348, 0.00349, 0.00350, 0.00351, 0.00352, 0.00353,
0.00354, 0.00355, 0.00356, 0.00357, 0.00358, 0.00359, 0.00360, 0.00361, 0.00362, 0.00363,
0.00364, 0.00365, 0.00366, 0.00367, 0.00368, 0.00369, 0.00370, 0.00371, 0.00372, 0.00373,
0.00374, 0.00375, 0.00376, 0.00377, 0.00378, 0.00379, 0.00380, 0.00381, 0.00382, 0.00383,
0.00384, 0.00385, 0.00386, 0.00387, 0.00388, 0.00389, 0.00390, 0.00391, 0.00392, 0.00393,
0.00394, 0.00395, 0.00396, 0.00397, 0.00398, 0.00399, 0.00400, 0.00401, 0.00402, 0.00403,
0.00404, 0.00405, 0.00406, 0.00407, 0.00408, 0.00409, 0.00410, 0.00411, 0.00412, 0.00413,
0.00414, 0.00415, 0.00416, 0.00417, 0.00418, 0.00419, 0.00420, 0.00421, 0.00422, 0.00423,
0.00424, 0.00425, 0.00426, 0.00427, 0.00428, 0.00429, or 0.00430. In some embodiments,
the ratio of CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells toto neutrophils neutrophils inin the the sample sample isis from from about about 0.002 0.002 toto
about 0.003. In some embodiments, the ratio of CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells toto neutrophils neutrophils
in the sample is from about 0.0014 to about 0.0034, such as a ratio of CD34+ CD90+ CD34 CD90
CD45RA cells to neutrophils in the sample of about 0.00140, 0.00141, 0.00142, 0.00143,
0.00144, 0.00145, 0.00146, 0.00147, 0.00148, 0.00149, 0.00150, 0.00151, 0.00152, 0.00153,
0.00154, 0.00155, 0.00156, 0.00157, 0.00158, 0.00159, 0.00160, 0.00161, 0.00162, 0.00163,
0.00164, 0.00165, 0.00166, 0.00167, 0.00168, 0.00169, 0.00170, 0.00171, 0.00172, 0.00173,
0.00174, 0.00175, 0.00176, 0.00177, 0.00178, 0.00179, 0.00180, 0.00181, 0.00182, 0.00183,
0.00184, 0.00185, 0.00186, 0.00187, 0.00188, 0.00189, 0.00190, 0.00191, 0.00192, 0.00193,
0.00194, 0.00195, 0.00196, 0.00197, 0.00198, 0.00199, 0.00200, 0.00201, 0.00202, 0.00203,
0.00204, 0.00205, 0.00206, 0.00207, 0.00208, 0.00209, 0.00210, 0.00211, 0.00212, 0.00213,
0.00214, 0.00215, 0.00216, 0.00217, 0.00218, 0.00219, 0.00220, 0.00221, 0.00222, 0.00223,
0.00224, 0.00225, 0.00226, 0.00227, 0.00228, 0.00229, 0.00230, 0.00231, 0.00232, 0.00233,
0.00234, 0.00235, 0.00236, 0.00237, 0.00238, 0.00239, 0.00240, 0.00241, 0.00242, 0.00243,
0.00244, 0.00245, 0.00246, 0.00247, 0.00248, 0.00249, 0.00250, 0.00251, 0.00252, 0.00253,
0.00254, 0.00255, 0.00256, 0.00257, 0.00258, 0.00259, 0.00260, 0.00261, 0.00262, 0.00263,
0.00264, 0.00265, 0.00266, 0.00267, 0.00268, 0.00269, 0.00270, 0.00271, 0.00272, 0.00273,
0.00274, 0.00275, 0.00276, 0.00277, 0.00278, 0.00279, 0.00280, 0.00281, 0.00282, 0.00283,
0.00284, 0.00285, 0.00286, 0.00287, 0.00288, 0.00289, 0.00290, 0.00291, 0.00292, 0.00293,
0.00294, 0.00295, 0.00296, 0.00297, 0.00298, 0.00299, 0.00300, 0.00300, 0.00301, 0.00302,
0.00303, 0.00304, 0.00305, 0.00306, 0.00307, 0.00308, 0.00309, 0.00310, 0.00311, 0.00312,
0.00313, 0.00314, 0.00315, 0.00316, 0.00317, 0.00318, 0.00319, 0.00320, 0.00321, 0.00322,
0.00323, 0.00324, 0.00325, 0.00326, 0.00327, 0.00328, 0.00329, 0.00330, 0.00331, 0.00332,
0.00333, 0.00334, 0.00335, 0.00336, 0.00337, 0.00338, 0.00339, or 0.00340. In some
embodiments, the ratio of CD34+ CD90 CD45RA CD34 CD90 CD45RA cells cells to to neutrophils neutrophils in in the the sample sample is is about about
0.0024.
[0027] In an additional aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to enrich the peripheral blood of the donor with
CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells relative relative toto neutrophils neutrophils byby a a ratio ratio ofof from from about about 3.5:1 3.5:1 toto about about
22.0:1 as assessed by comparing a sample of peripheral blood of the donor following
administration of the CXCR2 agonist and CXCR4 antagonist to a sample of peripheral blood
of the donor prior to administration of the CXCR2 agonist and CXCR4 antagonist. In some
embodiments, the peripheral blood of the donor may be enriched with CD34 CD90
CD45RA cells relative to neutrophils by a ratio of about 3.50:1, 3.55:1, 3.60:1, 3.65:1,
3.70:1, 3.75:1, 3.80:1, 3.85:1, 3.90:1, 3.95:1, 4.00:1, 4.05:1, 4.10:1, 4.15:1, 4.20:1, 4.25:1,
4.30:1, 4.35:1, 4.40:1, 4.45:1, 4.50:1, 4.55:1, 4.60:1, 4.65:1, 4.70:1, 4.75:1, 4.80:1, 4.85:1,
4.90:1, 4.95:1, 5.00:1, 5.05:1, 5.10:1, 5.15:1, 5.20:1, 5.25:1, 5.30:1, 5.35:1, 5.40:1, 5.45:1,
5.50:1, 5.55:1, 5.60:1, 5.65:1, 5.70:1, 5.75:1, 5.80:1, 5.85:1, 5.90:1, 5.95:1, 6.00:1, 6.05:1,
6.10:1, 6.15:1, 6.20:1, 6.25:1, 6.30:1, 6.35:1, 6.40:1, 6.45:1, 6.50:1, 6.55:1, 6.60:1, 6.65:1,
6.70:1, 6.75:1, 6.80:1, 6.85:1, 6.90:1, 6.95:1, 7.00:1, 7.05:1, 7.10:1, 7.15:1, 7.20:1, 7.25:1,
7.30:1, 7.35:1, 7.40:1, 7.45:1, 7.50:1, 7.55:1, 7.60:1, 7.65:1, 7.70:1, 7.75:1, 7.80:1, 7.85:1,
7.90:1, 7.95:1, 8.00:1, 8.05:1, 8.10:1, 8.15:1, 8.20:1, 8.25:1, 8.30:1, 8.35:1, 8.40:1, 8.45:1,
8.50:1, 8.55:1, 8.60:1, 8.65:1, 8.70:1, 8.75:1, 8.80:1, 8.85:1, 8.90:1, 8.95:1, 9.00:1, 9.05:1,
9.10:1, 9.15:1, 9.20:1, 9.25:1, 9.30:1, 9.35:1, 9.40:1, 9.45:1, 9.50:1, 9.55:1, 9.60:1, 9.65:1,
9.70:1, 9.75:1, 9.80:1, 9.85:1, 9.90:1, 9.95:1, 10.00:1, 10.05:1, 10.10:1, 10.15:1, 10.20:1,
10.25:1, 10.30:1, 10.35:1, 10.40:1, 10.45:1, 10.50:1, 10.55:1, 10.60:1, 10.65:1, 10.70:1,
10.75:1, 10.80:1, 10.85:1, 10.90:1, 10.95:1, 11.00:1, 11.05:1, 11.10:1, 11.15:1, 11.20:1,
11.25:1, 11.30:1, 11.35:1, 11.40:1, 11.45:1, 11.50:1, 11.55:1, 11.60:1, 11.65:1, 11.70:1,
11.75:1, 11.80:1, 11.85:1, 11.90:1, 11.95:1, 12.00:1, 12.05:1, 12.10:1, 12.15:1, 12.20:1,
12.25:1, 12.30:1, 12.35:1, 12.40:1, 12.45:1, 12.50:1, 12.55:1, 12.60:1, 12.65:1, 12.70:1,
26
WO wo 2019/113375 PCT/US2018/064335
12.75:1, 12.80:1, 12.85:1, 12.90:1, 12.95:1, 13.00:1, 13.05:1, 13.10:1, 13.15:1, 13.20:1,
13.25:1, 13.30:1, 13.35:1, 13.40:1, 13.45:1, 13.50:1, 13.55:1, 13.60:1, 13.65:1, 13.70:1,
13.75:1, 13.80:1, 13.85:1, 13.90:1, 13.95:1, 14.00:1, 14.05:1, 14.10:1, 14.15:1, 14.20:1,
14.25:1, 14.30:1, 14.35:1, 14.40:1, 14.45:1, 14.50:1, 14.55:1, 14.60:1, 14.65:1, 14.70:1,
14.75:1, 14.80:1, 14.85:1, 14.90:1, 14.95:1, 15.00:1, 15.05:1, 15.10:1, 15.15:1, 15.20:1,
15.25:1, 15.30:1, 15.35:1, 15.40:1, 15.45:1, 15.50:1, 15.55:1, 15.60:1, 15.65:1, 15.70:1,
15.75:1, 15.80:1, 15.85:1, 15.90:1, 15.95:1, 16.00:1, 16.05:1, 16.10:1, 16.15:1, 16.20:1,
16.25:1, 16.30:1, 16.35:1, 16.40:1, 16.45:1, 16.50:1, 16.55:1, 16.60:1, 16.65:1, 16.70:1,
16.75:1, 16.80:1, 16.85:1, 16.90:1, 16.95:1, 17.00:1, 17.05:1, 17.10:1, 17.15:1, 17.20:1,
17.25:1, 17.30:1, 17.35:1, 17.40:1, 17.45:1, 17.50:1, 17.55:1, 17.60:1, 17.65:1, 17.70:1,
17.75:1, 17.80:1, 17.85:1, 17.90:1, 17.95:1, 18.00:1, 18.05:1, 18.10:1, 18.15:1, 18.20:1,
18.25:1, 18.30:1, 18.35:1, 18.40:1, 18.45:1, 18.50:1, 18.55:1, 18.60:1, 18.65:1, 18.70:1,
18.75:1, 18.80:1, 18.85:1, 18.90:1, 18.95:1, 19.00:1, 19.05:1, 19.10:1, 19.15:1, 19.20:1,
19.25:1, 19.30:1, 19.35:1, 19.40:1, 19.45:1, 19.50:1, 19.55:1, 19.60:1, 19.65:1, 19.70:1,
19.75:1, 19.80:1, 19.85:1, 19.90:1, 19.95:1, 20.00:1, 20.05:1, 20.10:1, 20.15:1, 20.20:1,
20.25:1, 20.30:1, 20.35:1, 20.40:1, 20.45:1, 20.50:1, 20.55:1, 20.60:1, 20.65:1, 20.70:1,
20.75:1, 20.80:1, 20.85:1, 20.90:1, 20.95:1, 21.00:1, 21.05:1, 21.10:1, 21.15:1, 21.20:1,
21.25:1, 21.30:1, 21.35:1, 21.40:1, 21.45:1, 21.50:1, 21.55:1, 21.60:1, 21.65:1, 21.70:1,
21.75:1, 21.80:1, 21.85:1, 21.90:1, 21.95:1, or 22.00:1. In some embodiments, the peripheral
blood of the donor is enriched with CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells relative relative toto neutrophils neutrophils byby a a
ratio of about from about 7.0:1 to about 10:1. In some embodiments, the peripheral blood of
the donor is enriched with CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells relative relative toto neutrophils neutrophils byby a a ratio ratio ofof
from about 7.00:1 to about 9.00:1, such as a ratio of about 7.00:1, 7.05:1, 7.10:1, 7.15:1,
7.20:1, 7.25:1, 7.30:1, 7.35:1, 7.40:1, 7.45:1, 7.50:1, 7.55:1, 7.60:1, 7.65:1, 7.70:1, 7.75:1,
7.80:1, 7.85:1, 7.90:1, 7.95:1, 8.00:1, 8.05:1, 8.10:1, 8.15:1, 8.20:1, 8.25:1, 8.30:1, 8.35:1,
8.40:1, 8.45:1, 8.50:1, 8.55:1, 8.60:1, 8.65:1, 8.70:1, 8.75:1, 8.80:1, 8.85:1, 8.90:1, 8.95:1, or
9.00:1. In some embodiments, the peripheral blood of the donor is enriched with CD34+ CD34
CD90 CD45RA cells relative to neutrophils by a ratio of about 8.2:1.
[0028] In another aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density
of CD34 CD90+ CD45RAcells CD90 CD45RA cellsof ofat atleast leastabout about16,000 16,000cells/ml, cells/ml,such suchas asaadensity densityof offrom from
WO wo 2019/113375 PCT/US2018/064335
about 20,000 cells/ml to about 75,000 cells/ml, about 25,000 cells/ml to about 70,000
cells/ml, about 30,000 cells/ml to about 65,000 cells/ml, about 35,000 cells/ml to about
60,000 cells/ml, about 40,000 cells/ml to about 55,000 cells/ml, or about 45,000 cells/ml to
about 50,000 cells/ml (e.g., about 16,000 cells/ml, 17,000 cells/ml, 18,000 cells/ml, 19,000
cells/ml, 20,000 cells/ml, 21,000 cells/ml, 22,000 cells/ml, 23,000 cells/ml, 24,000 cells/ml,
25,000 cells/ml, 26,000 cells/ml, 27,000 cells/ml, 28,000 cells/ml, 29,000 cells/ml, 30,000
cells/ml, 31,000 cells/ml, 32,000 cells/ml, 33,000 cells/ml, 34,000 cells/ml, 35,000 cells/ml,
36,000 cells/ml, 37,000 cells/ml, 38,000 cells/ml, 39,000 cells/ml, 40,000 cells/ml, 41,000
cells/ml, 42,000 cells/ml, 43,000 cells/ml, 44,000 cells/ml, 45,000 cells/ml, 46,000 cells/ml,
47,000 cells/ml, 48,000 cells/ml, 49,000 cells/ml, 50,000 cells/ml, 51,000 cells/ml, 52,000
cells/ml, 53,000 cells/ml, 54,000 cells/ml, 55,000 cells/ml, 56,000 cells/ml, 57,000 cells/ml,
58,000 cells/ml, 59,000 cells/ml, 60,000 cells/ml, 61,000 cells/ml, 62,000 cells/ml, 63,000
cells/ml, 64,000 cells/ml, 65,000 cells/ml, 66,000 cells/ml, 67,000 cells/ml, 68,000 cells/ml,
69,000 cells/ml, 70,000 cells/ml, 71,000 cells/ml, 72,000 cells/ml, 73,000 cells/ml, 74,000
cells/ml, 75,000 cells/ml, 76,000 cells/ml, 77,000 cells/ml, or more), and having a density of
neutrophils neutrophils ofof no no more more thanthan about about 2.5 x2.5 107 cells/ml, 10 cells/ml, such assuch as a density a density of neutrophils of neutrophils of about of about
1 1 xX 107 10 cells/ml cells/mltotoabout 2.52.5 about x 107 cells/ml, X 10 aboutabout cells/ml, 1.3 x 1.3 107 X cells/ml to about 10 cells/ml to2 about X 107 cells/ml, 2 X cells/ml,
or about 1.5 107 X 10cells/ml cells/mlto toabout about1.9 1.9x X107 10 cells/ml (e.g., about 2.5x 2.5 x107 10 cells/ml, 2.4 x 107 10
cells/ml, 2.3 x X 107 cells/ml, 2.2 10 cells/ml, 2.2 Xx o07 cells/ml, cells/ml, 2.1 2.1 X x 10107 cells/ml, cells/ml, 2 X2 10 x 107 cells/ml, cells/ml, 1.9 1.9 X 10x 107
cells/ml, 1.8 x X 107 cells/ml, 1.7 10 cells/ml, 1.7 xx 10 107 cells/ml, cells/ml, 1.6 1.6 X x 10107 cells/ml, cells/ml, 1.51.5x x 10107 cells/ml cells/ml 1.41.4 x 107 x 10
cells/ml, 1.3 x X 107 cells/ml,1.2 10 cells/ml, 1.2: X 107 cells/ml,1.1 10 cells/ml, 1.1Xx10 107 cells/ml, cells/ml, 1 X 107 cells/ml,or 10 cells/ml, orless). less).In In
some embodiments, the method includes administering to the donor a CXCR2 agonist and a
CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of
CD34+ CD90 CD45RA CD34 CD90 CD45RA cells cells of of from from about about 20,000 20,000 cells/ml cells/ml to to about about 75,000 75,000 cells/ml, cells/ml, and and
having a density of neutrophils of from about X 1 107 x 10cells/ml cells/mlto toabout about2.5 2.5X x107 10 cells/ml. In
some embodiments, the method includes administering to the donor a CXCR2 agonist and a
CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of
CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells ofof from from about about 30,000 30,000 cells/ml cells/ml toto about about 60,000 60,000 cells/ml, cells/ml, and and
having a density of neutrophils of from about 1.3 107 X 10cells/ml cells/mlto toabout about2.3 2.3XX107 10 cells/ml.
In some embodiments, the method includes administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density
of CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells ofof from from about about 40,000 40,000 cells/ml cells/ml toto about about 50,000 50,000 cells/ml, cells/ml, and and
having a density of neutrophils of from about 1.5 107 x 10cells/ml cells/mlto toabout about2 2X x107 10 cells/ml.
WO wo 2019/113375 PCT/US2018/064335
[0029] Inyet
[0029] In yetanother another aspect, aspect, the invention the invention features features a methoda of method of mobilizing mobilizing a of a population population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to produce a population of cells having a ratio of
CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells toto lymphocytes lymphocytes ofof from from about about 0.0008 0.0008 toto about about 0.0069 0.0069 inin a a
sample of peripheral blood of the donor following administration of the CXCR2 agonist and
CXCR4 CXCR4 antagonist. antagonist.In In some embodiments, some the ratio embodiments, of CD34+ the ratio of CD90+ CD34 CD45RA cells to CD90 CD45RA cells to
lymphocytes in the sample may be about 0.00080, 0.00081, 0.00082, 0.00083, 0.00084,
0.00085, 0.00086, 0.00087, 0.00088, 0.00089, 0.00090, 0.00091, 0.00092, 0.00093, 0.00094,
0.00095, 0.00096, 0.00097, 0.00098, 0.00099, 0.00100, 0.00101, 0.00102, 0.00103, 0.00104,
0.00105, 0.00106, 0.00107, 0.00108, 0.00109, 0.00110, 0.00111, 0.00112, 0.00113, 0.00114,
0.00115, 0.00116, 0.00117, 0.00118, 0.00119, 0.00120, 0.00121, 0.00122, 0.00123, 0.00124,
0.00125, 0.00126, 0.00127, 0.00128, 0.00129, 0.00130, 0.00131, 0.00132, 0.00133, 0.00134,
0.00135, 0.00136, 0.00137, 0.00138, 0.00139, 0.00140, 0.00141, 0.00142, 0.00143, 0.00144,
0.00145, 0.00146, 0.00147, 0.00148, 0.00149, 0.00150, 0.00151, 0.00152, 0.00153, 0.00154,
0.00155, 0.00156, 0.00157, 0.00158, 0.00159, 0.00160, 0.00161, 0.00162, 0.00163, 0.00164,
0.00165, 0.00166, 0.00167, 0.00168, 0.00169, 0.00170, 0.00171, 0.00172, 0.00173, 0.00174,
0.00175, 0.00176, 0.00178, 0.00179, 0.00180, 0.00181, 0.00182, 0.00183, 0.00184, 0.00185,
0.00186, 0.00187, 0.00188, 0.00189, 0.00190, 0.00191, 0.00192, 0.00193, 0.00194, 0.00195,
0.00196, 0.00197, 0.00198, 0.00199, 0.00200, 0.00201, 0.00202, 0.00203, 0.00204, 0.00205,
0.00206, 0.00207, 0.00208, 0.00209, 0.00210, 0.00211, 0.00212, 0.00213, 0.00214, 0.00215,
0.00216, 0.00217, 0.00218, 0.00219, 0.00220, 0.00221, 0.00222, 0.00223, 0.00224, 0.00225,
0.00226, 0.00227, 0.00228, 0.00229, 0.00230, 0.00231, 0.00232, 0.00233, 0.00234, 0.00235,
0.00236, 0.00237, 0.00238, 0.00239, 0.00240, 0.00241, 0.00242, 0.00243, 0.00244, 0.00245,
0.00246, 0.00247, 0.00248, 0.00249, 0.00250, 0.00251, 0.00252, 0.00253, 0.00254, 0.00255,
0.00256, 0.00257, 0.00258, 0.00259, 0.00260, 0.00261, 0.00262, 0.00263, 0.00264, 0.00265,
0.00266, 0.00267, 0.00268, 0.00269, 0.00270, 0.00271, 0.00272, 0.00273, 0.00274, 0.00275,
0.00276, 0.00278, 0.00279, 0.00280, 0.00281, 0.00282, 0.00283, 0.00284, 0.00285, 0.00286,
0.00287, 0.00288, 0.00289, 0.00290, 0.00291, 0.00292, 0.00293, 0.00294, 0.00295, 0.00296,
0.00297, 0.00298, 0.00299, 0.00300, 0.00301, 0.00302, 0.00303, 0.00304, 0.00305, 0.00306,
0.00307, 0.00308, 0.00309, 0.00310, 0.00311, 0.00312, 0.00313, 0.00314, 0.00315, 0.00316,
0.00317, 0.00318, 0.00319, 0.00320, 0.00321, 0.00322, 0.00323, 0.00324, 0.00325, 0.00326,
0.00327, 0.00328, 0.00329, 0.00330, 0.00331, 0.00332, 0.00333, 0.00334, 0.00335, 0.00336,
0.00337, 0.00338, 0.00339, 0.00340, 0.00341, 0.00342, 0.00343, 0.00344, 0.00345, 0.00346,
0.00347, 0.00348, 0.00349, 0.00350, 0.00351, 0.00352, 0.00353, 0.00354, 0.00355, 0.00356,
0.00357, 0.00357, 0.00358, 0.00358, 0.00359, 0.00359, 0.00360, 0.00360, 0.00361, 0.00361, 0.00362, 0.00362, 0.00363, 0.00363, 0.00364, 0.00364, 0.00365, 0.00365, 0.00366, 0.00366,
0.00367, 0.00367, 0.00368, 0.00368, 0.00369, 0.00369, 0.00370, 0.00370, 0.00371, 0.00371, 0.00372, 0.00372, 0.00373, 0.00373, 0.00374, 0.00374, 0.00375, 0.00375, 0.00376, 0.00376,
0.00378, 0.00378, 0.00379, 0.00379, 0.00380, 0.00380, 0.00381, 0.00381, 0.00382, 0.00382, 0.00383, 0.00383, 0.00384, 0.00384, 0.00385, 0.00385, 0.00386, 0.00386, 0.00387, 0.00387,
0.00388, 0.00388, 0.00389, 0.00389, 0.00390, 0.00390, 0.00391, 0.00391, 0.00392, 0.00392, 0.00393, 0.00393, 0.00394, 0.00394, 0.00395, 0.00395, 0.00396, 0.00396, 0.00397, 0.00397,
0.00398, 0.00399, 0.00401, 0.00402, 0.00403, 0.00404, 0.00405, 0.00406, 0.00407, 0.00408,
0.00409, 0.00410, 0.00411, 0.00412, 0.00413, 0.00414, 0.00415, 0.00416, 0.00417, 0.00418,
0.00419, 0.00420, 0.00421, 0.00422, 0.00423, 0.00424, 0.00425, 0.00426, 0.00427, 0.00428,
0.00429, 0.00430, 0.00431, 0.00432, 0.00433, 0.00434, 0.00435, 0.00436, 0.00437, 0.00438,
0.00439, 0.00440, 0.00441, 0.00442, 0.00443, 0.00444, 0.00445, 0.00446, 0.00447, 0.00448,
0.00449, 0.00449, 0.00450, 0.00450, 0.00451, 0.00451, 0.00452, 0.00452, 0.00453, 0.00453, 0.00454, 0.00454, 0.00455, 0.00455, 0.00456, 0.00456, 0.00457, 0.00457, 0.00458, 0.00458,
0.00459, 0.00460, 0.00461, 0.00462, 0.00463, 0.00464, 0.00465, 0.00466, 0.00467, 0.00468,
0.00469, 0.00469, 0.00470, 0.00470, 0.00471, 0.00471, 0.00472, 0.00472, 0.00473, 0.00473, 0.00474, 0.00474, 0.00475, 0.00475, 0.00476, 0.00476, 0.00478, 0.00478, 0.00479, 0.00479,
0.00480, 0.00480, 0.00481, 0.00481, 0.00482, 0.00482, 0.00483, 0.00483, 0.00484, 0.00484, 0.00485, 0.00485, 0.00486, 0.00486, 0.00487, 0.00487, 0.00488, 0.00488, 0.00489, 0.00489,
0.00490, 0.00491, 0.00492, 0.00493, 0.00494, 0.00495, 0.00496, 0.00497, 0.00498, 0.00499,
0.00500, 0.00500, 0.00501, 0.00501, 0.00502, 0.00502, 0.00503, 0.00503, 0.00504, 0.00504, 0.00505, 0.00505, 0.00506, 0.00506, 0.00507, 0.00507, 0.00508, 0.00508, 0.00509, 0.00509,
0.00510, 0.00511, 0.00512, 0.00513, 0.00514, 0.00515, 0.00516, 0.00517, 0.00518, 0.00519,
0.00520, 0.00521, 0.00522, 0.00523, 0.00524, 0.00525, 0.00526, 0.00527, 0.00528, 0.00529,
0.00530, 0.00531, 0.00532, 0.00533, 0.00534, 0.00535, 0.00536, 0.00537, 0.00538, 0.00539,
0.00540, 0.00541, 0.00542, 0.00543, 0.00544, 0.00545, 0.00546, 0.00547, 0.00548, 0.00549,
0.00550, 0.00551, 0.00552, 0.00553, 0.00554, 0.00555, 0.00556, 0.00557, 0.00558, 0.00559,
0.00560, 0.00561, 0.00562, 0.00563, 0.00564, 0.00565, 0.00566, 0.00567, 0.00568, 0.00569,
0.00570, 0.00570, 0.00571, 0.00571, 0.00572, 0.00572, 0.00573, 0.00573, 0.00574, 0.00574, 0.00575, 0.00575, 0.00576, 0.00576, 0.00578, 0.00578, 0.00579, 0.00579, 0.00580, 0.00580,
0.00581, 0.00581, 0.00582, 0.00582, 0.00583, 0.00583, 0.00584, 0.00584, 0.00585, 0.00585, 0.00586, 0.00586, 0.00587, 0.00587, 0.00588, 0.00588, 0.00589, 0.00589, 0.00590, 0.00590,
0.00591, 0.00592, 0.00593, 0.00594, 0.00595, 0.00596, 0.00597, 0.00598, 0.00599, 0.00600,
0.00601, 0.00601, 0.00602, 0.00602, 0.00603, 0.00603, 0.00604, 0.00604, 0.00605, 0.00605, 0.00606, 0.00606, 0.00607, 0.00607, 0.00608, 0.00608, 0.00609, 0.00609, 0.00610, 0.00610,
0.00611, 0.00612, 0.00613, 0.00614, 0.00615, 0.00616, 0.00617, 0.00618, 0.00619, 0.00620,
0.00621, 0.00622, 0.00623, 0.00624, 0.00625, 0.00626, 0.00627, 0.00628, 0.00629, 0.00630,
0.00631, 0.00632, 0.00633, 0.00634, 0.00635, 0.00636, 0.00637, 0.00638, 0.00639, 0.00640,
0.00641, 0.00642, 0.00643, 0.00644, 0.00645, 0.00646, 0.00647, 0.00648, 0.00649, 0.00650,
0.00651, 0.00652, 0.00653, 0.00654, 0.00655, 0.00656, 0.00657, 0.00658, 0.00659, 0.00660,
0.00661, 0.00662, 0.00663, 0.00664, 0.00665, 0.00666, 0.00667, 0.00668, 0.00669, 0.00670,
0.00671, 0.00672, 0.00673, 0.00674, 0.00675, 0.00676, 0.00678, 0.00679, 0.00680, 0.00681,
0.00682, 0.00683, 0.00684, 0.00685, 0.00686, 0.00687, 0.00688, 0.00689, or 0.00690. In
some embodiments, the ratio of CD34+ CD90 CD45RA CD34 CD90 CD45RA cells cells to to lymphocytes lymphocytes in in the the sample sample
is from about 0.0011 to about 0.0031, such as a ratio of CD34+ CD90 CD45RA CD34 CD90 CD45RA cells cells to to
lymphocytes lymphocytes in in the the sample sample of of about about 0.00110, 0.00110, 0.00111, 0.00111, 0.00112, 0.00112, 0.00113, 0.00113, 0.00114, 0.00114, 0.00115, 0.00115,
0.00116, 0.00117, 0.00118, 0.00119, 0.00120, 0.00121, 0.00122, 0.00123, 0.00124, 0.00125,
0.00126, 0.00127, 0.00128, 0.00129, 0.00130, 0.00131, 0.00132, 0.00133, 0.00134, 0.00135,
0.00136, 0.00137, 0.00138, 0.00139, 0.00140, 0.00141, 0.00142, 0.00143, 0.00144, 0.00145,
0.00146, 0.00147, 0.00148, 0.00149, 0.00150, 0.00151, 0.00152, 0.00153, 0.00154, 0.00155,
0.00156, 0.00157, 0.00158, 0.00159, 0.00160, 0.00161, 0.00162, 0.00163, 0.00164, 0.00165,
0.00166, 0.00167, 0.00168, 0.00169, 0.00170, 0.00171, 0.00172, 0.00173, 0.00174, 0.00175,
0.00176, 0.00178, 0.00179, 0.00180, 0.00181, 0.00182, 0.00183, 0.00184, 0.00185, 0.00186,
0.00187, 0.00188, 0.00189, 0.00190, 0.00191, 0.00192, 0.00193, 0.00194, 0.00195, 0.00196,
0.00197, 0.00198, 0.00199, 0.00200, 0.00201, 0.00202, 0.00203, 0.00204, 0.00205, 0.00206,
0.00207, 0.00208, 0.00209, 0.00210, 0.00211, 0.00212, 0.00213, 0.00214, 0.00215, 0.00216,
0.00217, 0.00218, 0.00219, 0.00220, 0.00221, 0.00222, 0.00223, 0.00224, 0.00225, 0.00226,
0.00227, 0.00228, 0.00229, 0.00230, 0.00231, 0.00232, 0.00233, 0.00234, 0.00235, 0.00236,
0.00237, 0.00238, 0.00239, 0.00240, 0.00241, 0.00242, 0.00243, 0.00244, 0.00245, 0.00246,
0.00247, 0.00248, 0.00249, 0.00250, 0.00251, 0.00252, 0.00253, 0.00254, 0.00255, 0.00256,
0.00257, 0.00258, 0.00259, 0.00260, 0.00261, 0.00262, 0.00263, 0.00264, 0.00265, 0.00266,
0.00267, 0.00268, 0.00269, 0.00270, 0.00271, 0.00272, 0.00273, 0.00274, 0.00275, 0.00276,
0.00278, 0.00279, 0.00280, 0.00281, 0.00282, 0.00283, 0.00284, 0.00285, 0.00286, 0.00287,
0.00288, 0.00289, 0.00290, 0.00291, 0.00292, 0.00293, 0.00294, 0.00295, 0.00296, 0.00297,
0.00298, 0.00299, 0.00300, 0.00301, 0.00302, 0.00303, 0.00304, 0.00305, 0.00306, 0.00307,
0.00308, 0.00308,0.00309, 0.00309,or or 0.00310. In some 0.00310. embodiments, In some the ratio embodiments, the of CD34+of ratio CD90+ CD34CD45RA CD90 CD45RA
cells to lymphocytes in the sample is about 0.0021.
[0030] In an additional aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to enrich the peripheral blood of the donor with
CD34+ CD90CD45RA CD34 CD90 CD45RAcells cellsrelative relativeto tolymphocytes lymphocytesby byaaratio ratioof offrom fromabout about5.6:1 5.6:1to toabout about
37.0:1 as assessed by comparing a sample of peripheral blood of the donor following
administration of the CXCR2 agonist and CXCR4 antagonist to a sample of peripheral blood of the donor prior to administration of the CXCR2 agonist and CXCR4 antagonist. In some embodiments, the peripheral blood of the donor may be enriched with CD34+ CD90 CD34 CD90
CD45RA cells relative to lymphocytes by a ratio of about 5.60:1, 5.65:1, 5.70:1, 5.75:1,
5.80:1, 5.85:1, 5.90:1, 5.95:1, 6.00:1, 6.05:1, 6.10:1, 6.15:1, 6.20:1, 6.25:1, 6.30:1, 6.35:1,
6.40:1, 6.45:1, 6.50:1, 6.55:1, 6.60:1, 6.65:1, 6.70:1, 6.75:1, 6.80:1, 6.85:1, 6.90:1, 6.95:1,
7.00:1, 7.05:1, 7.10:1, 7.15:1, 7.20:1, 7.25:1, 7.30:1, 7.35:1, 7.40:1, 7.45:1, 7.50:1, 7.55:1,
7.60:1, 7.65:1, 7.70:1, 7.75:1, 7.80:1, 7.85:1, 7.90:1, 7.95:1, 8.00:1, 8.05:1, 8.10:1, 8.15:1,
8.20:1, 8.25:1, 8.30:1, 8.35:1, 8.40:1, 8.45:1, 8.50:1, 8.55:1, 8.60:1, 8.65:1, 8.70:1, 8.75:1,
8.80:1, 8.85:1, 8.90:1, 8.95:1, 9.00:1, 9.05:1, 9.10:1, 9.15:1, 9.20:1, 9.25:1, 9.30:1, 9.35:1,
9.40:1, 9.45:1, 9.50:1, 9.55:1, 9.60:1, 9.65:1, 9.70:1, 9.75:1, 9.80:1, 9.85:1, 9.90:1, 9.95:1,
10.00:1, 10.05:1, 10.10:1, 10.15:1, 10.20:1, 10.25:1, 10.30:1, 10.35:1, 10.40:1, 10.45:1,
10.50:1, 10.55:1, 10.60:1, 10.65:1, 10.70:1, 10.75:1, 10.80:1, 10.85:1, 10.90:1, 10.95:1,
11.00:1, 11.05:1, 11.10:1, 11.15:1, 11.20:1, 11.25:1, 11.30:1, 11.35:1, 11.40:1, 11.45:1,
11.50:1, 11.55:1, 11.60:1, 11.65:1, 11.70:1, 11.75:1, 11.80:1, 11.85:1, 11.90:1, 11.95:1,
12.00:1, 12.05:1, 12.10:1, 12.15:1, 12.20:1, 12.25:1, 12.30:1, 12.35:1, 12.40:1, 12.45:1,
12.50:1, 12.55:1, 12.60:1, 12.65:1, 12.70:1, 12.75:1, 12.80:1, 12.85:1, 12.90:1, 12.95:1,
13.00:1, 13.00:1, 13.05:1, 13.05:1, 13.10:1, 13.10:1, 13.15:1, 13.15:1, 13.20:1, 13.20:1, 13.25:1, 13.25:1, 13.30:1, 13.30:1, 13.35:1, 13.35:1, 13.40:1, 13.40:1, 13.45:1, 13.45:1,
13.50:1, 13.55:1, 13.60:1, 13.65:1, 13.70:1, 13.75:1, 13.80:1, 13.85:1, 13.90:1, 13.95:1,
14.00:1, 14.05:1, 14.10:1, 14.15:1, 14.20:1, 14.25:1, 14.30:1, 14.35:1, 14.40:1, 14.45:1,
14.50:1, 14.55:1, 14.60:1, 14.65:1, 14.70:1, 14.75:1, 14.80:1, 14.85:1, 14.90:1, 14.95:1,
15.00:1, 15.05:1, 15.10:1, 15.15:1, 15.20:1, 15.25:1, 15.30:1, 15.35:1, 15.40:1, 15.45:1,
15.50:1, 15.55:1, 15.60:1, 15.65:1, 15.70:1, 15.75:1, 15.80:1, 15.85:1, 15.90:1, 15.95:1,
16.00:1, 16.05:1, 16.10:1, 16.15:1, 16.20:1, 16.25:1, 16.30:1, 16.35:1, 16.40:1, 16.45:1,
16.50:1, 16.55:1, 16.60:1, 16.65:1, 16.70:1, 16.75:1, 16.80:1, 16.85:1, 16.90:1, 16.95:1,
17.00:1, 17.05:1, 17.10:1, 17.15:1, 17.20:1, 17.25:1, 17.30:1, 17.35:1, 17.40:1, 17.45:1,
17.50:1, 17.55:1, 17.60:1, 17.65:1, 17.70:1, 17.75:1, 17.80:1, 17.85:1, 17.90:1, 17.95:1,
18.00:1, 18.05:1, 18.10:1, 18.15:1, 18.20:1, 18.25:1, 18.30:1, 18.35:1, 18.40:1, 18.45:1,
18.50:1, 18.50:1, 18.55:1, 18.55:1, 18.60:1, 18.60:1, 18.65:1, 18.65:1, 18.70:1, 18.70:1, 18.75:1, 18.75:1, 18.80:1, 18.80:1, 18.85:1, 18.85:1, 18.90:1, 18.90:1, 18.95:1, 18.95:1,
19.00:1, 19.05:1, 19.10:1, 19.15:1, 19.20:1, 19.25:1, 19.30:1, 19.35:1, 19.40:1, 19.45:1,
19.50:1, 19.55:1, 19.60:1, 19.65:1, 19.70:1, 19.75:1, 19.80:1, 19.85:1, 19.90:1, 19.95:1,
20.00:1, 20.05:1, 20.10:1, 20.15:1, 20.20:1, 20.25:1, 20.30:1, 20.35:1, 20.40:1, 20.45:1,
20.50:1, 20.55:1, 20.60:1, 20.65:1, 20.70:1, 20.75:1, 20.80:1, 20.85:1, 20.90:1, 20.95:1,
21.00:1, 21.05:1, 21.10:1, 21.15:1, 21.20:1, 21.25:1, 21.30:1, 21.35:1, 21.40:1, 21.45:1,
WO 2019/113375 2019/11337 OM PCT/US2018/064335
21.50:1, 21.55:1, 21.60:1, 21.65:1, 21.70:1, 21.75:1, 21.80:1, 21.85:1, 21.90:1, 21.95:1,
22.00:1, 22.05:1, 22.10:1, 22.15:1, 22.20:1, 22.25:1, 22.30:1, 22.35:1, 22.40:1, 22.45:1,
22.50:1, 22.55:1, 22.60:1, 22.65:1, 22.70:1, 22.75:1, 22.80:1, 22.85:1, 22.90:1, 22.95:1,
23.00, 23.05:1, 23.10:1, 23.15:1, 23.20:1, 23.25:1, 23.30:1, 23.35:1, 23.40:1, 23.45:1,
S 23.50:1, 23.55:1, 23.60:1, 23.65:1, 23.70:1, 23.75:1, 23.80:1, 23.85:1, 23.90:1, 23.95:1,
24.00:1, 24.05:1, 24.10:1, 24.15:1, 24.20:1, 24.25:1, 24.30:1, 24.35:1, 24.40:1, 24.45:1,
24.50:1, 24.55:1, 24.60:1, 24.65:1, 24.70:1, 24.75:1, 24.80:1, 24.85:1, 24.90:1, 24.95:1,
25.05:1, 25.10:1, 25.15:1, 25.20:1, 25.25:1, 25.30:1, 25.35:1, 25.40:1, 25.45:1, 25.50:1,
25.55:1, 25.60:1, 25.65:1, 25.70:1, 25.75:1, 25.80:1, 25.85:1, 25.90:1, 25.95:1, 26.00:1,
OI 26.05:1, 26.10:1, 26.15:1, 26.20:1, 26.25:1, 26.30:1, 26.35:1, 26.40:1, 26.45:1, 26.50:1,
26.55:1, 26.60:1, 26.65:1, 26.70:1, 26.75:1, 26.80:1, 26.85:1, 26.90:1, 26.95:1, 27.00:1,
27.05:1, 27.10:1, 27.15:1, 27.20:1, 27.25:1, 27.30:1, 27.35:1, 27.40:1, 27.45:1, 27.50:1,
27.55:1, 27.60:1, 27.65:1, 27.70:1, 27.75:1, 27.80:1, 27.85:1, 27.90:1, 27.95:1, 28.00:1,
28.05:1, 28.10:1, 28.15:1, 28.20:1, 28.25:1, 28.30:1, 28.35:1, 28.40:1, 28.45:1, 28.50:1,
28.55:1, 28.60:1, 28.65:1, 28.70:1, 28.75:1, 28.80:1, 28.85:1, 28.90:1, 28.95:1, 29.00:1,
29.05:1, 29.10:1, 29.15:1, 29.20:1, 29.25:1, 29.30:1, 29.35:1, 29.40:1, 29.45:1, 29.50:1,
29.55:1, 29.60:1, 29.65:1, 29.70:1, 29.75:1, 29.80:1, 29.85:1, 29.90:1, 29.95:1, 30.00:1,
30.05:1, 30.10:1, 30.15:1, 30.20:1, 30.25:1, 30.30:1, 30.35:1, 30.40:1, 30.45:1, 30.50:1,
30.55:1, 30.60:1, 30.65:1, 30.70:1, 30.75:1, 30.80:1, 30.85:1, 30.90:1, 30.95:1, 31.00:1,
31.05:1, 31.10:1, 31.15:1, 31.20:1, 31.25:1, 31.30:1, 31.35:1, 31.40:1, 31.45:1, 31.50:1,
31.55:1, 31.60:1, 31.65:1, 31.70:1, 31.75:1, 31.80:1, 31.85:1, 31.90:1, 31.95:1, 32.00:1,
32.05:1, 32.10:1, 32.15:1, 32.20:1, 32.25:1, 32.30:1, 32.35:1, 32.40:1, 32.45:1, 32.50:1,
32.55:1, 32.60:1, 32.65:1, 32.70:1, 32.75:1, 32.80:1, 32.85:1, 32.90:1, 32.95:1, 33.00:1,
33.05:1, 33.10:1, 33.15:1, 33.20:1, 33.25:1, 33.30:1, 33.35:1, 33.40:1, 33.45:1, 33.50:1,
33.55:1, 33.60:1, 33.65:1, 33.70:1, 33.75:1, 33.80:1, 33.85:1, 33.90:1, 33.95:1, 34.00:1,
34.05:1, 34.10:1, 34.15:1, 34.20:1, 34.25:1, 34.30:1, 34.35:1, 34.40:1, 34.45:1, 34.50:1,
34.55:1, 34.60:1, 34.65:1, 34.70:1, 34.75:1, 34.80:1, 34.85:1, 34.90:1, 34.95:1, 35.00:1,
35.05:1, 35.10:1, 35.15:1, 35.20:1, 35.25:1, 35.30:1, 35.35:1, 35.40:1, 35.45:1, 35.50:1,
35.55:1, 35.60:1, 35.65:1, 35.70:1, 35.75:1, 35.80:1, 35.85:1, 35.90:1, 35.95:1, 36.00:1,
0£ 36.05:1, 36.10:1, 36.15:1, 36.20:1, 36.25:1, 36.30:1, 36.35:1, 36.40:1, 36.45:1, 36.50:1,
36.55:1, 36.60:1, 36.65:1, 36.70:1, 36.75:1, 36.80:1, 36.85:1, 36.90:1, 36.95:1, or 37.00. In
some embodiments, the peripheral blood of the donor is enriched with CD34 CD34+CD90 CD90
CD45RA cells relative to lymphocytes by a ratio of about from about 8.0:1 to about 10.0:1,
33 EE
WO wo 2019/113375 PCT/US2018/064335 PCT/US2018/064335
such as a ratio of about 8.00:1, 8.05:1, 8.10:1, 8.15:1, 8.20:1, 8.25:1, 8.30:1, 8.35:1, 8.40:1,
8.45:1, 8.50:1, 8.55:1, 8.60:1, 8.65:1, 8.70:1, 8.75:1, 8.80:1, 8.85:1, 8.90:1, 8.95:1, 9.00:1,
9.05:1, 9.10:1, 9.15:1, 9.20:1, 9.25:1, 9.30:1, 9.35:1, 9.40:1, 9.45:1, 9.50:1, 9.55:1, 9.60:1,
9.65:1, 9.70:1, 9.75:1, 9.80:1, 9.85:1, 9.90:1, 9.95:1, or 10.00:1. In some embodiments, the
peripheral blood of the donor is enriched with CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells relative relative toto
lymphocytes by a ratio of about 9.3:1.
[0031] In another aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density
of CD34+ CD90 CD45RA CD34 CD90 CD45RA cells cells of of at at least least about about 16,000 16,000 cells/ml, cells/ml, such such as as aa density density of of from from
about 20,000 cells/ml to about 75,000 cells/ml, about 25,000 cells/ml to about 70,000
cells/ml, about 30,000 cells/ml to about 65,000 cells/ml, about 35,000 cells/ml to about
60,000 cells/ml, about 40,000 cells/ml to about 55,000 cells/ml, or about 45,000 cells/ml to
about 50,000 cells/ml (e.g., about 16,000 cells/ml, 17,000 cells/ml, 18,000 cells/ml, 19,000
cells/ml, 20,000 cells/ml, 21,000 cells/ml, 22,000 cells/ml, 23,000 cells/ml, 24,000 cells/ml,
25,000 cells/ml, 26,000 cells/ml, 27,000 cells/ml, 28,000 cells/ml, 29,000 cells/ml, 30,000
cells/ml, 31,000 cells/ml, 32,000 cells/ml, 33,000 cells/ml, 34,000 cells/ml, 35,000 cells/ml,
36,000 cells/ml, 37,000 cells/ml, 38,000 cells/ml, 39,000 cells/ml, 40,000 cells/ml, 41,000
cells/ml, 42,000 cells/ml, 43,000 cells/ml, 44,000 cells/ml, 45,000 cells/ml, 46,000 cells/ml,
47,000 cells/ml, 48,000 cells/ml, 49,000 cells/ml, 50,000 cells/ml, 51,000 cells/ml, 52,000
cells/ml, 53,000 cells/ml, 54,000 cells/ml, 55,000 cells/ml, 56,000 cells/ml, 57,000 cells/ml,
58,000 cells/ml, 59,000 cells/ml, 60,000 cells/ml, 61,000 cells/ml, 62,000 cells/ml, 63,000
cells/ml, 64,000 cells/ml, 65,000 cells/ml, 66,000 cells/ml, 67,000 cells/ml, 68,000 cells/ml,
69,000 cells/ml, 70,000 cells/ml, 71,000 cells/ml, 72,000 cells/ml, 73,000 cells/ml, 74,000
cells/ml, 75,000 cells/ml, 76,000 cells/ml, 77,000 cells/ml, or more), and having a density of
lymphocytes of no more than about 2.4x 2.4 x107 10 cells/ml, such as a density of lymphocytes of
about 1 x 107 cells/ml to 10 cells/ml to about about 2.3 2.3 XX 10 10 cells/ml, cells/ml, about about 1.3 1.3 XX 10 107 cells/ml cells/ml toto about about 2.1 2.1 X X
cells/ml, or cells/ml, or about about 1.5 1.5 xX 107 10 cells/ml cells/ml to to about about 1.9 1.9 Xx 10 107cells/ml cells/ml(e.g., (e.g.,about about2.4 2.4x x10107
cells/ml, 2.3 X x 107 cells/ml, 2.2 10 cells/ml, 2.2 xX 007 cells/ml, cells/ml, 2.1 2.1 x x 10107 cells/ml, cells/ml, 2 XX 10 107 cells/ml, cells/ml, 1.9 1.9 X x 10107
cells/ml, 1.8 x X 107 cells/ml, 1.7 10 cells/ml, 1.7 Xx 10 107 cells/ml, cells/ml, 1.6x 1.6 107 X 10 cells/ml, cells/ml, 1.5 1.5 X x 10107 cells/ml cells/ml 1.41.4 x 107 x 10
cells/ml, 1.3 x 107 cells/ml,1.2 10 cells/ml, 1.2xx10 107 cells/ml, cells/ml, 1.1 1.1 x x 10107 cells/ml, cells/ml, 1 x107 10 cells/ml, or less, 0.9
X x 107 cells/ml, 0.8 10 cells/ml, 0.8X X107 10cells/ml, cells/ml,or or less). In some less). embodiments, In some the method embodiments, the includes method includes
WO wo 2019/113375 PCT/US2018/064335
administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient
to produce a population of cells having a density of CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells ofof from from
about 20,000 cells/ml to about 75,000 cells/ml, and having a density of lymphocytes of from
about 1 X x 107 cells/ml to 10 cells/ml to about about 2.3 2.3 XX 10 107 cells/ml. cells/ml. InIn some some embodiments, embodiments, the the method method includes includes
administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient
to produce a population of cells having a density of CD34 CD90+ CD45RA cells CD90 CD45RA cells of of from from
about 30,000 cells/ml to about 60,000 cells/ml, and having a density of lymphocytes of from
about 1.3 X 107 cells/ml to 10 cells/ml to about about 2.3 2.3 XX 10 107 cells/ml. cells/ml. InIn some some embodiments, embodiments, the the method method
includes administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts
sufficient to produce a population of cells having a density of CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells
of from about 40,000 cells/ml to about 50,000 cells/ml, and having a density of lymphocytes
of of from fromabout about1.51.5 X 107 cells/ml X 10 to about cells/ml 2 X 107 to about 2 Xcells/ml. 10 cells/ml.
[0032] In another aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to produce a population of cells having a ratio of
CD34+ CD90 CD45RA- CD34 CD90 CD45RA- cells cells to to monocytes monocytes of of from from about about 0.0028 0.0028 to to about about 0.0130 0.0130 in in aa sample sample
of peripheral blood of the donor following administration of the CXCR2 agonist. In some
embodiments, embodiments,the ratio the of of ratio CD34+ CD90 CD34 CD45RA CD90 cellscells CD45RA to monocytes in the in to monocytes sample the may be sample may be
about 0.00280, 0.00281, 0.00282, 0.00283, 0.00284, 0.00285, 0.00286, 0.00287, 0.00288,
0.00289, 0.00290, 0.00291, 0.00292, 0.00293, 0.00294, 0.00295, 0.00296, 0.00297, 0.00298,
0.00299, 0.00300, 0.00301, 0.00302, 0.00303, 0.00304, 0.00305, 0.00306, 0.00307, 0.00308,
0.00309, 0.00310, 0.00311, 0.00312, 0.00313, 0.00314, 0.00315, 0.00316, 0.00317, 0.00318,
0.00319, 0.00320, 0.00321, 0.00322, 0.00323, 0.00324, 0.00325, 0.00326, 0.00327, 0.00328,
0.00329, 0.00330, 0.00331, 0.00332, 0.00333, 0.00334, 0.00335, 0.00336, 0.00337, 0.00338,
0.00339, 0.00340, 0.00341, 0.00342, 0.00343, 0.00344, 0.00345, 0.00346, 0.00347, 0.00348,
0.00349, 0.00350, 0.00351, 0.00352, 0.00353, 0.00354, 0.00355, 0.00356, 0.00357, 0.00358,
0.00359, 0.00360, 0.00361, 0.00362, 0.00363, 0.00364, 0.00365, 0.00366, 0.00367, 0.00368,
0.00369, 0.00370, 0.00371, 0.00372, 0.00373, 0.00374, 0.00375, 0.00376, 0.00378, 0.00379,
0.00380, 0.00381, 0.00382, 0.00383, 0.00384, 0.00385, 0.00386, 0.00387, 0.00388, 0.00389,
0.00390, 0.00391, 0.00392, 0.00393, 0.00394, 0.00395, 0.00396, 0.00397, 0.00398, 0.00399,
0.00401, 0.00402, 0.00403, 0.00404, 0.00405, 0.00406, 0.00407, 0.00408, 0.00409, 0.00410,
0.00411, 0.00412, 0.00413, 0.00414, 0.00415, 0.00416, 0.00417, 0.00418, 0.00419, 0.00420,
WO wo 2019/113375 PCT/US2018/064335
0.00421, 0.00422, 0.00423, 0.00424, 0.00425, 0.00426, 0.00427, 0.00428, 0.00429, 0.00430,
0.00431, 0.00432, 0.00433, 0.00434, 0.00435, 0.00436, 0.00437, 0.00438, 0.00439, 0.00440,
0.00441, 0.00442, 0.00443, 0.00444, 0.00445, 0.00446, 0.00447, 0.00448, 0.00449, 0.00450,
0.00451, 0.00452, 0.00453, 0.00454, 0.00455, 0.00456, 0.00457, 0.00458, 0.00459, 0.00460,
0.00461, 0.00462, 0.00463, 0.00464, 0.00465, 0.00466, 0.00467, 0.00468, 0.00469, 0.00470,
0.00471, 0.00472, 0.00473, 0.00474, 0.00475, 0.00476, 0.00478, 0.00479, 0.00480, 0.00481,
0.00482, 0.00483, 0.00484, 0.00485, 0.00486, 0.00487, 0.00488, 0.00489, 0.00490, 0.00491,
0.00492, 0.00493, 0.00494, 0.00495, 0.00496, 0.00497, 0.00498, 0.00499, 0.00500, 0.00501,
0.00502, 0.00503, 0.00504, 0.00505, 0.00506, 0.00507, 0.00508, 0.00509, 0.00510, 0.00511,
0.00512, 0.00513, 0.00514, 0.00515, 0.00516, 0.00517, 0.00518, 0.00519, 0.00520, 0.00521,
0.00522, 0.00523, 0.00524, 0.00525, 0.00526, 0.00527, 0.00528, 0.00529, 0.00530, 0.00531,
0.00532, 0.00533, 0.00534, 0.00535, 0.00536, 0.00537, 0.00538, 0.00539, 0.00540, 0.00541,
0.00542, 0.00543, 0.00544, 0.00545, 0.00546, 0.00547, 0.00548, 0.00549, 0.00550, 0.00551,
0.00552, 0.00553, 0.00554, 0.00555, 0.00556, 0.00557, 0.00558, 0.00559, 0.00560, 0.00561,
0.00562, 0.00563, 0.00564, 0.00565, 0.00566, 0.00567, 0.00568, 0.00569, 0.00570, 0.00571,
0.00572, 0.00573, 0.00574, 0.00575, 0.00576, 0.00578, 0.00579, 0.00580, 0.00581, 0.00582,
0.00583, 0.00584, 0.00585, 0.00586, 0.00587, 0.00588, 0.00589, 0.00590, 0.00591, 0.00592,
0.00593, 0.00594, 0.00595, 0.00596, 0.00597, 0.00598, 0.00599, 0.00600, 0.00601, 0.00602,
0.00603, 0.00604, 0.00605, 0.00606, 0.00607, 0.00608, 0.00609, 0.00610, 0.00611, 0.00612,
0.00613, 0.00614, 0.00615, 0.00616, 0.00617, 0.00618, 0.00619, 0.00620, 0.00621, 0.00622,
0.00623, 0.00624, 0.00625, 0.00626, 0.00627, 0.00628, 0.00629, 0.00630, 0.00631, 0.00632,
0.00633, 0.00634, 0.00635, 0.00636, 0.00637, 0.00638, 0.00639, 0.00640, 0.00641, 0.00642,
0.00643, 0.00644, 0.00645, 0.00646, 0.00647, 0.00648, 0.00649, 0.00650, 0.00651, 0.00652,
0.00653, 0.00654, 0.00655, 0.00656, 0.00657, 0.00658, 0.00659, 0.00660, 0.00661, 0.00662,
0.00663, 0.00664, 0.00665, 0.00666, 0.00667, 0.00668, 0.00669, 0.00670, 0.00671, 0.00672,
0.00673, 0.00673, 0.00674, 0.00674, 0.00675, 0.00675, 0.00676, 0.00676, 0.00678, 0.00678, 0.00679, 0.00679, 0.00680, 0.00680, 0.00681, 0.00681, 0.00682, 0.00682, 0.00683, 0.00683,
0.00684, 0.00685, 0.00686, 0.00687, 0.00688, 0.00689, 0.00690, 0.00691, 0.00692, 0.00693,
0.00694, 0.00695, 0.00696, 0.00697, 0.00698, 0.00699, 0.00700, 0.00701, 0.00702, 0.00703,
0.00704, 0.00705, 0.00706, 0.00707, 0.00708, 0.00709, 0.00710, 0.00711, 0.00712, 0.00713,
0.00714, 0.00715, 0.00716, 0.00717, 0.00718, 0.00719, 0.00720, 0.00721, 0.00722, 0.00723,
0.00724, 0.00725, 0.00726, 0.00727, 0.00728, 0.00729, 0.00730, 0.00731, 0.00732, 0.00733,
0.00734, 0.00735, 0.00736, 0.00737, 0.00738, 0.00739, 0.00740, 0.00741, 0.00742, 0.00743,
0.00744, 0.00745, 0.00746, 0.00747, 0.00748, 0.00749, 0.00750, 0.00751, 0.00752, 0.00753,
WO wo 2019/113375 PCT/US2018/064335
0.00754, 0.00755, 0.00756, 0.00757, 0.00758, 0.00759, 0.00760, 0.00761, 0.00762, 0.00763,
0.00764, 0.00765, 0.00766, 0.00767, 0.00768, 0.00769, 0.00770, 0.00771, 0.00772, 0.00773,
0.00774, 0.00775, 0.00776, 0.00777, 0.00778, 0.00779, 0.00780, 0.00781, 0.00782, 0.00783,
0.00784, 0.00785, 0.00786, 0.00787, 0.00788, 0.00789, 0.00790, 0.00791, 0.00792, 0.00793,
0.00794, 0.00795, 0.00796, 0.00797, 0.00798, 0.00799, 0.00800, 0.00801, 0.00802, 0.00803,
0.00804, 0.00805, 0.00806, 0.00807, 0.00808, 0.00809, 0.00810, 0.00811, 0.00812, 0.00813,
0.00814, 0.00815, 0.00816, 0.00817, 0.00818, 0.00819, 0.00820, 0.00821, 0.00822, 0.00823,
0.00824, 0.00825, 0.00826, 0.00827, 0.00828, 0.00829, 0.00830, 0.00831, 0.00832, 0.00833,
0.00834, 0.00835, 0.00836, 0.00837, 0.00838, 0.00839, 0.00840, 0.00841, 0.00842, 0.00843,
0.00844, 0.00845, 0.00846, 0.00847, 0.00848, 0.00849, 0.00850, 0.00851, 0.00852, 0.00853,
0.00854, 0.00855, 0.00856, 0.00857, 0.00858, 0.00859, 0.00860, 0.00861, 0.00862, 0.00863,
0.00864, 0.00865, 0.00866, 0.00867, 0.00868, 0.00869, 0.00870, 0.00871, 0.00872, 0.00873,
0.00874, 0.00875, 0.00876, 0.00877, 0.00878, 0.00879, 0.00880, 0.00881, 0.00882, 0.00883,
0.00884, 0.00884, 0.00885, 0.00885, 0.00886, 0.00886, 0.00887, 0.00887, 0.00888, 0.00888, 0.00889, 0.00889, 0.00890, 0.00890, 0.00891, 0.00891, 0.00892, 0.00892, 0.00893, 0.00893,
0.00894, 0.00894, 0.00895, 0.00895, 0.00896, 0.00896, 0.00897, 0.00897, 0.00898, 0.00898, 0.00899, 0.00899, 0.00900, 0.00900, 0.00901, 0.00901, 0.00902, 0.00902, 0.00903, 0.00903,
0.00904, 0.00905, 0.00906, 0.00907, 0.00908, 0.00909, 0.00910, 0.00911, 0.00912, 0.00913,
0.00914, 0.00915, 0.00916, 0.00917, 0.00918, 0.00919, 0.00920, 0.00921, 0.00922, 0.00923,
0.00924, 0.00925, 0.00926, 0.00927, 0.00928, 0.00929, 0.00930, 0.00931, 0.00932, 0.00933,
0.00934, 0.00935, 0.00936, 0.00937, 0.00938, 0.00939, 0.00940, 0.00941, 0.00942, 0.00943,
0.00944, 0.00945, 0.00946, 0.00947, 0.00948, 0.00949, 0.00950, 0.00951, 0.00952, 0.00953,
0.00954, 0.00955, 0.00956, 0.00957, 0.00958, 0.00959, 0.00960, 0.00961, 0.00962, 0.00963,
0.00964, 0.00965, 0.00966, 0.00967, 0.00968, 0.00969, 0.00970, 0.00971, 0.00972, 0.00973,
0.00974, 0.00974, 0.00975, 0.00975, 0.00976, 0.00976, 0.00977, 0.00977, 0.00978, 0.00978, 0.00979, 0.00979, 0.00980, 0.00980, 0.00981, 0.00981, 0.00982, 0.00982, 0.00983, 0.00983,
0.00984, 0.00984, 0.00985, 0.00985, 0.00986, 0.00986, 0.00987, 0.00987, 0.00988, 0.00988, 0.00989, 0.00989, 0.00990, 0.00990, 0.00991, 0.00991, 0.00992, 0.00992, 0.00993, 0.00993,
0.00994, 0.00995, 0.00996, 0.00997, 0.00998, 0.00999, 0.0100, 0.0101, 0.0103, 0.0104,
0.0105, 0.0106, 0.0107, 0.0108, 0.0109, 0.0110, 0.0111, 0.0112, 0.0113, 0.0114, 0.0115,
0.0116, 0.0117, 0.0118, 0.0119, 0.0120, 0.0121, 0.0122, 0.0123, 0.0124, 0.0125, 0.0126,
0.0127, 0.0128, 0.0129, or 0.0130. In some embodiments, the ratio of CD34+ CD90+ CD34 CD90
CD45RA cells to monocytes in the sample is from about 0.0063 to about 0.0083, such as a
ratio of CD34 CD90+ CD45RA cells CD90 CD45RA cells to to monocytes monocytes in in the the sample sample of of about about 0.00630, 0.00630, 0.00631, 0.00631,
0.00632, 0.00633, 0.00634, 0.00635, 0.00636, 0.00637, 0.00638, 0.00639, 0.00640, 0.00641,
0.00642, 0.00643, 0.00644, 0.00645, 0.00646, 0.00647, 0.00648, 0.00649, 0.00650, 0.00651,
0.00652, 0.00653, 0.00654, 0.00655, 0.00656, 0.00657, 0.00658, 0.00659, 0.00660, 0.00661,
0.00662, 0.00663, 0.00664, 0.00665, 0.00666, 0.00667, 0.00668, 0.00669, 0.00670, 0.00671,
0.00672, 0.00673, 0.00674, 0.00675, 0.00676, 0.00678, 0.00679, 0.00680, 0.00681, 0.00682,
0.00683, 0.00684, 0.00685, 0.00686, 0.00687, 0.00688, 0.00689, 0.00690, 0.00691, 0.00692,
0.00693, 0.00694, 0.00695, 0.00696, 0.00697, 0.00698, 0.00699, 0.00700, 0.00701, 0.00702,
0.00703, 0.00704, 0.00705, 0.00706, 0.00707, 0.00708, 0.00709, 0.00710, 0.00711, 0.00712,
0.00713, 0.00714, 0.00715, 0.00716, 0.00717, 0.00718, 0.00719, 0.00720, 0.00721, 0.00722,
0.00723, 0.00724, 0.00725, 0.00726, 0.00727, 0.00728, 0.00729, 0.00730, 0.00731, 0.00732,
0.00733, 0.00734, 0.00735, 0.00736, 0.00737, 0.00738, 0.00739, 0.00740, 0.00741, 0.00742,
0.00743, 0.00744, 0.00745, 0.00746, 0.00747, 0.00748, 0.00749, 0.00750, 0.00751, 0.00752,
0.00753, 0.00754, 0.00755, 0.00756, 0.00757, 0.00758, 0.00759, 0.00760, 0.00761, 0.00762,
0.00763, 0.00764, 0.00765, 0.00766, 0.00767, 0.00768, 0.00769, 0.00770, 0.00771, 0.00772,
0.00773, 0.00774, 0.00775, 0.00776, 0.00777, 0.00778, 0.00779, 0.00780, 0.00781, 0.00782,
0.00783, 0.00784, 0.00785, 0.00786, 0.00787, 0.00788, 0.00789, 0.00790, 0.00791, 0.00792,
0.00793, 0.00794, 0.00795, 0.00796, 0.00797, 0.00798, 0.00799, 0.00800, 0.00801, 0.00802,
0.00803, 0.00804, 0.00805, 0.00806, 0.00807, 0.00808, 0.00809, 0.00810, 0.00811, 0.00812,
0.00813, 0.00814, 0.00815, 0.00816, 0.00817, 0.00818, 0.00819, 0.00820, 0.00821, 0.00822,
0.00823, 0.00824, 0.00825, 0.00826, 0.00827, 0.00828, 0.00829, 0.00830. In some
embodiments, the ratio of CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells toto monocytes monocytes inin the the sample sample isis about about
0.0073.
[0033] In an additional aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to enrich the peripheral blood of the donor with
CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells relative relative toto monocytes monocytes byby a a ratio ratio ofof from from about about 1.5:1 1.5:1 toto about about
8.5:1 8.5:1 as as assessed assessed by by comparing comparing aa sample sample of of peripheral peripheral blood blood of of the the donor donor following following
administration of the CXCR2 agonist and CXCR4 antagonist to a sample of peripheral blood
of the donor prior to administration of the CXCR2 agonist and CXCR4 antagonist. In some
embodiments, the peripheral blood of the donor may be enriched with CD34 CD90
CD45RA cells relative to monocytes by a ratio of about 1.50:1, 1.55:1, 1.60:1, 1.65:1,
1.70:1, 1.75:1, 1.80:1, 1.85:1, 1.90:1, 1.95:1, 2.00:1, 2.05:1, 2.10:1, 2.15:1, 2.20:1, 2.25:1,
2.30:1, 2.35:1, 2.40:1, 2.45:1, 2.50:1, 2.55:1, 2.60:1, 2.65:1, 2.70:1, 2.75:1, 2.80:1, 2.85:1,
2.90:1, 2.95:1, 3.00:1, 3.05:1, 3.10:1, 3.15:1, 3.20:1, 3.25:1, 3.30:1, 3.35:1, 3.40:1, 3.45:1,
3.50:1, 3.55:1, 3.60:1, 3.65:1, 3.70:1, 3.75:1, 3.80:1, 3.85:1, 3.90:1, 3.95:1, 4.00:1, 4.05:1,
WO wo 2019/113375 PCT/US2018/064335 PCT/US2018/064335
4.10:1, 4.15:1, 4.20:1, 4.25:1, 4.30:1, 4.35:1, 4.40:1, 4.45:1, 4.50:1, 4.55:1, 4.60:1, 4.65:1,
4.70:1, 4.75:1, 4.80:1, 4.85:1, 4.90:1, 4.95:1, 5.00:1, 5.05:1, 5.10:1, 5.15:1, 5.20:1, 5.25:1,
5.30:1, 5.35:1, 5.40:1, 5.45:1, 5.50:1, 5.55:1, 5.60:1, 5.65:1, 5.70:1, 5.75:1, 5.80:1, 5.85:1,
5.90:1, 5.95:1, 6.00:1, 6.05:1, 6.10:1, 6.15:1, 6.20:1, 6.25:1, 6.30:1, 6.35:1, 6.40:1, 6.45:1,
6.50:1, 6.55:1, 6.60:1, 6.65:1, 6.70:1, 6.75:1, 6.80:1, 6.85:1, 6.90:1, 6.95:1, 7.00:1, 7.05:1,
7.10:1, 7.15:1, 7.20:1, 7.25:1, 7.30:1, 7.35:1, 7.40:1, 7.45:1, 7.50:1, 7.55:1, 7.60:1, 7.65:1,
7.70:1, 7.75:1, 7.80:1, 7.85:1, 7.90:1, 7.95:1, 8.00:1, 8.05:1, 8.10:1, 8.15:1, 8.20:1, 8.25:1,
8.30:1, 8.35:1, 8.40:1, 8.45:1, or 8.50:1. In some embodiments, the peripheral blood of the
donor is enriched with CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells relative relative toto monocytes monocytes byby a a ratio ratio ofof
about 1.9:1.
[0034] In another aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density
of CD34+ CD90 CD45RA CD34 CD90 CD45RA cells cells of of at at least least about about 16,000 16,000 cells/ml, cells/ml, such such as as aa density density of of from from
about 20,000 cells/ml to about 75,000 cells/ml, about 25,000 cells/ml to about 70,000
cells/ml, about 30,000 cells/ml to about 65,000 cells/ml, about 35,000 cells/ml to about
60,000 cells/ml, about 40,000 cells/ml to about 55,000 cells/ml, or about 45,000 cells/ml to
about 50,000 cells/ml (e.g., about 16,000 cells/ml, 17,000 cells/ml, 18,000 cells/ml, 19,000
cells/ml, 20,000 cells/ml, 21,000 cells/ml, 22,000 cells/ml, 23,000 cells/ml, 24,000 cells/ml,
25,000 cells/ml, 26,000 cells/ml, 27,000 cells/ml, 28,000 cells/ml, 29,000 cells/ml, 30,000
cells/ml, 31,000 cells/ml, 32,000 cells/ml, 33,000 cells/ml, 34,000 cells/ml, 35,000 cells/ml,
36,000 cells/ml, 37,000 cells/ml, 38,000 cells/ml, 39,000 cells/ml, 40,000 cells/ml, 41,000
cells/ml, 42,000 cells/ml, 43,000 cells/ml, 44,000 cells/ml, 45,000 cells/ml, 46,000 cells/ml,
47,000 cells/ml, 48,000 cells/ml, 49,000 cells/ml, 50,000 cells/ml, 51,000 cells/ml, 52,000
cells/ml, 53,000 cells/ml, 54,000 cells/ml, 55,000 cells/ml, 56,000 cells/ml, 57,000 cells/ml,
58,000 cells/ml, 59,000 cells/ml, 60,000 cells/ml, 61,000 cells/ml, 62,000 cells/ml, 63,000
cells/ml, 64,000 cells/ml, 65,000 cells/ml, 66,000 cells/ml, 67,000 cells/ml, 68,000 cells/ml,
69,000 cells/ml, 70,000 cells/ml, 71,000 cells/ml, 72,000 cells/ml, 73,000 cells/ml, 74,000
cells/ml, 75,000 cells/ml, 76,000 cells/ml, 77,000 cells/ml, or more), and having a density of
monocytes of no more than about 6 X x 106 cells/ml, such 10 cells/ml, such as as aa density density of of monocytes monocytes of of from from 3.4 3.4
X x 106 cells/ml to 10 cells/ml to about about 5.9 5.9 xx 10 106 cells/ml, cells/ml, about about 3.5 3.5 X x 10106 cells/ml cells/ml to to about about 5.75.7 X 106 X 10 cells/ml, cells/ml,
or about 4 X x 106 cells/ml to 10 cells/ml to about about 55 xX 10 106 cells/ml cells/ml (e.g., (e.g., 5.9 5.9 X 106 cells/ml, 5.8 10 cells/ml, 5.8 XX 10 106 cells/ml, cells/ml,
WO wo 2019/113375 PCT/US2018/064335
5.7 5.7 xx 106 cells/ml, 5.6 10 cells/ml, 5.6 Xx 10 106cells/ml, cells/ml,5.5 5.5X x10106 cells/ml, cells/ml, 5.45.4 x 106 x 10 cells/ml, cells/ml, 5.3 5.3 x 10x cells/ml, 106 cells/ml,
5.2 106 X 10cells/ml, cells/ml,5.1 5.1x X106 10 cells/ml, 5 x X 106 cells/ml, 4.9 10 cells/ml, 4.9 Xx 10 106 cells/ml, cells/ml, 4.8 4.8 X x 10106 cells/ml, cells/ml,
4.7 x 106 cells/ml, 4.6 10 cells/ml, 4.6 Xx 10 106 cells/ml, cells/ml, 4.5 4.5 X x 1010 cells/ml, cells/ml, 4.4 4.4 X x 10106 cells/ml, cells/ml, 4.34.3 x 106 X 10 cells/ml, cells/ml,
4.2 106 x 10cells/ml, cells/ml,4.1 4.1x X106 10 cells/ml, 4 x 106 cells/ml, 3.9 10 cells/ml, 3.9 Xx 10 106 cells/ml, cells/ml, 3.8 3.8 x 106 cells/ml, 10 cells/ml,
3.7 X x 106 cells/ml, 3.6 10 cells/ml, 3.6 Xx 10 106 cells/ml, cells/ml, 3.5 3.5 X x 10106 cells/ml, cells/ml, 3.43.4 x 106 X 10 cells/ml, cells/ml, or less). or less). In some In some
embodiments, embodiments, the the method method includes includes administering administering to to the the donor donor aa CXCR2 CXCR2 agonist agonist and and aa
CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of
CD34 CD90 CD45RA cells of from about 20,000 cells/ml to about 75,000 cells/ml, and
having a density of monocytes of from about 3.4 x X 106 cells/ml to 10 cells/ml to about about 66 XX 10 106 cells/ml. cells/ml. InIn
some embodiments, the method includes administering to the donor a CXCR2 agonist and a
CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of
CD34+ CD90 CD45RA CD34 CD90 CD45RA cells cells of of from from about about 30,000 30,000 cells/ml cells/ml to to about about 60,000 60,000 cells/ml, cells/ml, and and
4 106 having a density of monocytes of from about X X 10cells/ml cells/mlto toabout about5.5 5.5X x106 10 cells/ml. In
some embodiments, the method includes administering to the donor a CXCR2 agonist and a
CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of
CD34 CD90+ CD45RA cells CD90 CD45RA cells of of from from about about 40,000 40,000 cells/ml cells/ml to to about about 50,000 50,000 cells/ml, cells/ml, and and
having having a adensity densityof of monocytes of from monocytes about about of from 4 X 1064 cells/ml to aboutto X 10 cells/ml 5 X 106 cells/ml. about 5 X 10 cells/ml.
[0035] In another aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to produce a population of cells having a ratio of
CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells toto CD34+ CD34 cells cells of of from from about about 0.393 0.393 to to about about 0.745 0.745 in in a sample a sample
of peripheral blood of the donor following administration of the CXCR2 agonist. In some
embodiments, embodiments,the ratio the of of ratio CD34+ CD90+ CD34 CD45RA CD90 cells CD45RA to CD34+ cells cells to CD34 in the cells insample may be may be the sample
about 0.393, 0.394, 0.395, 0.396, 0.397, 0.398, 0.399, 0.401, 0.402, 0.403, 0.404, 0.405,
0.406, 0.407, 0.408, 0.409, 0.410, 0.411, 0.412, 0.413, 0.414, 0.415, 0.416, 0.417, 0.418,
0.419, 0.420, 0.421, 0.422, 0.423, 0.424, 0.425, 0.426, 0.427, 0.428, 0.429, 0.430, 0.431,
0.432, 0.433, 0.434, 0.435, 0.436, 0.437, 0.438, 0.439, 0.440, 0.441, 0.442, 0.443, 0.444,
0.445, 0.446, 0.447, 0.448, 0.449, 0.450, 0.451, 0.452, 0.453, 0.454, 0.455, 0.456, 0.457,
0.458, 0.459, 0.460, 0.461, 0.462, 0.463, 0.464, 0.465, 0.466, 0.467, 0.468, 0.469, 0.470,
0.471, 0.472, 0.473, 0.474, 0.475, 0.476, 0.478, 0.479, 0.480, 0.481, 0.482, 0.483, 0.484,
0.485, 0.486, 0.487, 0.488, 0.489, 0.490, 0.491, 0.492, 0.493, 0.494, 0.495, 0.496, 0.497,
0.498, 0.499, 0.500, 0.501, 0.502, 0.503, 0.504, 0.505, 0.506, 0.507, 0.508, 0.509, 0.510,
WO wo 2019/113375 PCT/US2018/064335
0.511,0.512, 0.511, 0.512,0.513, 0.513,0.514, 0.514,0.515, 0.515,0.516, 0.516,0.517, 0.517,0.518, 0.518,0.519, 0.519,0.520, 0.520,0.521, 0.521,0.522, 0.522,0.523, 0.523,
0.524, 0.525, 0.526, 0.527, 0.528, 0.529, 0.530, 0.531, 0.532, 0.533, 0.534, 0.535, 0.536,
0.537, 0.538, 0.539, 0.540, 0.541, 0.542, 0.543, 0.544, 0.545, 0.546, 0.547, 0.548, 0.549,
0.550, 0.551, 0.552, 0.553, 0.554, 0.555, 0.556, 0.557, 0.558, 0.559, 0.560, 0.561, 0.562,
0.563, 0.564, 0.565, 0.566, 0.567, 0.568, 0.569, 0.570, 0.571, 0.572, 0.573, 0.574, 0.575,
0.576, 0.578, 0.579, 0.580, 0.581, 0.582, 0.583, 0.584, 0.585, 0.586, 0.587, 0.588, 0.589,
0.590, 0.591, 0.592, 0.593, 0.594, 0.595, 0.596, 0.597, 0.598, 0.599, 0.600, 0.601, 0.602,
0.603, 0.604, 0.605, 0.606, 0.607, 0.608, 0.609, 0.610, 0.611, 0.612, 0.613, 0.614, 0.615,
0.616, 0.617, 0.618, 0.619, 0.620, 0.621, 0.622, 0.623, 0.624, 0.625, 0.626, 0.627, 0.628,
0.629, 0.630, 0.631, 0.632, 0.633, 0.634, 0.635, 0.636, 0.637, 0.638, 0.639, 0.640, 0.641,
0.642, 0.643, 0.644, 0.645, 0.646, 0.647, 0.648, 0.649, 0.650, 0.651, 0.652, 0.653, 0.654,
0.655, 0.656, 0.657, 0.658, 0.659, 0.660, 0.661, 0.662, 0.663, 0.664, 0.665, 0.666, 0.667,
0.668, 0.669, 0.670, 0.671, 0.672, 0.673, 0.674, 0.675, 0.676, 0.678, 0.679, 0.680, 0.681,
0.682, 0.683, 0.684, 0.685, 0.686, 0.687, 0.688, 0.689, 0.690, 0.691, 0.692, 0.693, 0.694,
0.695, 0.696, 0.697, 0.698, 0.699, 0.700, 0.701, 0.702, 0.703, 0.704, 0.705, 0.706, 0.707,
0.708, 0.709, 0.710, 0.711, 0.712, 0.713, 0.714, 0.715, 0.716, 0.717, 0.718, 0.719, 0.720,
0.721, 0.722, 0.723, 0.724, 0.725, 0.726, 0.727, 0.728, 0.729, 0.730, 0.731, 0.732, 0.733,
0.734, 0.735, 0.736, 0.737, 0.738, 0.739, 0.740, 0.741, 0.742, 0.743, 0.744, or 0.745. In some
embodiments, the ratio of CD34+ CD90 CD45RA CD34 CD90 CD45RA cells cells to to CD34 CD34 cells cells in in the the sample sample is is from from
about 0.625 to about 0.725, such as a ratio of CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells toto CD34+ CD34 cells cells in in
the sample of about 0.625, 0.626, 0.627, 0.628, 0.629, 0.630, 0.631, 0.632, 0.633, 0.634,
0.635, 0.636, 0.637, 0.638, 0.639, 0.640, 0.641, 0.642, 0.643, 0.644, 0.645, 0.646, 0.647,
0.648, 0.649, 0.650, 0.651, 0.652, 0.653, 0.654, 0.655, 0.656, 0.657, 0.658, 0.659, 0.660,
0.661, 0.662, 0.663, 0.664, 0.665, 0.666, 0.667, 0.668, 0.669, 0.670, 0.671, 0.672, 0.673,
0.674, 0.675, 0.676, 0.678, 0.679, 0.680, 0.681, 0.682, 0.683, 0.684, 0.685, 0.686, 0.687,
0.688, 0.689, 0.690, 0.691, 0.692, 0.693, 0.694, 0.695, 0.696, 0.697, 0.698, 0.699, 0.700,
0.701, 0.702, 0.703, 0.704, 0.705, 0.706, 0.707, 0.708, 0.709, 0.710, 0.711, 0.712, 0.713,
0.714, 0.715, 0.716, 0.717, 0.718, 0.719, 0.720, 0.721, 0.722, 0.723, 0.724, or 0.725. In some
embodiments, the ratio of CD34 CD90+ CD45RA cells CD90 CD45RA cells to to CD34 CD34+ cells cells inin the the sample sample isis
about 0.676.
[0036] In an additional aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
WO wo 2019/113375 PCT/US2018/064335
a CXCR4 antagonist in amounts sufficient to enrich the peripheral blood of the donor with
CD34 cells CD34 CD90 CD45RA cells relative to CD34+ cells by by aa ratio ratio of of from from about about 1.1:1 1.1:1 to to about about
4.8:1 as assessed by comparing a sample of peripheral blood of the donor following
administration of the CXCR2 agonist and CXCR4 antagonist to a sample of peripheral blood
of the donor prior to administration of the CXCR2 agonist and CXCR4 antagonist. In some
embodiments, the peripheral blood of the donor may be enriched with CD34+ CD90+ CD34 CD90
CD45RA cells relative to monocytes by a ratio of about 1.10:1, 1.15:1, 1.20:1, 1.25:1,
1.30:1, 1.35:1, 1.40:1, 1.45:1, 1.50:1, 1.55:1, 1.60:1, 1.65:1, 1.70:1, 1.75:1, 1.80:1, 1.85:1,
1.90:1, 1.95:1, 2.00:1, 2.05:1, 2.10:1, 2.15:1, 2.20:1, 2.25:1, 2.30:1, 2.35:1, 2.40:1, 2.45:1,
2.50:1, 2.55:1, 2.60:1, 2.65:1, 2.70:1, 2.75:1, 2.80:1, 2.85:1, 2.90:1, 2.95:1, 3.00:1, 3.05:1,
3.10:1, 3.15:1, 3.20:1, 3.25:1, 3.30:1, 3.35:1, 3.40:1, 3.45:1, 3.50:1, 3.55:1, 3.60:1, 3.65:1,
3.70:1, 3.75:1, 3.80:1, 3.85:1, 3.90:1, 3.95:1, 4.00:1, 4.05:1, 4.10:1, 4.15:1, 4.20:1, 4.25:1,
4.30:1, 4.35:1, 4.40:1, 4.45:1, 4.50:1, 4.55:1, 4.60:1, 4.65:1, 4.70:1, 4.75:1, or 4.80:1. In
CD34 CD90 some embodiments, the peripheral blood of the donor is enriched with CD34+ CD90+
CD45RA cells relative to CD34+ cells by CD34 cells by aa ratio ratio of of about about 1.2:1. 1.2:1.
[0037] In another aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to produce a population of cells having a
frequency ofofCD34+ frequency CD34CD90+ CD90 CD45RA CD45RAcells of of cells from about from 0.020% about to about 0.020% 0.110% 0.110% to about in a in a
sample of peripheral blood of the donor following administration of the CXCR2 agonist and
CXCR4 antagonist. In some embodiments, the population of cells may have a frequency of
CD34+ CD90 CD45RA CD34 CD90 CD45RA cells cells of of about about 0.020%, 0.020%, 0.021%, 0.021%, 0.022%, 0.022%, 0.023%, 0.023%, 0.024%, 0.024%, 0.025%, 0.025%,
0.026%, 0.027%, 0.028%, 0.029%, 0.030%, 0.031%, 0.032%, 0.033%, 0.034%, 0.035%,
0.036%, 0.037%, 0.038%, 0.039%, 0.040%, 0.041%, 0.042%, 0.043%, 0.044%, 0.045%,
0.046%, 0.047%, 0.048%, 0.049%, 0.050%, 0.051%, 0.052%, 0.053%, 0.054%, 0.055%,
0.056%, 0.057%, 0.058%, 0.059%, 0.060%, 0.061%, 0.062%, 0.063%, 0.064%, 0.065%,
0.066%, 0.067%, 0.068%, 0.069%, 0.070%, 0.071%, 0.072%, 0.073%, 0.074%, 0.075%,
0.076%, 0.077%, 0.078%, 0.079%, 0.080%, 0.081%, 0.082%, 0.083%, 0.084%, 0.085%,
0.086%, 0.087%, 0.088%, 0.089%, 0.090%, 0.091%, 0.092%, 0.093%, 0.094%, 0.095%,
0.096%, 0.097%, 0.098%, 0.099%, 0.100%, 0.101%, 0.102%, 0.103%, 0.104%, 0.105%,
0.106%, 0.107%, 0.108%, 0.109%, or 0.110%. In some embodiments, the population of cells
has has aa frequency frequencyofof CD34+ CD34CD90 CD45RA CD90 cells CD45RA of from cells about about of from 0.046%0.046% to about to0.086%, about 0.086%,
WO wo 2019/113375 PCT/US2018/064335
such as a frequency of hematopoietic stem cells of about 0.046%, 0.047%, 0.048%, 0.049%,
0.050%, 0.051%, 0.052%, 0.053%, 0.054%, 0.055%, 0.056%, 0.057%, 0.058%, 0.059%,
0.060%, 0.061%, 0.062%, 0.063%, 0.064%, 0.065%, 0.066%, 0.067%, 0.068%, 0.069%,
0.070%, 0.071%, 0.072%, 0.073%, 0.074%, 0.075%, 0.076%, 0.077%, 0.078%, 0.079%,
0.080%, 0.081%, 0.082%, 0.083%, 0.084%, 0.085%, or 0.086%. In some embodiments, the
population of cells has a frequency of CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells ofof about about 0.066%. 0.066%.
[0038] In an additional aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
into peripheral blood, the method including administering to the donor a CXCR2 agonist and
a CXCR4 antagonist in amounts sufficient to induce an increase in the frequency of CD34+ CD34
CD90 CD45RA cells in the peripheral blood of the donor by at least 3-fold as assessed by
comparing a sample of peripheral blood of the donor following administration of the CXCR2
agonist and CXCR4 antagonist to a sample of peripheral blood of the donor prior to
administration of the CXCR2 agonist and CXCR4 antagonist (e.g., by from about 5.1-fold to
about 25.7-fold, such as by about 5.1-fold, 5.2-fold, 5.3-fold, 5.4-fold, 5.5-fold, 5.6-fold, 5.7-
fold, 5.8-fold, 5.9-fold, 6.0-fold, 6.1-fold, 6.2-fold, 6.3-fold, 6.4-fold, 6.5-fold, 6.6-fold, 6.7-
fold, 6.8-fold, 6.9-fold, 7.0-fold, 7.1-fold, 7.2-fold, 7.3-fold, 7.4-fold, 7.5-fold, 7.6-fold, 7.7-
fold, 7.8-fold, 7.9-fold, 8.0-fold, 8.1-fold, 8.2-fold, 8.3-fold, 8.4-fold, 8.5-fold, 8.6-fold, 8.7-
fold, fold, 8.8-fold, 8.8-fold, 8.9-fold, 8.9-fold, 9.0-fold, 9.0-fold, 9.1-fold, 9.1-fold, 9.2-fold, 9.2-fold, 9.3-fold, 9.3-fold, 9.4-fold, 9.4-fold, 9.5-fold, 9.5-fold, 9.6-fold, 9.6-fold, 9.7- 9.7-
fold, 9.8-fold, 9.9-fold, 10.0-fold, 10.1-fold, 10.2-fold, 10.3-fold, 10.4-fold, 10.5-fold, 10.6-
fold, 10.7-fold, 10.8-fold, 10.9-fold, 11.0-fold, 11.1-fold, 11.2-fold, 11.3-fold, 11.4-fold,
11.5-fold, 11.5-fold, 11.6-fold, 11.6-fold, 11.7-fold, 11.7-fold, 11.8-fold, 11.8-fold, 11.9-fold, 11.9-fold, 12.0-fold, 12.0-fold, 12.1-fold, 12.1-fold, 12.2-fold, 12.2-fold, 12.3-fold, 12.3-fold,
12.4-fold, 12.5-fold, 12.6-fold, 12.7-fold, 12.8-fold, 12.9-fold, 13.0-fold, 13.1-fold, 13.2-fold,
13.3-fold, 13.4-fold, 13.5-fold, 13.6-fold, 13.7-fold, 13.8-fold, 13.9-fold, 14.0-fold, 14.1-fold,
14.2-fold, 14.3-fold, 14.4-fold, 14.5-fold, 14.6-fold, 14.7-fold, 14.8-fold, 14.9-fold, 15.0-fold,
15.1-fold, 15.1-fold, 15.2-fold, 15.2-fold, 15.3-fold, 15.3-fold, 15.4-fold, 15.4-fold, 15.5-fold, 15.5-fold, 15.6-fold, 15.6-fold, 15.7-fold, 15.7-fold, 15.8-fold, 15.8-fold, 15.9-fold, 15.9-fold,
16.0-fold, 16.1-fold, 16.2-fold, 16.3-fold, 16.4-fold, 16.5-fold, 16.6-fold, 16.7-fold, 16.8-fold,
16.9-fold, 16.9-fold, 17.0-fold, 17.0-fold, 17.1-fold, 17.1-fold, 17.2-fold, 17.2-fold, 17.3-fold, 17.3-fold, 17.4-fold, 17.4-fold, 17.5-fold, 17.5-fold, 17.6-fold, 17.6-fold, 17.7-fold, 17.7-fold,
17.8-fold, 17.8-fold, 17.9-fold, 17.9-fold, 18.0-fold, 18.0-fold, 18.1-fold, 18.1-fold, 18.2-fold, 18.2-fold, 18.3-fold, 18.3-fold, 18.4-fold, 18.4-fold, 18.5-fold, 18.5-fold, 18.6-fold, 18.6-fold,
18.7-fold, 18.7-fold, 18.8-fold, 18.8-fold, 18.9-fold, 18.9-fold, 19.0-fold, 19.0-fold, 19.1-fold, 19.1-fold, 19.2-fold, 19.2-fold, 19.3-fold, 19.3-fold, 19.4-fold, 19.4-fold, 19.5-fold, 19.5-fold,
19.6-fold, 19.7-fold, 19.8-fold, 19.9-fold, 20.0-fold, 20.1-fold, 20.2-fold, 20.3-fold, 20.4-fold,
20.5-fold, 20.6-fold, 20.7-fold, 20.8-fold, 20.9-fold, 21.0-fold, 21.1-fold, 21.2-fold, 21.3-fold,
21.4-fold, 21.5-fold, 21.6-fold, 21.7-fold, 21.8-fold, 21.9-fold, 22.0-fold, 22.1-fold, 22.2-fold,
WO wo 2019/113375 PCT/US2018/064335
22.3-fold, 22.4-fold, 22.5-fold, 22.6-fold, 22.7-fold, 22.8-fold, 22.9-fold, 23.0-fold, 23.1-fold,
23.2-fold, 23.3-fold, 23.4-fold, 23.5-fold, 23.6-fold, 23.7-fold, 23.8-fold, 23.9-fold, 24.0-fold,
24.1-fold, 24.2-fold, 24.3-fold, 24.4-fold, 24.5-fold, 24.6-fold, 24.7-fold, 24.8-fold, 24.9-fold,
25.0-fold, 25.1-fold, 25.2-fold, 25.3-fold, 25.4-fold, 25.5-fold, 25.6-fold, or 25.7-fold. In
some embodiments, the frequency of CD34+ CD90CD45RA CD34 CD90 CD45RAcells cellsin inthe theperipheral peripheralblood bloodof of
the the donor donorisisincreased by from increased aboutabout by from 5.1-fold to about 5.1-fold to 7.1-fold following following about 7.1-fold administration of administration of
the CXCR2 agonist and CXCR4 antagonist, such as by about 5.1-fold, 5.2-fold, 5.3-fold, 5.4-
fold, 5.5-fold, 5.6-fold, 5.7-fold, 5.8-fold, 5.9-fold, 6.0-fold, 6.1-fold, 6.2-fold, 6.3-fold, 6.4-
fold, 6.5-fold, 6.6-fold, 6.7-fold, 6.8-fold, 6.9-fold, 7.0-fold, or 7.1-fold. In some
embodiments, the frequency of CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells inin the the peripheral peripheral blood blood ofof the the
donor is increased by about 5.8-fold.
[0039] In another aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells, from the bone marrow of a mammalian donor into peripheral blood,
the method comprising administering to the donor mobilizing amounts of a CXCR2 agonist
and a CXCR4 antagonist; acquiring an input value for each of one or more parameters in
TABLE 2 characterizing a sample of peripheral blood of the donor, and releasing the sample
for ex vivo expansion of the hematopoietic stem cells or for use in the treatment of one or
more stem cell disorders in a mammalian patient if the input value for each of the one or
more parameters meets the corresponding reference criterion for each of the one or more
parameters. In some embodiments, the one or more reference parameters are a set of
parameters listed in any one of TABLES 3-6 herein.
[0040] In some embodiments of any of the above aspects of the invention, the sample is
isolated from the donor at from about 3 hours to about 5 hours following administration of
the CXCR2 agonist and CXCR4 antagonist (e.g., at about 3 hours, 3.1 hours, 3.2 hours, 3.3
hours, 3.4 hours, 3.5 hours, 3.6 hours, 3.7 hours, 3.8 hours, 3.9 hours, 4.0 hours, 4.1 hours,
4.2 hours, 4.3 hours, 4.4 hours, 4.5 hours, 4.6 hours, 4.7 hours, 4.8 hours, 4.9 hours, or 5.0
hours following administration of the CXCR2 agonist and CXCR4 antagonist). In some
embodiments, the sample is isolated from the donor at about 4 hours following administration
of the CXCR2 agonist and CXCR4 antagonist.
[0041] In some embodiments of any of the above aspects of the invention, the CXCR2
agonist is Gro-B Gro-ß T or a variant thereof. In some embodiments, the CXCR2 agonist may be a
peptide having at least about 85% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%,
44
WO wo 2019/113375 PCT/US2018/064335 PCT/US2018/064335
99%, or more) sequence identity to the amino acid sequence of SEQ ID NO: 2. In some
embodiments, the CXCR2 agonist is a peptide having from about 85% to 100% sequence
identity to the amino acid sequence of SEQ ID NO: 2, such as a peptide having from about
86% to about 100%, from about 87% to about 99%, about 88% to about 98%, about 89%, to
about 97%, about 90% to about 96%, or about 91% to about 95% sequence identity to the
amino acid sequence of SEQ ID NO: 2. In some embodiments, the CXCR2 agonist is a
peptide having an amino acid sequence that differs from that of SEQ ID NO: 2 only by way
of one or more conservative amino acid substitutions (e.g., only by way of from 1 to 10
conservative amino acid substitutions, from 1 to 5 conservative amino acid substitutions, or
from 1 to 3 conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
conservative amino acid substitutions). In some embodiments, the CXCR2 agonist is Gro-B Gro-ß
T. In some embodiments, the Gro-B Gro-ß T is not covalently modified. In some embodiments, the
Gro-B Gro-ß T is not covalently modified with a polyalkylene glycol moiety, such as a polyethylene
glycol moiety.
[0042] In some embodiments of any of the above aspects of the invention, the CXCR2
agonist is Gro-B Gro-ß or a variant thereof. In some embodiments, the CXCR2 agonist may be a
peptide having at least about 85% (e.g., about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
more) sequence identity to the amino acid sequence of SEQ ID NO: 1. In some
embodiments, the CXCR2 agonist is a peptide having from about 85% to 100% sequence
identity to the amino acid sequence of SEQ ID NO: 1, such as a peptide having from about
86% to about 100%, from about 87% to about 99%, about 88% to about 98%, about 89%, to
about 97%, about 90% to about 96%, or about 91% to about 95% sequence identity to the
amino acid sequence of SEQ ID NO: 1. In some embodiments, the CXCR2 agonist is a
peptide having an amino acid sequence that differs from that of SEQ ID NO: 1 only by way
of one or more conservative amino acid substitutions (e.g., only by way of from 1 to 10
conservative amino acid substitutions, from 1 to 5 conservative amino acid substitutions, or
from 1 to 3 conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
conservative amino acid substitutions). In some embodiments, the CXCR2 agonist is Gro-ß.
In some embodiments, the Gro-B Gro-ß T is not covalently modified. In some embodiments, the
Gro-B Gro-ß is not covalently modified with a polyalkylene glycol moiety, such as a polyethylene
glycol moiety.
[0043] In some embodiments, the CXCR2 agonist (e.g., Gro-B Gro-ß or Gro-B Gro-ß T, such as
unmodified Gro-B Gro-ß or Gro-B Gro-ß T) is administered to the donor at a dose of from about 50 ug/kg µg/kg
WO 2019/113375 2019/11337 OM PCT/US2018/064335
to about 1 mg/kg, such as a dose of about 50 ug/kg, µg/kg, 55 ug/kg, µg/kg, 60 ug/kg, µg/kg, 65 ug/kg, µg/kg, 70 ug/kg, µg/kg,
ug/kg, 80 µg/kg, 75 µg/kg, ug/kg, 85 µg/kg, ug/kg, 90 µg/kg, ug/kg, 95 µg/kg, ug/kg, 100 µg/kg, ug/kg, 105 µg/kg, ug/kg, 110 µg/kg, ug/kg, 115
ug/kg, µg/kg, 120 ug/kg, µg/kg, 125 ug/kg, µg/kg, 130 ug/kg, µg/kg, 135 ug/kg, µg/kg, 140 ug/kg, µg/kg, 145 ug/kg, µg/kg, 150 ug/kg, µg/kg, 155
ug/kg, µg/kg, 160 ug/kg, µg/kg, 165 ug/kg, µg/kg, 170 ug/kg, µg/kg, 175 ug/kg, µg/kg, 180 ug/kg, µg/kg, 185 ug/kg, µg/kg, 190 ug/kg, µg/kg, 195
ug/kg, 200 µg/kg, µg/kg, ug/kg, 205 µg/kg, ug/kg, 210 µg/kg, ug/kg, 215 µg/kg, ug/kg, 220 µg/kg, ug/kg, 225 µg/kg, ug/kg, 230 µg/kg, ug/kg, 235
ug/kg, 240 µg/kg, µg/kg, ug/kg, 245 µg/kg, ug/kg, 250 µg/kg, ug/kg, 255 µg/kg, ug/kg, 260 µg/kg, ug/kg, 265 µg/kg, ug/kg, 270 µg/kg, ug/kg, 275
ug/kg, µg/kg, 280 ug/kg, µg/kg, 285 ug/kg, µg/kg, 290 ug/kg, µg/kg, 295 ug/kg, µg/kg, 300 ug/kg, µg/kg, 305 ug/kg, µg/kg, 310 ug/kg, µg/kg, 315
ug/kg, 320 µg/kg, µg/kg, ug/kg, 325 µg/kg, ug/kg, 330 µg/kg, ug/kg, 335 µg/kg, ug/kg, 340 µg/kg, ug/kg, 345 µg/kg, ug/kg, 350 µg/kg, ug/kg, 355
ug/kg, µg/kg, 360 ug/kg, µg/kg, 365 ug/kg, µg/kg, 370 ug/kg, µg/kg, 375 ug/kg, µg/kg, 380 ug/kg, µg/kg, 400 ug/kg, µg/kg, 405 ug/kg, µg/kg, 410
OI ug/kg, µg/kg, 415 ug/kg, µg/kg, 425 ug/kg, µg/kg, 430 ug/kg, µg/kg, 435 ug/kg, µg/kg, 440 ug/kg, µg/kg, 445 ug/kg, µg/kg, 450 ug/kg, µg/kg, 210
ug/kg, 300 µg/kg, µg/kg, ug/kg, 400 µg/kg, ug/kg, 405 µg/kg, ug/kg, 410 µg/kg, ug/kg, 415 µg/kg, ug/kg, 420 µg/kg, ug/kg, 425 µg/kg, ug/kg, 430
ug/kg, µg/kg, 435 ug/kg, µg/kg, 440 ug/kg, µg/kg, 445 ug/kg, µg/kg, 450 ug/kg, µg/kg, 455 ug/kg, µg/kg, 460 ug/kg, µg/kg, 465 ug/kg, µg/kg, 470
ug/kg, µg/kg, 475 ug/kg, µg/kg, 480 ug/kg, µg/kg, 485 ug/kg, µg/kg, 490 ug/kg, µg/kg, 495 ug/kg, µg/kg, 500 ug/kg, µg/kg, 505 ug/kg, µg/kg, 510
ug/kg, 505 µg/kg, µg/kg, ug/kg, 515 µg/kg, ug/kg, 520 µg/kg, ug/kg, 525 µg/kg, ug/kg, 530 µg/kg, ug/kg, 545 µg/kg, ug/kg, 550 µg/kg, ug/kg, 555
ug/kg, 560 µg/kg, µg/kg, ug/kg, 565 µg/kg, ug/kg, 570 µg/kg, ug/kg, 575 µg/kg, ug/kg, 580 µg/kg, ug/kg, 585 µg/kg, ug/kg, 590 µg/kg, ug/kg, 595
ug/kg, 600 µg/kg, µg/kg, ug/kg, 605 µg/kg, ug/kg, 610 µg/kg, ug/kg, 615 µg/kg, ug/kg, 620 µg/kg, ug/kg, 625 µg/kg, ug/kg, 630 µg/kg, ug/kg, 635
ug/kg, µg/kg, 640 ug/kg, µg/kg, 645 ug/kg, µg/kg, 650 ug/kg, µg/kg, 655 ug/kg, µg/kg, 660 ug/kg, µg/kg, 665 ug/kg, µg/kg, 670 ug/kg, µg/kg, 675
ug/kg, µg/kg, 680 ug/kg, µg/kg, 685 ug/kg, µg/kg, 690 ug/kg, µg/kg, 695 ug/kg, µg/kg, 700 ug/kg, µg/kg, 705 ug/kg, µg/kg, 710 ug/kg, µg/kg, 715
ug/kg, 720 µg/kg, µg/kg, ug/kg, 725 µg/kg, ug/kg, 730 µg/kg, ug/kg, 735 µg/kg, ug/kg, 740 µg/kg, ug/kg, 745 µg/kg, ug/kg, 750 µg/kg, ug/kg, 755
ug/kg, 760 µg/kg, µg/kg, ug/kg, 765 µg/kg, ug/kg, 770 µg/kg, ug/kg, 775 µg/kg, ug/kg, 780 µg/kg, ug/kg, 785 µg/kg, ug/kg, 790 µg/kg, ug/kg, 795
ug/kg, 800 µg/kg, µg/kg, ug/kg, 805 µg/kg, ug/kg, 810 µg/kg, ug/kg, 815 µg/kg, ug/kg, 820 µg/kg, ug/kg, 825 µg/kg, ug/kg, 830 µg/kg, ug/kg, 835
ug/kg, µg/kg, 840 ug/kg, µg/kg, 845 ug/kg, µg/kg, 850 ug/kg, µg/kg, 855 ug/kg, µg/kg, 860 ug/kg, µg/kg, 865 ug/kg, µg/kg, 870 ug/Kg, µg/Kg, 875
ug/kg, 880 µg/kg, µg/kg, ug/kg, 885 µg/kg, ug/kg, 890 µg/kg, ug/kg, 895 µg/kg, ug/kg, 900 µg/kg, ug/kg, 905 µg/kg, ug/kg, 910 µg/kg, ug/kg, 915
ug/kg, µg/kg, 920 ug/kg, µg/kg, 925 ug/kg, µg/kg, 930 ug/kg, µg/kg, 935 ug/kg, µg/kg, 940 ug/kg, µg/kg, 945 ug/kg, µg/kg, 950 ug/kg, µg/kg, 955
ug/kg, µg/kg, 960 ug/kg, µg/kg, 965 ug/kg, µg/kg, 970 ug/kg, µg/kg, 975 ug/kg, µg/kg, 980 ug/kg, µg/kg, 985 ug/kg, µg/kg, 990 ug/kg, µg/kg, 995
ug/kg, µg/kg, or 1,000 ug/kg. µg/kg. In some embodiments, the CXCR2 agonist (e.g., Gro-B Gro-ß or Gro-B Gro-ß T,
Gro-B or Gro-ß such as unmodified Gro-ß Gro-B T) is administered to the donor at a dose of from about 50
ug/kg to about 300 µg/kg, µg/kg ug/kg, such as a dose of from about 100 µg/kg ug/kg to about 250 µg/kg, ug/kg, or
ug/kg to about 225 µg/kg. from about 125 µg/kg ug/kg. In some embodiments, the CXCR2 agonist (e.g.,
0£ Gro-B Gro-ß or Gro-B Gro-ß T, such as unmodified Gro-B Gro-ß or Gro-B Gro-ß T) is administered to the donor at a
dose of about 150 µg/kg. ug/kg.
[0044] In another aspect, the invention features a method of mobilizing a population of
hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor)
46
WO 2019/113375 2019/11337 OM PCT/US2018/064335 PCT/US2018/064335
into peripheral blood, the method including administering to the donor a CXCR2 agonist
Gro-B, Gro-ß selected from the group consisting of Gro-ß, Gro-B T, and variants thereof at a dose of from
about 50 ug/kg µg/kg to about 1 mg/kg (e.g., a dose of 50 ug/kg, µg/kg, 55 ug/kg, µg/kg, 60 ug/kg, µg/kg, 65 ug/kg, µg/kg, 70
ug/kg, 75 µg/kg, µg/kg, ug/kg, 80 µg/kg, ug/kg, 85 µg/kg, ug/kg, 90 µg/kg, ug/kg, 95 µg/kg, ug/kg, 100 µg/kg, ug/kg, 105 µg/kg, ug/kg, 110 µg/kg, ug/kg,
S ug/kg, 120 µg/kg, 115 µg/kg, ug/kg, 125 µg/kg, ug/kg, 130 µg/kg, ug/kg, 135 µg/kg, ug/kg, 140 µg/kg, ug/kg, 145 µg/kg, ug/kg, 150 µg/kg, ug/kg,
155 ug/kg, µg/kg, 160 ug/kg, µg/kg, 165 ug/kg, µg/kg, 170 ug/kg, µg/kg, 175 ug/kg, µg/kg, 180 ug/kg, µg/kg, 185 ug/kg, µg/kg, 190 ug/kg, µg/kg,
195 ug/kg, µg/kg, 200 ug/kg, µg/kg, 205 ug/kg, µg/kg, 210 ug/kg, µg/kg, 215 ug/kg, µg/kg, 220 ug/kg, µg/kg, 225 ug/kg, µg/kg, 230 ug/kg, µg/kg,
235 ug/kg, µg/kg, 240 ug/kg, µg/kg, 245 ug/kg, µg/kg, 250 ug/kg, µg/kg, 255 ug/kg, µg/kg, 260 ug/kg, µg/kg, 265 ug/kg, µg/kg, 270 ug/kg, µg/kg,
275 ug/kg, µg/kg, 280 ug/kg, µg/kg, 285 ug/kg, µg/kg, 290 ug/kg, µg/kg, 295 ug/kg, µg/kg, 300 ug/kg, µg/kg, 305 ug/kg, µg/kg, 310 ug/kg, µg/kg,
OI 315 µg/kg, ug/kg, 320 µg/kg, ug/kg, 325 µg/kg, ug/kg, 330 µg/kg, ug/kg, 335 µg/kg, ug/kg, 340 µg/kg, ug/kg, 345 µg/kg, ug/kg, 350 µg/kg, ug/kg,
ug/kg, 360 µg/kg, 355 µg/kg, ug/kg, 365 µg/kg, ug/kg, 370 µg/kg, ug/kg, 375 µg/kg, ug/kg, 380 µg/kg, ug/kg, 400 µg/kg, ug/kg, 405 µg/kg, ug/kg,
410 ug/kg, µg/kg, 415 ug/kg, µg/kg, 425 ug/kg, µg/kg, 430 ug/kg, µg/kg, 435 ug/kg, µg/kg, 440 ug/kg, µg/kg, 445 ug/kg, µg/kg, 450 ug/kg, µg/kg,
ug/kg, 300 µg/kg, 210 µg/kg, ug/kg, 400 µg/kg, ug/kg, 405 µg/kg, ug/kg, 410 µg/kg, ug/kg, 415 µg/kg, ug/kg, 420 µg/kg, ug/kg, 425 µg/kg, ug/kg,
430 ug/kg, µg/kg, 435 ug/kg, µg/kg, 440 ug/kg, µg/kg, 445 ug/kg, µg/kg, 450 ug/kg, µg/kg, 455 ug/kg, µg/kg, 460 ug/kg, µg/kg, 465 ug/kg, µg/kg,
470 ug/kg, µg/kg, 475 ug/kg, µg/kg, 480 ug/kg, µg/kg, 485 ug/kg, µg/kg, 490 ug/kg, µg/kg, 495 ug/kg, µg/kg, 500 ug/kg, µg/kg, 505 ug/kg, µg/kg,
510 ug/kg, µg/kg, 505 ug/kg, µg/kg, 515 ug/kg, µg/kg, 520 ug/kg, µg/kg, 525 ug/kg, µg/kg, 530 ug/kg, µg/kg, 545 ug/kg, µg/kg, 550 ug/kg, µg/kg,
555 ug/kg, µg/kg, 560 ug/kg, µg/kg, 565 ug/kg, µg/kg, 570 ug/kg, µg/kg, 575 ug/kg, µg/kg, 580 ug/kg, µg/kg, 585 ug/kg, µg/kg, 590 ug/kg, µg/kg,
595 ug/kg, µg/kg, 600 ug/kg, µg/kg, 605 ug/kg, µg/kg, 610 ug/kg, µg/kg, 615 ug/kg, µg/kg, 620 ug/kg, µg/kg, 625 ug/kg, µg/kg, 630 ug/kg, µg/kg,
635 ug/kg, µg/kg, 640 ug/kg, µg/kg, 645 ug/kg, µg/kg, 650 ug/kg, µg/kg, 655 ug/kg, µg/kg, 660 ug/kg, µg/kg, 665 ug/kg, µg/kg, 670 ug/kg, µg/kg,
675 ug/kg, µg/kg, 680 ug/kg, µg/kg, 685 ug/kg, µg/kg, 690 ug/kg, µg/kg, 695 ug/kg, µg/kg, 700 ug/kg, µg/kg, 705 ug/kg, µg/kg, 710 ug/kg, µg/kg,
715 µg/kg, ug/kg, 720 µg/kg, ug/kg, 725 µg/kg, ug/kg, 730 µg/kg, ug/kg, 735 µg/kg, ug/kg, 740 µg/kg, ug/kg, 745 µg/kg, ug/kg, 750 µg/kg, ug/kg,
ug/kg, 760 µg/kg, 755 µg/kg, ug/kg, 765 µg/kg, ug/kg, 770 µg/kg, ug/kg, 775 µg/kg, ug/kg, 780 µg/kg, ug/kg, 785 µg/kg, ug/kg, 790 µg/kg, ug/kg,
795 ug/kg, µg/kg, 800 ug/kg, µg/kg, 805 ug/kg, µg/kg, 810 ug/kg, µg/kg, 815 ug/kg, µg/kg, 820 ug/kg, µg/kg, 825 ug/kg, µg/kg, 830 ug/kg, µg/kg,
ug/kg, 840 µg/kg, 835 µg/kg, ug/kg, 845 µg/kg, ug/kg, 850 µg/kg, ug/kg, 855 µg/kg, ug/kg, 860 µg/kg, ug/kg, 865 µg/kg, ug/kg, 870 µg/Kg, ug/Kg,
ug/kg, 880 µg/kg, 875 µg/kg, ug/kg, 885 µg/kg, ug/kg, 890 µg/kg, ug/kg, 895 µg/kg, ug/kg, 900 µg/kg, ug/kg, 905 µg/kg, ug/kg, 910 µg/kg, ug/kg,
915 ug/kg, µg/kg, 920 ug/kg, µg/kg, 925 ug/kg, µg/kg, 930 ug/kg, µg/kg, 935 ug/kg, µg/kg, 940 ug/kg, µg/kg, 945 ug/kg, µg/kg, 950 ug/kg, µg/kg,
955 ug/kg, 960 ug/kg, 965 ug/kg, 970 ug/kg, 975 ug/kg, 980 ug/kg, 985 ug/kg, 990 ug/kg, 066 $86 086 SL6 0L6 $96 096 SS6 995 ug/kg, µg/kg, or 1,000 ug/kg). µg/kg). In some embodiments, the method further includes
administering a CXCR4 antagonist to the donor.
0£ [0045] In some embodiments of any of the above aspects of the invention, the CXCR2
Gro-B or Gro-ß agonist (e.g., Gro-ß Gro-B T, such as unmodified Gro-ß Gro-B or Gro-ß Gro-B T) is administered to the
ug/kg to about 300 µg/kg, donor at a dose of from about 50 µg/kg ug/kg, such as a dose of about 50 µg/kg, ug/kg,
ug/kg, 60 µg/kg, 55 µg/kg, ug/kg, 65 µg/kg, ug/kg, 70 µg/kg, ug/kg, 75 µg/kg, ug/kg, 80 µg/kg, ug/kg, 85 µg/kg, ug/kg, 90 µg/kg, ug/kg, 95 µg/kg, ug/kg,
100 ug/kg, µg/kg, 105 ug/kg, µg/kg, 110 ug/kg, µg/kg, 115 ug/kg, µg/kg, 120 ug/kg, µg/kg, 125 ug/kg, µg/kg, 130 ug/kg, µg/kg, 135 ug/kg, µg/kg,
140 ug/kg, µg/kg, 145 ug/kg, µg/kg, 150 ug/kg, µg/kg, 155 ug/kg, µg/kg, 160 ug/kg, µg/kg, 165 ug/kg, µg/kg, 170 ug/kg, µg/kg, 175 ug/kg, µg/kg,
180 ug/kg, µg/kg, 185 ug/kg, µg/kg, 190 ug/kg, µg/kg, 195 ug/kg, µg/kg, 200 ug/kg, µg/kg, 205 ug/kg, µg/kg, 210 ug/kg, µg/kg, 215 ug/kg, µg/kg,
220 ug/kg, µg/kg, 225 ug/kg, µg/kg, 230 ug/kg, µg/kg, 235 ug/kg, µg/kg, 240 ug/kg, µg/kg, 245 ug/kg, µg/kg, 250 ug/kg, µg/kg, 255 ug/kg, µg/kg,
260 ug/kg, µg/kg, 265 ug/kg, µg/kg, 270 ug/kg, µg/kg, 275 ug/kg, µg/kg, 280 ug/kg, µg/kg, 285 ug/kg, µg/kg, 290 ug/kg, µg/kg, 295 ug/kg, µg/kg, or
300 ug/kg. µg/kg.
[0046] In some embodiments of any of the above aspects of the invention, the CXCR2
agonist (e.g., Gro-B Gro-ß or Gro-B Gro-ß T, such as unmodified Gro-B Gro-ß or Gro-B Gro-ß T) is administered to the
donor at a dose of from about 100 ug/kg µg/kg to about 250 ug/kg, µg/kg, such as a dose of about 100
ug/kg, µg/kg, 105 ug/kg, µg/kg, 110 ug/kg, µg/kg, 115 ug/kg, µg/kg, 120 ug/kg, µg/kg, 125 ug/kg, µg/kg, 130 ug/kg, µg/kg, 135 ug/kg, µg/kg, 140
ug/kg, µg/kg, 145 ug/kg, µg/kg, 150 ug/kg, µg/kg, 155 ug/kg, µg/kg, 160 ug/kg, µg/kg, 165 ug/kg, µg/kg, 170 ug/kg, µg/kg, 175 ug/kg, µg/kg, 180
ug/kg, µg/kg, 185 ug/kg, µg/kg, 190 ug/kg, µg/kg, 195 ug/kg, µg/kg, 200 ug/kg, µg/kg, 205 ug/kg, µg/kg, 210 ug/kg, µg/kg, 215 ug/kg, µg/kg, 220
ug/kg, µg/kg, 225 ug/kg, µg/kg, 230 ug/kg, µg/kg, 235 ug/kg, µg/kg, 240 ug/kg, µg/kg, 245 ug/kg, µg/kg, or 250 ug/kg. µg/kg.
[0047] In some embodiments of any of the above aspects of the invention, the CXCR2
agonist (e.g., Gro-B Gro-ß or Gro-B Gro-ß T, such as unmodified Gro-B Gro-ß or Gro-B Gro-ß T) is administered to the
donor at a dose of about 150 ug/kg. µg/kg. For example, in some embodiments, the CXCR2 agonist
(e.g., Gro-B Gro-ß or Gro-B Gro-ß T, such as unmodified Gro-B Gro-ß or Gro-B Gro-ß T) is administered to the donor at
a dose of from about 50 ug/kg µg/kg per day to about 1 mg/kg per day, such as a dose of about 50
ug/kg µg/kg per day, 55 ug/kg µg/kg per day, 60 ug/kg µg/kg per day, 65 ug/kg µg/kg per day, 70 ug/kg µg/kg per day, 75
ug/kg µg/kg per day, 80 ug/kg µg/kg per day, 85 ug/kg µg/kg per day, 90 ug/kg µg/kg per day, 95 ug/kg µg/kg per day, 100
ug/kg µg/kg per day, 105 ug/kg µg/kg per day, 110 ug/kg µg/kg per day, 115 ug/kg µg/kg per day, 120 ug/kg µg/kg per day,
125 ug/kg µg/kg per day, 130 ug/kg µg/kg per day, 135 ug/kg µg/kg per day, 140 ug/kg µg/kg per day, 145 ug/kg µg/kg per
day, 150 ug/kg µg/kg per day, 155 ug/kg µg/kg per day, 160 ug/kg µg/kg per day, 165 ug/kg µg/kg per day, 170 ug/kg µg/kg
per day, 175 ug/kg µg/kg per day, 180 ug/kg µg/kg per day, 185 ug/kg µg/kg per day, 190 ug/kg µg/kg per day, 195
ug/kg µg/kg per day, 200 ug/kg µg/kg per day, 205 ug/kg µg/kg per day, 210 ug/kg µg/kg per day, 215 ug/kg µg/kg per day,
220 ug/kg µg/kg per day, 225 ug/kg µg/kg per day, 230 ug/kg µg/kg per day, 235 ug/kg µg/kg per day, 240 ug/kg µg/kg per
day, 245 ug/kg µg/kg per day, 250 ug/kg µg/kg per day, 255 ug/kg µg/kg per day, 260 ug/kg µg/kg per day, 265 ug/kg µg/kg
per day, 270 ug/kg µg/kg per day, 275 ug/kg µg/kg per day, 280 ug/kg µg/kg per day, 285 ug/kg µg/kg per day, 290
ug/kg µg/kg per day, 295 ug/kg µg/kg per day, 300 ug/kg µg/kg per day, 305 ug/kg µg/kg per day, 310 ug/kg µg/kg per day,
315 ug/kg µg/kg per day, 320 ug/kg µg/kg per day, 325 ug/kg µg/kg per day, 330 ug/kg µg/kg per day, 335 ug/kg µg/kg per
day, 340 ug/kg µg/kg per day, 345 ug/kg µg/kg per day, 350 ug/kg µg/kg per day, 355 ug/kg µg/kg per day, 360 ug/kg µg/kg
per day, 365 ug/kg µg/kg per day, 370 ug/kg µg/kg per day, 375 ug/kg µg/kg per day, 380 ug/kg µg/kg per day, 400
ug/kg µg/kg per day, 405 ug/kg µg/kg per day, 410 ug/kg µg/kg per day, 415 ug/kg µg/kg per day, 425 ug/kg µg/kg per day,
WO wo 2019/113375 PCT/US2018/064335
430 ug/kg µg/kg per day, 435 ug/kg µg/kg per day, 440 ug/kg µg/kg per day, 445 ug/kg µg/kg per day, 450 ug/kg µg/kg per
day, 210 ug/kg µg/kg per day, 300 ug/kg µg/kg per day, 400 ug/kg µg/kg per day, 405 ug/kg µg/kg per day, 410 ug/kg µg/kg
per day, 415 ug/kg µg/kg per day, 420 ug/kg µg/kg per day, 425 ug/kg µg/kg per day, 430 ug/kg µg/kg per day, 435
ug/kg µg/kg per day, 440 ug/kg µg/kg per day, 445 ug/kg µg/kg per day, 450 ug/kg µg/kg per day, 455 ug/kg µg/kg per day,
460 ug/kg µg/kg per day, 465 ug/kg µg/kg per day, 470 ug/kg µg/kg per day, 475 ug/kg µg/kg per day, 480 ug/kg µg/kg per
day, 485 ug/kg µg/kg per day, 490 ug/kg µg/kg per day, 495 ug/kg µg/kg per day, 500 ug/kg µg/kg per day, 505 ug/kg µg/kg
per day, 510 ug/kg µg/kg per day, 505 ug/kg µg/kg per day, 515 ug/kg µg/kg per day, 520 ug/kg µg/kg per day, 525
ug/kg µg/kg per day, 530 ug/kg µg/kg per day, 545 ug/kg µg/kg per day, 550 ug/kg µg/kg per day, 555 ug/kg µg/kg per day,
560 ug/kg µg/kg per day, 565 ug/kg µg/kg per day, 570 ug/kg µg/kg per day, 575 ug/kg µg/kg per day, 580 ug/kg µg/kg per
day, 585 ug/kg µg/kg per day, 590 ug/kg µg/kg per day, 595 ug/kg µg/kg per day, 600 ug/kg µg/kg per day, 605 ug/kg µg/kg
per day, 610 ug/kg µg/kg per day, 615 ug/kg µg/kg per day, 620 ug/kg µg/kg per day, 625 ug/kg µg/kg per day, 630
ug/kg µg/kg per day, 635 ug/kg µg/kg per day, 640 ug/kg µg/kg per day, 645 ug/kg µg/kg per day, 650 ug/kg µg/kg per day,
655 ug/kg µg/kg per day, 660 ug/kg µg/kg per day, 665 ug/kg µg/kg per day, 670 ug/kg µg/kg per day, 675 ug/kg µg/kg per
day, 680 ug/kg µg/kg per day, 685 ug/kg µg/kg per day, 690 ug/kg µg/kg per day, 695 ug/kg µg/kg per day, 700 ug/kg µg/kg
per day, 705 ug/kg µg/kg per day, 710 ug/kg µg/kg per day, 715 ug/kg µg/kg per day, 720 ug/kg µg/kg per day, 725
ug/kg µg/kg per day, 730 ug/kg µg/kg per day, 735 ug/kg µg/kg per day, 740 ug/kg µg/kg per day, 745 ug/kg µg/kg per day,
750 ug/kg µg/kg per day, 755 ug/kg µg/kg per day, 760 ug/kg µg/kg per day, 765 ug/kg µg/kg per day, 770 ug/kg µg/kg per
day, 775 ug/kg µg/kg per day, 780 ug/kg µg/kg per day, 785 ug/kg µg/kg per day, 790 ug/kg µg/kg per day, 795 ug/kg µg/kg
per day, 800 ug/kg µg/kg per day, 805 ug/kg µg/kg per day, 810 ug/kg µg/kg per day, 815 ug/kg µg/kg per day, 820
ug/kg µg/kg per day, 825 ug/kg µg/kg per day, 830 ug/kg µg/kg per day, 835 ug/kg µg/kg per day, 840 ug/kg µg/kg per day,
845 ug/kg µg/kg per day, 850 ug/kg µg/kg per day, 855 ug/kg µg/kg per day, 860 ug/kg µg/kg per day, 865 ug/kg µg/kg per
day, 870 ug/kg µg/kg per day, 875 ug/kg µg/kg per day, 880 ug/kg µg/kg per day, 885 ug/kg µg/kg per day, 890 ug/kg µg/kg
per day, 895 ug/kg µg/kg per day, 900 ug/kg µg/kg per day, 905 ug/kg µg/kg per day, 910 ug/kg µg/kg per day, 915
ug/kg µg/kg per day, 920 ug/kg µg/kg per day, 925 ug/kg µg/kg per day, 930 ug/kg µg/kg per day, 935 ug/kg µg/kg per day,
940 ug/kg µg/kg per day, 945 ug/kg µg/kg per day, 950 ug/kg µg/kg per day, 955 ug/kg µg/kg per day, 960 ug/kg µg/kg per
day, 965 ug/kg µg/kg per day, 970 ug/kg µg/kg per day, 975 ug/kg µg/kg per day, 980 ug/kg µg/kg per day, 985 ug/kg µg/kg
per day, 990 ug/kg µg/kg per day, 995 ug/kg µg/kg per day, or 1,000 ug/kg µg/kg per day. In some
embodiments, the CXCR2 agonist (e.g., Gro-B Gro-ß or Gro-B Gro-ß T, such as unmodified Gro-B Gro-ß or Gro-
B ß T) is administered to the donor at a dose of from about 50 ug/kg µg/kg per day to about 300 ug/kg µg/kg
per day, such as a dose of from about 100 ug/kg µg/kg per day to about 250 ug/kg µg/kg per day, or from
about 125 ug/kg µg/kg per day to about 225 ug/kg µg/kg per day, or from about 125 ug/kg µg/kg per day to
about 175 ug/kg µg/kg per day. In some embodiments, the CXCR2 agonist (e.g., Gro-B Gro-ß or Gro-B Gro-ß T,
such as unmodified Gro-ß or Gro-ß T) is administered to the donor at a dose of about 150
WO wo 2019/113375 PCT/US2018/064335
ug/kg µg/kg per day. In some embodiments, the CXCR2 agonist may be administered as a single
dose. In other embodiments, the CXCR2 agonist may be administered as two or more doses.
[0048] In some embodiments, a human equivalent dose (HED) may be derived from animal
dosage data using a conversion factor. For example, Nair and Jacob, J. Basic Clin. Pharma.
(2016) 7:27-31 disclose methods extrapolation of dose between species. For instance, in one
non-limiting example, HED may be derived from rhesus monkey dose by multiplying the
rhesus monkey dose by about 0.324.
[0049] In some embodiments of any of the above aspects of the invention, the CXCR2
agonist (e.g., Gro-B Gro-ß or Gro-B Gro-ß T, such as unmodified Gro-B Gro-ß or Gro-B Gro-ß T) is administered
intravenously to the donor.
[0050] In some embodiments of any of the above aspects of the invention, the CXCR4
antagonist is a compound represented by formula (I)
Z - linker - Z' (I)
or a pharmaceutically acceptable salt thereof, wherein Z is:
(i) a cyclic polyamine containing from 9 to 32 ring members, wherein
from 2 to 8 of the ring members are nitrogen atoms separated from one
another by 2 or more carbon atoms; or
(ii) (ii) an amine represented by formula (IA)
A B NY N (IA)
wherein A includes a monocyclic or bicyclic fused ring system including at least one nitrogen
atom and atom andB BisisH H or or a substituent of from a substituent of 1from to 20 1 atoms; to 20 atoms;
and wherein Z' is:
(i) a cyclic polyamine containing from 9 to 32 ring members, wherein
from 2 to 8 of the ring members are nitrogen atoms separated from one
another by 2 or more carbon atoms;
(ii) an amine represented by formula (IB)
WO wo 2019/113375 PCT/US2018/064335
A' A' N B' B' (IB)
wherein A' includes a monocyclic or bicyclic fused ring system including at least one
nitrogen atom and B' is H or a substituent of from 1 to 20 atoms; or
(iii) (iii) a asubstituent substituentrepresented representedbybyformula formula(IC) (IC)
- - N(R) - (CR2)n -N(R)- (CR)- -X X(IC) (IC)
wherein whereineach eachR R is is independently H or HC1-C6 independently alkyl, or C-C n is n alkyl, 1 or is 2, and 2, 1 or X is an Xaryl and is or an heteroaryl aryl or heteroaryl
group or a mercaptan;
wherein whereinthe thelinker is is linker a bond, optionally a bond, substituted optionally C1-C6 alkylene, substituted optionally C-C alkylene, optionally
substituted substitutedC1-C6 C-C heteroalkylene, heteroalkylene, optionally substituted optionally C2-C6 C-C substituted alkenylene, optionally alkenylene, optionally
substituted C2-C6 heteroalkenylene, C-C heteroalkenylene, optionally optionally substituted substituted C2-C6 C-C alkynylene, alkynylene, optionally optionally
substituted substitutedC2-C6 C-C heteroalkynylene, heteroalkynylene, optionally substituted optionally cycloalkylene, substituted optionally cycloalkylene, optionally
substituted heterocycloalkylene, optionally substituted arylene, or optionally substituted
heteroarylene. heteroarylene.
[0051] In some embodiments, Z and Z' are each independently a cyclic polyamine containing
from 9 to 32 ring members, of which from 2 to 8 are nitrogen atoms separated from one
another by 2 or more carbon atoms. Z and Z' may be identical substituents. In some
embodiments, Z and/or Z' is a cyclic polyamine including from 10 to 24 ring members, such
as a cyclic polyamine including 14 ring members. In some embodiments, Z includes 4
nitrogen atoms. Z and/or Z' may be, for example, 1,4,8,11-tetraazocyclotetradecane. ,4,8,11-tetraazocyclotetradecane.
[0052] In some embodiments, the linker is represented by formula (ID)
Y-| IX D (ID)
wherein ring D is an optionally substituted aryl group, an optionally substituted
heteroaryl group, an optionally substituted cycloalkyl group, or an optionally substituted
heterocycloalkyl group; and
X and Y are each independently optionally substituted C1-C6 alkylene, C-C alkylene, optionally optionally
substituted substitutedC1-C6 C-C heteroalkylene, heteroalkylene, optionally substituted optionally C2-C6 C-C substituted alkenylene, optionally alkenylene, optionally
PCT/US2018/064335
substituted substitutedC2-C6 C-C heteroalkenylene, heteroalkenylene, optionally substituted optionally C2-C6 alkynylene, substituted or optionally C-C alkynylene, or optionally
substituted C2-C6 heteroalkynylene. C-C heteroalkynylene.
In some embodiments, the linker is represented by formula (IE)
X X (IE).
wherein ring D is an optionally substituted aryl group, an optionally substituted
heteroaryl group, an optionally substituted cycloalkyl group, or an optionally substituted
heterocycloalkyl group; and
X and Y are each independently optionally substituted C1-C6 alkylene, C-C alkylene, optionally optionally
substituted substitutedC1-C6 C-C heteroalkylene, heteroalkylene, optionally substituted optionally C2-C6 C-C substituted alkenylene, optionally alkenylene, optionally
substituted substitutedC2-C6 C-C heteroalkenylene, heteroalkenylene, optionally substituted optionally C2-C6 alkynylene, substituted or optionally C-C alkynylene, or optionally
substituted C2-C6 heteroalkynylene. C-C heteroalkynylene.
[0053] In some embodiments, X and Y are each independently optionally substituted C1-C6 C-C
alkylene. In some embodiments, X and Y are identical substituents, such as identical
alkylene substituents (e.g., methylene, ethylene, propylene, or butylene substituents).
[0054] In some embodiments, the CXCR4 antagonist is plerixafor or a pharmaceutically
acceptable salt thereof. In some embodiments, the CXCR4 antagonist (e.g., plerixafor or a
pharmaceutically acceptable salt thereof) is administered subcutaneously to the donor. In
some embodiments, the CXCR4 antagonist (e.g., plerixafor or a pharmaceutically acceptable
salt thereof) is administered to the donor at a dose of from about 50 ug/kg µg/kg to about 500
ug/kg, µg/kg, such as a dose of about 50 ug/kg, µg/kg, 55 ug/kg, µg/kg, 60 ug/kg, µg/kg, 65 ug/kg, µg/kg, 70 ug/kg, µg/kg, 75 ug/kg, µg/kg,
80 ug/kg, µg/kg, 85 ug/kg, µg/kg, 90 ug/kg, µg/kg, 95 ug/kg, µg/kg, 100 ug/kg, µg/kg, 105 ug/kg, µg/kg, 110 ug/kg, µg/kg, 115 ug/kg, µg/kg, 120
ug/kg, µg/kg, 125 ug/kg, µg/kg, 130 ug/kg, µg/kg, 135 ug/kg, µg/kg, 140 ug/kg, µg/kg, 145 ug/kg, µg/kg, 150 ug/kg, µg/kg, 155 ug/kg, µg/kg, 160
ug/kg, µg/kg, 165 ug/kg, µg/kg, 170 ug/kg, µg/kg, 175 ug/kg, µg/kg, 180 ug/kg, µg/kg, 185 ug/kg, µg/kg, 190 ug/kg, µg/kg, 195 ug/kg, µg/kg, 200
ug/kg, µg/kg, 205 ug/kg, µg/kg, 210 ug/kg, µg/kg, 215 ug/kg, µg/kg, 220 ug/kg, µg/kg, 225 ug/kg, µg/kg, 230 ug/kg, µg/kg, 235 ug/kg, µg/kg, 240
ug/kg, µg/kg, 245 ug/kg, µg/kg, 250 ug/kg, µg/kg, 255 ug/kg, µg/kg, 260 ug/kg, µg/kg, 265 ug/kg, µg/kg, 270 ug/kg, µg/kg, 275 ug/kg, µg/kg, 280
ug/kg, µg/kg, 285 ug/kg, µg/kg, 290 ug/kg, µg/kg, 295 ug/kg, µg/kg, 300 ug/kg, µg/kg, 305 ug/kg, µg/kg, 310 ug/kg, µg/kg, 315 ug/kg, µg/kg, 320
ug/kg, µg/kg, 325 ug/kg, µg/kg, 330 ug/kg, µg/kg, 335 ug/kg, µg/kg, 340 ug/kg, µg/kg, 345 ug/kg, µg/kg, 350 ug/kg, µg/kg, 355 ug/kg, µg/kg, 360
ug/kg, µg/kg, 365 ug/kg, µg/kg, 370 ug/kg, µg/kg, 375 ug/kg, µg/kg, 380 ug/kg, µg/kg, 385 ug/kg, µg/kg, 390 ug/kg, µg/kg, 395 ug/kg, µg/kg, 400
ug/kg, µg/kg, 405 ug/kg, µg/kg, 410 ug/kg, µg/kg, 415 ug/kg, µg/kg, 420 ug/kg, µg/kg, 425 ug/kg, µg/kg, 430 ug/kg, µg/kg, 435 ug/kg, µg/kg, 440
ug/kg, µg/kg, 445 ug/kg, µg/kg, 450 ug/kg, µg/kg, 455 ug/kg, µg/kg, 460 ug/kg, µg/kg, 465 ug/kg, µg/kg, 470 ug/kg, µg/kg, 475 ug/kg, µg/kg, 480
WO wo 2019/113375 PCT/US2018/064335
ug/kg, µg/kg, 485 ug/kg, µg/kg, 490 ug/kg, µg/kg, 495 ug/kg, µg/kg, or 500 ug/kg. µg/kg. In some embodiments, the CXCR4
antagonist (e.g., plerixafor or a pharmaceutically acceptable salt thereof) is administered to
the donor at a dose of from about 200 ug/kg µg/kg to about 300 ug/kg, µg/kg, such as a dose of about 240
ug/kg. µg/kg.
[0055] For example, in some embodiments, the CXCR4 antagonist (e.g., plerixafor or a
pharmaceutically acceptable salt thereof) is administered to the donor at a dose of from about
50 ug/kg µg/kg per day to about 500 ug/kg µg/kg per day, such as a dose of about 50 ug/kg µg/kg per day, 55
ug/kg µg/kg per day, 60 ug/kg µg/kg per day, 65 ug/kg µg/kg per day, 70 ug/kg µg/kg per day, 75 ug/kg µg/kg per day, 80
ug/kg µg/kg per day, 85 ug/kg µg/kg per day, 90 ug/kg µg/kg per day, 95 ug/kg µg/kg per day, 100 ug/kg µg/kg per day, 105
ug/kg µg/kg per day, 110 ug/kg µg/kg per day, 115 ug/kg µg/kg per day, 120 ug/kg µg/kg per day, 125 ug/kg µg/kg per day,
130 ug/kg µg/kg per day, 135 ug/kg µg/kg per day, 140 ug/kg µg/kg per day, 145 ug/kg µg/kg per day, 150 ug/kg µg/kg per
day, 155 ug/kg µg/kg per day, 160 ug/kg µg/kg per day, 165 ug/kg µg/kg per day, 170 ug/kg µg/kg per day, 175 ug/kg µg/kg
per day, 180 ug/kg µg/kg per day, 185 ug/kg µg/kg per day, 190 ug/kg µg/kg per day, 195 ug/kg µg/kg per day, 200
ug/kg µg/kg per day, 205 ug/kg µg/kg per day, 210 ug/kg µg/kg per day, 215 ug/kg µg/kg per day, 220 ug/kg µg/kg per day,
225 ug/kg µg/kg per day, 230 ug/kg µg/kg per day, 235 ug/kg µg/kg per day, 240 ug/kg µg/kg per day, 245 ug/kg µg/kg per
day, 250 ug/kg µg/kg per day, 255 ug/kg µg/kg per day, 260 ug/kg µg/kg per day, 265 ug/kg µg/kg per day, 270 ug/kg µg/kg
per day, 275 ug/kg µg/kg per day, 280 ug/kg µg/kg per day, 285 ug/kg µg/kg per day, 290 ug/kg µg/kg per day, 295
ug/kg µg/kg per day, 300 ug/kg µg/kg per day, 305 ug/kg µg/kg per day, 310 ug/kg µg/kg per day, 315 ug/kg µg/kg per day,
320 ug/kg µg/kg per day, 325 ug/kg µg/kg per day, 330 ug/kg µg/kg per day, 335 ug/kg µg/kg per day, 340 ug/kg µg/kg per
day, 345 ug/kg µg/kg per day, 350 ug/kg µg/kg per day, 355 ug/kg µg/kg per day, 360 ug/kg µg/kg per day, 365 ug/kg µg/kg
per day, 370 ug/kg µg/kg per day, 375 ug/kg µg/kg per day, 380 ug/kg µg/kg per day, 385 ug/kg µg/kg per day, 390
ug/kg µg/kg per day, 395 ug/kg µg/kg per day, 400 ug/kg µg/kg per day, 405 ug/kg µg/kg per day, 410 ug/kg µg/kg per day,
415 ug/kg µg/kg per day, 420 ug/kg µg/kg per day, 425 ug/kg µg/kg per day, 430 ug/kg µg/kg per day, 435 ug/kg µg/kg per
day, 440 ug/kg µg/kg per day, 445 ug/kg µg/kg per day, 450 ug/kg µg/kg per day, 455 ug/kg µg/kg per day, 460 ug/kg µg/kg
per day, 465 ug/kg µg/kg per day, 470 ug/kg µg/kg per day, 475 ug/kg µg/kg per day, 480 ug/kg µg/kg per day, 485
ug/kg µg/kg per day, 490 ug/kg µg/kg per day, 495 ug/kg µg/kg per day, or 500 ug/kg µg/kg per day. In some
embodiments, the CXCR4 antagonist (e.g., plerixafor or a pharmaceutically acceptable salt
thereof) is administered to the donor at a dose of from about 200 ug/kg µg/kg per day to about 300
ug/kg µg/kg per day, such as a dose of about 240 ug/kg µg/kg per day. In some embodiments, the
CXCR4 antagonist may be administered as a single dose. In other embodiments, the CXCR4
antagonist may be administered as two or more doses.
[0056] In some embodiments of any of the above aspects of the invention, the CXCR2
agonist and the CXCR4 antagonist are administered to the donor concurrently. In some
WO wo 2019/113375 PCT/US2018/064335
embodiments, the CXCR4 antagonist is administered to the donor prior to administration of
the CXCR2 agonist. In some embodiments, the CXCR4 antagonist may be administered to
the donor from about 1 minute to about 180 minutes prior to administration of the CXCR2
agonist, such as from about 15 minutes to about 180 minutes, about 30 minutes to about 180
minutes, about 40 minutes to about 160 minutes, about 50 minutes to about 150 minutes,
about 60 minutes to about 140 minutes, about 70 minutes to about 130 minutes, about 60
minutes to about 120 minutes, about 70 minutes to about 110 minutes, or about 80 minutes to
about 100 minutes (e.g., about 30 minutes, about 35 minutes, about 40 minutes, about 45
minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70
minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95
minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, about
120 minutes, about 125 minutes, about 130 minutes, about 135 minutes, about 140 minutes,
about 145 minutes, about 150 minutes, about 155 minutes, about 160 minutes, about 165
minutes, about 170 minutes, about 175 minutes, or about 180 minutes prior to administration
of the CXCR2 agonist). In some embodiments, the CXCR4 antagonist is administered to the
donor from about 30 minutes to about 60 minutes prior to administration of the CXCR2
agonist (e.g., about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about
50 minutes, about 55 minutes, or about 60 minutes prior to administration of the CXCR2
agonist). In some embodiments, the CXCR4 antagonist may be administered to the donor
about 45 minutes prior to administration of the CXCR2 agonist.
[0057] In a further aspect, the invention features a pharmaceutical composition including a
population of hematopoietic stem cells or progeny thereof isolated from a mammalian donor
(e.g., a human donor), wherein the ratio of CD34+ cells to CD34 cells to leukocytes leukocytes in in the the population population is is
from about 0.0008 to about 0.0021. In some embodiments, the ratio of CD34+ cells to CD34 cells to
leukocytes may be about 0.00080, 0.00081, 0.00082, 0.00083, 0.00084, 0.00085, 0.00086,
0.00087, 0.00088, 0.00089, 0.00090, 0.00091, 0.00092, 0.00093, 0.00094, 0.00095, 0.00096,
0.00097, 0.00098, 0.00099, 0.00100, 0.00101, 0.00102, 0.00103, 0.00104, 0.00105, 0.00106,
0.00107, 0.00108, 0.00109, 0.00110, 0.00111, 0.00112, 0.00113, 0.00114, 0.00115, 0.00116,
0.00117, 0.00118, 0.00119, 0.00120, 0.00121, 0.00122, 0.00123, 0.00124, 0.00125, 0.00126,
0.00127, 0.00128, 0.00129, 0.00130, 0.00131, 0.00132, 0.00133, 0.00134, 0.00135, 0.00136,
0.00137, 0.00138, 0.00139, 0.00140, 0.00141, 0.00142, 0.00143, 0.00144, 0.00145, 0.00146,
0.00147, 0.00148, 0.00149, 0.00150, 0.00151, 0.00152, 0.00153, 0.00154, 0.00155, 0.00156,
0.00157, 0.00158, 0.00159, 0.00160, 0.00161, 0.00162, 0.00163, 0.00164, 0.00165, 0.00166,
PCT/US2018/064335
0.00167, 0.00168, 0.00169, 0.00170, 0.00171, 0.00172, 0.00173, 0.00174, 0.00175, 0.00176,
0.00178, 0.00179, 0.00180, 0.00181, 0.00182, 0.00183, 0.00184, 0.00185, 0.00186, 0.00187,
0.00188, 0.00189, 0.00190, 0.00191, 0.00192, 0.00193, 0.00194, 0.00195, 0.00196, 0.00197,
0.00198, 0.00199, 0.00200, 0.00201, 0.00202, 0.00203, 0.00204, 0.00205, 0.00206, 0.00207,
0.00208, 0.00209, 0.00210, 0.00211, 0.00212, 0.00213, 0.00214, 0.00215, 0.00216, 0.00217,
0.00218, 0.00219, 0.00220, 0.00221, 0.00222, 0.00223, 0.00224, or 0.00225. In some
embodiments, the ratio of CD34+ cells to CD34 cells to leukocytes leukocytes is is from from about about 0.0010 0.0010 to to about about 0.0018, 0.0018,
such as a ratio of CD34+ cells to CD34 cells to leukocytes leukocytes of of about about 0.00100, 0.00100, 0.00101, 0.00101, 0.00102, 0.00102, 0.00103, 0.00103,
0.00104, 0.00105, 0.00106, 0.00107, 0.00108, 0.00109, 0.00110, 0.00111, 0.00112, 0.00113,
0.00114, 0.00115, 0.00116, 0.00117, 0.00118, 0.00119, 0.00120, 0.00121, 0.00122, 0.00123,
0.00124, 0.00125, 0.00126, 0.00127, 0.00128, 0.00129, 0.00130, 0.00131, 0.00132, 0.00133,
0.00134, 0.00135, 0.00136, 0.00137, 0.00138, 0.00139, 0.00140, 0.00141, 0.00142, 0.00143,
0.00144, 0.00145, 0.00146, 0.00147, 0.00148, 0.00149, 0.00150, 0.00151, 0.00152, 0.00153,
0.00154, 0.00155, 0.00156, 0.00157, 0.00158, 0.00159, 0.00160, 0.00161, 0.00162, 0.00163,
0.00164, 0.00165, 0.00166, 0.00167, 0.00168, 0.00169, 0.00170, 0.00171, 0.00172, 0.00173,
0.00174, 0.00175, 0.00176, 0.00178, 0.00179, or 0.00180. In some embodiments, the ratio of
CD34+ cells to CD34 cells to leukocytes leukocytes is is about about 0.0014. 0.0014.
[0058] In an additional aspect, the invention features a pharmaceutical composition including
a population of hematopoietic stem cells or progeny thereof isolated from a mammalian
donor donor (e.g., (e.g.,a a human donor), human wherein donor), the ratio wherein of CD34+ the ratio ofcells CD34 to neutrophils cells in the population to neutrophils in the population
is from about 0.0018 to about 0.0058. In some embodiments, the ratio of CD34+ cells to CD34 cells to
neutrophils may be about 0.00180, 0.00181, 0.00182, 0.00183, 0.00184, 0.00185, 0.00186,
0.00187, 0.00188, 0.00189, 0.00190, 0.00191, 0.00192, 0.00193, 0.00194, 0.00195, 0.00196,
0.00197, 0.00198, 0.00199, 0.00200, 0.00201, 0.00202, 0.00203, 0.00204, 0.00205, 0.00206,
0.00207, 0.00208, 0.00209, 0.00210, 0.00211, 0.00212, 0.00213, 0.00214, 0.00215, 0.00216,
0.00217, 0.00218, 0.00219, 0.00220, 0.00221, 0.00222, 0.00223, 0.00224, 0.00225, 0.00226,
0.00227, 0.00228, 0.00229, 0.00230, 0.00231, 0.00232, 0.00233, 0.00234, 0.00235, 0.00236,
0.00237, 0.00238, 0.00239, 0.00240, 0.00241, 0.00242, 0.00243, 0.00244, 0.00245, 0.00246,
0.00247, 0.00248, 0.00249, 0.00250, 0.00251, 0.00252, 0.00253, 0.00254, 0.00255, 0.00256,
0.00257, 0.00258, 0.00259, 0.00260, 0.00261, 0.00262, 0.00263, 0.00264, 0.00265, 0.00266,
0.00267, 0.00268, 0.00269, 0.00270, 0.00271, 0.00272, 0.00273, 0.00274, 0.00275, 0.00276,
0.00277, 0.00278, 0.00279, 0.00280, 0.00281, 0.00282, 0.00283, 0.00284, 0.00285, 0.00286,
0.00287, 0.00288, 0.00289, 0.00290, 0.00291, 0.00292, 0.00293, 0.00294, 0.00295, 0.00296,
WO wo 2019/113375 PCT/US2018/064335
0.00297, 0.00297, 0.00298, 0.00298, 0.00299, 0.00299, 0.00300, 0.00300, 0.00300, 0.00300, 0.00301, 0.00301, 0.00302, 0.00302, 0.00303, 0.00303, 0.00304, 0.00304, 0.00305, 0.00305,
0.00306, 0.00306, 0.00307, 0.00307, 0.00308, 0.00308, 0.00309, 0.00309, 0.00310, 0.00310, 0.00311, 0.00311, 0.00312, 0.00312, 0.00313, 0.00313, 0.00314, 0.00314, 0.00315, 0.00315,
0.00316, 0.00316, 0.00317, 0.00317, 0.00318, 0.00318, 0.00319, 0.00319, 0.00320, 0.00320, 0.00321, 0.00321, 0.00322, 0.00322, 0.00323, 0.00323, 0.00324, 0.00324, 0.00325, 0.00325,
0.00326, 0.00326, 0.00327, 0.00327, 0.00328, 0.00328, 0.00329, 0.00329, 0.00330, 0.00330, 0.00331, 0.00331, 0.00332, 0.00332, 0.00333, 0.00333, 0.00334, 0.00334, 0.00335, 0.00335,
0.00336, 0.00336, 0.00337, 0.00337, 0.00338, 0.00338, 0.00339, 0.00339, 0.00340, 0.00340, 0.00341, 0.00341, 0.00342, 0.00342, 0.00343, 0.00343, 0.00344, 0.00344, 0.00345, 0.00345,
0.00346, 0.00347, 0.00346, 0.00347, 0.00348, 0.00348, 0.00349, 0.00349, 0.00350, 0.00350, 0.00351, 0.00351, 0.00352, 0.00352, 0.00353, 0.00353, 0.00354, 0.00354, 0.00355, 0.00355,
0.00356, 0.00356, 0.00357, 0.00357, 0.00358, 0.00358, 0.00359, 0.00359, 0.00360, 0.00360, 0.00361, 0.00361, 0.00362, 0.00362, 0.00363, 0.00363, 0.00364, 0.00364, 0.00365, 0.00365,
0.00366, 0.00367, 0.00368, 0.00369, 0.00370, 0.00371, 0.00372, 0.00373, 0.00374, 0.00375,
0.00376, 0.00377, 0.00378, 0.00379, 0.00380, 0.00381, 0.00382, 0.00383, 0.00384, 0.00385,
0.00386, 0.00387, 0.00388, 0.00389, 0.00390, 0.00391, 0.00392, 0.00393, 0.00394, 0.00395,
0.00396, 0.00396, 0.00397, 0.00397, 0.00398, 0.00398, 0.00399, 0.00399, 0.00400, 0.00400, 0.00401, 0.00401, 0.00402, 0.00402, 0.00403, 0.00403, 0.00404, 0.00404, 0.00405, 0.00405,
0.00406, 0.00406, 0.00407, 0.00407, 0.00408, 0.00408, 0.00409, 0.00409, 0.00410, 0.00410, 0.00411, 0.00411, 0.00412, 0.00412, 0.00413, 0.00413, 0.00414, 0.00414, 0.00415, 0.00415,
0.00416, 0.00416, 0.00417, 0.00417, 0.00418, 0.00418, 0.00419, 0.00419, 0.00420, 0.00420, 0.00421, 0.00421, 0.00422, 0.00422, 0.00423, 0.00423, 0.00424, 0.00424, 0.00425, 0.00425,
0.00426, 0.00426, 0.00427, 0.00427, 0.00428, 0.00428, 0.00429, 0.00429, 0.00430, 0.00430, 0.00431, 0.00431, 0.00432, 0.00432, 0.00433, 0.00433, 0.00434, 0.00434, 0.00435, 0.00435,
0.00436, 0.00436, 0.00437, 0.00437, 0.00438, 0.00438, 0.00439, 0.00439, 0.00440, 0.00440, 0.00441, 0.00441, 0.00442, 0.00442, 0.00443, 0.00443, 0.00444, 0.00444, 0.00445, 0.00445,
0.00446, 0.00446, 0.00447, 0.00447, 0.00448, 0.00448, 0.00449, 0.00449, 0.00450, 0.00450, 0.00451, 0.00451, 0.00452, 0.00452, 0.00453, 0.00453, 0.00454, 0.00454, 0.00455, 0.00455,
0.00456, 0.00456, 0.00457, 0.00457, 0.00458, 0.00458, 0.00459, 0.00459, 0.00460, 0.00460, 0.00461, 0.00461, 0.00462, 0.00462, 0.00463, 0.00463, 0.00464, 0.00464, 0.00465, 0.00465,
0.00466, 0.00466, 0.00467, 0.00467, 0.00468, 0.00468, 0.00469, 0.00469, 0.00470, 0.00470, 0.00471, 0.00471, 0.00472, 0.00472, 0.00473, 0.00473, 0.00474, 0.00474, 0.00475, 0.00475,
0.00476, 0.00476, 0.00477, 0.00477, 0.00478, 0.00478, 0.00479, 0.00479, 0.00480, 0.00480, 0.00481, 0.00481, 0.00482, 0.00482, 0.00483, 0.00483, 0.00484, 0.00484, 0.00485, 0.00485,
0.00486, 0.00487, 0.00488, 0.00489, 0.00490, 0.00491, 0.00492, 0.00493, 0.00494, 0.00495,
0.00496, 0.00497, 0.00498, 0.00499, 0.00500, 0.00501, 0.00502, 0.00503, 0.00504, 0.00505,
0.00506, 0.00506, 0.00507, 0.00507, 0.00508, 0.00508, 0.00509, 0.00509, 0.00510, 0.00510, 0.00511, 0.00511, 0.00512, 0.00512, 0.00513, 0.00513, 0.00514, 0.00514, 0.00515, 0.00515,
0.00516, 0.00516, 0.00517, 0.00517, 0.00518, 0.00518, 0.00519, 0.00519, 0.00520, 0.00520, 0.00521, 0.00521, 0.00522, 0.00522, 0.00523, 0.00523, 0.00524, 0.00524, 0.00525, 0.00525,
0.00526, 0.00526, 0.00527, 0.00527, 0.00528, 0.00528, 0.00529, 0.00529, 0.00530, 0.00530, 0.00531, 0.00531, 0.00532, 0.00532, 0.00533, 0.00533, 0.00534, 0.00534, 0.00535, 0.00535,
0.00536, 0.00536, 0.00537, 0.00537, 0.00538, 0.00538, 0.00539, 0.00539, 0.00540, 0.00540, 0.00541, 0.00541, 0.00542, 0.00542, 0.00543, 0.00543, 0.00544, 0.00544, 0.00545, 0.00545,
0.00546, 0.00546, 0.00547, 0.00547, 0.00548, 0.00548, 0.00549, 0.00549, 0.00550, 0.00550, 0.00551, 0.00551, 0.00552, 0.00552, 0.00553, 0.00553, 0.00554, 0.00554, 0.00555, 0.00555,
0.00556, 0.00556, 0.00557, 0.00557, 0.00558, 0.00558, 0.00559, 0.00559, 0.00560, 0.00560, 0.00561, 0.00561, 0.00562, 0.00562, 0.00563, 0.00563, 0.00564, 0.00564, 0.00565, 0.00565,
0.00566, 0.00567, 0.00566, 0.00567, 0.00568, 0.00568, 0.00569, 0.00569, 0.00570, 0.00570, 0.00571, 0.00571, 0.00572, 0.00572, 0.00573, 0.00573, 0.00574, 0.00574, 0.00575, 0.00575,
0.00576, 0.00577, 0.00578, 0.00579, or 0.00580. In some embodiments, the ratio of CD34+ CD34
cells to neutrophils is from about 0.0026 to about 0.0046, such as a ratio of CD34+ cells to CD34 cells to
neutrophils neutrophils of of about about 0.00260, 0.00260, 0.00261, 0.00261, 0.00262, 0.00262, 0.00263, 0.00263, 0.00264, 0.00264, 0.00265, 0.00265, 0.00266, 0.00266,
0.00267, 0.00267, 0.00268, 0.00268, 0.00269, 0.00269, 0.00270, 0.00270, 0.00271, 0.00271, 0.00272, 0.00272, 0.00273, 0.00273, 0.00274, 0.00274, 0.00275, 0.00275, 0.00276, 0.00276,
0.00277, 0.00277, 0.00278, 0.00278, 0.00279, 0.00279, 0.00280, 0.00280, 0.00281, 0.00281, 0.00282, 0.00282, 0.00283, 0.00283, 0.00284, 0.00284, 0.00285, 0.00285, 0.00286, 0.00286,
0.00287, 0.00288, 0.00289, 0.00290, 0.00291, 0.00292, 0.00293, 0.00294, 0.00295, 0.00296,
0.00297, 0.00298, 0.00299, 0.00300, 0.00300, 0.00301, 0.00302, 0.00303, 0.00304, 0.00305,
0.00306, 0.00307, 0.00308, 0.00309, 0.00310, 0.00311, 0.00312, 0.00313, 0.00314, 0.00315,
0.00316, 0.00317, 0.00318, 0.00319, 0.00320, 0.00321, 0.00322, 0.00323, 0.00324, 0.00325,
0.00326, 0.00327, 0.00328, 0.00329, 0.00330, 0.00331, 0.00332, 0.00333, 0.00334, 0.00335,
0.00336, 0.00337, 0.00338, 0.00339, 0.00340, 0.00341, 0.00342, 0.00343, 0.00344, 0.00345,
0.00346, 0.00347, 0.00348, 0.00349, 0.00350, 0.00351, 0.00352, 0.00353, 0.00354, 0.00355,
0.00356, 0.00357, 0.00358, 0.00359, 0.00360, 0.00361, 0.00362, 0.00363, 0.00364, 0.00365,
0.00366, 0.00367, 0.00368, 0.00369, 0.00370, 0.00371, 0.00372, 0.00373, 0.00374, 0.00375,
0.00376, 0.00377, 0.00378, 0.00379, 0.00380, 0.00381, 0.00382, 0.00383, 0.00384, 0.00385,
0.00386, 0.00387, 0.00388, 0.00389, 0.00390, 0.00391, 0.00392, 0.00393, 0.00394, 0.00395,
0.00396, 0.00397, 0.00398, 0.00399, 0.00400, 0.00401, 0.00402, 0.00403, 0.00404, 0.00405,
0.00406, 0.00407, 0.00408, 0.00409, 0.00410, 0.00411, 0.00412, 0.00413, 0.00414, 0.00415,
0.00416, 0.00417, 0.00418, 0.00419, 0.00420, 0.00421, 0.00422, 0.00423, 0.00424, 0.00425,
0.00426, 0.00427, 0.00428, 0.00429, 0.00430, 0.00431, 0.00432, 0.00433, 0.00434, 0.00435,
0.00436, 0.00437, 0.00438, 0.00439, 0.00440, 0.00441, 0.00442, 0.00443, 0.00444, 0.00445,
0.00446, 0.00447, 0.00448, 0.00449, 0.00450, 0.00451, 0.00452, 0.00453, 0.00454, 0.00455,
0.00456, 0.00457, 0.00458, 0.00459, or 0.00460. In some embodiments, the ratio of CD34+ CD34
cells to neutrophils is about 0.0036.
[0059] In another aspect, the invention features a pharmaceutical composition including a
population of hematopoietic stem cells or progeny thereof isolated from a mammalian donor
(e.g., (e.g.,a ahuman humandonor), wherein donor), the ratio wherein of CD34+ the ratio of cells CD34 to lymphocytes cells in the population to lymphocytes is in the population is
from about 0.0021 to about 0.0094. In some embodiments, the ratio of CD34+ cells to CD34 cells to
lymphocytes may be about 0.00210, 0.00211, 0.00212, 0.00213, 0.00214, 0.00215, 0.00216,
0.00217, 0.00218, 0.00219, 0.00220, 0.00221, 0.00222, 0.00223, 0.00224, 0.00225, 0.00226,
0.00227, 0.00228, 0.00229, 0.00230, 0.00231, 0.00232, 0.00233, 0.00234, 0.00235, 0.00236,
0.00237, 0.00238, 0.00239, 0.00240, 0.00241, 0.00242, 0.00243, 0.00244, 0.00245, 0.00246,
0.00247, 0.00248, 0.00249, 0.00250, 0.00251, 0.00252, 0.00253, 0.00254, 0.00255, 0.00256,
0.00257, 0.00258, 0.00259, 0.00260, 0.00261, 0.00262, 0.00263, 0.00264, 0.00265, 0.00266,
0.00267, 0.00268, 0.00269, 0.00270, 0.00271, 0.00272, 0.00273, 0.00274, 0.00275, 0.00276,
0.00277, 0.00278, 0.00279, 0.00280, 0.00281, 0.00282, 0.00283, 0.00284, 0.00285, 0.00286,
0.00287, 0.00288, 0.00289, 0.00290, 0.00291, 0.00292, 0.00293, 0.00294, 0.00295, 0.00296,
0.00297, 0.00298, 0.00299, 0.00300, 0.00300, 0.00301, 0.00302, 0.00303, 0.00304, 0.00305,
0.00306, 0.00307, 0.00308, 0.00309, 0.00310, 0.00311, 0.00312, 0.00313, 0.00314, 0.00315,
0.00316, 0.00317, 0.00318, 0.00319, 0.00320, 0.00321, 0.00322, 0.00323, 0.00324, 0.00325,
0.00326, 0.00327, 0.00328, 0.00329, 0.00330, 0.00331, 0.00332, 0.00333, 0.00334, 0.00335,
0.00336, 0.00337, 0.00338, 0.00339, 0.00340, 0.00341, 0.00342, 0.00343, 0.00344, 0.00345,
0.00346, 0.00347, 0.00348, 0.00349, 0.00350, 0.00351, 0.00352, 0.00353, 0.00354, 0.00355,
0.00356, 0.00357, 0.00358, 0.00359, 0.00360, 0.00361, 0.00362, 0.00363, 0.00364, 0.00365,
0.00366, 0.00367, 0.00368, 0.00369, 0.00370, 0.00371, 0.00372, 0.00373, 0.00374, 0.00375,
0.00376, 0.00377, 0.00378, 0.00379, 0.00380, 0.00381, 0.00382, 0.00383, 0.00384, 0.00385,
0.00386, 0.00387, 0.00388, 0.00389, 0.00390, 0.00391, 0.00392, 0.00393, 0.00394, 0.00395,
0.00396, 0.00397, 0.00398, 0.00399, 0.00400, 0.00401, 0.00402, 0.00403, 0.00404, 0.00405,
0.00406, 0.00407, 0.00408, 0.00409, 0.00410, 0.00411, 0.00412, 0.00413, 0.00414, 0.00415,
0.00416, 0.00416, 0.00417, 0.00417, 0.00418, 0.00418, 0.00419, 0.00419, 0.00420, 0.00420, 0.00421, 0.00421, 0.00422, 0.00422, 0.00423, 0.00423, 0.00424, 0.00424, 0.00425, 0.00425,
0.00426, 0.00427, 0.00428, 0.00429, 0.00430, 0.00431, 0.00432, 0.00433, 0.00434, 0.00435,
0.00436, 0.00436, 0.00437, 0.00437, 0.00438, 0.00438, 0.00439, 0.00439, 0.00440, 0.00440, 0.00441, 0.00441, 0.00442, 0.00442, 0.00443, 0.00443, 0.00444, 0.00444, 0.00445, 0.00445,
0.00446, 0.00447, 0.00448, 0.00449, 0.00450, 0.00451, 0.00452, 0.00453, 0.00454, 0.00455,
0.00456, 0.00457, 0.00458, 0.00459, 0.00460, 0.00461, 0.00462, 0.00463, 0.00464, 0.00465,
0.00466, 0.00467, 0.00468, 0.00469, 0.00470, 0.00471, 0.00472, 0.00473, 0.00474, 0.00475,
0.00476, 0.00477, 0.00478, 0.00479, 0.00480, 0.00481, 0.00482, 0.00483, 0.00484, 0.00485,
0.00486, 0.00487, 0.00488, 0.00489, 0.00490, 0.00491, 0.00492, 0.00493, 0.00494, 0.00495,
0.00496, 0.00497, 0.00498, 0.00499, 0.00500, 0.00501, 0.00502, 0.00503, 0.00504, 0.00505,
0.00506, 0.00507, 0.00508, 0.00509, 0.00510, 0.00511, 0.00512, 0.00513, 0.00514, 0.00515,
0.00516, 0.00517, 0.00518, 0.00519, 0.00520, 0.00521, 0.00522, 0.00523, 0.00524, 0.00525,
0.00526, 0.00527, 0.00528, 0.00529, 0.00530, 0.00531, 0.00532, 0.00533, 0.00534, 0.00535,
0.00536, 0.00536, 0.00537, 0.00537, 0.00538, 0.00538, 0.00539, 0.00539, 0.00540, 0.00540, 0.00541, 0.00541, 0.00542, 0.00542, 0.00543, 0.00543, 0.00544, 0.00544, 0.00545, 0.00545,
0.00546, 0.00546, 0.00547, 0.00547, 0.00548, 0.00548, 0.00549, 0.00549, 0.00550, 0.00550, 0.00551, 0.00551, 0.00552, 0.00552, 0.00553, 0.00553, 0.00554, 0.00554, 0.00555, 0.00555,
0.00556, 0.00556, 0.00557, 0.00557, 0.00558, 0.00558, 0.00559, 0.00559, 0.00560, 0.00560, 0.00561, 0.00561, 0.00562, 0.00562, 0.00563, 0.00563, 0.00564, 0.00564, 0.00565, 0.00565,
0.00566, 0.00567, 0.00568, 0.00569, 0.00570, 0.00571, 0.00572, 0.00573, 0.00574, 0.00575,
0.00576, 0.00577, 0.00578, 0.00579, 0.00580, 0.00581, 0.00582, 0.00583, 0.00584, 0.00585,
0.00586, 0.00587, 0.00588, 0.00589, 0.00590, 0.00591, 0.00592, 0.00593, 0.00594, 0.00595,
0.00596, 0.00597, 0.00598, 0.00599, 0.00600, 0.00601, 0.00602, 0.00603, 0.00604, 0.00605,
0.00606, 0.00607, 0.00608, 0.00609, 0.00610, 0.00611, 0.00612, 0.00613, 0.00614, 0.00615,
0.00616, 0.00617, 0.00618, 0.00619, 0.00620, 0.00621, 0.00622, 0.00623, 0.00624, 0.00625,
0.00626, 0.00627, 0.00628, 0.00629, 0.00630, 0.00631, 0.00632, 0.00633, 0.00634, 0.00635,
PCT/US2018/064335
0.00636, 0.00637, 0.00638, 0.00639, 0.00640, 0.00641, 0.00642, 0.00643, 0.00644, 0.00645,
0.00646, 0.00647, 0.00648, 0.00649, 0.00650, 0.00651, 0.00652, 0.00653, 0.00654, 0.00655,
0.00656, 0.00657, 0.00658, 0.00659, 0.00660, 0.00661, 0.00662, 0.00663, 0.00664, 0.00665,
0.00666, 0.00667, 0.00668, 0.00669, 0.00670, 0.00671, 0.00672, 0.00673, 0.00674, 0.00675,
0.00676, 0.00677, 0.00678, 0.00679, 0.00680, 0.00681, 0.00682, 0.00683, 0.00684, 0.00685,
0.00686, 0.00687, 0.00688, 0.00689, 0.00690, 0.00691, 0.00692, 0.00693, 0.00694, 0.00695,
0.00696, 0.00697, 0.00698, 0.00699, 0.00700, 0.00701, 0.00702, 0.00703, 0.00704, 0.00705,
0.00706, 0.00707, 0.00708, 0.00709, 0.00710, 0.00711, 0.00712, 0.00713, 0.00714, 0.00715,
0.00716, 0.00717, 0.00718, 0.00719, 0.00720, 0.00721, 0.00722, 0.00723, 0.00724, 0.00725,
0.00726, 0.00727, 0.00728, 0.00729, 0.00730, 0.00731, 0.00732, 0.00733, 0.00734, 0.00735,
0.00736, 0.00737, 0.00738, 0.00739, 0.00740, 0.00741, 0.00742, 0.00743, 0.00744, 0.00745,
0.00746, 0.00747, 0.00748, 0.00749, 0.00750, 0.00751, 0.00752, 0.00753, 0.00754, 0.00755,
0.00756, 0.00757, 0.00758, 0.00759, 0.00760, 0.00761, 0.00762, 0.00763, 0.00764, 0.00765,
0.00766, 0.00767, 0.00768, 0.00769, 0.00770, 0.00771, 0.00772, 0.00773, 0.00774, 0.00775,
0.00776, 0.00777, 0.00778, 0.00779, 0.00780, 0.00781, 0.00782, 0.00783, 0.00784, 0.00785,
0.00786, 0.00787, 0.00788, 0.00789, 0.00790, 0.00791, 0.00792, 0.00793, 0.00794, 0.00795,
0.00796, 0.00797, 0.00798, 0.00799, 0.00800, 0.00801, 0.00802, 0.00803, 0.00804, 0.00805,
0.00806, 0.00807, 0.00808, 0.00809, 0.00810, 0.00811, 0.00812, 0.00813, 0.00814, 0.00815,
0.00816, 0.00817, 0.00818, 0.00819, 0.00820, 0.00821, 0.00822, 0.00823, 0.00824, 0.00825,
0.00826, 0.00827, 0.00828, 0.00829, 0.00830, 0.00831, 0.00832, 0.00833, 0.00834, 0.00835,
0.00836, 0.00837, 0.00838, 0.00839, 0.00840, 0.00841, 0.00842, 0.00843, 0.00844, 0.00845,
0.00846, 0.00847, 0.00848, 0.00849, 0.00850, 0.00851, 0.00852, 0.00853, 0.00854, 0.00855,
0.00856, 0.00857, 0.00858, 0.00859, 0.00860, 0.00861, 0.00862, 0.00863, 0.00864, 0.00865,
0.00866, 0.00867, 0.00868, 0.00869, 0.00870, 0.00871, 0.00872, 0.00873, 0.00874, 0.00875,
0.00876, 0.00877, 0.00878, 0.00879, 0.00880, 0.00881, 0.00882, 0.00883, 0.00884, 0.00885,
0.00886, 0.00887, 0.00888, 0.00889, 0.00890, 0.00891, 0.00892, 0.00893, 0.00894, 0.00895,
0.00896, 0.00897, 0.00898, 0.00899, 0.00900, 0.00901, 0.00902, 0.00903, 0.00904, 0.00905,
0.00906, 0.00907, 0.00908, 0.00909, 0.00910, 0.00911, 0.00912, 0.00913, 0.00914, 0.00915,
0.00916, 0.00917, 0.00918, 0.00919, 0.00920, 0.00921, 0.00922, 0.00923, 0.00924, 0.00925,
0.00926, 0.00927, 0.00928, 0.00929, 0.00930, 0.00931, 0.00932, 0.00933, 0.00934, 0.00935,
0.00936, 0.00937, 0.00938, 0.00939, or 0.00940. In some embodiments, the ratio of CD34+ CD34
cells cells to tolymphocytes lymphocytesis from aboutabout is from 0.0025 to about 0.0025 to 0.0035, such as asuch about 0.0035, ratioasofa CD34+ ratiocells to of CD34 cells to
lymphocytes of about 0.00250, 0.00251, 0.00252, 0.00253, 0.00254, 0.00255, 0.00256,
0.00257, 0.00258, 0.00259, 0.00260, 0.00261, 0.00262, 0.00263, 0.00264, 0.00265, 0.00266,
0.00267, 0.00268, 0.00269, 0.00270, 0.00271, 0.00272, 0.00273, 0.00274, 0.00275, 0.00276,
0.00277, 0.00278, 0.00279, 0.00280, 0.00281, 0.00282, 0.00283, 0.00284, 0.00285, 0.00286,
0.00287, 0.00288, 0.00289, 0.00290, 0.00291, 0.00292, 0.00293, 0.00294, 0.00295, 0.00296,
0.00297, 0.00298, 0.00299, 0.00300, 0.00300, 0.00301, 0.00302, 0.00303, 0.00304, 0.00305,
0.00306, 0.00307, 0.00308, 0.00309, 0.00310, 0.00311, 0.00312, 0.00313, 0.00314, 0.00315,
0.00316, 0.00317, 0.00318, 0.00319, 0.00320, 0.00321, 0.00322, 0.00323, 0.00324, 0.00325,
0.00326, 0.00327, 0.00328, 0.00329, 0.00330, 0.00331, 0.00332, 0.00333, 0.00334, 0.00335,
0.00336, 0.00337, 0.00338, 0.00339, 0.00340, 0.00341, 0.00342, 0.00343, 0.00344, 0.00345,
0.00346, 0.00347, 0.00348, 0.00349, or 0.00350. In some embodiments, the ratio of CD34+ CD34
cells to cells tolymphocytes lymphocytesis is about 0.0031. about 0.0031.
[0060] In a further aspect, the invention features a pharmaceutical composition including a
population of hematopoietic stem cells or progeny thereof isolated from a mammalian donor
(e.g., a human donor), wherein the ratio of CD34+ cells to CD34 cells to monocytes monocytes in in the the population population is is
from about 0.0071 to about 0.0174. In some embodiments, the ratio of CD34+ cells to CD34 cells to
monocytes may be about 0.00710, 0.00711, 0.00712, 0.00713, 0.00714, 0.00715, 0.00716,
0.00717, 0.00718, 0.00719, 0.00720, 0.00721, 0.00722, 0.00723, 0.00724, 0.00725, 0.00726,
0.00727, 0.00728, 0.00729, 0.00730, 0.00731, 0.00732, 0.00733, 0.00734, 0.00735, 0.00736,
0.00737, 0.00738, 0.00739, 0.00740, 0.00741, 0.00742, 0.00743, 0.00744, 0.00745, 0.00746,
0.00747, 0.00748, 0.00749, 0.00750, 0.00751, 0.00752, 0.00753, 0.00754, 0.00755, 0.00756,
0.00757, 0.00758, 0.00759, 0.00760, 0.00761, 0.00762, 0.00763, 0.00764, 0.00765, 0.00766,
0.00767, 0.00768, 0.00769, 0.00770, 0.00771, 0.00772, 0.00773, 0.00774, 0.00775, 0.00776,
0.00777, 0.00778, 0.00779, 0.00780, 0.00781, 0.00782, 0.00783, 0.00784, 0.00785, 0.00786,
0.00787, 0.00788, 0.00789, 0.00790, 0.00791, 0.00792, 0.00793, 0.00794, 0.00795, 0.00796,
0.00797, 0.00798, 0.00799, 0.00800, 0.00801, 0.00802, 0.00803, 0.00804, 0.00805, 0.00806,
0.00807, 0.00808, 0.00809, 0.00810, 0.00811, 0.00812, 0.00813, 0.00814, 0.00815, 0.00816,
0.00817, 0.00818, 0.00819, 0.00820, 0.00821, 0.00822, 0.00823, 0.00824, 0.00825, 0.00826,
0.00827, 0.00828, 0.00829, 0.00830, 0.00831, 0.00832, 0.00833, 0.00834, 0.00835, 0.00836,
0.00837, 0.00838, 0.00839, 0.00840, 0.00841, 0.00842, 0.00843, 0.00844, 0.00845, 0.00846,
0.00847, 0.00848, 0.00849, 0.00850, 0.00851, 0.00852, 0.00853, 0.00854, 0.00855, 0.00856,
0.00857, 0.00858, 0.00859, 0.00860, 0.00861, 0.00862, 0.00863, 0.00864, 0.00865, 0.00866,
0.00867, 0.00868, 0.00869, 0.00870, 0.00871, 0.00872, 0.00873, 0.00874, 0.00875, 0.00876,
0.00877, 0.00878, 0.00879, 0.00880, 0.00881, 0.00882, 0.00883, 0.00884, 0.00885, 0.00886,
WO wo 2019/113375 PCT/US2018/064335
0.00887, 0.00888, 0.00889, 0.00890, 0.00891, 0.00892, 0.00893, 0.00894, 0.00895, 0.00896,
0.00897, 0.00898, 0.00899, 0.00900, 0.00901, 0.00902, 0.00903, 0.00904, 0.00905, 0.00906,
0.00907, 0.00908, 0.00909, 0.00910, 0.00911, 0.00912, 0.00913, 0.00914, 0.00915, 0.00916,
0.00917, 0.00918, 0.00919, 0.00920, 0.00921, 0.00922, 0.00923, 0.00924, 0.00925, 0.00926,
0.00927, 0.00928, 0.00929, 0.00930, 0.00931, 0.00932, 0.00933, 0.00934, 0.00935, 0.00936,
0.00937, 0.00938, 0.00939, 0.00940, 0.00941, 0.00942, 0.00943, 0.00944, 0.00945, 0.00946,
0.00947, 0.00948, 0.00949, 0.00950, 0.00951, 0.00952, 0.00953, 0.00954, 0.00955, 0.00956,
0.00957, 0.00958, 0.00959, 0.00960, 0.00961, 0.00962, 0.00963, 0.00964, 0.00965, 0.00966,
0.00967, 0.00968, 0.00969, 0.00970, 0.00971, 0.00972, 0.00973, 0.00974, 0.00975, 0.00976,
0.00977, 0.00978, 0.00979, 0.00980, 0.00981, 0.00982, 0.00983, 0.00984, 0.00985, 0.00986,
0.00987, 0.00988, 0.00989, 0.00990, 0.00991, 0.00992, 0.00993, 0.00994, 0.00995, 0.00996,
0.00997, 0.00998, 0.00999, 0.0100, 0.0101, 0.0103, 0.0104, 0.0105, 0.0106, 0.0107, 0.0108,
0.0109, 0.0110, 0.0111, 0.0112, 0.0113, 0.0114, 0.0115, 0.0116, 0.0117, 0.0118, 0.0119,
0.0120, 0.0121, 0.0122, 0.0123, 0.0124, 0.0125, 0.0126, 0.0127, 0.0128, 0.0129, 0.0130,
0.0131, 0.0132, 0.0133, 0.0134, 0.0135, 0.0136, 0.0137, 0.0138, 0.0139, 0.0140, 0.0141,
0.0142, 0.0143, 0.0144, 0.0145, 0.0146, 0.0147, 0.0148, 0.0149, 0.0150, 0.0151, 0.0152,
0.0153, 0.0154, 0.0155, 0.0156, 0.0157, 0.0158, 0.0159, 0.0160, 0.0161, 0.0162, 0.0163,
0.0164, 0.0165, 0.0166, 0.0167, 0.0168, 0.0169, 0.0170, 0.0171, 0.0172, 0.0173, or 0.0174.
CD34 cells In some embodiments, the ratio of CD34+ cellsto tomonocytes monocytesis isfrom fromabout about0.0100 0.0100to toabout about
0.0140, such as a ratio of CD34+ cellsto CD34 cells tomonocytes monocytesof ofabout about0.0100, 0.0100,0.0101, 0.0101,0.0103, 0.0103,0.0104, 0.0104,
0.0105, 0.0106, 0.0107, 0.0108, 0.0109, 0.0110, 0.0111, 0.0112, 0.0113, 0.0114, 0.0115,
0.0116, 0.0117, 0.0118, 0.0119, 0.0120, 0.0121, 0.0122, 0.0123, 0.0124, 0.0125, 0.0126,
0.0127, 0.0128, 0.0129, 0.0130, 0.0131, 0.0132, 0.0133, 0.0134, 0.0135, 0.0136, 0.0137,
0.0138, 0.0139, or 0.0140. In some embodiments, the ratio of CD34+ cells to CD34 cells to monocytes monocytes is is
about 0.0118.
[0061] In a further aspect, the invention features a pharmaceutical composition including a
population of hematopoietic stem cells or progeny thereof isolated from a mammalian donor
(e.g., a human donor), wherein the frequency of CD34+ cellsin CD34 cells inthe thepopulation populationis isfrom fromabout about
0.051% to about 0.140%. In some embodiments, the population of cells may have a
frequency of CD34 CD34+cells cellsof ofabout about0.051%, 0.051%,0.052%, 0.052%,0.053%, 0.053%,0.054%, 0.054%,0.055%, 0.055%,0.056%, 0.056%,
0.057%, 0.058%, 0.059%, 0.060%, 0.061%, 0.062%, 0.063%, 0.064%, 0.065%, 0.066%,
0.067%, 0.068%, 0.069%, 0.070%, 0.071%, 0.072%, 0.073%, 0.074%, 0.075%, 0.076%,
0.077%, 0.078%, 0.079%, 0.080%, 0.081%, 0.082%, 0.083%, 0.084%, 0.085%, 0.086%,
WO wo 2019/113375 PCT/US2018/064335
0.087%, 0.088%, 0.089%, 0.090%, 0.091%, 0.092%, 0.093%, 0.094%, 0.095%, 0.096%,
0.097%, 0.098%, 0.099%, 0.100%, 0.101%, 0.102%, 0.103%, 0.104%, 0.105%, 0.106%,
0.107%, 0.108%, 0.109%, 0.110%, 0.111%, 0.112%, 0.113%, 0.114%, 0.115%, 0.116%,
0.117%, 0.118%, 0.119%, 0.120%, 0.121%, 0.122%, 0.123%, 0.124%, 0.125%, 0.126%,
0.127%, 0.128%, 0.129%, 0.130%, 0.131%, 0.132%, 0.133%, 0.134%, 0.135%, 0.136%,
0.137%, 0.138%, 0.139%, or 0.140%. In some embodiments, the population of cells has a
frequency of CD34+ cells of CD34 cells of from from about about 0.080% 0.080% to to about about 0.120%, 0.120%, such such as as aa frequency frequency of of
CD34+ cells of CD34 cells of about about 0.080%, 0.080%, 0.081%, 0.081%, 0.082%, 0.082%, 0.083%, 0.083%, 0.084%, 0.084%, 0.085%, 0.085%, 0.086%, 0.086%, 0.087%, 0.087%,
0.088%, 0.089%, 0.090%, 0.091%, 0.092%, 0.093%, 0.094%, 0.095%, 0.096%, 0.097%,
0.098%, 0.099%, 0.100%, 0.101%, 0.102%, 0.103%, 0.104%, 0.105%, 0.106%, 0.107%,
0.108%, 0.109%, 0.110%, 0.111%, 0.112%, 0.113%, 0.114%, 0.115%, 0.116%, 0.117%,
0.118%, 0.119%, or 0.120%. In some embodiments, the population of cells has a frequency
of CD34+ cells of CD34 cells of about about 0.097%. 0.097%.
[0062] In a further aspect, the invention features a pharmaceutical composition including a
population of hematopoietic stem cells or progeny thereof isolated from a mammalian donor
(e.g., a human donor), wherein the ratio of CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells toto leukocytes leukocytes inin the the
population is from about 0.0003 to about 0.0016. In some embodiments, the ratio of CD34+ CD34
CD90 CD45RA cells to leukocytes may be about 0.00030, 0.00031, 0.00032, 0.00033,
0.00034, 0.00035, 0.00036, 0.00037, 0.00038, 0.00039, 0.00040, 0.00041, 0.00042, 0.00043,
0.00044, 0.00045, 0.00046, 0.00047, 0.00048, 0.00049, 0.00050, 0.00051, 0.00052, 0.00053,
0.00054, 0.00055, 0.00056, 0.00057, 0.00058, 0.00059, 0.00060, 0.00061, 0.00062, 0.00063,
0.00064, 0.00065, 0.00066, 0.00067, 0.00068, 0.00069, 0.00070, 0.00071, 0.00072, 0.00073,
0.00074, 0.00075, 0.00076, 0.00077, 0.00078, 0.00079, 0.00080, 0.00081, 0.00082, 0.00083,
0.00084, 0.00085, 0.00086, 0.00087, 0.00088, 0.00089, 0.00090, 0.00091, 0.00092, 0.00093,
0.00094, 0.00095, 0.00096, 0.00097, 0.00098, 0.00099, 0.00100, 0.00101, 0.00102, 0.00103,
0.00104, 0.00105, 0.00106, 0.00107, 0.00108, 0.00109, 0.00110, 0.00111, 0.00112, 0.00113,
0.00114, 0.00115, 0.00116, 0.00117, 0.00118, 0.00119, 0.00120, 0.00121, 0.00122, 0.00123,
0.00124, 0.00125, 0.00126, 0.00127, 0.00128, 0.00129, 0.00130, 0.00131, 0.00132, 0.00133,
0.00134, 0.00135, 0.00136, 0.00137, 0.00138, 0.00139, 0.00140, 0.00141, 0.00142, 0.00143,
0.00144, 0.00145, 0.00146, 0.00147, 0.00148, 0.00149, 0.00150, 0.00151, 0.00152, 0.00153,
0.00154, 0.00155, 0.00156, 0.00157, 0.00158, 0.00159, or 0.00160. In some embodiments,
the ratio of CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells toto leukocytes leukocytes isis from from about about 0.0006 0.0006 toto about about
0.0012, such as a ratio of CD34 CD90 CD45RA cells to leukocytes of about 0.00060,
0.00061, 0.00062, 0.00063, 0.00064, 0.00065, 0.00066, 0.00067, 0.00068, 0.00069, 0.00070,
0.00071, 0.00072, 0.00073, 0.00074, 0.00075, 0.00076, 0.00077, 0.00078, 0.00079, 0.00080,
0.00081, 0.00082, 0.00083, 0.00084, 0.00085, 0.00086, 0.00087, 0.00088, 0.00089, 0.00090,
0.00091, 0.00092, 0.00093, 0.00094, 0.00095, 0.00096, 0.00097, 0.00098, 0.00099, 0.00100,
0.00101, 0.00102, 0.00103, 0.00104, 0.00105, 0.00106, 0.00107, 0.00108, 0.00109, 0.00110,
0.00111, 0.00112, 0.00113, 0.00114, 0.00115, 0.00116, 0.00117, 0.00118, 0.00119, or
0.00120. In some embodiments, the ratio of CD34 CD90+ CD45RA cells CD90 CD45RA cells to to leukocytes leukocytes is is
about 0.0009.
[0063] In an additional aspect, the invention features a pharmaceutical composition including
a population of hematopoietic stem cells or progeny thereof isolated from a mammalian
donor (e.g., a human donor), wherein the ratio of CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells toto
neutrophils in the population is from about 0.0007 to about 0.0043. In some embodiments,
the ratio of CD34+ CD90 CD45RA CD34 CD90 CD45RA cells cells to to neutrophils neutrophils may may be be about about 0.00070, 0.00070, 0.00071, 0.00071,
0.00072, 0.00073, 0.00074, 0.00075, 0.00076, 0.00077, 0.00078, 0.00079, 0.00080, 0.00081,
0.00082, 0.00083, 0.00084, 0.00085, 0.00086, 0.00087, 0.00088, 0.00089, 0.00090, 0.00091,
0.00092, 0.00093, 0.00094, 0.00095, 0.00096, 0.00097, 0.00098, 0.00099, 0.00100, 0.00101,
0.00102, 0.00103, 0.00104, 0.00105, 0.00106, 0.00107, 0.00108, 0.00109, 0.00110, 0.00111,
0.00112, 0.00113, 0.00114, 0.00115, 0.00116, 0.00117, 0.00118, 0.00119, 0.00120, 0.00121,
0.00122, 0.00123, 0.00124, 0.00125, 0.00126, 0.00127, 0.00128, 0.00129, 0.00130, 0.00131,
0.00132, 0.00133, 0.00134, 0.00135, 0.00136, 0.00137, 0.00138, 0.00139, 0.00140, 0.00141,
0.00142, 0.00143, 0.00144, 0.00145, 0.00146, 0.00147, 0.00148, 0.00149, 0.00150, 0.00151,
0.00152, 0.00153, 0.00154, 0.00155, 0.00156, 0.00157, 0.00158, 0.00159, 0.00160, 0.00161,
0.00162, 0.00163, 0.00164, 0.00165, 0.00166, 0.00167, 0.00168, 0.00169, 0.00170, 0.00171,
0.00172, 0.00173, 0.00174, 0.00175, 0.00176, 0.00177, 0.00178, 0.00179, 0.00180, 0.00181,
0.00182, 0.00183, 0.00184, 0.00185, 0.00186, 0.00187, 0.00188, 0.00189, 0.00190, 0.00191,
0.00192, 0.00193, 0.00194, 0.00195, 0.00196, 0.00197, 0.00198, 0.00199, 0.00200, 0.00201,
0.00202, 0.00203, 0.00204, 0.00205, 0.00206, 0.00207, 0.00208, 0.00209, 0.00210, 0.00211,
0.00212, 0.00213, 0.00214, 0.00215, 0.00216, 0.00217, 0.00218, 0.00219, 0.00220, 0.00221,
0.00222, 0.00223, 0.00224, 0.00225, 0.00226, 0.00227, 0.00228, 0.00229, 0.00230, 0.00231,
0.00232, 0.00233, 0.00234, 0.00235, 0.00236, 0.00237, 0.00238, 0.00239, 0.00240, 0.00241,
0.00242, 0.00243, 0.00244, 0.00245, 0.00246, 0.00247, 0.00248, 0.00249, 0.00250, 0.00251,
0.00252, 0.00253, 0.00254, 0.00255, 0.00256, 0.00257, 0.00258, 0.00259, 0.00260, 0.00261,
0.00262, 0.00263, 0.00264, 0.00265, 0.00266, 0.00267, 0.00268, 0.00269, 0.00270, 0.00271,
WO wo 2019/113375 PCT/US2018/064335 PCT/US2018/064335
0.00272, 0.00273, 0.00274, 0.00275, 0.00276, 0.00277, 0.00278, 0.00279, 0.00280, 0.00281,
0.00282, 0.00283, 0.00284, 0.00285, 0.00286, 0.00287, 0.00288, 0.00289, 0.00290, 0.00291,
0.00292, 0.00293, 0.00294, 0.00295, 0.00296, 0.00297, 0.00298, 0.00299, 0.00300, 0.00300,
0.00301, 0.00302, 0.00303, 0.00304, 0.00305, 0.00306, 0.00307, 0.00308, 0.00309, 0.00310,
0.00311, 0.00312, 0.00313, 0.00314, 0.00315, 0.00316, 0.00317, 0.00318, 0.00319, 0.00320,
0.00321, 0.00322, 0.00323, 0.00324, 0.00325, 0.00326, 0.00327, 0.00328, 0.00329, 0.00330,
0.00331, 0.00332, 0.00333, 0.00334, 0.00335, 0.00336, 0.00337, 0.00338, 0.00339, 0.00340,
0.00341, 0.00342, 0.00343, 0.00344, 0.00345, 0.00346, 0.00347, 0.00348, 0.00349, 0.00350,
0.00351, 0.00352, 0.00353, 0.00354, 0.00355, 0.00356, 0.00357, 0.00358, 0.00359, 0.00360,
0.00361, 0.00362, 0.00363, 0.00364, 0.00365, 0.00366, 0.00367, 0.00368, 0.00369, 0.00370,
0.00371, 0.00372, 0.00373, 0.00374, 0.00375, 0.00376, 0.00377, 0.00378, 0.00379, 0.00380,
0.00381, 0.00381, 0.00382, 0.00382, 0.00383, 0.00383, 0.00384, 0.00384, 0.00385, 0.00385, 0.00386, 0.00386, 0.00387, 0.00387, 0.00388, 0.00388, 0.00389, 0.00389, 0.00390, 0.00390,
0.00391, 0.00392, 0.00393, 0.00394, 0.00395, 0.00396, 0.00397, 0.00398, 0.00399, 0.00400,
0.00401, 0.00402, 0.00403, 0.00404, 0.00405, 0.00406, 0.00407, 0.00408, 0.00409, 0.00410,
0.00411, 0.00412, 0.00413, 0.00414, 0.00415, 0.00416, 0.00417, 0.00418, 0.00419, 0.00420,
0.00421, 0.00422, 0.00423, 0.00424, 0.00425, 0.00426, 0.00427, 0.00428, 0.00429, or
0.00430. In some embodiments, the ratio of CD34+ CD90CD45RA CD34 CD90 CD45RAcells cellsto toneutrophils neutrophilsis is
from about 0.0014 to about 0.0034, such as a ratio of CD34+ CD90 CD45RA CD34 CD90 CD45RA cells cells to to
neutrophils of about 0.00140, 0.00141, 0.00142, 0.00143, 0.00144, 0.00145, 0.00146,
0.00147, 0.00148, 0.00149, 0.00150, 0.00151, 0.00152, 0.00153, 0.00154, 0.00155, 0.00156,
0.00157, 0.00158, 0.00159, 0.00160, 0.00161, 0.00162, 0.00163, 0.00164, 0.00165, 0.00166,
0.00167, 0.00168, 0.00169, 0.00170, 0.00171, 0.00172, 0.00173, 0.00174, 0.00175, 0.00176,
0.00177, 0.00178, 0.00179, 0.00180, 0.00181, 0.00182, 0.00183, 0.00184, 0.00185, 0.00186,
0.00187, 0.00188, 0.00189, 0.00190, 0.00191, 0.00192, 0.00193, 0.00194, 0.00195, 0.00196,
0.00197, 0.00198, 0.00199, 0.00200, 0.00201, 0.00202, 0.00203, 0.00204, 0.00205, 0.00206,
0.00207, 0.00208, 0.00209, 0.00210, 0.00211, 0.00212, 0.00213, 0.00214, 0.00215, 0.00216,
0.00217, 0.00218, 0.00219, 0.00220, 0.00221, 0.00222, 0.00223, 0.00224, 0.00225, 0.00226,
0.00227, 0.00228, 0.00229, 0.00230, 0.00231, 0.00232, 0.00233, 0.00234, 0.00235, 0.00236,
0.00237, 0.00238, 0.00239, 0.00240, 0.00241, 0.00242, 0.00243, 0.00244, 0.00245, 0.00246,
0.00247, 0.00248, 0.00249, 0.00250, 0.00251, 0.00252, 0.00253, 0.00254, 0.00255, 0.00256,
0.00257, 0.00258, 0.00259, 0.00260, 0.00261, 0.00262, 0.00263, 0.00264, 0.00265, 0.00266,
0.00267, 0.00268, 0.00269, 0.00270, 0.00271, 0.00272, 0.00273, 0.00274, 0.00275, 0.00276,
0.00277, 0.00278, 0.00279, 0.00280, 0.00281, 0.00282, 0.00283, 0.00284, 0.00285, 0.00286,
PCT/US2018/064335
0.00287, 0.00288, 0.00289, 0.00290, 0.00291, 0.00292, 0.00293, 0.00294, 0.00295, 0.00296,
0.00297, 0.00298, 0.00299, 0.00300, 0.00300, 0.00301, 0.00302, 0.00303, 0.00304, 0.00305,
0.00306, 0.00307, 0.00308, 0.00309, 0.00310, 0.00311, 0.00312, 0.00313, 0.00314, 0.00315,
0.00316, 0.00317, 0.00318, 0.00319, 0.00320, 0.00321, 0.00322, 0.00323, 0.00324, 0.00325,
0.00326, 0.00327, 0.00328, 0.00329, 0.00330, 0.00331, 0.00332, 0.00333, 0.00334, 0.00335,
0.00336, 0.00337, 0.00338, 0.00339, or 0.00340. In some embodiments, the ratio of CD34+ CD34
CD90 CD45RA cells to neutrophils is about 0.0024.
[0064] In another aspect, the invention features a pharmaceutical composition including a
population of hematopoietic stem cells or progeny thereof isolated from a mammalian donor
(e.g., a human donor), wherein the ratio of CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells toto lymphocytes lymphocytes inin
the population is from about 0.0008 to about 0.0069. In some embodiments, the ratio of
CD34+ CD90+ CD45RA CD34 CD90 CD45RA cells cells to tolymphocytes lymphocytesmaymay be about 0.00080, be about 0.00081, 0.00080, 0.00082, 0.00081, 0.00082,
0.00083, 0.00084, 0.00085, 0.00086, 0.00087, 0.00088, 0.00089, 0.00090, 0.00091, 0.00092,
0.00093, 0.00094, 0.00095, 0.00096, 0.00097, 0.00098, 0.00099, 0.00100, 0.00101, 0.00102,
0.00103, 0.00104, 0.00105, 0.00106, 0.00107, 0.00108, 0.00109, 0.00110, 0.00111, 0.00112,
0.00113, 0.00114, 0.00115, 0.00116, 0.00117, 0.00118, 0.00119, 0.00120, 0.00121, 0.00122,
0.00123, 0.00124, 0.00125, 0.00126, 0.00127, 0.00128, 0.00129, 0.00130, 0.00131, 0.00132,
0.00133, 0.00134, 0.00135, 0.00136, 0.00137, 0.00138, 0.00139, 0.00140, 0.00141, 0.00142,
0.00143, 0.00144, 0.00145, 0.00146, 0.00147, 0.00148, 0.00149, 0.00150, 0.00151, 0.00152,
0.00153, 0.00154, 0.00155, 0.00156, 0.00157, 0.00158, 0.00159, 0.00160, 0.00161, 0.00162,
0.00163, 0.00164, 0.00165, 0.00166, 0.00167, 0.00168, 0.00169, 0.00170, 0.00171, 0.00172,
0.00173, 0.00174, 0.00175, 0.00176, 0.00178, 0.00179, 0.00180, 0.00181, 0.00182, 0.00183,
0.00184, 0.00185, 0.00186, 0.00187, 0.00188, 0.00189, 0.00190, 0.00191, 0.00192, 0.00193,
0.00194, 0.00195, 0.00196, 0.00197, 0.00198, 0.00199, 0.00200, 0.00201, 0.00202, 0.00203,
0.00204, 0.00205, 0.00206, 0.00207, 0.00208, 0.00209, 0.00210, 0.00211, 0.00212, 0.00213,
0.00214, 0.00215, 0.00216, 0.00217, 0.00218, 0.00219, 0.00220, 0.00221, 0.00222, 0.00223,
0.00224, 0.00225, 0.00226, 0.00227, 0.00228, 0.00229, 0.00230, 0.00231, 0.00232, 0.00233,
0.00234, 0.00235, 0.00236, 0.00237, 0.00238, 0.00239, 0.00240, 0.00241, 0.00242, 0.00243,
0.00244, 0.00245, 0.00246, 0.00247, 0.00248, 0.00249, 0.00250, 0.00251, 0.00252, 0.00253,
0.00254, 0.00255, 0.00256, 0.00257, 0.00258, 0.00259, 0.00260, 0.00261, 0.00262, 0.00263,
0.00264, 0.00265, 0.00266, 0.00267, 0.00268, 0.00269, 0.00270, 0.00271, 0.00272, 0.00273,
0.00274, 0.00275, 0.00276, 0.00278, 0.00279, 0.00280, 0.00281, 0.00282, 0.00283, 0.00284,
0.00285, 0.00286, 0.00287, 0.00288, 0.00289, 0.00290, 0.00291, 0.00292, 0.00293, 0.00294,
0.00295, 0.00296, 0.00297, 0.00298, 0.00299, 0.00300, 0.00301, 0.00302, 0.00303, 0.00304,
0.00305, 0.00306, 0.00307, 0.00308, 0.00309, 0.00310, 0.00311, 0.00312, 0.00313, 0.00314,
0.00315, 0.00316, 0.00317, 0.00318, 0.00319, 0.00320, 0.00321, 0.00322, 0.00323, 0.00324,
0.00325, 0.00326, 0.00327, 0.00328, 0.00329, 0.00330, 0.00331, 0.00332, 0.00333, 0.00334,
0.00335, 0.00336, 0.00337, 0.00338, 0.00339, 0.00340, 0.00341, 0.00342, 0.00343, 0.00344,
0.00345, 0.00346, 0.00347, 0.00348, 0.00349, 0.00350, 0.00351, 0.00352, 0.00353, 0.00354,
0.00355, 0.00356, 0.00357, 0.00358, 0.00359, 0.00360, 0.00361, 0.00362, 0.00363, 0.00364,
0.00365, 0.00366, 0.00367, 0.00368, 0.00369, 0.00370, 0.00371, 0.00372, 0.00373, 0.00374,
0.00375, 0.00376, 0.00378, 0.00379, 0.00380, 0.00381, 0.00382, 0.00383, 0.00384, 0.00385,
0.00386, 0.00387, 0.00388, 0.00389, 0.00390, 0.00391, 0.00392, 0.00393, 0.00394, 0.00395,
0.00396, 0.00397, 0.00398, 0.00399, 0.00401, 0.00402, 0.00403, 0.00404, 0.00405, 0.00406,
0.00407, 0.00408, 0.00409, 0.00410, 0.00411, 0.00412, 0.00413, 0.00414, 0.00415, 0.00416,
0.00417, 0.00418, 0.00419, 0.00420, 0.00421, 0.00422, 0.00423, 0.00424, 0.00425, 0.00426,
0.00427, 0.00428, 0.00429, 0.00430, 0.00431, 0.00432, 0.00433, 0.00434, 0.00435, 0.00436,
0.00437, 0.00438, 0.00439, 0.00440, 0.00441, 0.00442, 0.00443, 0.00444, 0.00445, 0.00446,
0.00447, 0.00448, 0.00449, 0.00450, 0.00451, 0.00452, 0.00453, 0.00454, 0.00455, 0.00456,
0.00457, 0.00458, 0.00459, 0.00460, 0.00461, 0.00462, 0.00463, 0.00464, 0.00465, 0.00466,
0.00467, 0.00468, 0.00469, 0.00470, 0.00471, 0.00472, 0.00473, 0.00474, 0.00475, 0.00476,
0.00478, 0.00479, 0.00480, 0.00481, 0.00482, 0.00483, 0.00484, 0.00485, 0.00486, 0.00487,
0.00488, 0.00489, 0.00490, 0.00491, 0.00492, 0.00493, 0.00494, 0.00495, 0.00496, 0.00497,
0.00498, 0.00499, 0.00500, 0.00501, 0.00502, 0.00503, 0.00504, 0.00505, 0.00506, 0.00507,
0.00508, 0.00509, 0.00510, 0.00511, 0.00512, 0.00513, 0.00514, 0.00515, 0.00516, 0.00517,
0.00518, 0.00519, 0.00520, 0.00521, 0.00522, 0.00523, 0.00524, 0.00525, 0.00526, 0.00527,
0.00528, 0.00529, 0.00530, 0.00531, 0.00532, 0.00533, 0.00534, 0.00535, 0.00536, 0.00537,
0.00538, 0.00538, 0.00539, 0.00539, 0.00540, 0.00540, 0.00541, 0.00541, 0.00542, 0.00542, 0.00543, 0.00543, 0.00544, 0.00544, 0.00545, 0.00545, 0.00546, 0.00546, 0.00547, 0.00547,
0.00548, 0.00549, 0.00550, 0.00551, 0.00552, 0.00553, 0.00554, 0.00555, 0.00556, 0.00557,
0.00558, 0.00559, 0.00560, 0.00561, 0.00562, 0.00563, 0.00564, 0.00565, 0.00566, 0.00567,
0.00568, 0.00569, 0.00570, 0.00571, 0.00572, 0.00573, 0.00574, 0.00575, 0.00576, 0.00578,
0.00579, 0.00580, 0.00581, 0.00582, 0.00583, 0.00584, 0.00585, 0.00586, 0.00587, 0.00588,
0.00589, 0.00590, 0.00591, 0.00592, 0.00593, 0.00594, 0.00595, 0.00596, 0.00597, 0.00598,
0.00599, 0.00600, 0.00601, 0.00602, 0.00603, 0.00604, 0.00605, 0.00606, 0.00607, 0.00608,
0.00609, 0.00610, 0.00611, 0.00612, 0.00613, 0.00614, 0.00615, 0.00616, 0.00617, 0.00618,
0.00619, 0.00620, 0.00621, 0.00622, 0.00623, 0.00624, 0.00625, 0.00626, 0.00627, 0.00628,
WO wo 2019/113375 PCT/US2018/064335
0.00629, 0.00630, 0.00631, 0.00632, 0.00633, 0.00634, 0.00635, 0.00636, 0.00637, 0.00638,
0.00639, 0.00640, 0.00641, 0.00642, 0.00643, 0.00644, 0.00645, 0.00646, 0.00647, 0.00648,
0.00649, 0.00650, 0.00651, 0.00652, 0.00653, 0.00654, 0.00655, 0.00656, 0.00657, 0.00658,
0.00659, 0.00660, 0.00661, 0.00662, 0.00663, 0.00664, 0.00665, 0.00666, 0.00667, 0.00668,
0.00669, 0.00670, 0.00671, 0.00672, 0.00673, 0.00674, 0.00675, 0.00676, 0.00678, 0.00679,
0.00680, 0.00681, 0.00682, 0.00683, 0.00684, 0.00685, 0.00686, 0.00687, 0.00688, 0.00689,
or or 0.00690. 0.00690.InInsome embodiments, some the ratio embodiments, of CD34+ the ratio CD90+ CD90 of CD34 CD45RA cells cells CD45RA to lymphocytes to lymphocytes
is from about 0.0011 to about 0.0031, such as a ratio of CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells toto
lymphocytes of about 0.00110, 0.00111, 0.00112, 0.00113, 0.00114, 0.00115, 0.00116,
0.00117, 0.00118, 0.00119, 0.00120, 0.00121, 0.00122, 0.00123, 0.00124, 0.00125, 0.00126,
0.00127, 0.00128, 0.00129, 0.00130, 0.00131, 0.00132, 0.00133, 0.00134, 0.00135, 0.00136,
0.00137, 0.00138, 0.00139, 0.00140, 0.00141, 0.00142, 0.00143, 0.00144, 0.00145, 0.00146,
0.00147, 0.00148, 0.00149, 0.00150, 0.00151, 0.00152, 0.00153, 0.00154, 0.00155, 0.00156,
0.00157, 0.00158, 0.00159, 0.00160, 0.00161, 0.00162, 0.00163, 0.00164, 0.00165, 0.00166,
0.00167, 0.00168, 0.00169, 0.00170, 0.00171, 0.00172, 0.00173, 0.00174, 0.00175, 0.00176,
0.00178, 0.00179, 0.00180, 0.00181, 0.00182, 0.00183, 0.00184, 0.00185, 0.00186, 0.00187,
0.00188, 0.00189, 0.00190, 0.00191, 0.00192, 0.00193, 0.00194, 0.00195, 0.00196, 0.00197,
0.00198, 0.00199, 0.00200, 0.00201, 0.00202, 0.00203, 0.00204, 0.00205, 0.00206, 0.00207,
0.00208, 0.00209, 0.00210, 0.00211, 0.00212, 0.00213, 0.00214, 0.00215, 0.00216, 0.00217,
0.00218, 0.00219, 0.00220, 0.00221, 0.00222, 0.00223, 0.00224, 0.00225, 0.00226, 0.00227,
0.00228, 0.00229, 0.00230, 0.00231, 0.00232, 0.00233, 0.00234, 0.00235, 0.00236, 0.00237,
0.00238, 0.00239, 0.00240, 0.00241, 0.00242, 0.00243, 0.00244, 0.00245, 0.00246, 0.00247,
0.00248, 0.00249, 0.00250, 0.00251, 0.00252, 0.00253, 0.00254, 0.00255, 0.00256, 0.00257,
0.00258, 0.00259, 0.00260, 0.00261, 0.00262, 0.00263, 0.00264, 0.00265, 0.00266, 0.00267,
0.00268, 0.00269, 0.00270, 0.00271, 0.00272, 0.00273, 0.00274, 0.00275, 0.00276, 0.00278,
0.00279, 0.00280, 0.00281, 0.00282, 0.00283, 0.00284, 0.00285, 0.00286, 0.00287, 0.00288,
0.00289, 0.00290, 0.00291, 0.00292, 0.00293, 0.00294, 0.00295, 0.00296, 0.00297, 0.00298,
0.00299, 0.00300, 0.00301, 0.00302, 0.00303, 0.00304, 0.00305, 0.00306, 0.00307, 0.00308,
0.00309, or 0.00310. In some embodiments, the ratio of CD34+ CD90+ CD34 CD90 CD45RA-cells CD45RA'- cells to
lymphocytes is about 0.0021.
[0065] In a further aspect, the invention features a pharmaceutical composition including a
population of hematopoietic stem cells or progeny thereof isolated from a mammalian donor
(e.g., a human donor), wherein the ratio of CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells toto monocytes monocytes inin the the
WO wo 2019/113375 PCT/US2018/064335
population is from about 0.0028 to about 0.0130. In some embodiments, the ratio of CD34+ CD34
CD90 CD45RA cells to monocytes may be about 0.00280, 0.00281, 0.00282, 0.00283,
0.00284, 0.00285, 0.00286, 0.00287, 0.00288, 0.00289, 0.00290, 0.00291, 0.00292, 0.00293,
0.00294, 0.00295, 0.00296, 0.00297, 0.00298, 0.00299, 0.00300, 0.00301, 0.00302, 0.00303,
0.00304, 0.00305, 0.00306, 0.00307, 0.00308, 0.00309, 0.00310, 0.00311, 0.00312, 0.00313,
0.00314, 0.00315, 0.00316, 0.00317, 0.00318, 0.00319, 0.00320, 0.00321, 0.00322, 0.00323,
0.00324, 0.00325, 0.00326, 0.00327, 0.00328, 0.00329, 0.00330, 0.00331, 0.00332, 0.00333,
0.00334, 0.00335, 0.00336, 0.00337, 0.00338, 0.00339, 0.00340, 0.00341, 0.00342, 0.00343,
0.00344, 0.00345, 0.00346, 0.00347, 0.00348, 0.00349, 0.00350, 0.00351, 0.00352, 0.00353,
0.00354, 0.00355, 0.00356, 0.00357, 0.00358, 0.00359, 0.00360, 0.00361, 0.00362, 0.00363,
0.00364, 0.00365, 0.00366, 0.00367, 0.00368, 0.00369, 0.00370, 0.00371, 0.00372, 0.00373,
0.00374, 0.00375, 0.00376, 0.00378, 0.00379, 0.00380, 0.00381, 0.00382, 0.00383, 0.00384,
0.00385, 0.00386, 0.00387, 0.00388, 0.00389, 0.00390, 0.00391, 0.00392, 0.00393, 0.00394,
0.00395, 0.00396, 0.00397, 0.00398, 0.00399, 0.00401, 0.00402, 0.00403, 0.00404, 0.00405,
0.00406, 0.00407, 0.00408, 0.00409, 0.00410, 0.00411, 0.00412, 0.00413, 0.00414, 0.00415,
0.00416, 0.00417, 0.00418, 0.00419, 0.00420, 0.00421, 0.00422, 0.00423, 0.00424, 0.00425,
0.00426, 0.00427, 0.00428, 0.00429, 0.00430, 0.00431, 0.00432, 0.00433, 0.00434, 0.00435,
0.00436, 0.00437, 0.00438, 0.00439, 0.00440, 0.00441, 0.00442, 0.00443, 0.00444, 0.00445,
0.00446, 0.00447, 0.00448, 0.00449, 0.00450, 0.00451, 0.00452, 0.00453, 0.00454, 0.00455,
0.00456, 0.00457, 0.00458, 0.00459, 0.00460, 0.00461, 0.00462, 0.00463, 0.00464, 0.00465,
0.00466, 0.00467, 0.00468, 0.00469, 0.00470, 0.00471, 0.00472, 0.00473, 0.00474, 0.00475,
0.00476, 0.00478, 0.00479, 0.00480, 0.00481, 0.00482, 0.00483, 0.00484, 0.00485, 0.00486,
0.00487, 0.00488, 0.00489, 0.00490, 0.00491, 0.00492, 0.00493, 0.00494, 0.00495, 0.00496,
0.00497, 0.00498, 0.00499, 0.00500, 0.00501, 0.00502, 0.00503, 0.00504, 0.00505, 0.00506,
0.00507, 0.00508, 0.00509, 0.00510, 0.00511, 0.00512, 0.00513, 0.00514, 0.00515, 0.00516,
0.00517, 0.00518, 0.00519, 0.00520, 0.00521, 0.00522, 0.00523, 0.00524, 0.00525, 0.00526,
0.00527, 0.00528, 0.00529, 0.00530, 0.00531, 0.00532, 0.00533, 0.00534, 0.00535, 0.00536,
0.00537, 0.00538, 0.00539, 0.00540, 0.00541, 0.00542, 0.00543, 0.00544, 0.00545, 0.00546,
0.00547, 0.00548, 0.00549, 0.00550, 0.00551, 0.00552, 0.00553, 0.00554, 0.00555, 0.00556,
0.00557, 0.00558, 0.00559, 0.00560, 0.00561, 0.00562, 0.00563, 0.00564, 0.00565, 0.00566,
0.00567, 0.00568, 0.00569, 0.00570, 0.00571, 0.00572, 0.00573, 0.00574, 0.00575, 0.00576,
0.00578, 0.00579, 0.00580, 0.00581, 0.00582, 0.00583, 0.00584, 0.00585, 0.00586, 0.00587,
0.00588, 0.00589, 0.00590, 0.00591, 0.00592, 0.00593, 0.00594, 0.00595, 0.00596, 0.00597,
WO wo 2019/113375 PCT/US2018/064335 PCT/US2018/064335
0.00598, 0.00599, 0.00600, 0.00601, 0.00602, 0.00603, 0.00604, 0.00605, 0.00606, 0.00607,
0.00608, 0.00609, 0.00610, 0.00611, 0.00612, 0.00613, 0.00614, 0.00615, 0.00616, 0.00617,
0.00618, 0.00619, 0.00620, 0.00621, 0.00622, 0.00623, 0.00624, 0.00625, 0.00626, 0.00627,
0.00628, 0.00629, 0.00630, 0.00631, 0.00632, 0.00633, 0.00634, 0.00635, 0.00636, 0.00637,
0.00638, 0.00639, 0.00640, 0.00641, 0.00642, 0.00643, 0.00644, 0.00645, 0.00646, 0.00647,
0.00648, 0.00649, 0.00650, 0.00651, 0.00652, 0.00653, 0.00654, 0.00655, 0.00656, 0.00657,
0.00658, 0.00659, 0.00660, 0.00661, 0.00662, 0.00663, 0.00664, 0.00665, 0.00666, 0.00667,
0.00668, 0.00669, 0.00670, 0.00671, 0.00672, 0.00673, 0.00674, 0.00675, 0.00676, 0.00678,
0.00679, 0.00680, 0.00681, 0.00682, 0.00683, 0.00684, 0.00685, 0.00686, 0.00687, 0.00688,
0.00689, 0.00690, 0.00691, 0.00692, 0.00693, 0.00694, 0.00695, 0.00696, 0.00697, 0.00698,
0.00699, 0.00700, 0.00701, 0.00702, 0.00703, 0.00704, 0.00705, 0.00706, 0.00707, 0.00708,
0.00709, 0.00710, 0.00711, 0.00712, 0.00713, 0.00714, 0.00715, 0.00716, 0.00717, 0.00718,
0.00719, 0.00720, 0.00721, 0.00722, 0.00723, 0.00724, 0.00725, 0.00726, 0.00727, 0.00728,
0.00729, 0.00729, 0.00730, 0.00730, 0.00731, 0.00731, 0.00732, 0.00732, 0.00733, 0.00733, 0.00734, 0.00734, 0.00735, 0.00735, 0.00736, 0.00736, 0.00737, 0.00737, 0.00738, 0.00738,
0.00739, 0.00740, 0.00741, 0.00742, 0.00743, 0.00744, 0.00745, 0.00746, 0.00747, 0.00748,
0.00749, 0.00750, 0.00751, 0.00752, 0.00753, 0.00754, 0.00755, 0.00756, 0.00757, 0.00758,
0.00759, 0.00760, 0.00761, 0.00762, 0.00763, 0.00764, 0.00765, 0.00766, 0.00767, 0.00768,
0.00769, 0.00770, 0.00771, 0.00772, 0.00773, 0.00774, 0.00775, 0.00776, 0.00777, 0.00778,
0.00779, 0.00780, 0.00781, 0.00782, 0.00783, 0.00784, 0.00785, 0.00786, 0.00787, 0.00788,
0.00789, 0.00790, 0.00791, 0.00792, 0.00793, 0.00794, 0.00795, 0.00796, 0.00797, 0.00798,
0.00799, 0.00800, 0.00801, 0.00802, 0.00803, 0.00804, 0.00805, 0.00806, 0.00807, 0.00808,
0.00809, 0.00810, 0.00811, 0.00812, 0.00813, 0.00814, 0.00815, 0.00816, 0.00817, 0.00818,
0.00819, 0.00820, 0.00821, 0.00822, 0.00823, 0.00824, 0.00825, 0.00826, 0.00827, 0.00828,
0.00829, 0.00830, 0.00831, 0.00832, 0.00833, 0.00834, 0.00835, 0.00836, 0.00837, 0.00838,
0.00839, 0.00839, 0.00840, 0.00840, 0.00841, 0.00841, 0.00842, 0.00842, 0.00843, 0.00843, 0.00844, 0.00844, 0.00845, 0.00845, 0.00846, 0.00846, 0.00847, 0.00847, 0.00848, 0.00848,
0.00849, 0.00850, 0.00851, 0.00852, 0.00853, 0.00854, 0.00855, 0.00856, 0.00857, 0.00858,
0.00859, 0.00860, 0.00861, 0.00862, 0.00863, 0.00864, 0.00865, 0.00866, 0.00867, 0.00868,
0.00869, 0.00870, 0.00871, 0.00872, 0.00873, 0.00874, 0.00875, 0.00876, 0.00877, 0.00878,
0.00879, 0.00880, 0.00881, 0.00882, 0.00883, 0.00884, 0.00885, 0.00886, 0.00887, 0.00888,
0.00889, 0.00890, 0.00891, 0.00892, 0.00893, 0.00894, 0.00895, 0.00896, 0.00897, 0.00898,
0.00899, 0.00900, 0.00901, 0.00902, 0.00903, 0.00904, 0.00905, 0.00906, 0.00907, 0.00908,
0.00909, 0.00910, 0.00911, 0.00912, 0.00913, 0.00914, 0.00915, 0.00916, 0.00917, 0.00918,
0.00919, 0.00920, 0.00921, 0.00922, 0.00923, 0.00924, 0.00925, 0.00926, 0.00927, 0.00928,
WO wo 2019/113375 PCT/US2018/064335 PCT/US2018/064335
0.00929, 0.00930, 0.00931, 0.00932, 0.00933, 0.00934, 0.00935, 0.00936, 0.00937, 0.00938,
0.00939, 0.00940, 0.00941, 0.00942, 0.00943, 0.00944, 0.00945, 0.00946, 0.00947, 0.00948,
0.00949, 0.00950, 0.00951, 0.00952, 0.00953, 0.00954, 0.00955, 0.00956, 0.00957, 0.00958,
0.00959, 0.00960, 0.00961, 0.00962, 0.00963, 0.00964, 0.00965, 0.00966, 0.00967, 0.00968,
0.00969, 0.00970, 0.00971, 0.00972, 0.00973, 0.00974, 0.00975, 0.00976, 0.00977, 0.00978,
0.00979, 0.00980, 0.00981, 0.00982, 0.00983, 0.00984, 0.00985, 0.00986, 0.00987, 0.00988,
0.00989, 0.00990, 0.00991, 0.00992, 0.00993, 0.00994, 0.00995, 0.00996, 0.00997, 0.00998,
0.00999, 0.0100, 0.0101, 0.0103, 0.0104, 0.0105, 0.0106, 0.0107, 0.0108, 0.0109, 0.0110,
0.0111, 0.0112, 0.0113, 0.0114, 0.0115, 0.0116, 0.0117, 0.0118, 0.0119, 0.0120, 0.0121,
0.0122, 0.0122, 0.0123, 0.0123, 0.0124, 0.0124, 0.0125, 0.0125, 0.0126, 0.0126, 0.0127, 0.0127, 0.0128, 0.0128, 0.0129, 0.0129, or or 0.0130. 0.0130. In In some some
embodiments, the ratio of CD34+ CD90 CD45RA CD34 CD90 CD45RA cells cells to to monocytes monocytes is is from from about about 0.0063 0.0063
to about 0.0083, such as a ratio of CD34+ CD90 CD45RA CD34 CD90 CD45RA cells cells to to monocytes monocytes of of about about
0.00630, 0.00631, 0.00632, 0.00633, 0.00634, 0.00635, 0.00636, 0.00637, 0.00638, 0.00639,
0.00640, 0.00641, 0.00642, 0.00643, 0.00644, 0.00645, 0.00646, 0.00647, 0.00648, 0.00649,
0.00650, 0.00651, 0.00652, 0.00653, 0.00654, 0.00655, 0.00656, 0.00657, 0.00658, 0.00659,
0.00660, 0.00661, 0.00662, 0.00663, 0.00664, 0.00665, 0.00666, 0.00667, 0.00668, 0.00669,
0.00670, 0.00671, 0.00672, 0.00673, 0.00674, 0.00675, 0.00676, 0.00678, 0.00679, 0.00680,
0.00681, 0.00682, 0.00683, 0.00684, 0.00685, 0.00686, 0.00687, 0.00688, 0.00689, 0.00690,
0.00691, 0.00692, 0.00693, 0.00694, 0.00695, 0.00696, 0.00697, 0.00698, 0.00699, 0.00700,
0.00701, 0.00702, 0.00703, 0.00704, 0.00705, 0.00706, 0.00707, 0.00708, 0.00709, 0.00710,
0.00711, 0.00712, 0.00713, 0.00714, 0.00715, 0.00716, 0.00717, 0.00718, 0.00719, 0.00720,
0.00721, 0.00722, 0.00723, 0.00724, 0.00725, 0.00726, 0.00727, 0.00728, 0.00729, 0.00730,
0.00731, 0.00732, 0.00733, 0.00734, 0.00735, 0.00736, 0.00737, 0.00738, 0.00739, 0.00740,
0.00741, 0.00742, 0.00743, 0.00744, 0.00745, 0.00746, 0.00747, 0.00748, 0.00749, 0.00750,
0.00751, 0.00752, 0.00753, 0.00754, 0.00755, 0.00756, 0.00757, 0.00758, 0.00759, 0.00760,
0.00761, 0.00762, 0.00763, 0.00764, 0.00765, 0.00766, 0.00767, 0.00768, 0.00769, 0.00770,
0.00771, 0.00772, 0.00773, 0.00774, 0.00775, 0.00776, 0.00777, 0.00778, 0.00779, 0.00780,
0.00781, 0.00782, 0.00783, 0.00784, 0.00785, 0.00786, 0.00787, 0.00788, 0.00789, 0.00790,
0.00791, 0.00792, 0.00793, 0.00794, 0.00795, 0.00796, 0.00797, 0.00798, 0.00799, 0.00800,
0.00801, 0.00802, 0.00803, 0.00804, 0.00805, 0.00806, 0.00807, 0.00808, 0.00809, 0.00810,
0.00811, 0.00812, 0.00813, 0.00814, 0.00815, 0.00816, 0.00817, 0.00818, 0.00819, 0.00820,
0.00821, 0.00822, 0.00823, 0.00824, 0.00825, 0.00826, 0.00827, 0.00828, 0.00829, or
WO wo 2019/113375 PCT/US2018/064335
0.00830. 0.00830.InInsome embodiments, some the the embodiments, ratioratio of CD34+ CD90+CD90 of CD34 CD45RA cells cells CD45RA to monocytes is to monocytes is
about 0.0073.
[0066] In a further aspect, the invention features a pharmaceutical composition including a
population of hematopoietic stem cells or progeny thereof isolated from a mammalian donor
(e.g., a human donor), wherein the ratio of CD34+ CD90 CD45RA CD34 CD90 CD45RA cells cells to to CD34 CD34+ cells cells inin
CD34 the population is from about 0.393 to about 0.745. In some embodiments, the ratio of CD34+
CD90+ CD45RAcells CD90 CD45RA cellsto toCD34 CD34+ cells cells may may bebe about about 0.393, 0.393, 0.394, 0.394, 0.395, 0.395, 0.396, 0.396, 0.397, 0.397, 0.398, 0.398,
0.399, 0.401, 0.402, 0.403, 0.404, 0.405, 0.406, 0.407, 0.408, 0.409, 0.410, 0.411, 0.412,
0.413, 0.414, 0.415, 0.416, 0.417, 0.418, 0.419, 0.420, 0.421, 0.422, 0.423, 0.424, 0.425,
0.426, 0.427, 0.428, 0.429, 0.430, 0.431, 0.432, 0.433, 0.434, 0.435, 0.436, 0.437, 0.438,
0.439, 0.440, 0.441, 0.442, 0.443, 0.444, 0.445, 0.446, 0.447, 0.448, 0.449, 0.450, 0.451,
0.452, 0.453, 0.454, 0.455, 0.456, 0.457, 0.458, 0.459, 0.460, 0.461, 0.462, 0.463, 0.464,
0.465, 0.466, 0.467, 0.468, 0.469, 0.470, 0.471, 0.472, 0.473, 0.474, 0.475, 0.476, 0.478,
0.479, 0.480, 0.481, 0.482, 0.483, 0.484, 0.485, 0.486, 0.487, 0.488, 0.489, 0.490, 0.491,
0.492, 0.493, 0.494, 0.495, 0.496, 0.497, 0.498, 0.499, 0.500, 0.501, 0.502, 0.503, 0.504,
0.505, 0.506, 0.507, 0.508, 0.509, 0.510, 0.511, 0.512, 0.513, 0.514, 0.515, 0.516, 0.517,
0.518, 0.519, 0.520, 0.521, 0.522, 0.523, 0.524, 0.525, 0.526, 0.527, 0.528, 0.529, 0.530,
0.531, 0.532, 0.533, 0.534, 0.535, 0.536, 0.537, 0.538, 0.539, 0.540, 0.541, 0.542, 0.543,
0.544, 0.545, 0.546, 0.547, 0.548, 0.549, 0.550, 0.551, 0.552, 0.553, 0.554, 0.555, 0.556,
0.557, 0.558, 0.559, 0.560, 0.561, 0.562, 0.563, 0.564, 0.565, 0.566, 0.567, 0.568, 0.569,
0.570, 0.571, 0.572, 0.573, 0.574, 0.575, 0.576, 0.578, 0.579, 0.580, 0.581, 0.582, 0.583,
0.584, 0.585, 0.586, 0.587, 0.588, 0.589, 0.590, 0.591, 0.592, 0.593, 0.594, 0.595, 0.596,
0.597, 0.598, 0.599, 0.600, 0.601, 0.602, 0.603, 0.604, 0.605, 0.606, 0.607, 0.608, 0.609,
0.610, 0.611, 0.612, 0.613, 0.614, 0.615, 0.616, 0.617, 0.618, 0.619, 0.620, 0.621, 0.622,
0.623, 0.624, 0.625, 0.626, 0.627, 0.628, 0.629, 0.630, 0.631, 0.632, 0.633, 0.634, 0.635,
0.636, 0.637, 0.638, 0.639, 0.640, 0.641, 0.642, 0.643, 0.644, 0.645, 0.646, 0.647, 0.648,
0.649, 0.650, 0.651, 0.652, 0.653, 0.654, 0.655, 0.656, 0.657, 0.658, 0.659, 0.660, 0.661,
0.662, 0.663, 0.664, 0.665, 0.666, 0.667, 0.668, 0.669, 0.670, 0.671, 0.672, 0.673, 0.674,
0.675, 0.676, 0.678, 0.679, 0.680, 0.681, 0.682, 0.683, 0.684, 0.685, 0.686, 0.687, 0.688,
0.689, 0.690, 0.691, 0.692, 0.693, 0.694, 0.695, 0.696, 0.697, 0.698, 0.699, 0.700, 0.701,
0.702, 0.703, 0.704, 0.705, 0.706, 0.707, 0.708, 0.709, 0.710, 0.711, 0.712, 0.713, 0.714,
0.715, 0.716, 0.717, 0.718, 0.719, 0.720, 0.721, 0.722, 0.723, 0.724, 0.725, 0.726, 0.727,
0.728, 0.729, 0.730, 0.731, 0.732, 0.733, 0.734, 0.735, 0.736, 0.737, 0.738, 0.739, 0.740,
WO wo 2019/113375 PCT/US2018/064335
CD34+CD90 0.741, 0.742, 0.743, 0.744, or 0.745. In some embodiments, the ratio of CD34 CD90+
CD45RA cells to CD34+ cells is CD34 cells is from from about about 0.625 0.625 to to about about 0.725, 0.725, such such as as aa ratio ratio of of CD34 CD34+
CD90+ CD45RAcells CD90 CD45RA cellsto toCD34 CD34+ cells cells ofof about about 0.625, 0.625, 0.626, 0.626, 0.627, 0.627, 0.628, 0.628, 0.629, 0.629, 0.630, 0.630,
0.631, 0.632, 0.633, 0.634, 0.635, 0.636, 0.637, 0.638, 0.639, 0.640, 0.641, 0.642, 0.643,
0.644, 0.645, 0.646, 0.647, 0.648, 0.649, 0.650, 0.651, 0.652, 0.653, 0.654, 0.655, 0.656,
0.657, 0.658, 0.659, 0.660, 0.661, 0.662, 0.663, 0.664, 0.665, 0.666, 0.667, 0.668, 0.669,
0.670, 0.671, 0.672, 0.673, 0.674, 0.675, 0.676, 0.678, 0.679, 0.680, 0.681, 0.682, 0.683,
0.684, 0.685, 0.686, 0.687, 0.688, 0.689, 0.690, 0.691, 0.692, 0.693, 0.694, 0.695, 0.696,
0.697, 0.698, 0.699, 0.700, 0.701, 0.702, 0.703, 0.704, 0.705, 0.706, 0.707, 0.708, 0.709,
0.710, 0.711, 0.712, 0.713, 0.714, 0.715, 0.716, 0.717, 0.718, 0.719, 0.720, 0.721, 0.722,
0.723, 0.724, or 0.725. In some embodiments, the ratio of CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells toto
CD34+ cells is CD34 cells is about about 0.676. 0.676.
[0067] In a further aspect, the invention features a pharmaceutical composition including a
population of hematopoietic stem cells or progeny thereof isolated from a mammalian donor
(e.g., a human donor), wherein the frequency of CD34+ CD90 CD45RA CD34 CD90 CD45RA cells cells in in the the
population is from about 0.020% to about 0.110%. In some embodiments, the population of
cells cells may mayhave havea a frequency of CD34+ frequency CD90+ of CD34 CD45RA CD90 cells CD45RA of about cells 0.020%, of about 0.021%,0.021%, 0.020%,
0.022%, 0.023%, 0.024%, 0.025%, 0.026%, 0.027%, 0.028%, 0.029%, 0.030%, 0.031%,
0.032%, 0.033%, 0.034%, 0.035%, 0.036%, 0.037%, 0.038%, 0.039%, 0.040%, 0.041%,
0.042%, 0.043%,0.044%, 0.042%, 0.043%, 0.044%, 0.045%, 0.045%, 0.046%, 0.046%, 0.047%, 0.047%, 0.048%, 0.048%, 0.049%,0.049%, 0.050%, 0.050%, 0.051%, 0.051%,
0.052%, 0.053%, 0.054%, 0.055%, 0.056%, 0.057%, 0.058%, 0.059%, 0.060%, 0.061%,
0.062%, 0.063%, 0.064%, 0.065%, 0.066%, 0.067%, 0.068%, 0.069%, 0.070%, 0.071%,
0.072%, 0.073%, 0.074%, 0.075%, 0.076%, 0.077%, 0.078%, 0.079%, 0.080%, 0.081%,
0.082%, 0.083%, 0.084%, 0.085%, 0.086%, 0.087%, 0.088%, 0.089%, 0.090%, 0.091%,
0.092%, 0.093%, 0.094%, 0.095%, 0.096%, 0.097%, 0.098%, 0.099%, 0.100%, 0.101%,
0.102%, 0.103%, 0.104%, 0.105%, 0.106%, 0.107%, 0.108%, 0.109%, or 0.110%. In some
embodiments, the population of cells has a frequency of CD34+ CD90CD45RA CD34 CD90 CD45RA-cells cellsof of
from about 0.046% to about 0.086%, such as a frequency of hematopoietic stem cells of
about 0.046%, 0.047%, 0.048%, 0.049%, 0.050%, 0.051%, 0.052%, 0.053%, 0.054%,
0.055%, 0.056%, 0.057%, 0.058%, 0.059%, 0.060%, 0.061%, 0.062%, 0.063%, 0.064%,
0.065%, 0.066%, 0.067%, 0.068%, 0.069%, 0.070%, 0.071%, 0.072%, 0.073%, 0.074%,
0.075%, 0.076%, 0.077%, 0.078%, 0.079%, 0.080%, 0.081%, 0.082%, 0.083%, 0.084%,
WO wo 2019/113375 PCT/US2018/064335
CD34 0.085%, or 0.086%. In some embodiments, the population of cells has a frequency of CD34+
CD90 CD45RA CD90+ CD45RAcells cellsof ofabout about0.066%. 0.066%.
[0068] In another aspect, the invention features a method of treating a stem cell disorder in a
mammalian patient (e.g., a human patient), the method including mobilizing a population of
hematopoietic stem cells in a mammalian donor (e.g., a human donor) in accordance with any
of the above-described methods, and infusing a therapeutically effective amount of the
hematopoietic stem cells, or progeny thereof, into the patient.
[0069] In a further aspect, the invention features a method of treating a stem cell disorder in a
mammalian patient mammalian patient(e.g., a human (e.g., patient), a human the method patient), including the method infusing into including the patient infusing a into the patient a
therapeutically effective amount of the hematopoietic stem cells mobilized by any of the
above-described methods, or progeny thereof.
[0070] In another aspect, the invention features a method of treating a stem cell disorder in a
mammalian patient (e.g., a human patient), the method including administering to the patient
any one or more of the above-described pharmaceutical compositions.
[0071] In some embodiments of any of the three preceding aspects, the stem cell disorder is a
hemoglobinopathy disorder, such as sickle cell anemia, thalassemia, Fanconi anemia, aplastic
anemia, and Wiskott-Aldrich syndrome. In some embodiments, the stem cell disorder is a
myelodysplastic disorder. The stem cell disorder may be an immunodeficiency disorder,
such as a congenital immunodeficiency or an acquired immunodeficiency, for example,
human immunodeficiency virus or acquired immune deficiency syndrome. In some
embodiments, the stem cell disorder is a metabolic disorder, such as a metabolic disorder
selected from glycogen storage diseases, mucopolysaccharidoses, Gaucher's Disease, Hurlers
Disease, sphingolipidoses, and metachromatic leukodystrophy.
[0072] In some embodiments, the stem cell disorder is cancer. The cancer may be, for
example, leukemia, lymphoma, multiple myeloma, and neuroblastoma. In some
embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is
acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic
lymphoid leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's
lymphoma.
[0073] In some embodiments, the stem cell disorder is a disorder selected from the group
consisting of adenosine deaminase deficiency and severe combined immunodeficiency, hyper
immunoglobulin M syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis,
WO wo 2019/113375 PCT/US2018/064335
osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, systemic
sclerosis, systemic lupus erythematosus, multiple sclerosis, and juvenile rheumatoid arthritis.
[0074] In some embodiments, the stem cell disorder is an autoimmune disorder, such as an
autoimmune disorder selected from multiple sclerosis, human systemic lupus, rheumatoid
arthritis, inflammatory bowel disease, treating psoriasis, Type 1 diabetes mellitus, acute
disseminated encephalomyelitis, Addison's disease, alopecia universalis, ankylosing
spondylitis, antiphospholipid antibody syndrome, aplastic anemia, autoimmune hemolytic
anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune
lymphoproliferative syndrome, autoimmune oophoritis, Balo disease, Behcet's disease,
bullous pemphigoid, cardiomyopathy, Chagas' disease, chronic fatigue immune dysfunction
syndrome, chronic inflammatory demyelinating polyneuropathy, Crohn's disease, cicatricial
pemphigoid, coeliac sprue-dermatitis herpetiformis, cold agglutinin disease, CREST
syndrome, Degos disease, discoid lupus, dysautonomia, endometriosis, essential mixed
cryoglobulinemia, fibromyalgia-fibromyositis, Goodpasture's Goodpasture' Ssyndrome, syndrome,Grave's Grave'sdisease, disease,
S thyroiditis, Guillain-Barre syndrome, Hashimoto' thyroiditis, Hidradenitis Hidradenitis suppurativa, suppurativa, idiopathic idiopathic
and/or acute thrombocytopenic purpura, idiopathic pulmonary fibrosis, IgA neuropathy,
interstitial cystitis, juvenile arthritis, Kawasaki's disease, lichen planus, Lyme disease,
Meniere disease, mixed connective tissue disease, myasthenia gravis, neuromyotonia,
opsoclonus myoclonus syndrome, optic neuritis, Ord's thyroiditis, pemphigus vulgaris,
pernicious anemia, polychondritis, polymyositis and dermatomyositis, primary biliary
cirrhosis, polyarteritis nodosa, polyglandular syndromes, polymyalgia rheumatica, primary
agammaglobulinemia, Raynaud phenomenon, Reiter' S syndrome, rheumatic fever,
sarcoidosis, scleroderma, Sjögren's syndrome, stiff person syndrome, Takayasu's arteritis,
temporal arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, vulvodynia, and Wegener's
granulomatosis.
[0075] In some embodiments, the hematopoietic stem cells are autologous with respect to the
patient. In some embodiments, the hematopoietic stem cells are allogeneic with respect to the
patient, and may be, for example, HLA-matched with respect to the patient.
[0076] In some embodiments, the hematopoietic stem cells have been genetically modified to
disrupt an endogenous gene, such as a gene encoding a major histocompatibility complex
protein. The hematopoietic stem cells may be genetically modified to disrupt an endogenous
by way of, for example, a CRISPR-associated protein, such as caspase 9, or another nuclease
WO wo 2019/113375 PCT/US2018/064335
described herein, such as a transcription activator-like effector nuclease, a meganuclease, or a
zinc finger nuclease.
[0077] In some embodiments, the hematopoietic stem cells, or progeny thereof, maintain
hematopoietic stem cell functional potential after two or more days following infusion of the
hematopoietic stem cells, or progeny thereof, into the patient. In some embodiments, the
hematopoietic stem cells, or progeny thereof, localize to hematopoietic tissue and/or
reestablish hematopoiesis following infusion of the hematopoietic stem cells, or progeny
thereof, into the patient. In some embodiments, upon infusion into the patient, the
hematopoietic stem cells, or progeny thereof, give rise to recovery of a population of cells
selected from the group consisting of megakaryocytes, thrombocytes, platelets, erythrocytes,
mast cells, myeoblasts, basophils, neutrophils, eosinophils, microglia, granulocytes,
monocytes, osteoclasts, antigen-presenting cells.
[0078] In another aspect, the disclosure relates to a method of mobilizing CD34di CD34im cells from
the bone marrow of a human donor into peripheral blood, the method comprising
administering to the donor (i) a CXCR2 agonist selected from the group consisting of Gro-B, Gro-ß,
Gro-ß T, and variants thereof at a dose of from about 50 ug/kg µg/kg to about 1,000 ug/kg µg/kg and (ii) a
CXCR4 antagonist.
[0079] In another aspect, the disclosure relates to a method of performing an allogeneic
hematopoietic stem cell transplant in a patient in need thereof, the method comprising
infusing into the patient a therapeutically effective amount of allogeneic hematopoietic stem
cells, wherein the hematopoietic stem cells were mobilized from bone marrow of a human
donor into peripheral blood of the human donor by a method comprising administering to the
donor (i) a CXCR2 agonist selected from the group consisting of Gro-B, Gro-ß, Gro-B Gro-ß T, and
variants thereof at a dose of from about 50 ug/kg µg/kg to about 1,000 ug/kg µg/kg and (ii) a CXCR4
antagonist.
[0080] In another aspect, the disclosure relates to a method of preventing, reducing the risk of
developing, or reducing the severity of a post-transplant infection in a patient in need thereof,
the method comprising infusing into the patient a therapeutically effective amount of
hematopoietic stem cells, wherein the hematopoietic stem cells were mobilized from bone
marrow of a human donor into peripheral blood of the human donor by a method comprising
administering to the human donor (i) a CXCR2 agonist selected from the group consisting of
WO wo 2019/113375 PCT/US2018/064335
Gro-B, Gro-ß, Gro-B Gro-ß T, and variants thereof at a dose of from about 50 ug/kg µg/kg to about 1,000 ug/kg µg/kg
and (ii) a CXCR4 antagonist.
[0081] In another aspect, the disclosure relates to a method of preventing, reducing the risk of
developing, or reducing the severity of graft versus host disease (GVHD) in a patient in need
thereof, the method comprising infusing into the patient a therapeutically effective amount of
hematopoietic stem cells, wherein the hematopoietic stem cells were mobilized from bone
marrow of a human donor into peripheral blood of the human donor by a method comprising
administering to the human donor (i) a CXCR2 agonist selected from the group consisting of
Gro-B, Gro-ß, Gro-B Gro-ß T, and variants thereof at a dose of from about 50 ug/kg µg/kg to about 1,000 ug/kg µg/kg
and (ii) a CXCR4 antagonist.
[0082] In
[0082] In certain certain embodiments, embodiments, the the CD34 CD34icells cellsare arepresent presentin inaahigher higheramount amountin inthe the
peripheral blood than if the hematopoietic stem cells were mobilized using the CXCR4
CD34i cells antagonist alone. In certain embodiments, the CD34dim are cells capable are ofof capable suppressing suppressing
alloreactive T lymphocyte proliferation when administered to a recipient.
[0083] In certain embodiments, the CXCR2 agonist is Gro-B Gro-ß T. In certain embodiments, the
CXCR2 agonist is administered to the donor at a dose of from about 100 ug/kg µg/kg to about 250
ug/kg. µg/kg. In certain embodiments, the CXCR2 agonist is administered to the donor at a dose of
from about 125 ug/kg µg/kg to about 225 ug/kg. µg/kg. In certain embodiments, the CXCR2 agonist is
administered to the donor at a dose of about 150 ug/kg. µg/kg. In certain embodiments, the CXCR2
agonist is administered intravenously to the donor.
[0084] In certain embodiments, the CXCR4 antagonist is administered subcutaneously to the
donor. In certain embodiments, the CXCR4 antagonist is plerixafor or a pharmaceutically
acceptable acceptablesalt thereof. salt In certain thereof. embodiments, In certain the plerixafor embodiments, or pharmaceutically the plerixafor or pharmaceutically
acceptable salt thereof is administered to the donor at a dose of from about 50 ug/kg µg/kg to about
500 ug/kg. µg/kg. In certain embodiments, the plerixafor or pharmaceutically acceptable salt
thereof is administered to the donor at a dose of from about 200 ug/kg µg/kg to about 300 ug/kg. µg/kg. In
certain embodiments, the plerixafor or pharmaceutically acceptable salt thereof is
administered to the donor at a dose of about 240 ug/kg. µg/kg.
[0085] In certain embodiments, the method further includes testing a sample of peripheral
blood for the presence of CD34dim cells CD34i cells and and releasing releasing the the sample sample for for exex vivo vivo expansion expansion ofof the the
CD34i CD34di cells. cells.
WO wo 2019/113375 PCT/US2018/064335
[0086] In
[0086] In certain certain embodiments, embodiments, the the disclosure disclosure relates relates to to the the population population of of CD34' CD34i cells cells
derived from any of the above methods, or a composition comprising the same.
Brief Description of the Figures
Gro-B T is co-administered with
[0087] FIG. 1A provides a graph showing that, when Gro-ß
plerixafor (AMD3100) in mice, a synergistic increase in mobilization results, and grafts are
enriched in highly engraftable, long-term hematopoietic stem cells (LT-HSC = Lin-c-kit Lin- c-kit
Sca-1 CD150+ CD48*). CD150 CD48). FIG. FIG. 1B1B provides provides a a graph graph showing showing that that grafts grafts containing containing cells cells
mobilized by Gro-B Gro-ß T and plerixaflor led to higher relative numbers of competitive
repopulating units (CRU) at week 16 than did grafts containing cells mobilized by G-CSF
alone.
[0088] FIG. 2A is a graph demonstrating the pharmacokinetic profile of various dosages of
Gro-B Gro-ß T when administered intravenously to Rhesus monkeys. FIG. 2B is a graph
Gro-B T when administered demonstrating the pharmacokinetic profile of various dosages of Gro-ß
subcutaneously to Rhesus monkeys. In all experiments, Gro-B Gro-ß T was administered to
subjects concurrently with plerixafor.
[0089] FIG. 3A shows a series of graphs demonstrating the mobilization response of
leukocytes (white blood cells, "WBCs") to various dosages of Gro-ß Gro-B T upon intravenous
administration to Rhesus monkeys. Leukocyte response is shown both in terms of the
quantity of cells mobilized (top) and the fold change in leukocyte density relative to baseline
leukocyte density prior to administration (bottom). FIG. 3B shows a series of graphs
demonstrating the mobilization response of leukocytes (white blood cells, "WBCs") to
various dosages of Gro-B Gro-ß T upon subcutaneous administration to Rhesus monkeys.
Leukocyte response is shown both in terms of the quantity of cells mobilized (top) and the
fold change in leukocyte density relative to baseline leukocyte density prior to administration
(bottom). In all experiments, Gro-B Gro-ß T was administered to subjects concurrently with
plerixafor.
[0090] FIG. 4A shows a series of graphs demonstrating the mobilization response of
neutrophils to various dosages of Gro-B Gro-ß T upon intravenous administration to Rhesus
monkeys. Neutrophil response is shown both in terms of the quantity of cells mobilized (top)
and the fold change in neutrophil density relative to baseline neutrophil density prior to
administration (bottom). FIG. 4B shows a series of graphs demonstrating the mobilization
WO wo 2019/113375 PCT/US2018/064335
response of neutrophils to various dosages of Gro-B Gro-ß T upon subcutaneous administration to
Rhesus monkeys. Neutrophil response is shown both in terms of the quantity of cells
mobilized (top) and the fold change in neutrophil density relative to baseline neutrophil
density prior to administration (bottom). In all experiments, Gro-B Gro-ß T was administered to
subjects concurrently with plerixafor.
[0091] FIG. 5A shows a series of graphs demonstrating the mobilization response of
lymphocytes to various dosages of Gro-B Gro-ß T upon intravenous administration to Rhesus
monkeys. Lymphocyte response is shown both in terms of the quantity of cells mobilized
(top) and the fold change in lymphocyte density relative to baseline lymphocyte density prior
to administration (bottom). FIG. 5B shows a series of graphs demonstrating the mobilization
response of lymphocytes to various dosages of Gro-ß T upon subcutaneous administration to
Rhesus monkeys. Lymphocyte response is shown both in terms of the quantity of cells
mobilized (top) and the fold change in lymphocyte density relative to baseline lymphocyte
density prior to administration (bottom). In all experiments, Gro-B Gro-ß T was administered to
subjects concurrently with plerixafor.
[0092] FIG. 6A shows a series of graphs demonstrating the mobilization response of
monocytes to various dosages of Gro-ß T upon intravenous administration to Rhesus
monkeys. Monocyte response is shown both in terms of the quantity of cells mobilized (top)
and the fold change in monocyte density relative to baseline monocyte density prior to
administration (bottom). FIG. 6B shows a series of graphs demonstrating the mobilization
response of monocytes to various dosages of Gro-B Gro-ß T upon subcutaneous administration to
Rhesus monkeys. Monocyte response is shown both in terms of the quantity of cells
mobilized (top) and the fold change in monocyte density relative to baseline monocyte
density prior to administration (bottom). In all experiments, Gro-B Gro-ß T was administered to
subjects concurrently with plerixafor.
[0093] FIG. 7A shows a series of graphs demonstrating the mobilization response of CD34+ CD34
cells to various dosages of Gro-B Gro-ß T upon intravenous administration to Rhesus monkeys.
CD34 cell CD34+ cellresponse responseis isshown shownboth bothin interms termsof ofthe thefrequency frequencyof ofCD34 cells CD34+ inin cells the sample the sample
obtained from peripheral blood of the subjects (top) and the fold change in CD34+ cell CD34 cell
frequency relative to baseline CD34+ cellfrequency CD34 cell frequencyprior priorto toadministration administration(bottom). (bottom).FIG. FIG.
7B shows a series of graphs demonstrating the mobilization response of CD34+ CD34 cells cells to to
various dosages of Gro-B Gro-ß T upon subcutaneous administration to Rhesus monkeys. CD34
WO wo 2019/113375 PCT/US2018/064335
cell response is shown both in terms of the frequency of CD34+ cellsin CD34 cells inthe thesample sampleobtained obtained
CD34 cell from peripheral blood of the subjects (top) and the fold change in CD34+ cell frequency frequency
CD34 cell relative to baseline CD34+ cell frequency frequency prior prior to to administration administration (bottom). (bottom). In In all all
experiments, Gro-B Gro-ß T was administered to subjects concurrently with plerixafor.
CD34+
[0094] FIG. 8A shows a series of graphs demonstrating the mobilization response of CD34
cells to various dosages of Gro-B Gro-ß T upon intravenous administration to Rhesus monkeys.
CD34+ cell response CD34 cell responseisis shown both shown in terms both of the in terms ofquantity of cellsofmobilized the quantity (top) and the cells mobilized (top) and the
fold change in CD34 cell density relative to baseline CD34+ celldensity CD34 cell densityprior priorto to
administration (bottom). FIG. 8B shows a series of graphs demonstrating the mobilization
CD34 cells response of CD34+ cells to to various various dosages dosages of of Gro-ß Gro-B TT upon upon subcutaneous subcutaneous administration administration to to
CD34+cell Rhesus monkeys. CD34 cellresponse responseis isshown shownboth bothin interms termsof ofthe thequantity quantityof ofcells cells
mobilized (top) and the fold change in CD34+ celldensity CD34 cell densityrelative relativeto tobaseline baselineCD34 CD34+ cell cell
density prior to administration (bottom). In all experiments, Gro-B Gro-ß T was administered to
subjects concurrently with plerixafor.
[0095] FIG. 9A shows a series of graphs demonstrating the mobilization response of
hematopoietic stem cells (CD34+ CD90CD45RA (CD34 CD90 CD45RAcells) cells)to tovarious variousdosages dosagesof ofGro-ß Gro-BTTupon upon
intravenous intravenousadministration to Rhesus administration monkeys. to Rhesus CD34+ CD34 monkeys. CD90 CD45RA cell response CD90 CD45RA is cell response is
shown both in terms of the frequency of CD34+ CD90+ CD34 CD90 CD45RA CD45RA cells cells inin the the sample sample
obtained from peripheral blood of the subjects (top) and the fold change in CD34+ CD90+ CD34 CD90
CD45RA cell frequency relative to baseline CD34+ CD90+ CD34 CD90 CD45RA CD45RA cell cell frequency frequency prior prior toto
administration (bottom). FIG. 9B shows a series of graphs demonstrating the mobilization
response of hematopoietic stem cells (CD34 CD90 CD45RA cells) to various dosages of
Gro-ß T upon subcutaneous administration to Rhesus monkeys. CD34+ Gro-B CD34 CD90 CD90CD45RA CD45RA
cell response is shown both in terms of the frequency of CD34 CD90+ CD45RAcells CD90 CD45RA cellsin inthe the
CD34+ sample obtained from peripheral blood of the subjects (top) and the fold change in CD34
CD90 CD45RA cell frequency relative to baseline CD34+ CD90+ CD34 CD90 CD45RA CD45RA cell cell frequency frequency
prior to administration (bottom). In all experiments, Gro-B Gro-ß T was administered to subjects
concurrently with plerixafor.
[0096] FIG. 10A shows a series of graphs demonstrating the mobilization response of
hematopoietic stem cells (CD34 CD90+ CD45RA*cells)to CD90 CD45RA`cells) tovarious variousdosages dosagesof ofGro-ß Gro-BTTupon upon
intravenous intravenousadministration to Rhesus administration monkeys. to Rhesus CD34+ CD34 monkeys. CD90+ CD90 CD45RA cell response CD45RA is cell response is
shown both in terms of the quantity of cells mobilized (top) and the fold change in CD34+ CD34
WO wo 2019/113375 PCT/US2018/064335
CD90+ CD45RA'celldensity CD90 CD45RA'ce density relative relative to tobaseline baselineCD34 CD90CD90 CD34 CD45RA`cell density prior CD45RA density priortoto
administration (bottom). FIG. 10B shows a series of graphs demonstrating the mobilization
response of hematopoietic stem cells (CD34 CD90+ CD45RA cells) CD90 CD45RA cells) to to various various dosages dosages of of
Gro-B Gro-ß T upon subcutaneous administration to Rhesus monkeys. CD34+ cell response CD34 cell response is is
shown both in terms of the quantity of cells mobilized (top) and the fold change in CD34+ CD34
CD90 CD45RA cell density relative to baseline CD34+ CD90CD45RA CD34 CD90 CD45RAcell celldensity densityprior prior
to administration (bottom). In all experiments, Gro-B Gro-ß T was administered to subjects
concurrently with plerixafor.
[0097] FIG. 11 shows a series of graphs demonstrating the increase in the quantity of
colony-forming units (CFU) of hematopoietic stem cells achieved by the intravenous
administration of various dosages of Gro-B Gro-ß T to Rhesus monkeys. CFR response is shown
both in terms of the concentration of CFUs (top) and the fold change in CFU concentration
relative to baseline CFU concentration prior to administration (bottom). In all experiments,
Gro-ß T was administered to subjects concurrently with plerixafor.
[0098] FIG. 12A shows a series of graphs demonstrating the response of plasma matrix
metalloproteinase 9 (MMP9) to various dosages of Gro-B Gro-ß T upon intravenous administration
to Rhesus monkeys. Plasma MMP9 response is shown both in terms of absolute
concentration (top) and the fold change in plasma MMP9 concentration relative to baseline
MMP9 concentration prior to administration (bottom). FIG. 12B shows a series of graphs
demonstrating the response of plasma MMP9 to various dosages of Gro-B Gro-ß T upon
subcutaneous administration to Rhesus monkeys. Plasma MMP9 response is shown both in
terms of absolute concentration (top) and the fold change in plasma MMP9 concentration
relative to baseline MMP9 concentration prior to administration (bottom). In all experiments,
Gro-B Gro-ß T was administered to subjects concurrently with plerixafor.
[0099] FIG. 13A shows a series of graphs demonstrating the response of plasma tissue
inhibitor of matrix metalloproteinase 1 (TIMP-1) to various dosages of Gro-B Gro-ß T upon
intravenous administration to Rhesus monkeys. Plasma TIMP-1 response is shown both in
terms of absolute concentration (top) and the fold change in plasma TIMP-1 concentration
relative to baseline TIMP-1 concentration prior to administration (bottom). FIG. 13B shows
a series of graphs demonstrating the response of plasma TIMP-1 to various dosages of Gro-B Gro-ß
T upon subcutaneous administration to Rhesus monkeys. Plasma TIMP-1 response is shown
both in terms of absolute concentration (top) and the fold change in plasma TIMP-1 wo 2019/113375 WO PCT/US2018/064335 concentration relative to baseline TIMP-1 concentration prior to administration (bottom). In all experiments, Gro-B Gro-ß T was administered to subjects concurrently with plerixafor.
[0100] FIG. 14A is a graph showing the response of the molar ratio of plasma MMP9 to
plasma TIMP-1 to various dosages of Gro-B Gro-ß T upon intravenous administration to Rhesus
monkeys. FIG. 14B is a graph showing the response of the molar ratio of plasma MMP9 to
plasma TIMP-1 to various dosages of Gro-B Gro-ß T upon subcutaneous administration to Rhesus
monkeys. In all experiments, Gro-B Gro-ß T was administered to subjects concurrently with
plerixafor.
[0101] FIG. 15 provides representative flow plots from blood samples taken four hours post-
mobilization from Rhesus monkeys. Intravenous administration of 450 ug/kg µg/kg Gro-B Gro-ß T and
subcutaneous administration of 1 mg/kg plerixafor (AMD3100) leads to the mobilization of a
CD34im cells. population of CD34dim cells.
[0102] FIG. 16 provides representative flow plots from blood samples taken at baseline
versus four hours post-mobilization from Rhesus monkeys. Mobilization was induced by (1)
intravenous administration of 450 ug/kg µg/kg Gro-B Gro-ß T and subcutaneous administration of 1
mg/kg plerixafor (AMD3100) or (2) subcutaneous administration of 1 mg/kg plerixafor
(AMD3100). The combination of Gro-B Gro-ß T and plerixafor (as compared to plerixafor alone)
leads leads to toenhanced enhancedmobilization of CD34 mobilization dim cells. of CD34i cells.
CD34i cells
[0103] FIG. 17 is a graph quantifying the concentration of CD34dim inin cells peripheral blood peripheral blood
from untreated Rhesus monkeys ("Unmobilized"), Rhesus monkeys that have been treated
with intravenous administration of 450 ug/kg µg/kg Gro-B Gro-ß T and subcutaneous administration of 1
mg/kg plerixafor ("Gro-B ("Gro-ß T + plerixafor"), Rhesus monkeys that have been treated with
subcutaneous administration of 1 mg/kg plerixafor ("plerixafor") and Rhesus monkeys that
have been treated with subcutaneous administration of 50 ug/kg µg/kg (q.d. X 5) G-CSF ("G-CSF").
CD34i cells CD34dim were cells present were atat present a significantly higher a significantly concentration higher inin concentration blood mobilized blood using mobilized using
Gro-B Gro-ß T plus plerixafor.
[0104] FIG. 18 is a graph depicting the composition of unmobilized cells and grafts
mobilized by G-CSF, Gro-B Gro-ß T and AMD3100 and AMD3100 alone. As shown, grafts Gro-ß T and AMD3100 show a 3 fold increase in CD34dim mobilized using Gro-B CD34i cells and cells a a and 3 3 fold fold
increase in T-cells as compared to grafts mobilized using G-CSF.
[0105] FIG.
[0105] FIG.1919provides provides graphs graphs showing showing that that Gro-B Gro-ß T and AMD3100 T and AMD3100 mobilized mobilized CD34dim CD34i
cells suppressed T-cell proliferation as measured by carboxyfluorescein succinimidyl ester
WO wo 2019/113375 PCT/US2018/064335
(CFSE) staining after four days in culture. "Beads" indicates stimulation of T-cells using
anti-CD2/CD3/CD28 coated microbeads.
[0106] FIG. 20 provides a survival curve showing that by day 24, all mice (13/13)
transplanted with unmobilized PBMCs had died of aGVHD compared to 5/16 mice
transplanted with AMD3100 mobilized peripheral blood, 3/16 mice transplanted with G-CSF
mobilized PBMCs and none of the mice transplanted with Gro-B Gro-ß T and AMD3100 mobilized
PBMCs. At day 60 post-transplant, 15/16 mice transplanted with Gro-B Gro-ß T and AMD3100
remained alive, whereas only 10/16 mice transplanted with AMD3100 mobilized PBMCs and
11/16 mice transplanted with G-CSF mobilized PBMCs remained alive. p<0.0001
B T and AMD3100 V. unmobilized) and p<0.05 (comparing Gro- ß (comparing Gro- ß B T and
AMD3100 V. AMD3100 alone).
[0107] FIG. 21A shows rhesus CD45+CD3+ T-cell numbers in mice at day 14 post-
transplant with unmobilized PBMCs, PBMCs mobilized with Gro-B Gro-ß T and plerixafor (i.e.,
AMD3100), PBMCs mobilized with plerixafor alone, and PBMCs mobilized with G-CSF
alone. FIG. 21B shows T-cell numbers in mice at day 14 post-transplant with unmobilized
Gro-B T and plerixafor (i.e., AMD3100) mobilized PBMCs, and Gro-ß PBMCs, Gro-ß Gro-B T and
plerixafor mobilized PBMCs that have been depleted of CD34dim cells. FIG. 21C provides
a survival curve of mice transplanted with unmobilzied PBMCs, Gro-B Gro-ß T and AMD3100
mobilized PBMCs, and Gro-B Gro-ß T and AMD3100 mobilized PBMCs that have been depleted of
CD34dim cells.
Detailed Description
[0108] The present invention provides compositions and methods for mobilizing
hematopoietic stem and progenitor cells in a subject. For example, the subject may be a
hematopoietic stem and progenitor cell donor (i.e., a donor), such as a mammalian donor
(e.g., a human donor). The compositions and methods described herein can additionally be
used for the treatment of one or more stem cell disorders in a patient, such as a human
patient. Using the compositions and methods described herein, a C-X-C chemokine receptor
type 2 (CXCR2) agonist, such as Gro-B Gro-ß or a variant thereof, such as a truncated form of Gro-
ß (e.g., Gro-B Gro-ß T), as described herein, optionally in combination with a C-X-C chemokine
receptor type 4 (CXCR4) antagonist, such as 1,1'-[1,4-phenylenebis(methylene)]-bis 1,1'-[1,4-phenylenebis(methylene)]-bis-
1,4,8,11-tetra-azacyclotetradecane 1,4,8,11-tetra-azacyclotetradecane or or aa variant variant thereof, thereof, may may be be administered administered to to aa donor, donor, as as
WO wo 2019/113375 PCT/US2018/064335
described herein, in amounts sufficient to mobilize hematopoietic stem and progenitor cells.
The compositions and methods described herein are capable of mobilizing hematopoietic
stem and progenitor cells from a stem cell niche within a donor into circulating peripheral
blood while reducing the mobilization of other cells of the hematopoietic lineage, such as
leukocytes, neutrophils, lymphocytes, and monocytes. The compositions and methods
described herein thus enable the selective mobilization of hematopoietic stem and progenitor
cells in a donor, which may then be isolated from a donor for therapeutic use.
[0109] The invention is based, in part, on the discovery that administration of a CXCR2
agonist, such as Gro-B, Gro-ß, Gro-B Gro-ß T, or a variant thereof, optionally in combination with a
CXCR4 antagonist, such as plerixafor or a pharmaceutically acceptable salt thereof, at
particular doses can provide the important clinical benefit of mobilizing populations of cells
that are enriched in hematopoietic stem cells relative to other cell types, such as leukocytes,
neutrophils, and monocytes. This ability is advantageous, as these other cell types may be
undesirable for administration to a human patient undergoing hematopoietic stem cell
transplant therapy. Thus, the populations of mobilized hematopoietic stem and progenitor
cells produced using the compositions and methods described herein are particularly suitable
for hematopoietic stem cell transplantation therapy.
[0110] Following mobilization, the hematopoietic stem or progenitor cells may be isolated
for ex vivo expansion and/or for therapeutic use. In some embodiments, upon collection of
the mobilized hematopoietic stem and/or progenitor cells, the withdrawn cells may be infused
into a patient, such as the donor or another subject (e.g., a subject that is HLA-matched to the
donor) for the treatment of one or more pathologies of the hematopoietic system.
Additionally or alternatively, the mobilized cells may be withdrawn and then expanded ex
vivo, such as by contacting the cells with an aryl hydrocarbon receptor antagonist, SO so as to
produce a population of hematopoietic stem cells having a sufficient quantity of cells for
transplantation.
[0111] As described herein, hematopoietic stem cells are capable of differentiating into a
multitude of cell types in the hematopoietic lineage, and can thus be administered to a patient
in order to populate or repopulate a cell type that is defective or deficient in the patient. The
patient may be one, for example, that is suffering from one or more blood disorders, such as
an autoimmune disease, cancer, hemoglobinopathy, or other hematopoietic pathology, and is
therefore in need of hematopoietic stem cell transplantation. The invention thus provides
WO wo 2019/113375 PCT/US2018/064335
methods of treating a variety of hematopoietic conditions, such as sickle cell anemia,
thalassemia, Fanconi anemia, Wiskott-Aldrich syndrome, adenosine deaminase deficiency-
severe combined immunodeficiency, metachromatic leukodystrophy, Diamond-Blackfan
anemia and Schwachman-Diamond syndrome, human immunodeficiency virus infection, and
acquired immune deficiency syndrome, as well as cancers and autoimmune diseases, among
others.
[0112] The sections that follow provide a description of CXCR4 antagonists and CXCR2
agonists that can be administered to a donor SO so as to induce mobilization of a population of
hematopoietic stem or progenitor cells from a stem cell niche into peripheral blood, from
which the cells may subsequently be isolated and infused into a patient for the treatment, for
example, of one or more stem cell disorders, such as a cancer, autoimmune disease, of
metabolic disorder described herein. The following sections additionally describe methods of
determining whether populations of cells mobilized with a CXCR2 agonist and/or a CXCR
antagonist are suitable for release for ex vivo expansion and/or for therapeutic applications.
Definitions
[0113] As used herein, the term "about" refers to a value that is within 10% above or below
the value being described. For example, the term "about 5 nM" indicates a range of from 4.5
nM to 5.5 nM.
[0114] As used herein, the terms "acquire" and "acquiring" means obtaining possession of a
physical entity, or a value, such as a numerical value, directly acquiring or indirectly
acquiring the physical entity or value. "Directly acquiring" means performing a process (e.g.,
performing an assay or test on a sample or analyzing a sample) to obtain the physical entity
or value. "Indirectly acquiring" refers to receiving the physical entity or value from another
party or source (e.g., a third party laboratory that directly acquired the physical entity or
value). Directly acquiring a physical entity includes performing a process, e.g., analyzing a
sample, such as a sample of hematopoietic cells isolated from a donor that has undergone or
is undergoing a hematopoietic stem cell mobilization regimen described herein. Directly
acquiring a value includes performing a process, such as an assay, on a sample or another
substance, e.g., performing an analytical process which includes determining the quantity of
hematopoietic stem cells in a sample, the ratio of hematopoietic stem cells to cells of another
WO wo 2019/113375 PCT/US2018/064335
type within the hematopoietic lineage, or the frequency of hematopoietic stem cells among
the total quantity of cells in a sample.
[0115] As used herein, the term "affinity" refers to the strength of the non-covalent
interaction between two or more molecules, such as two or more proteins (e.g., a
metalloproteinase and an endogenous inhibitor thereof as described herein). Affinity can be
expressed quantitatively, for example, as an equilibrium dissociation constant (Kd) or, in
cases in which one of the binding partners is an enzyme, as an inhibition constant (Ki). (K).
Binding affinity can be determined using standard techniques, such as enzyme-linked
immunosorbent assays (ELISA), surface plasmon resonance assays, and isothermal titration
calorimetry assays, among others.
[0116] As used herein, the term "antibody" refers to an immunoglobulin molecule that
specifically binds to, or is immunologically reactive with, a particular antigen, and includes
polyclonal, monoclonal, genetically engineered, and otherwise modified forms of antibodies,
including but not limited to chimeric antibodies, humanized antibodies, heteroconjugate
antibodies (e.g., bi- tri- and quad-specific antibodies, diabodies, triabodies, and tetrabodies),
and antigen binding fragments of antibodies, including, for example, Fab', F(ab')2, Fab, Fv, F(ab'), Fab, Fv,
rlgG, and scFv fragments. Unless otherwise indicated, the term "monoclonal antibody"
(mAb) is meant to include both intact molecules, as well as antibody fragments (including,
for example, Fab and F(ab')2 fragments) that F(ab') fragments) that are are capable capable of of specifically specifically binding binding to to aa target target
protein. protein.AsAsused herein, used the the herein, Fab and Fab F(ab')2 fragments and F(ab') refer to fragments antibody refer fragments fragments to antibody that lack that lack
the Fc fragment of an intact antibody. Examples of these antibody fragments are described
herein.
[0117] The term "antigen-binding fragment," as used herein, refers to one or more fragments
of an antibody that retain the ability to specifically bind to a target antigen. The antigen-
binding function of an antibody can be performed by fragments of a full-length antibody.
The antibody fragments can be, for example, a Fab, F(ab')2, scFv,diabody, F(ab'), scFv, diabody,aatriabody, triabody,an an
affibody, a nanobody, an aptamer, or a domain antibody. Examples of binding fragments
encompassed of the term "antigen-binding fragment" of an antibody include, but are not
limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CHI CH1
domains; (ii) a F(ab')2 fragment, aa bivalent F(ab') fragment, bivalent fragment fragment containing containing two two Fab Fab fragments fragments linked linked by by
a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1
domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an
WO wo 2019/113375 PCT/US2018/064335
antibody, (v) a dAb including VH and VL domains; (vi) V domains; (vi) aa dAb dAb fragment fragment that that consists consists of of aa VH VH
domain (see, e.g., Ward et al. (1989) Nature 341:544-546); (vii) a dAb which consists of a
VH or a VL domain; (viii) an isolated complementarity determining region (CDR); and (ix) a
combination of two or more (e.g., two, three, four, five, or six) isolated CDRs which may
optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv
fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant
methods, by a linker that enables them to be made as a single protein chain in which the VL
and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, for
example, Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad.
Sci. USA 85:5879-5883). These antibody fragments can be obtained using conventional
techniques known to those of skill in the art, and the fragments can be screened for utility in
the same manner as intact antibodies. Antigen-binding fragments can be produced by
recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins,
or, in certain cases, by chemical peptide synthesis procedures known in the art.
[0118] As used herein, the term "bispecific antibody" refers to, for example, a monoclonal,
often a human or humanized antibody that is capable of binding at least two different
antigens.
[0119] As used herein the term "CD34dim cells" refers to a population of cells, of which at
least a portion of the population expresses the markers CD34, CD11b, and CD45 and does
not substantially express the markers CD3, CD8, or CD20, wherein CD34 and CD45 are
expressed at a relatively low level. This cell population exhibits characteristics of
monocytes, for example, the ability to suppress alloreactive T lymphocyte proliferation.
(D'Aveni (D' Aveni et et al. al. (2015) (2015) Science Science Translational Translational Medicine Medicine 7(281):1-12. In some 7(281): In some embodiments, embodiments, a a
population populationofofCD34dim CD34i cells cellsmay maybebe CD14+. CD14+.
CD34i cell
[0120] The person of skill can readily recognize a CD34dim population cell when population viewing when a a viewing
flow flow cytometry cytometryplot as as plot a population of cells a population that isthat of cells CD34+isbut has but CD34 a brightness between thebetween the has a brightness
base level of fluorescence (e.g., autofluorescence) in the cell population being examined and
CD34 bright the brightness of a CD34+ brightcell cell(e.g., (e.g.,aahematopoietic hematopoieticstem stemcell celland/or and/oraaCD34 CD90 CD34+ CD90
CD34i cell cell) population. For example, in certain embodiments, a CD34dim population cell exhibits population exhibits
CD34 bright between 5% and 95% of the brightness of a CD34+ brightcell cell(e.g., (e.g.,aahematopoietic hematopoieticstem stem
cell) population, but is brighter than a CD34 cell population.
WO wo 2019/113375 PCT/US2018/064335
[0121] In certain embodiments, CD34dim cells CD34i cells exhibit exhibit between between 10% 10% and and 90%, 90%, 10% 10% and and 80%, 80%,
10% and 70%, 10% and 60%, 10% and 50%, 10% and 40%, 10% and 30%, 10% and 20%,
20% and 90%, 20% and 80%, 20% and 70%, 20% and 60%, 20% and 50%, 20% and 40%,
20% and 30%, 30% and 90%, 30% and 80%, 30% and 70%, 30% and 60%, 30% and 50%,
30% and 40%, 40% and 90%, 40% and 80%, 40% and 70%, 40% and 60%, 40% and 50%,
50% and 90%, 50% and 80%, 50% and 70%, 50% and 60%, 60% and 90%, 60% and 80%,
60% 60% and and 70%, 70%,70% and 70% 90%, and 70% 70% 90%, and and 80%, 80%, or 80% orand 80%90%, andof90%, the of brightness of CD34+ of CD34 the brightness
bright cells (e.g., hematopoietic stem cells), but is brighter than CD34 cells. In certain
embodiments, embodiments,the CD34dim the CD34imcells are are cells at least 5% brighter, at least at least 5% brighter, at10% brighter, least at least 20% 10% brighter, at least 20%
brighter or at least 30% brighter than CD34 cells, but is less bright than CD34+ bright cells CD34 bright cells
(e.g., a hematopoietic stem cells).
CD34i cells
[0122] In certain embodiments, CD34dim are cells identified are inin identified a a cell sample cell byby sample using flow using flow
cytometry with magnetic beads instead of fluorescence. The magnetic beads will selectively
CD34im cells pull down CD34 bright cells, leaving CD34dim cellsin inthe thecell cellsample. sample.
CD34i cells
[0123] In certain embodiments, CD34dim are cells identified are byby identified measuring the measuring number the ofof number
copies of CD34 expressed by the cells. For example, CD34dim cells CD34i cells can can exhibit exhibit between between 5%5%
and 95% of the number of copies of CD34 as compared to CD34+ brightcells CD34 bright cells(e.g., (e.g.,
hematopoietic stem cells), or between 10% and 90%, 10% and 80%, 10% and 70%, 10% and
60%, 10% and 50%, 10% and 40%, 10% and 30%, 10% and 20%, 20% and 90%, 20% and
80%, 20% and 70%, 20% and 60%, 20% and 50%, 20% and 40%, 20% and 30%, 30% and
90%, 30% and 80%, 30% and 70%, 30% and 60%, 30% and 50%, 30% and 40%, 40% and
90%, 40% and 80%, 40% and 70%, 40% and 60%, 40% and 50%, 50% and 90%, 50% and
80%, 50% and 70%, 50% and 60%, 60% and 90%, 60% and 80%, 60% and 70%, 70% and
90%, 70% and 80%, or 80% and 90% of the number of copies of CD34 as compared to
CD34+ bright cells CD34 bright cells(e.g., hematopoietic (e.g., stem stem hematopoietic cells). cells).
[0124] As used herein, the term "complementarity determining region" (CDR) refers to a
hypervariable region found both in the light chain and the heavy chain variable domains of an
antibody. The more highly conserved portions of variable domains are referred to as
framework regions (FRs). The amino acid positions that delineate a hypervariable region of
an antibody can vary, depending on the context and the various definitions known in the art.
Some positions within a variable domain may be viewed as hybrid hypervariable positions in
that these positions can be deemed to be within a hypervariable region under one set of
WO wo 2019/113375 PCT/US2018/064335
criteria while being deemed to be outside a hypervariable region under a different set of
criteria. One or more of these positions can also be found in extended hypervariable regions.
The antibodies described herein may contain modifications in these hybrid hypervariable
positions. The variable domains of native heavy and light chains each contain four
framework regions that primarily adopt a B-sheet ß-sheet configuration, connected by three CDRs,
which form loops that connect, and in some cases form part of, the B-sheet ß-sheet structure. The
CDRs in each chain are held together in close proximity by the framework regions in the
order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and, with the CDRs from the other antibody
chains, contribute to the formation of the target binding site of antibodies (see Kabat et al.,
Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda,
MD., 1987). As used herein, numbering of immunoglobulin amino acid residues is
performed according to the immunoglobulin amino acid residue numbering system of Kabat
et al., unless otherwise indicated.
[0125] As used herein in the context of the administration of one or more agents to a subject,
the term "completion of administration" refers to the point in time by which the one or more
agents have been administered to the subject in their entirety. In some embodiments, an
agent as described herein, such as a CXCR4 antagonist (e.g., plerixafor or a variant thereof)
and/or a CXCR2 agonist (e.g., Gro-B Gro-ß or a variant or truncation thereof, such as Gro-B Gro-ß T) can
be administered to a subject over a period of time, for example, by intravenous or
subcutaneous injection. An agent is considered to have "completed administration" once the
prescribed dosage of the agent has been administered to the subject in its entirety. In the case
of the administration of multiple agents to a subject, such as both a CXCR4 antagonist (e.g.,
plerixafor or a variant thereof) and a CXCR2 agonist (e.g., Gro-B Gro-ß or a variant or truncation
thereof, such as Gro-B Gro-ß T), the agents are considered to have "completed administration" once
the prescribed dosages of all agents in a particular regimen have been administered to the
subject in their entirety.
[0126] As used herein, the terms "conservative mutation," "conservative substitution," or
"conservative amino acid substitution" refer to a substitution of one or more amino acids for
one or more different amino acids that exhibit similar physicochemical properties, such as
polarity, electrostatic charge, and steric volume. These properties are summarized for each of
the twenty naturally-occurring amino acids in TABLE 1 below.
TABLE 1. Representative physicochemical properties of naturally-occurring amino acids
Amino Acid 3 Letter 1 Letter Side-chain Electrostatic Steric
Code Code Polarity character at Volume* Volume physiological
pH (7.4)
Alanine Alanine Ala nonpolar neutral small A Arginine polar cationic large large Arg R Asparagine polar neutral intermediate Asn N Aspartic acid Asp polar anionic intermediate D Cysteine Cys nonpolar neutral intermediate Cys C Glutamic acid Glu polar anionic intermediate E Glutamine Gln polar neutral intermediate Q Glycine Gly nonpolar neutral small G Histidine His polar Both neutral large H and cationic
forms in
equilibrium
at pH 7.4
Isoleucine Ile I nonpolar nonpolar neutral large
Leucine nonpolar neutral large Leu L Lysine polar cationic large large Lys K Methionine Met nonpolar neutral large Met M large Phenylalanine Phe F nonpolar neutral large
Proline Pro P non-polar neutral intermediate
Serine Ser S polar neutral small
Threonine Thr polar neutral intermediate T Tryptophan Trp nonpolar nonpolar neutral bulky
Tyrosine W polar neutral large large Tyr Y Valine Val nonpolar neutral intermediate V based *basedononvolume volumeininA3: A3:50-100 50-100isissmall, small,100-150 100-150isisintermediate, intermediate,150-200 150-200isislarge, large,and and>200 >200
is bulky
[0127] From this table it is appreciated that the conservative amino acid families include,
e.g., (i) G, A, V, L, I, P, and M; (ii) D and E; (iii) C, S and T; (iv) H, K and R; (v) N and Q;
and (vi) F, Y and W. A conservative mutation or substitution is therefore one that substitutes
WO wo 2019/113375 PCT/US2018/064335
one amino acid for a member of the same amino acid family (e.g., a substitution of Ser for
Thr Thr or or Lys Lysfor Arg). for Arg).
[0128] As used herein, "CRU (competitive repopulating unit)" refers to a unit of measure of
long-term engrafting stem cells, which can be detected after in-vivo transplantation.
[0129] As used herein, the term "donor" refers to a subject, such as a mammalian subject
(e.g., a human subject) from which one or more cells are isolated prior to administration of
the cells, or progeny thereof, into a recipient. The one or more cells may be, for example, a
population of hematopoietic stem or progenitor cells.
[0130] As used herein, the term "diabody" refers to a bivalent antibody containing two
polypeptide chains, in which each polypeptide chain includes VH and VL domains joined by a
linker that is too short (e.g., a linker composed of five amino acids) to allow for
intramolecular association of VH and VL domains on the same peptide chain. This
configuration forces each domain to pair with a complementary domain on another
polypeptide chain SO so as to form a homodimeric structure. Accordingly, the term "triabody"
refers to trivalent antibodies containing three peptide chains, each of which contains one VH
domain and one VL domain joined by a linker that is exceedingly short (e.g., a linker
composed of 1-2 amino acids) to permit intramolecular association of VH and VL domains
within the same peptide chain. In order to fold into their native structures, peptides
configured in this way typically trimerize SO so as to position the VH and VL domains of
neighboring peptide chains spatially proximal to one another (see, for example, Holliger et al.
(1993) Proc. Natl. Acad. Sci. USA 90:6444-48).
[0131] As used herein, the term "disrupt" with respect to a gene refers to preventing the
formation of a functional gene product. A gene product is functional only if it fulfills its
normal (wild-type) functions. Disruption of the gene prevents expression of a functional
factor encoded by the gene and comprises an insertion, deletion, or substitution of one or
more bases in a sequence encoded by the gene and/or a promoter and/or an operator that is
necessary for expression of the gene in the animal. The disrupted gene may be disrupted by,
e.g., removal of at least a portion of the gene from a genome of the animal, alteration of the
gene to prevent expression of a functional factor encoded by the gene, an interfering RNA, or
expression of a dominant negative factor by an exogenous gene. Materials and methods of
genetically modifying hematopoietic stem/progenitor cells are detailed in US 8,518,701 8,518,701;US US
2010/0251395; and US 2012/0222143, the disclosures of each of which are incorporated
90
WO wo 2019/113375 PCT/US2018/064335
herein by reference in their entirety (in case of conflict, the instant specification is
controlling).
[0132] Various techniques known in the art can be used to inactivate genes to make knock-
out animals and/or to introduce nucleic acid constructs into animals to produce founder
animals and to make animal lines, in which the knockout or nucleic acid construct is
integrated into the genome. Such techniques include, without limitation, pronuclear
microinjection (U.S. Pat. No. 4,873,191), retrovirus mediated gene transfer into germ lines
(Van der Putten et al. (1985) Proc. Natl. Acad. Sci. USA, 82:6148-6152), gene targeting into
embryonic stem cells (Thompson et al. (1989) Cell, 56:313-321), electroporation of embryos
(Lo (1983) Mol. Cell. Biol., 3:1803-1814), sperm-mediated gene transfer (Lavitrano et al.
(2002) Proc. Natl. Acad. Sci. USA, 99:14230-14235; Lavitrano et al. (2006) Reprod. Fert.
Develop., 18:19-23), and in vitro transformation of somatic cells, such as cumulus or
mammary cells, or adult, fetal, or embryonic stem cells, followed by nuclear transplantation
(Wilmut et al. (1997) Nature, 385:810-813; and Wakayama et al. (1998) Nature, 394:369-
374). Pronuclear microinjection, sperm mediated gene transfer, and somatic cell nuclear
transfer are particularly useful techniques. An animal that is genomically modified is an
animal wherein all of its cells have the genetic modification, including its germ line cells.
When methods are used that produce an animal that is mosaic in its genetic modification, the
animals may be inbred and progeny that are genomically modified may be selected. Cloning,
for example, may be used to make a mosaic animal if its cells are modified at the blastocyst
state, or genomic modification can take place when a single-cell is modified. Animals that are
modified SO so they do not sexually mature can be homozygous or heterozygous for the
modification, depending on the specific approach that is used. If a particular gene is
inactivated by a knock out modification, homozygosity would normally be required. If a
particular gene is inactivated by an RNA interference or dominant negative strategy, then
heterozygosity is often adequate.
[0133] As used herein, a "dual variable domain immunoglobulin" ("DVD-Ig") refers to an
antibody that combines the target-binding variable domains of two monoclonal antibodies via
linkers to create a tetravalent, dual-targeting single agent (see, for example, Gu et al. (2012)
Meth. Enzymol., 502:25-41).
[0134] As used herein, the term "endogenous" describes a substance, such as a molecule,
cell, tissue, or organ (e.g., a hematopoietic stem cell or a cell of hematopoietic lineage, such
91 as a megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeoblast, basophil, neutrophil, eosinophil, microglial cell, granulocyte, monocyte, osteoclast, antigen-presenting cell, macrophage, dendritic cell, natural killer cell, T-lymphocyte, or B-lymphocyte) that is found naturally in a particular organism, such as a human patient.
[0135] As used herein, the term "engraftment potential" is used to refer to the ability of
hematopoietic stem and progenitor cells to repopulate a tissue, whether such cells are
naturally circulating or are provided by transplantation. The term encompasses all events
surrounding or leading up to engraftment, such as tissue homing of cells and colonization of
cells within the tissue of interest. The engraftment efficiency or rate of engraftment can be
evaluated or quantified using any clinically acceptable parameter as known to those of skill in
the art and can include, for example, assessment of competitive repopulating units (CRU);
incorporation or expression of a marker in tissue(s) into which stem cells have homed,
colonized, or become engrafted; or by evaluation of the progress of a subject through disease
progression, survival of hematopoietic stem and progenitor cells, or survival of a recipient.
Engraftment can also be determined by measuring white blood cell counts in peripheral blood
during a post-transplant period. Engraftment can also be assessed by measuring recovery of
marrow cells by donor cells in a bone marrow aspirate sample.
[0136] As used herein, the term "exogenous" describes a substance, such as a molecule, cell,
tissue, or organ (e.g., a hematopoietic stem cell or a cell of hematopoietic lineage, such as a
megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeoblast, basophil, neutrophil,
eosinophil, microglial cell, granulocyte, monocyte, osteoclast, antigen-presenting cell,
macrophage, dendritic cell, natural killer cell, T-lymphocyte, or B-lymphocyte) that is not
found naturally in a particular organism, such as a human patient. Exogenous substances
include those that are provided from an external source to an organism or to cultured matter
extracted therefrom.
[0137] As used herein, the term "framework region" or "FW region" includes amino acid
residues that are adjacent to the CDRs of an antibody or antigen-binding fragment thereof.
FW region residues may be present in, for example, human antibodies, humanized antibodies,
monoclonal antibodies, antibody fragments, Fab fragments, single chain antibody fragments,
scFv fragments, antibody domains, and bispecific antibodies, among others.
[0138] As used herein, the term "hematopoietic progenitor cells" includes pluripotent cells
capable of differentiating into several cell types of the hematopoietic system, including,
WO wo 2019/113375 PCT/US2018/064335
without limitation, granulocytes, monocytes, erythrocytes, megakaryocytes, B-cells and T-
cells, among others. Hematopoietic progenitor cells are committed to the hematopoietic cell
lineage and generally do not self-renew. Hematopoietic progenitor cells can be identified, for
example, by expression patterns of cell surface antigens, and include cells having the
following immunophenotype: Lin KLS Flk2 CD34+ CD34. Hematopoietic progenitor cells
include short-term hematopoietic stem cells, multi-potent progenitor cells, common myeloid
progenitor cells, granulocyte-monocyte progenitor cells, and megakaryocyte-erythrocyte
progenitor cells. The presence of hematopoietic progenitor cells can be determined
functionally, for example, by detecting colony-forming unit cells, e.g., in complete
methylcellulose assays, or phenotypically through the detection of cell surface markers using
flow cytometry and cell sorting assays described herein and known in the art.
[0139] As used herein, the term "hematopoietic stem cells" ("HSCs") refers to immature
blood cells having the capacity to self-renew and to differentiate into mature blood cells
containing diverse lineages including but not limited to granulocytes (e.g., promyelocytes,
neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes),
thrombocytes (e.g., megakaryoblasts, platelet producing megakaryocytes, platelets),
monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and
lymphocytes (e.g., NK cells, B-cells and T-cells). Such cells may include CD34+ cells. CD34 cells.
CD34+ cellsare CD34 cells areimmature immaturecells cellsthat thatexpress expressthe theCD34 CD34cell cellsurface surfacemarker. marker.In Inhumans, humans,
CD34+ cells are CD34 cells are believed believed to to include include aa subpopulation subpopulation of of cells cells with with the the stem stem cell cell properties properties
defined above, whereas in mice, HSCs are CD34-. In addition, HSCs also refer to long term
repopulating HSCs (LT-HSC) and short term repopulating HSCs (ST-HSC). LT-HSCs and
ST-HSCs are differentiated, based on functional potential and on cell surface marker
expression. For example, human HSCs are CD34+, CD38`, CD34, CD38; CD45RA*, CD45RA; CD90+, CD90, CD49F*, CD49F, and and
lin (negative for mature lineage markers including CD2, CD3, CD4, CD7, CD8, CD10,
CD11B, CD19, CD20, CD56, CD235A). In mice, bone marrow LT-HSCs are CD34-, SCA-
1+, C-kit+, CD135-, Slamfl/CD150+, CD48-, and lin- (negative for mature lineage markers
including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, IL7ra), whereas ST-HSCs are
CD34+, SCA-1*, C-kit, CD34, SCA-1, C-kit*, CD135; CD135`, Slamfl/CD150, Slamfl/CD150t, and and lin lin(negative (negativeforfor mature lineage mature lineage
markers including Ter119, CD11b, Grl, Gr1, CD3, CD4, CD8, B220, IL7ra). In addition, ST-
HSCs are less quiescent and more proliferative than LT-HSCs under homeostatic conditions.
However, LT-HSC have greater self-renewal potential (i.e., they survive throughout
adulthood, and can be serially transplanted through successive recipients), whereas ST-HSCs
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have limited self-renewal (i.e., they survive for only a limited period of time, and do not
possess serial transplantation potential). Any of these HSCs can be used in the methods
described herein. ST-HSCs are particularly useful because they are highly proliferative and
thus, can more quickly give rise to differentiated progeny.
[0140] As used herein, the term "hematopoietic stem cell functional potential" refers to the
functional properties of hematopoietic stem cells which include 1) multi-potency (which
refers to the ability to differentiate into multiple different blood lineages including, but not
limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils),
erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet
producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic
cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B-cells and T-cells), 2) self-
renewal (which refers to the ability of hematopoietic stem cells to give rise to daughter cells
that have equivalent potential as the mother cell, and further that this ability can repeatedly
occur throughout the lifetime of an individual without exhaustion), and 3) the ability of
hematopoietic stem cells or progeny thereof to be reintroduced into a transplant recipient
whereupon they home to the hematopoietic stem cell niche and re-establish productive and
sustained hematopoiesis.
[0141] As used herein, the terms "Major histocompatibility complex antigens" ("MHC", also
referred to as "human leukocyte antigens" ("HLA") in the context of humans) refer to
proteins expressed on the cell surface that confer a unique antigenic identity to a cell.
MHC/HLA antigens are target molecules that are recognized by T cells and NK cells as being
derived from the same source of hematopoietic stem cells as the immune effector cells
("self") or as being derived from another source of hematopoietic reconstituting cells ("non-
self"). Two main classes of HLA antigens are recognized: HLA class I and HLA class II.
HLA class I antigens (A, B, and C in humans) render each cell recognizable as "self,"
whereas HLA class II antigens (DR, DP, and DQ in humans) are involved in reactions
between lymphocytes and antigen presenting cells. Both have been implicated in the
rejection of transplanted organs. An important aspect of the HLA gene system is its
polymorphism. Each gene, MHC class I (A, B and C) and MHC class II (DP, DQ and DR)
exists in exists indifferent differentalleles. For example, alleles. two unrelated For example, individuals two unrelated may carry may individuals classcarry I HLA-B, class IHLA-B,
genes B5, and Bw41, respectively. Allelic gene products differ in one or more amino acids in
the the aand/or and/or domain(s). ß domain(s). Large Large panels panels of specific of specific antibodies antibodies or nucleic or nucleic acid are acid reagents reagents used are used
to type HLA haplotypes of individuals, using leukocytes that express class I and class II
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molecules. The genes commonly used for HLA typing are the six MHC Class I and Class II
proteins, two alleles for each of HLA- A; HLA-B and HLA-DR. The HLA genes are
clustered in a "super-locus" present on chromosome position 6p21, which encodes the six
classical transplantation HLA genes and at least 132 protein coding genes that have important
roles in the regulation of the immune system as well as some other fundamental molecular
and cellular processes. The complete locus measures roughly 3.6 Mb, with at least 224 gene
loci. One effect of this clustering is that "haplotypes", i.e. the set of alleles present on a single
chromosome, which is inherited from one parent, tend to be inherited as a group. The set of
alleles inherited from each parent forms a haplotype, in which some alleles tend to be
associated together. Identifying a patient's haplotypes can help predict the probability of
finding matching donors and assist in developing a search strategy, because some alleles and
haplotypes are more common than others and they are distributed at different frequencies in
different racial and ethnic groups.
[0142] As used herein, the term "HLA-matched" refers to a donor-recipient pair in which
none of the HLA antigens are mismatched between the donor and recipient, such as a donor
providing a hematopoietic stem cell graft to a recipient in need of hematopoietic stem cell
transplant therapy. HLA-matched (i.e., where all of the 6 alleles are matched) donor-
recipient pairs have a decreased risk of graft rejection, as endogenous T cells and NK cells
are less likely to recognize the incoming graft as foreign, and are thus less likely to mount an
immune response against the transplant.
[0143] As used herein, the term "HLA-mismatched" refers to a donor-recipient pair in which
at least one HLA antigen, in particular with respect to HLA-A, HLA-B and HLA-DR, is
mismatched between the donor and recipient, such as a donor providing a hematopoietic stem
cell graft to a recipient in need of hematopoietic stem cell transplant therapy. In some
embodiments, one haplotype is matched and the other is mismatched. HLA-mismatched
donor-recipient pairs may have an increased risk of graft rejection relative to HLA-matched
donor-recipient pairs, as endogenous T cells and NK cells are more likely to recognize the
incoming graft as foreign in the case of an HLA-mismatched donor-recipient pair, and such T
cells and NK cells are thus more likely to mount an immune response against the transplant.
[0144] As used herein, the term "human antibody" refers to an antibody in which
substantially every part of the protein (for example, all CDRs, framework regions, CL, CH
domains (e.g., CH1, CH2, CH3), hinge, and VL and VH domains) is substantially non-
WO wo 2019/113375 PCT/US2018/064335
immunogenic in humans, with only minor sequence changes or variations. A human
antibody can be produced in a human cell (for example, by recombinant expression) or by a
non-human animal or a prokaryotic or eukaryotic cell that is capable of expressing
functionally rearranged human immunoglobulin (such as heavy chain and/or light chain)
genes. When a human antibody is a single chain antibody, it can include a linker peptide that
is not found in native human antibodies. For example, an Fv can contain a linker peptide,
such as two to about eight glycine or other amino acid residues, which connects the variable
region of the heavy chain and the variable region of the light chain. Such linker peptides are
considered to be of human origin. Human antibodies can be made by a variety of methods
known in the art including phage display methods using antibody libraries derived from
human immunoglobulin sequences. Human antibodies can also be produced using transgenic
mice that are incapable of expressing functional endogenous immunoglobulins, but which can
express human immunoglobulin genes (see, for example, PCT Publication Nos. WO
1998/24893; WO 1992/01047; WO 1996/34096; WO 1996/33735; U.S. Patent Nos.
5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793;
5,916,771; and 5,939,598).
[0145] As used herein, the term "humanized" antibody refers to a non-human antibody that
contains minimal sequences derived from non-human immunoglobulin. In general, a
humanized antibody contains substantially all of at least one, and typically two, variable
domains, in which all or substantially all of the CDR regions correspond to those of a non-
human immunoglobulin. All or substantially all of the FW regions may also be those of a
human immunoglobulin sequence. The humanized antibody can also contain at least a portion
of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin
consensus sequence. Methods of antibody humanization are known in the art and have been
described, for example, in Riechmann et al. (1988) Nature 332:323-7; U.S. Patent Nos:
5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370.
[0146] As used herein, patients that are "in need of" a hematopoietic stem cell transplant
include patients that exhibit a defect or deficiency in one or more blood cell types, as well as
patients having a stem cell disorder, autoimmune disease, cancer, or other pathology
described herein. Hematopoietic stem cells generally exhibit 1) multi-potency, and can thus
differentiate into multiple different blood lineages including, but not limited to, granulocytes
(e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes,
erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producing megakaryocytes,
WO wo 2019/113375 PCT/US2018/064335
platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts,
and lymphocytes (e.g., NK cells, B-cells and T-cells), 2) self-renewal, and can thus give rise
to daughter cells that have equivalent potential as the mother cell, and 3) the ability to be
reintroduced into a transplant recipient whereupon they home to the hematopoietic stem cell
niche and re-establish productive and sustained hematopoiesis. Hematopoietic stem cells can
thus be administered to a patient defective or deficient in one or more cell types of the
hematopoietic lineage in order to re-constitute the defective or deficient population of cells in
vivo. For example, the patient may be suffering from cancer, and the deficiency may be
caused by administration of a chemotherapeutic agent or other medicament that depletes,
either selectively or non-specifically, the cancerous cell population. Additionally or
alternatively, the patient may be suffering from a hemoglobinopathy (e.g., a non-malignant
hemoglobinopathy), such as sickle cell anemia, thalassemia, Fanconi anemia, aplastic
anemia, and Wiskott-Aldrich syndrome. The subject may be one that is suffering from
adenosine deaminase severe combined immunodeficiency (ADA SCID), HIV/AIDS,
metachromatic leukodystrophy, Diamond-Blackfan anemia, and Schwachman-Diamond
syndrome. The subject may have or be affected by an inherited blood disorder (e.g., sickle
cell anemia) or an autoimmune disorder. Additionally or alternatively, the subject may have
or be affected by a malignancy, such as neuroblastoma or a hematologic cancer. In some
embodiments, the subject may have a leukemia, lymphoma, or myeloma. In some
embodiments, the subject has acute myeloid leukemia, acute lymphoid leukemia, chronic
myeloid leukemia, chronic lymphoid leukemia, multiple myeloma, diffuse large B-cell
lymphoma, or non-Hodgkin's lymphoma. In some embodiments, the subject has
myelodysplastic syndrome. In some embodiments, the subject has an autoimmune disease,
such as scleroderma, multiple sclerosis, ulcerative colitis, Crohn's disease, Type 1 diabetes,
or another autoimmune pathology described herein. In some embodiments, the subject is in
need of chimeric antigen receptor T-cell (CART) therapy. In some embodiments, the subject
has or is otherwise affected by a metabolic storage disorder. The subject may suffer or
otherwise be affected by a metabolic disorder selected from the group consisting of glycogen
storage diseases, mucopolysaccharidoses, Gaucher's Disease, Hurlers Disease,
sphingolipidoses, metachromatic leukodystrophy, or any other diseases or disorders which
may benefit from the treatments and therapies disclosed herein and including, without
limitation, severe combined immunodeficiency, Wiscott-Aldrich syndrome, hyper
immunoglobulin M (IgM) syndrome, Chediak-Higashi disease, hereditary
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lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia
major, sickle cell disease, systemic sclerosis, systemic lupus erythematosus, multiple
sclerosis, juvenile rheumatoid arthritis and those diseases, or disorders described in "Bone
Marrow Transplantation for Non-Malignant Disease," ASH Education Book, 1:319-338
(2000), the disclosure of which is incorporated herein by reference in its entirety as it pertains
to pathologies that may be treated by administration of hematopoietic stem cell transplant
therapy. Additionally or alternatively, a patient "in need of" a hematopoietic stem cell
transplant may one that is or is not suffering from one of the foregoing pathologies, but
nonetheless exhibits a reduced level (e.g., as compared to that of an otherwise healthy
subject) of one or more endogenous cell types within the hematopoietic lineage, such as
megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeoblasts, basophils,
neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting
cells, macrophages, dendritic cells, natural killer cells, T-lymphocytes, and B-lymphocytes.
One of skill in the art can readily determine whether one's level of one or more of the
foregoing cell types, or other blood cell type, is reduced with respect to an otherwise healthy
subject, for example, by way of flow cytometry and fluorescence activated cell sorting
(FACS) methods, among other procedures, known in the art.
[0147] As used herein, the term "leukocyte" refers to a heterogeneous group of nucleated
blood cell types, and excludes erythrocytes and platelets. Leukocytes can be divided into two
general groups: polymorphonucleocytes, which include neutrophils, eosinophils, and
basophils, and mononucleocytes, which include lymphocytes and monocytes.
Polymorphonucleocytes contain many cytoplasmic granules and a multilobed nucleus and
include the following: neutrophils, which are generally amoeboid in shape, phagocytic, and
stain with both basic and acidic dyes, and eosinophils and basophils, which contain
cytoplasmic granules that stain with acidic dyes and with basic dyes, respectively.
[0148] As used herein, the term "lymphocyte" refers to a mononuclear leukocyte that is
involved in the mounting of an immune response. In general, lymphocytes include B
lymphocytes, T lymphocytes, and NK cells.
[0149] As used herein, the terms "mobilize" and "mobilization" refer to processes by which a
population of hematopoietic stem or progenitor cells is released from a stem cell niche, such
as the bone marrow of a subject, into circulation in the peripheral blood. Mobilization of
hematopoietic stem and progenitor cells can be monitored, for example, by assessing the
WO wo 2019/113375 PCT/US2018/064335
quantity or concentration of hematopoietic stem or progenitor cells in a peripheral blood
sample isolated from a subject. For example, the peripheral blood sample may be withdrawn
from the subject, and the quantity or concentration of hematopoietic stem or progenitor cells
in the peripheral blood sample may subsequently be assessed, following the administration
of a hematopoietic stem or progenitor cell mobilization regimen to the subject. The
mobilization regimen may include, for example, a CXCR4 antagonist, such as a CXCR4
antagonist described herein (e.g., plerixafor or a variant thereof), and a CXCR2 agonist, such
as a CXCR2 agonist described herein (e.g., Gro-B Gro-ß or a variant thereof, such as a truncation of
Gro-B, Gro-ß, for example, Gro-B Gro-ß T). The quantity or concentration of hematopoietic stem or
progenitor cells in the peripheral blood sample isolated from the subject following
administration of the mobilization regimen may be compared to the quantity or concentration
of hematopoietic stem or progenitor cells in a peripheral blood sample isolated from the
subject prior to administration of the mobilization regimen. An observation that the quantity
or concentration of hematopoietic stem or progenitor cells has increased in the peripheral
blood of the subject following administration of the mobilization regimen is an indication that
the subject is responding to the mobilization regimen, and that hematopoietic stem and
progenitor cells have been released from one or more stem cell niches, such as the bone
marrow, into peripheral blood circulation. In some embodiments, an observation that the
quantity or concentration of hematopoietic stem or progenitor cells has increased in the
peripheral blood of the subject by 1%, 100%, 1,000%, or more (e.g., by 1%, 2%, 3%, 4%,
5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,
200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1,000%, or more) following
administration of the mobilization regimen is an indication that the subject is responding to
the mobilization regimen, and that hematopoietic stem and progenitor cells have been
released from one or more stem cell niches, such as the bone marrow, into peripheral blood
circulation. Methods for determining the quantity or concentration of hematopoietic stem or
progenitor cells are described herein and known in the art, and include, for example, flow
cytometry techniques that quantify hematopoietic stem or progenitor cells on the basis of the
antigen expression profile of such cells, which is described herein. For example, human
HSCs HSCs are areCD34+, CD34, CD38 CD45RA`, CD38; CD90+, CD45RA; CD49F*, CD90, CD49F,andand lin- (negative lin- for mature (negative lineage for mature lineage
markers including CD2, CD3, CD4, CD7, CD8, CD10, CD11B, CD19, CD20, CD56,
CD235A). Additional methods for determining the quantity or concentration of
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hematopoietic stem or progenitor cells in a peripheral blood sample isolated from a subject
include assays that quantify the number of colony-forming units (CFUs) in the sample, which
is a measure of the quantity of viable hematopoietic stem or progenitor cells that, upon
incubation with an appropriate culture medium, give rise to an individual population of
hematopoietic stem or progenitor cells.
[0150] As used herein, the term "mobilizing amount" refers to a quantity of one or more
agents, such as a quantity of a CXCR4 antagonist and/or a CXCR2 agonist described herein
Gro-B, or a (In some embodiments, a quantity of plerixafor, or a variant thereof, and/or Gro-ß,
variant thereof, such as a truncation of Gro-B, Gro-ß, for example, Gro-B Gro-ß T) that mobilizes a
population of hematopoietic stem or progenitor cells upon administration to a subject, such as
a mammalian subject (e.g., a human subject). Exemplary mobilizing amounts of these agents
include amounts sufficient to effectuate the release of a population of, for example, from
about 20 to about 40 CD34 cells/uL cells/µL of peripheral blood, such as from about 21 to about 39
CD34+ cells/uL of CD34 cells/µL of peripheral peripheral blood, blood, about about 22 22 to to about about 38 38 CD34 CD34+ cells/uL cells/µL ofof peripheral peripheral
CD34 cells/µL blood, about 23 to about 37 CD34+ cells/uLof ofperipheral peripheralblood, blood,about about24 24to toabout about36 36CD34 CD34+
cells/uL cells/µL of peripheral blood, about 25 to about 35 CD34+ cells/uL of CD34 cells/µL of peripheral peripheral blood, blood, about about
CD34 cells/µL 26 to about 34 CD34+ cells/uLof ofperipheral peripheralblood, blood,about about27 27to toabout about33 33CD34 CD34cells/µL cells/uLof of
CD34 cells/µL peripheral blood, about 28 to about 32 CD34+ cells/uL of of peripheral peripheral blood, blood, or or about about 29 29 to to
CD34 cells/µL about 31 CD34+ cells/uL of of peripheral peripheral blood blood (e.g., (e.g., about about 20 20 CD34 cells/µL CD34+ ofof cells/uL peripheral peripheral
blood, 21 CD34+ cells/uL of CD34 cells/µL of peripheral peripheral blood, blood, 22 22 CD34 CD34+ cells/uL cells/µL ofof peripheral peripheral blood, blood, 2323
CD34+ cells/uLof CD34 cells/µL ofperipheral peripheralblood, blood,24, 24,CD34 CD34+ cells/uL cells/µL ofof peripheral peripheral blood, blood, 2525 CD34+ CD34
cells/uL cells/µL of peripheral blood, 26 CD34+ cells/uL of CD34 cells/µL of peripheral peripheral blood, blood, 27 27 CD34 CD34+ cells/uL cells/µL ofof
peripheral blood, 28 CD34+ cells/uL of CD34 cells/µL of peripheral peripheral blood, blood, 29 29 CD34 CD34 cells/µL cells/uL of of peripheral peripheral
CD34 cells/µL blood, 30 CD34+ cells/uLof ofperipheral peripheralblood, blood,31 31CD34 cells/µL CD34+ ofof cells/uL peripheral blood, peripheral 3232 blood,
CD34 cells/µL CD34+ cells/uL of of peripheral peripheral blood blood 33 33 CD34 cells/µL CD34+ ofof cells/uL peripheral blood, peripheral 3434 blood, CD34 CD34+
cells/uL cells/µL of peripheral blood, 35 CD34+ cells/uLof CD34 cells/µL ofperipheral peripheralblood, blood,36 36CD34 CD34+ cells/uL cells/µL ofof
peripheral blood, 37 CD34+ cells/uL of CD34 cells/µL of peripheral peripheral blood, blood, 38 38 CD34 CD34+ cells/uL cells/µL ofof peripheral peripheral
blood, 39 CD34+ cells/uL of CD34 cells/µL of peripheral peripheral blood, blood, 40 40 CD34 CD34+ cells/uL cells/µL ofof peripheral peripheral blood, blood, oror
more. For instance, mobilizing amounts of a CXCR2 agonist, such as Gro-B Gro-ß T, include from
about 50 ug/kg µg/kg of recipient to about 1 mg/kg of recipient, such as from about 50 ug/kg µg/kg to
about 300 ug/kg, µg/kg, 100 ug/kg µg/kg to about 250 ug/kg, µg/kg, or about 150 ug/kg. µg/kg. Mobilizing amounts of of
a CXCR4 antagonist, such as plerixafor or a pharmaceutically acceptable salt thereof, include
WO wo 2019/113375 PCT/US2018/064335
from about 50 ug/kg µg/kg of recipient to about 500 ug/kg µg/kg of recipient, such as from about 200
ug/kg µg/kg to about 300 ug/kg, µg/kg, or about 240 ug/kg. µg/kg.
[0151] As used herein, the term "monoclonal antibody" refers to an antibody that is derived
from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the
method by which it is produced.
[0152] As used herein, the term "monocyte" refers to a CD14+ and CD34- CD14 and CD34- peripheral peripheral blood blood
mononuclear cell (PBMC), which is generally capable of differentiating into a macrophage
and/or dendritic cell upon activation by one or more foreign substances, such as, a microbial
product. In particular, a monocyte may express elevated levels of the CD14 surface antigen
marker, and may express at least one biomarker selected from CD64, CD93, CD180, CD328
(also known as sialic acid-binding Ig-like lectin 7 or Siglec7), and CD329 (sialic acid-binding
Ig-like lectin 9 or Siglec9), as well as the peanut agglutinin protein (PNA).
[0153] As used herein, a "peptide" refers to a single-chain polyamide containing a plurality
of amino acid residues, such as naturally-occurring and/or non-natural amino acid residues,
that are consecutively bound by amide bonds. Examples of peptides include shorter
fragments of full-length proteins, such as full-length naturally-occurring proteins.
[0154] As used herein, the term "recipient" refers to a patient that receives a transplant, such
as a transplant containing a population of hematopoietic stem cells. The transplanted cells
administered to a recipient may be, e.g., autologous, syngeneic, or allogeneic cells.
[0155] As used herein, the term "sample" refers to a specimen (e.g., blood, blood component
(e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g.,
placental or dermal), pancreatic fluid, chorionic villus sample, and cells) taken from a
subject. A sample may be, for example, withdrawn peripheral blood from a donor that is
undergoing or has undergone a hematopoietic stem or progenitor cell mobilization regimen
described herein.
[0156] As used herein, the term "scFv" refers to a single chain Fv antibody in which the
variable domains of the heavy chain and the light chain from an antibody have been joined to
form one chain. scFv fragments contain a single polypeptide chain that includes the variable
region of an antibody light chain (VL) (e.g., CDR-L1, CDR-L2, and/or CDR-L3) and the
variable region of an antibody heavy chain (VH) (e.g., CDR-H1, CDR-H2, and/or CDR-H3)
separated separatedbybya a linker. The The linker. linker that that linker joins joins the VL the and VH regions V and of a scFvoffragment VH regions a scFv can be a fragment can be a
peptide linker composed of proteinogenic amino acids. Alternative linkers can be used to SO so
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as to increase the resistance of the scFv fragment to proteolytic degradation (for example,
linkers containing D-amino acids), in order to enhance the solubility of the scFv fragment
(for example, hydrophilic linkers such as polyethylene glycol-containing linkers or
polypeptides containing repeating glycine and serine residues), to improve the biophysical
stability of the molecule (for example, a linker containing cysteine residues that form
intramolecular or intermolecular disulfide bonds), or to attenuate the immunogenicity of the
scFv fragment (for example, linkers containing glycosylation sites). It will also be
understood by one of ordinary skill in the art that the variable regions of the scFv molecules
described herein can be modified such that they vary in amino acid sequence from the
antibody molecule from which they were derived. For example, nucleotide or amino acid
substitutions leading to conservative substitutions or changes at amino acid residues can be
made (e.g., in CDR and/or framework residues) SO so as to preserve or enhance the ability of the
scFv to bind to the antigen recognized by the corresponding antibody.
[0157] As used herein, the phrase "stem cell disorder" broadly refers to any disease, disorder,
or condition that may be treated or cured by engrafting or transplanting a population of
hematopoietic stem or progenitor cells in a target tissue within a patient. For example, Type I
diabetes has been shown to be cured by hematopoietic stem cell transplant, along with
various other disorders. Exemplary diseases that can be treated by infusion of hematopoietic
stem or progenitor cells into a patient are sickle cell anemia, thalassemias, Fanconi anemia,
aplastic anemia, Wiskott-Aldrich syndrome, ADA SCID, HIV/AIDS, metachromatic
leukodystrophy, Diamond-Blackfan anemia, and Schwachman-Diamond syndrome.
Additional diseases that may be treated by transplantation of hematopoietic stem and
progenitor cells as described herein include blood disorders (e.g., sickle cell anemia) and
autoimmune disorders, such as scleroderma, multiple sclerosis, ulcerative colitis, and Crohn's
disease. Additional diseases that may be treated using hematopoietic stem and progenitor cell
transplant therapy include cancer, such as a cancer described herein. Exemplary stem cell
disorders are malignancies, such as a neuroblastoma or a hematologic cancers, such as
leukemia, lymphoma, and myeloma. In some embodiments, the cancer may be acute myeloid
leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia,
multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma. Additional
diseases treatable using hematopoietic stem or progenitor cell transplant therapy include
myelodysplastic syndrome. In some embodiments, the patient has or is otherwise affected by
a metabolic storage disorder. For example, the patient may suffer or otherwise be affected by
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a metabolic metabolicdisorder selected disorder from from selected the group consisting the group of glycogen consisting storage diseases, of glycogen storage diseases,
mucopolysaccharidoses, Gaucher's Disease, Hurlers Disease, sphingolipidoses,
metachromatic leukodystrophy, or any other diseases or disorders which may benefit from
the treatments and therapies disclosed herein and including, without limitation, severe
combined immunodeficiency, Wiscott-Aldrich syndrome, hyper immunoglobulin M (IgM)
syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis,
osteogenesis imperfecta, storage diseases, thalassemia major, sickle cell disease, systemic
sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis and
those diseases, or disorders described in "Bone Marrow Transplantation for Non-Malignant
Disease," ASH Education Book, 1:319-338 (2000), the disclosure of which is incorporated
herein by reference in its entirety as it pertains to pathologies that may be treated by
administration of hematopoietic stem or progenitor cell transplant therapy.
[0158] As used herein in the context of hematopoietic stem cell mobilization, the term "stem
cell niche" refers to a microenvironment within a donor, such as a mammalian donor (e.g., a
human donor) in which endogenous hematopoietic stem or progenitor cells reside. An
exemplary stem cell niche is bone marrow tissue.
[0159] As used herein, the terms "subject" and "patient" refer to an organism, such as a
human, that receives treatment for a particular disease or condition as described herein. In In
some embodiments, a patient, such as a human patient, that is in need of hematopoietic stem
cell transplantation may receive treatment that includes a population of hematopoietic stem
cells SO so as to treat a stem cell disorder, such as a cancer, autoimmune disease, or metabolic
disorder described herein. The hematopoietic stem cells that are transplanted into the patient
may be, for example, a population of hematopoietic stem cells that has been mobilized and
withdrawn from a donor in accordance with the compositions and methods described herein.
In some embodiments, the hematopoietic stem cells that are transplanted into the patient may
be mobilized within a donor by administration of a CXCR4 antagonist and/or a CXCR2
agonist to the donor.
[0160] As used herein, the term "transfection" refers to any of a wide variety of techniques
commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host
cell, such as electroporation, lipofection, calcium- phosphate precipitation, DEAE- dextran
transfection and the like.
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[0161] As used herein, the terms "treat" or "treatment" refer to therapeutic treatment, in
which the object is to prevent or slow down (lessen) an undesired physiological change or
disorder or to promote a beneficial phenotype in the patient being treated. Beneficial or
desired clinical results include, but are not limited to, promoting the engraftment of
exogenous hematopoietic cells in a patient following hematopoietic stem or progenitor cell
transplant therapy. In certain embodiments, the benefits include a more rapid engraftment of
transplanted cells, e.g., neutrophils and platelets. For example, in certain embodiments, using
the methods described herein, neutrophil recovery occurs within about 5-20 days post-
transplant, about 5-15 days post-transplant, about 5-10 days post-transplant, about 7-12 days
post-transplant, about 8-12 days post transplant, about 9-15 days post-transplant, about 10-15
days post-transplant, or about 10 days post-transplant. In certain embodiments, using the
methods described herein, platelet recovery occurs within about 10-20 days post-transplant,
about 10-15 days post-transplant, about 15-20 days post-transplant, about 12-18 days post-
transplant, about 12-17 days post transplant, about 13-18 days post-transplant, about 12-17
days post-transplant, or about 15 days post-transplant. Additional beneficial results include an
increase in the cell count or relative concentration of hematopoietic stem cells in a patient in
need of a hematopoietic stem or progenitor cell transplant following administration of an
exogenous hematopoietic stem or progenitor cell graft to the patient. Beneficial results of
therapy described herein may also include an increase in the cell count or relative
concentration of one or more cells of hematopoietic lineage, such as a megakaryocyte,
thrombocyte, platelet, erythrocyte, mast cell, myeoblast, basophil, neutrophil, eosinophil,
microglial cell, granulocyte, monocyte, osteoclast, antigen-presenting cell, macrophage,
dendritic cell, natural killer cell, T-lymphocyte, or B-lymphocyte, following and subsequent
hematopoietic stem cell transplant therapy. Additional beneficial results may include the
reduction in quantity of a disease-causing cell population, such as a population of cancer cells
or autoimmune cells.
[0162] As used herein, the terms "variant" and "derivative" are used interchangeably and
refer to naturally-occurring, synthetic, and semi-synthetic analogues of a compound, peptide,
protein, or other substance described herein. A variant or derivative of a compound, peptide,
protein, or other substance described herein may retain or improve upon the biological
activity of the original material.
[0163] As used herein, the term "vector" includes a nucleic acid vector, such as a plasmid, a
DNA vector, a plasmid, a RNA vector, virus, or other suitable replicon. Expression vectors
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described herein may contain a polynucleotide sequence as well as, for example, additional
sequence elements used for the expression of proteins and/or the integration of these
polynucleotide sequences into the genome of a mammalian cell. Certain vectors that can be
used for the expression of peptides and proteins, such as those described herein, include
plasmids that contain regulatory sequences, such as promoter and enhancer regions, which
direct gene transcription. Other useful vectors for expression of peptides and proteins
described herein contain polynucleotide sequences that enhance the rate of translation of
these genes or improve the stability or nuclear export of the mRNA that results from gene
transcription. These sequence elements may include, for example, 5' and 3' untranslated
regions and a polyadenylation signal site in order to direct efficient transcription of the gene
carried on the expression vector. The expression vectors described herein may also contain a
polynucleotide encoding a marker for selection of cells that contain such a vector. Examples
of a suitable marker include genes that encode resistance to antibiotics, such as ampicillin,
chloramphenicol, kanamycin, and nourseothricin.
[0164] As used herein, the term "alkyl" refers to a straight- or branched-chain alkyl group
having, for example, from 1 to 20 carbon atoms in the chain. Examples of alkyl groups
include methyl, ethyl, in-propyl, isopropyl, butyl, n-propyl, isopropyl, butyl, isobutyl, isobutyl, sec-butyl, sec-butyl, tert-butyl, tert-butyl, pentyl, pentyl,
isopentyl, tert-pentyl, hexyl, isohexyl, and the like.
[0165] As used herein, the term "alkylene" refers to a straight- or branched-chain divalent
alkyl group. alkyl group.The divalent The positions divalent may be positions on be may the on same theor same different atoms within or different the within atoms alkyl the alkyl
chain. Examples of alkylene include methylene, ethylene, propylene, isopropylene, and the
like. like.
[0166] As used herein, the term "heteroalkyl" refers to a straight or branched-chain alkyl
group having, for example, from 1 to 20 carbon atoms in the chain, and further containing
one or more heteroatoms (e.g., oxygen, nitrogen, or sulfur, among others) in the chain.
[0167] As used herein, the term "heteroalkylene" refers to a straight- or branched-chain
divalent heteroalkyl group. The divalent positions may be on the same or different atoms
within the heteroalkyl chain. The divalent positions may be one or more heteroatoms.
[0168] As used herein, the term "alkenyl" refers to a straight- or branched-chain alkenyl
group having, for example, from 2 to 20 carbon atoms in the chain. Examples of alkenyl
groups include vinyl, propenyl, isopropenyl, butenyl, tert-butylenyl, hexenyl, and the like.
WO wo 2019/113375 PCT/US2018/064335
[0169] As used herein, the term "alkenylene" refers to a straight- or branched-chain divalent
alkenyl group. The divalent positions may be on the same or different atoms within the
alkenyl chain. Examples of alkenylene include ethenylene, propenylene, isopropenylene,
butenylene, and the like.
[0170] As used herein, the term "heteroalkenyl" refers to a straight- or branched-chain
alkenyl group having, for example, from 2 to 20 carbon atoms in the chain, and further
containing one containing one or or more more heteroatoms heteroatoms (e.g., (e.g., oxygen, oxygen, nitrogen, nitrogen, or among or sulfur, sulfur, among others) in others) the in the
chain.
[0171] As used herein, the term "heteroalkenylene" refers to a straight- or branched-chain
divalent heteroalkenyl group. The divalent positions may be on the same or different atoms
within the heteroalkenyl chain. The divalent positions may be one or more heteroatoms.
[0172] As used herein, the term "alkynyl" refers to a straight- or branched-chain alkynyl
group having, for example, from 2 to 20 carbon atoms in the chain. Examples of alkynyl
groups include propargyl, butynyl, pentynyl, hexynyl, and the like.
[0173] As used herein, the term "alkynylene" refers to a straight- or branched-chain divalent
alkynyl group. The divalent positions may be on the same or different atoms within the
alkynyl chain.
[0174] As used herein, the term "heteroalkynyl" refers to a straight- or branched-chain
alkynyl group having, for example, from 2 to 20 carbon atoms in the chain, and further
containing one or more heteroatoms (e.g., oxygen, nitrogen, or sulfur, among others) in the
chain.
[0175] As used herein, the term "heteroalkynylene" refers to a straight- or branched-chain
divalent heteroalkynyl group. The divalent positions may be on the same or different atoms
within the heteroalkynyl chain. The divalent positions may be one or more heteroatoms.
[0176] As used herein, the term "cycloalkyl" refers to a monocyclic, or fused, bridged, or
spiro polycyclic ring structure that is saturated and has, for example, from 3 to 12 carbon ring
atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[3.1.0]hexane, and the like.
[0177] As used herein, the term "cycloalkylene" refers to a divalent cycloalkyl group. The
divalent positions may be on the same or different atoms within the ring structure. Examples
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of cycloalkylene include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and
the the like. like.
[0178] As used herein, the term "heterocycloalkyl" refers to a monocyclic, or fused, bridged,
or spiro polycyclic ring structure that is saturated and has, for example, from 3 to 12 ring
atoms per ring structure selected from carbon atoms and heteroatoms selected from, e.g.,
nitrogen, oxygen, and sulfur, among others. The ring structure may contain, for example, one
or more OXO groups on carbon, nitrogen, or sulfur ring members.
[0179] As used herein, the term "heterocycloalkylene" refers to a divalent heterocyclolalkyl
group. The divalent positions may be on the same or different atoms within the ring
structure.
[0180] As used herein, the term "aryl" refers to a monocyclic or multicyclic aromatic ring
system containing, for example, from 6 to 19 carbon atoms. Aryl groups include, but are not
limited to, phenyl, fluorenyl, naphthyl, and the like. The divalent positions may be one or
more heteroatoms.
[0181] As used herein, the term "arylene" refers to a divalent aryl group. The divalent
positions may be on the same or different atoms.
[0182] As used herein, the term "heteroaryl" refers to a monocyclic heteroaromatic, or a
bicyclic or a tricyclic fused-ring heteroaromatic group. Heteroaryl groups include pyridyl,
pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl,
1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadia-zolyl, 1,2,5-oxadiazolyl, 1,3,4-
oxadiazolyl, 1,3,4-triazinyl, 1,2,3-triazinyl, benzofuryl, [2,3-dihydro]benzofuryl,
isobenzofuryl, benzothienyl, benzotriazolyl, isobenzothienyl, indolyl, isoindolyl, 3H-indolyl,
benzimidazolyl, imidazo[1,2-a]pyridyl, benzothiazolyl, benzoxazolyl, quinolizinyl,
quinazolinyl, pthalazinyl, quinoxalinyl, cinnolinyl, napthyridinyl, pyrido[3,4-b]pyridyl,
pyrido[3,2-b]pyridyl, pyrido[3,2-b]pyridyl, pyrido[4,3-b]pyridyl, pyrido[4,3-b]pyridyl, quinolyl, quinolyl, isoquinolyl, isoquinolyl, tetrazolyl, tetrazolyl, 5,6,7,8- 5,6,7,8-
tetrahydroquinolyl, 5,6,7,8-tetrahydroisoquinolyl, purinyl, pteridinyl, carbazolyl, xanthenyl,
benzoquinolyl, and the like.
[0183] As used herein, the term "heteroarylene" refers to a divalent heteroaryl group. The
divalent positions may be on the same or different atoms. The divalent positions may be one
or more heteroatoms.
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[0184] Unless otherwise constrained by the definition of the individual substituent, the
foregoing chemical moieties, such as "alkyl", "alkylene", "heteroalkyl", "heteroalkylene",
"alkenyl", "alkenylene", "heteroalkenyl", "heteroalkenylene", "alkynyl", "alkynylene",
"heteroalkynyl", "heteroalkynylene", "cycloalkyl", "cycloalkylene", "heterocyclolalkyl",
heterocycloalkylene", "aryl," "arylene", "heteroaryl", and "heteroarylene" groups can
optionally be substituted. As used herein, the term "optionally substituted" refers to a
compound or moiety containing one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or
more) substituents, as permitted by the valence of the compound or moiety or a site thereof,
such as a substituent selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, alkyl aryl, alkyl heteroaryl, alkyl cycloalkyl, alkyl heterocycloalkyl, amino,
ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate,
aryl, heteroaryl, sulfinyl, sulfonyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano,
hydroxy, mercapto, nitro, and the like. The substitution may include situations in which
neighboring substituents have undergone ring closure, such as ring closure of vicinal
functional substituents, to form, for example, lactams, lactones, cyclic anhydrides, acetals,
hemiacetals, thioacetals, aminals, and hemiaminals, formed by ring closure, for example, to
furnish a protecting group.
Methods of Mobilizing Hematopoietic Stem and Progenitor Cells and Releasing Cells
for Expansion and Therapeutic Use
[0185] The present invention is based, in part, on the discovery that hematopoietic stem and
progenitor cells can be mobilized by administering particular doses of a CXCR2 agonist, such
as Gro-ß, Gro-B Gro-ß T, or a variant thereof, optionally in combination with a CXCR4 antagonist
to a mammalian donor (e.g., a human donor) while reducing the mobilization of other cell
types, such as leukocytes, neutrophils, lymphocytes, and monocytes. This property is
particularly beneficial in the context of hematopoietic stem cell transplant therapy, as
hematopoietic stem cells that are mobilized and isolated from a donor using the compositions
and method described herein have reduced quantities of cell types that are undesirable for
administration to a human patient suffering from a stem cell disorder.
[0186] Particularly, it has been discovered that CXCR2 agonists, such as Gro-B, Gro-ß, Gro-B Gro-ß T, or
a variant thereof, when administered intravenously at a dose of from about 50 ug/kg µg/kg to about
1 mg/kg, preferably from about 100 ug/kg µg/kg to about 250 ug/kg, µg/kg, and even more preferably at a
dose of about 150 ug/kg, µg/kg, exhibit the ability to rapidly mobilize hematopoietic stem and progenitor cells in a donor (e.g., a mammalian donor, such as a human donor) while reducing the mobilization of other cells of the hematopoietic lineage that may be undesirable for infusion into a patient (e.g., a mammalian patient, such as a human patient) that is undergoing hematopoietic stem cell transplant therapy. CXCR2 agonists, such as Gro-B, Gro-ß, Gro-B Gro-ß T, or a variant thereof, when administered at the above doses to a donor exhibit the ability to selectively mobilize hematopoietic stem cells as described in detail in Example 1, below.
[0187] When determining whether hematopoietic stem cells mobilized in a donor by
administration of a CXCR2 agonist, such as Gro-ß, Gro-B Gro-ß T, or a variant thereof, and
optionally, a CXCR4 antagonist, such as plerixafor or a pharmaceutically acceptable salt
thereof, are suitable for release for ex vivo expansion and/or for therapeutic use, one may
acquire an input value for each of one or more parameters set forth in TABLE 2 that
characterize a sample of peripheral blood of the donor. The one or more parameters may be
compared to the corresponding reference criterion for each parameter, and if the reference
criterion is satisfied by the ample of hematopoietic stem cells, the cells isolated from the
donor may be released for expansion ex vivo and/or for infusion into a patient for therapeutic
use (e.g., for the treatment of one or more stem cell disorders described herein).
[0188] Exemplary hematopoietic stem cell parameters and corresponding reference criteria
useful in conjunction with the compositions and methods described herein are set forth in
TABLE 2, below.
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TABLE 2. Hematopoietic stem cell population parameters and corresponding reference
criteria
Parameter Reference Parameter Category Parameter No. Criterion Ratio of CD34+ cells to 1 At least 0.0006 leukocytes Ratio of CD34+ cells to 2 At least 0.0009 leukocytes Ratio of CD34+ CD90 3 At least 0.0002 CD45RA cells to leukocytes Ratio of CD34+ CD90 4 At least 0.0003 CD45RA cells to leukocytes Ratio of CD34+ CD90 5 At least 0.0004 CD45RA cells to leukocytes Ratio of CD34+ cells to 6 At least 0.0011 neutrophils Ratio of CD34+ cells to 7 At least 0.0004 neutrophils Ratio of CD34+ cells Ratio of CD34+ CD90 (e.g., CD34+ CD90+ 8 At least 0.0006 CD45RA cells to neutrophils CD45RA cells) to CD90+ Ratio of CD34+ CD90 other cells of the 9 At least 0.0007 CD45RA cells to neutrophils hematopoietic Ratio of CD34+ cells to lineage within 10 At least 0.0020 lymphocytes peripheral blood Ratio of CD34+ cells to sample isolated from 11 11 At least 0.0025 lymphocytes mammalian donor Ratio of CD34+ CD90+ CD90 12 CD45RA cells to At least 0.0005 lymphocytes Ratio of CD34+ CD90 13 CD45RA cells to At least 0.0011
lymphocytes Ratio of CD34+ cells to 14 At least 0.0047 monocytes Ratio of CD34+ cells to 15 At least 0.0111 monocytes Ratio Ratio of of CD34+ CD34 CD90+ CD90 16 At least 0.0020 CD45RA cells to monocytes Ratio Ratio of of CD34+ CD34 CD90+ CD90 17 At least 0.0039 CD45RA cells to monocytes Frequency ofofCD34+ Frequency CD34cells cells Proportion ProportionofofCD34+ CD34 relative to total quantity of At least 0.051% 18 cells cells (e.g., (e.g.,CD34+ CD34 cells in sample isolated from
CD90 CD45RA donor cells) in peripheral Frequency of CD34 cells blood sample isolated relative to total quantity of 19 At least 0.097% from donor cells in sample isolated from
donor
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Frequency FrequencyofofCD34 CD34CD90+ CD90 CD45RA- cells relative to 20 At least 0.020% total quantity of cells in
sample isolated from donor Frequency of Frequency ofCD34+ CD34 CD90+ CD90 CD45RA cells relative to 21 At least 0.066% total quantity of cells in
sample isolated from donor
[0189] In selecting parameters for determining whether a population of hematopoietic stem
cells obtained from a donor (e.g., a mammalian donor, such as a human donor) is suitable for
release for ex vivo expansion or therapeutic use, one may select one or more input parameters
listed in TABLE 2. In some embodiments, one may select an individual parameter from
parameter numbers 1-21. Alternatively, one may select a combination of parameters, such as
a CD34+ cellratio CD34 cell ratioparameter parameter(e.g., (e.g.,one oneor ormore moreof ofparameter parameternumbers numbers1-17 1-17in inTABLE TABLE2) 2)and and
a frequency parameter (e.g., one or more of parameter numbers 18-21 listed in TABLE 2).
In some embodiments, the parameters used for determining whether a population of
hematopoietic stem cells obtained from a donor (e.g., a mammalian donor, such as a human
donor) is suitable for release for ex vivo expansion or therapeutic use are a combination of
parameters as set forth in any one of TABLES 3-6, below.
TABLE 3. Two-way combinations of hematopoietic stem cell population parameters for
assessment
First Parameter Second No. Parameter No. 1 6 1 7 1 8 1 9 2 6 2 7 2 8 2 9 3 6 3 7 3 8 3 9 4 6 4 7 4 8 4 9 5 6
First Parameter Second Second No. Parameter No. 5 7 7 5 8 5 9 1 10 1 11 1 12 1 13 2 10 2 11
2 12 2 13 3 10 3 11
3 12 3 13 13 4 10 4 11
4 12 4 13 5 10 5 11
5 12 5 13 1 14 1 15 1 16 1 17 2 14 2 15 2 16 2 17 3 3 14 3 15 3 16 3 17 4 14 4 15 4 16 4 17 5 14 5 15 5 16 5 17 1 18 1 19 1 20
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First Parameter Second Second No. Parameter No. 1 21 2 18 2 19 2 20 2 21 3 18 3 19 3 20 3 21 4 18 4 19 4 20 4 21 5 18 5 19 5 20 5 21 6 10 6 11
6 12 6 13 7 10 7 11
7 12 7 13 8 10 8 11
8 12 8 13 9 10 9 11
9 12 9 13 6 14 6 15 6 16 6 17 7 14 7 15 7 16 7 17 8 14 8 15 8 16 8 17 9 14
First Parameter Second No. Parameter No. 9 15 9 16 9 17 6 18 6 19 6 20 6 21 7 18 7 19 7 20 7 21 8 18 8 19 8 20 8 21 9 18 9 19 9 20 9 21 10 14 10 15 10 16 10 17 11 14 11 11 15 11 16 11 17 12 14 12 15 12 16 12 17 13 14 13 15 13 16 13 17 10 18 10 19 10 20 10 21 11 11 18 11 11 19 11 20 11 11 21 12 18 12 19 12 20
First Parameter Second No. Parameter No. 12 21 13 18 13 19 13 20 13 21 14 18 14 19 14 20 14 21 15 18 15 19 15 20 15 21 16 18 16 19 16 20 16 21 17 17 18 17 19 17 20 17 21
TABLE 4. Three-way combinations of hematopoietic stem cell population parameters for
assessment
First Parameter Second Second Parameter Parameter Third ThirdParameter Parameter No. No. No. 1 6 10 1 7 10 1 8 10 1 9 10 2 6 10 2 7 10 2 8 10 2 9 10 3 6 10 3 7 10 3 8 10 3 9 10 4 6 10 4 7 10 4 8 10 4 9 10 5 6 10
First Parameter Second Parameter Third Third Parameter Parameter No. No. No. 5 7 10 5 8 10 5 9 10 1 11 6 1 11 7 1 8 11 11 1 11 9 2 6 11
2 7 11
2 8 11
2 9 11 11
3 6 11 11
3 7 11
3 8 11
3 9 11
4 6 11 4 7 11 11
4 8 11
4 9 11
5 6 11
5 7 11 5 8 11
5 9 11 1 6 12 1 7 12 1 8 12 1 9 12 2 6 12 2 7 7 12 2 8 12 2 9 12 3 6 12 3 7 7 12 3 8 12 3 9 12 4 6 12 4 7 7 12 4 8 12 4 9 12 5 6 12 5 7 7 12 5 8 12 5 9 12 1 6 13 1 7 13 1 8 13
First Parameter Second Parameter Third Third Parameter Parameter No. No. No. 1 9 13 2 6 13 2 7 13 2 8 13 13 2 9 13 3 6 13 3 7 13 3 8 13 3 9 13 4 6 13 13 4 7 13 4 8 13 4 9 13 5 6 13 5 7 13 5 8 13 5 9 13 1 6 14 1 7 14 1 8 14 1 14 9 2 6 14 2 7 14 2 8 14 2 9 14 3 6 14 3 7 14 3 8 14 3 9 14 4 6 14 4 7 14 4 8 14 4 9 14 5 6 14 5 7 14 5 8 14 5 9 14 1 6 15 1 7 15 1 8 15 1 9 15 2 6 15 2 7 15 2 8 15 2 9 15 3 6 15
First Parameter Second Parameter Third Parameter No. No. No. 3 7 15 3 8 15 3 9 15 4 6 15 4 7 15 4 8 15 4 9 15 5 6 15 5 7 15 5 8 15 5 9 15 1 16 6 1 7 16 1 8 16 1 9 16 2 6 16 2 7 16 2 8 16 2 9 16 3 6 16 3 7 16 3 8 16 3 9 16 4 6 16 4 7 16 4 8 16 4 9 16 5 6 16 5 7 16 5 8 16 5 9 16 1 6 17 1 7 17 1 8 17 1 17 9 2 6 17 2 7 17 2 8 17 2 9 17 3 6 17 3 7 17 3 8 17 3 9 17 4 6 17 4 7 17 4 8 17
First Parameter Second Parameter Third Third Parameter Parameter No. No. No. 4 9 17 5 6 17 5 7 17 5 8 17 5 9 17 1 6 18 1 18 7 1 8 18 1 9 18 2 6 18 2 7 18 2 8 18 2 9 18 3 6 18 3 7 18 3 8 18 3 9 18 4 6 18 4 7 7 18 4 8 18 4 9 18 5 6 18 5 7 18 5 8 18 5 9 18 1 6 19 1 7 19 1 8 19 1 9 19 2 6 19 2 7 7 19 2 8 19 2 9 19 3 6 19 3 7 19 3 8 19 3 9 19 4 6 19 4 7 19 4 8 19 4 9 19 5 6 19 5 7 19 5 8 19 5 9 19 1 6 20
First Parameter Second Parameter Third Parameter No. No. No. 1 7 20 1 8 20 1 9 20 2 6 20 2 7 20 2 8 20 2 9 20 3 6 20 3 7 20 3 8 20 3 9 20 4 6 20 4 7 20 4 8 20 4 9 20 5 6 20 5 7 20 5 8 20 5 9 20 1 6 21 1 7 21 1 8 21 1 9 21 2 6 21 2 7 21 2 8 21 21 2 9 21 3 6 21 3 7 21 3 8 21 3 9 21 4 6 21 4 7 7 21 4 8 21 4 9 21 5 6 21 5 7 21 5 8 21 5 9 21 1 10 14 1 11 14 1 12 14 1 13 14 2 10 14 2 11 14 2 12 14
First Parameter Second Parameter Third Third Parameter Parameter No. No. No. 2 13 14 3 10 14 3 11 14 3 12 14 3 13 14 4 10 14 4 11 14 4 12 14 4 13 14 5 10 14 5 11 14 5 12 14 5 13 14 1 10 15 1 11 15 1 12 15 1 13 15 2 10 15 2 11 15 2 12 15 2 13 15 3 10 15 3 11 15 3 12 15 3 13 15 4 10 15 4 11 15 4 12 15 4 13 15 5 10 15 5 11 11 15 5 12 15 5 13 15 1 10 16 1 11 16 1 12 16 1 13 16 2 10 16 2 11 16 2 12 16 2 13 16 3 10 16 3 11 16 3 12 16 3 13 16 4 10 16
First Parameter Second Parameter Third Parameter No. No. No. 4 11 16 4 12 16 4 13 16 5 10 16 5 11 16 5 12 16 5 13 16 1 10 17 1 11 11 17 1 12 17 1 13 17 2 10 17 2 11 17 2 12 17 2 13 17 3 10 17 3 11 11 17 3 12 17 3 13 17 4 10 17 4 11 17 4 12 17 4 13 17 5 10 17 5 11 17 5 12 17 5 13 17 1 10 18 1 11 11 18 1 12 18 1 13 18 2 10 18 2 11 18 2 12 18 2 13 18 3 10 18 3 11 18 3 12 18 3 13 18 4 10 18 4 11 11 18 4 12 18 4 13 18 5 10 18 5 11 18 5 12 18
First Parameter Second Parameter Third Parameter No. No. No. 5 13 18 1 10 19 1 11 11 19 1 12 19 1 13 19 2 10 19 2 11 19 2 12 19 2 13 19 3 10 19 3 11 19 3 12 19 3 13 19 4 10 19 4 11 19 4 12 19 4 13 19 5 10 19 5 11 19 5 12 19 5 13 19 1 10 20 1 11 20 1 12 20 1 13 20 2 10 20 2 11 20 2 12 20 2 13 20 3 10 20 3 11 20 3 12 20 3 13 20 4 10 20 4 11 20 4 12 20 4 13 20 5 10 20 5 11 20 5 12 20 5 13 20 1 1 10 21 1 11 11 21 1 12 21 1 13 21 2 10 21
First Parameter Second Parameter Third Parameter No. No. No. 2 11 21 2 12 21 2 13 21 3 10 21 3 11 21 3 12 21 3 13 21 4 10 21 4 11 21 4 12 21 4 13 21 5 10 21 5 11 21 5 12 21 5 13 21 1 14 18 1 15 18 1 16 18 1 17 18 2 14 18 2 15 18 2 16 18 2 17 17 18 3 14 18 3 15 18 3 16 18 3 17 18 4 14 18 4 15 18 4 16 18 4 17 18 5 14 18 5 15 18 5 16 18 5 17 18 1 14 19 1 15 19 1 16 19 1 17 19 2 14 19 2 15 19 2 16 19 2 17 19 3 14 19 3 15 19 3 16 19
First Parameter Second Parameter Third Parameter No. No. No. 3 17 19 4 14 19 4 15 19 4 16 19 4 17 19 5 14 19 5 15 19 5 16 19 5 17 19 1 14 20 1 15 20 1 16 20 1 17 20 2 14 20 2 15 20 2 16 20 2 17 20 3 14 20 3 15 20 3 16 20 3 17 20 4 14 20 4 15 20 4 16 20 4 17 20 5 14 20 5 15 20 5 16 20 5 17 20 1 14 21 1 15 21 1 16 21 1 17 21 2 14 21 2 15 21 2 16 21 2 17 21 3 14 21 3 15 21 3 16 21 3 17 21 4 14 21 4 15 21 4 16 21 4 17 21 5 14 21
First Parameter Second Parameter Third Third Parameter Parameter No. No. No. 5 15 21 5 16 21 5 17 21 6 10 14 6 11 14 6 12 14 6 13 14 7 10 14 7 11 11 14 7 12 14 7 13 14 8 10 14 8 11 14 8 12 14 8 13 14 9 10 14 9 11 14 9 12 14 9 13 14 6 10 15 6 11 11 15 6 12 15 6 13 15 7 10 15 7 11 15 7 12 15 7 13 15 8 10 15 8 11 11 15 8 12 15 8 13 15 9 10 15 9 11 15 9 12 15 9 13 15 6 10 16 6 11 16 6 12 16 6 13 16 7 10 16 7 11 16 7 7 12 16 7 13 16 8 10 16 8 11 16 8 12 16
First Parameter Second Second Parameter Parameter Third ThirdParameter Parameter No. No. No. 8 13 16 9 10 16 9 11 16 9 12 16 9 13 16 6 10 17 6 11 17 6 12 12 17 6 13 17 7 7 10 17 7 11 17 7 7 12 17 7 13 17 8 10 17 8 11 17 8 12 17 8 13 17 9 10 17 9 11 17 9 12 17 9 13 17 6 10 18 6 11 18 6 12 18 6 13 18 7 10 18 7 11 18 7 12 18 7 7 13 18 8 10 18 8 11 18 8 12 18 8 13 18 9 10 18 9 11 18 9 12 18 9 13 18 6 10 19 6 11 19 6 12 19 6 13 19 7 7 10 19 7 11 19 7 12 19 7 13 19 8 10 19
First Parameter Second Second Parameter Parameter Third ThirdParameter Parameter No. No. No. 8 11 11 19 8 12 19 8 13 19 9 10 19 9 11 19 9 12 19 9 13 19 6 10 20 6 11 20 6 12 20 6 13 20 7 10 20 7 7 11 20 7 12 20 7 13 20 8 10 20 8 11 20 8 12 20 8 13 20 9 10 20 9 11 11 20 9 12 20 9 13 20 6 10 21 6 11 21 6 12 21 6 13 21 21 7 10 21 7 7 11 21 7 12 21 7 7 13 21 8 10 21 8 11 21 8 12 21 8 13 21 9 10 21 9 11 21 9 12 21 9 13 21 6 14 18 6 15 18 6 16 18 6 17 18 7 14 18 7 15 18 7 16 18
First Parameter Second Parameter Third Third Parameter Parameter No. No. No. 7 17 18 8 14 18 8 15 18 8 16 18 8 17 18 9 14 18 9 15 18 9 16 18 9 17 18 6 14 19 6 15 19 6 16 19 6 17 19 7 7 14 19 7 15 19 7 16 19 7 17 19 8 14 19 8 15 19 8 16 16 19 8 17 19 9 14 19 9 15 19 9 16 19 9 17 19 6 14 20 6 15 20 6 16 16 20 6 17 20 7 14 20 7 15 20 7 16 20 7 17 17 20 8 14 20 8 15 20 8 16 20 8 17 20 9 14 20 9 15 20 9 16 20 9 17 20 6 14 21 6 15 21 6 16 21 6 17 21 7 14 21
Third Parameter First Parameter Second Parameter Third Parameter No. No. No. 7 7 15 21 7 16 21 7 17 21 8 14 21 8 15 21 8 16 21 21 8 17 21 21 9 14 21 9 15 21 9 16 21 9 17 17 21 21 10 14 18 10 15 18 10 16 18 10 17 18 11 14 18 11 15 18 11 16 18 11 17 18 12 14 18 12 15 18 12 16 18 12 17 18 13 14 18 13 15 18 13 16 18 13 17 18 10 14 19 10 15 19 10 16 19 10 17 19 11 14 19 11 15 19 11 16 19 11 17 19 12 14 19 12 15 19 12 16 19 12 17 17 19 13 14 19 13 15 19 13 16 19 13 17 17 19 10 14 20 10 15 20 10 16 20
First Parameter Second Parameter Third Parameter No. No. No. 10 10 17 17 20 11 14 20 11 15 20 11 16 20 11 17 20 12 14 14 20 12 15 20 12 16 20 12 17 17 20 13 14 20 13 15 20 13 16 20 13 17 17 20 10 14 21 10 15 21 10 16 16 21 21 10 17 21 11 14 21 11 15 21 11 16 21 11 17 17 21 12 14 21 12 15 21 12 16 21 12 17 21 13 14 21 13 15 21 13 16 21 13 17 21
TABLE 5. Four-way combinations of hematopoietic stem cell population parameters for
assessment
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 1 6 10 14 1 7 10 14 1 8 10 14 1 9 10 14 2 6 10 14 2 7 10 14 2 8 10 14 2 9 10 10 14 3 6 10 14 3 7 10 14 3 8 10 14
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 3 9 10 14
4 6 10 14 4 7 10 10 14 4 8 10 14
4 9 10 14 5 6 10 14 5 7 10 14 5 8 10 14 5 9 10 14 1 6 11 11 14 1 1 11 11 14 7 1 8 11 11 14 1 9 11 14 2 6 11 14 2 7 11 14 2 8 11 11 14 2 9 11 11 14 3 6 11 14 3 7 11 14 3 8 11 11 14 3 9 11 14 4 6 11 11 14
4 7 11 14 4 8 11 14
4 9 11 11 14 5 6 11 14 5 7 11 11 14 5 8 11 14 5 9 11 11 14 1 12 14 6 1 7 12 14 1 8 12 14 1 14 9 12 2 6 12 14 2 7 12 14 2 8 12 14 2 9 12 14 3 6 12 14 3 7 12 14 3 8 12 14 3 9 12 14 4 6 12 14
4 7 12 14 4 8 12 14 4 9 12 14 5 6 12 14
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 5 7 12 14 5 8 12 14 5 9 12 14 1 6 13 14 1 7 13 14 1 8 13 14 1 13 14 9 2 6 13 14 2 7 13 14 2 8 13 14 2 9 13 14 3 6 13 14 3 7 13 14 3 8 13 14 3 9 13 14 4 6 13 14 4 7 13 14 4 8 13 14 4 9 13 14 5 6 13 14 5 7 13 14 5 8 13 14 5 9 13 14 1 6 10 15 1 7 10 15 1 8 10 15 1 10 15 9 2 6 10 15 2 7 10 15 2 8 10 15 2 9 10 15 3 6 10 15 3 7 10 15 3 8 10 15 3 9 10 15 4 6 10 15 4 7 10 15 4 8 10 15 4 9 10 15 5 6 10 15 5 7 10 15 5 8 10 15 5 9 10 15 1 11 6 11 15 1 11 7 11 15 1 8 11 11 15
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 1 11 9 15 2 6 11 11 15 2 7 11 15 2 8 11 15 2 9 11 11 15 3 6 11 15 3 7 11 11 15 3 8 11 15 3 9 11 15
4 6 11 11 15 4 7 11 15 4 8 11 11 15 4 9 11 11 15 5 6 11 11 15 5 7 11 15 5 8 11 11 15 5 9 11 11 15 1 6 12 15 1 7 12 15 1 8 12 15 1 12 15 9 2 6 12 15 2 7 12 15 2 8 12 15 2 9 12 15 3 6 12 15 3 7 12 15 3 8 12 15 3 9 12 15
4 6 12 15 4 7 12 15 4 8 12 15 4 9 12 15 5 6 12 15 5 7 12 15 5 8 12 15 5 9 12 15 1 6 13 15 1 7 13 15 1 8 13 15 1 13 15 9 2 6 13 15 2 7 13 15 2 8 13 13 15 2 9 13 15 3 6 13 15
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 3 7 13 15 3 8 13 15 3 9 13 15 4 6 13 15 4 7 13 15 4 8 13 15 4 9 13 15 5 6 13 15 5 7 13 15 5 8 13 15 5 9 13 15 1 10 16 6 1 7 10 16 1 8 10 16 1 10 16 9 2 6 10 16 2 7 10 16 2 8 10 16 2 9 10 16 3 6 10 16 3 7 10 16 3 8 10 16 3 9 10 16 4 6 10 16
4 7 10 16 4 8 10 16 4 9 10 16 5 6 10 16 5 7 10 16 5 8 10 16 5 9 10 16 1 11 6 16 1 11 7 16 1 8 11 11 16 1 11 11 16 9 2 6 11 16 2 7 11 16 2 8 11 16 2 9 11 11 16 3 6 11 11 16 3 7 11 16 3 8 11 11 16 3 9 11 16 4 6 11 11 16 4 7 11 11 16 4 8 11 11 16
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 4 9 11 16 5 6 11 16 5 7 11 11 16 5 8 11 11 16 5 9 11 11 16 1 6 12 16 1 12 16 7 1 8 12 16 1 12 16 9 2 6 12 16 2 7 12 16 2 8 12 16 2 9 12 16 3 6 12 16 3 7 12 16 3 8 12 16 3 9 12 16 4 6 12 16 4 7 12 16
4 8 12 16 4 9 12 16 5 6 12 16 5 7 12 16 5 8 12 16 5 9 12 16 1 6 13 16 1 13 16 7 1 8 13 16 11 13 16 9 2 6 13 16 2 7 13 16 2 8 13 16 2 9 13 16 3 6 13 16 3 7 13 16 3 8 13 16 3 9 13 16
4 6 13 16 4 7 13 16 4 8 13 16 4 9 13 16 5 6 13 16 5 7 13 16 5 8 13 16 5 9 13 13 16 1 6 10 17
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 1 17 7 10 1 10 17 8 10 1 10 17 9 17 2 6 10 17 2 7 10 17 2 8 10 17 2 9 10 17 3 6 10 17 3 7 10 17 3 8 10 17 3 9 10 17
4 6 10 10 17 4 7 10 17 4 8 10 17 4 9 10 17 5 6 10 17 5 7 10 17 5 8 10 17 5 9 10 17 1 11 17 6 1 11 17 7 1 8 11 17 1 11 17 9 2 6 11 17 2 7 11 11 17 2 8 11 17 2 9 11 11 17 3 6 11 17 3 7 11 17 3 8 11 17 3 9 11 17 4 6 11 17 17 4 7 11 11 17 4 8 11 17
4 9 11 17 5 6 11 11 17 5 7 11 11 17 5 8 11 11 17 5 9 11 11 17 1 12 17 6 1 12 17 7 1 8 12 17 1 12 17 9 2 6 12 17 2 7 12 17 2 8 12 17
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 2 9 12 17 3 6 12 17 3 7 12 17 3 8 12 17 3 9 12 17 4 6 12 17 4 7 12 17 4 8 12 17 4 9 12 17 5 6 12 17 5 7 12 17 5 8 12 17 5 9 12 17 1 13 17 6 1 13 17 7 1 13 17 8 1 13 17 9 2 6 13 17 2 7 13 17 2 8 13 17 2 9 13 17 3 6 13 17 3 7 13 17 3 8 13 17 3 9 13 17 4 6 13 17 4 7 13 17 4 8 13 17 4 9 13 17 5 6 13 17 5 7 13 17 5 8 13 17 5 9 13 17 1 6 10 18 1 10 18 7 1 8 10 18 1 10 18 9 2 6 10 18 2 7 10 18 2 8 10 18 2 9 10 18 3 6 10 18 3 7 10 18 3 8 10 18 3 9 10 18 4 6 10 10 18
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 4 4 7 10 18 18 4 8 10 10 18 4 9 10 18 5 6 10 18 5 7 10 18 5 8 10 18 18 5 9 10 18 1 6 11 18 11 7 11 18 18 11 8 11 18 1 9 11 18 2 6 11 18 2 7 11 18 2 8 11 18 2 9 11 18 3 6 11 18 3 7 11 18 3 8 11 18 18 3 9 11 18 18 4 6 11 18 18 4 7 11 18 18 4 4 8 11 18 4 9 11 18 18 5 6 11 18 5 7 11 18 18 5 8 11 18 5 9 11 18 1 12 18 6 11 12 18 7 1 8 12 18 18 1 9 12 18 2 6 12 18 2 7 12 12 18 2 8 12 18 2 9 12 18 18 3 6 12 18 3 7 12 18 3 8 12 18 3 9 12 18 18 4 6 12 18 18 4 7 12 18 4 8 12 18 4 9 12 18 5 6 12 18 18 5 7 12 18 18 5 8 12 18
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 5 9 12 18 1 6 13 18 1 7 13 18 11 8 13 18 11 13 18 9 2 6 13 18 2 7 13 18 2 8 13 18 2 9 13 18 3 6 13 18 3 7 13 18 3 8 13 18 3 9 13 18 4 6 13 18 4 7 13 18 4 8 13 18 4 9 13 18 5 6 13 18 5 7 13 18 5 8 13 18 5 9 13 18 1 10 19 6 1 10 19 7 1 8 10 19 1 1 10 19 9 2 6 10 19 2 7 10 19 2 8 10 19 2 9 10 19 3 6 10 19 3 7 10 10 19 3 8 10 19 3 9 10 19 4 6 10 19
4 7 10 19 4 8 10 10 19 4 9 10 19 5 6 10 19 5 7 10 19 5 8 10 19 5 9 10 19 1 11 11 19 6 1 11 11 19 7 1 11 19 8 1 11 19 9 2 6 11 19
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 2 7 11 19 19 2 8 11 19 2 9 11 19 3 6 11 19 3 7 11 19 3 8 11 19 3 9 11 19 4 6 11 19 4 7 11 19
4 8 11 19 4 4 9 11 19 5 6 11 19 5 7 11 19 5 8 11 19 5 9 11 19 11 12 19 6 1 12 19 7 1 8 12 19 1 9 12 19 2 6 12 19 2 7 12 19 2 8 12 19 2 9 12 19 3 6 12 19 3 7 12 19 3 8 12 19 3 9 12 19 4 6 12 19 4 7 12 19
4 8 12 19 4 9 12 19 5 6 12 19 5 7 12 12 19 5 8 12 19 5 9 12 19 11 6 13 19 1 7 13 19 1 8 13 13 19 1 1 13 19 19 9 2 6 13 19 2 7 13 19 2 8 13 19 2 9 13 19 3 6 13 19 3 7 13 19 3 8 13 19
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 3 9 13 19
4 6 13 19 4 7 13 19 4 8 13 19
4 9 13 19 5 6 13 19 5 7 13 19 5 8 13 19 5 9 13 19 11 6 10 20 1 1 10 7 20 20 1 8 10 20 1 10 9 20 2 6 10 20 2 7 10 20 2 8 10 20 2 9 10 20 3 6 10 20 3 7 10 20 3 8 10 20 3 9 10 20 4 6 10 20 4 7 10 20 4 8 10 20 4 9 10 20 5 6 10 20 5 7 10 20 5 8 10 20 5 9 10 20 1 11 11 6 20 1 7 11 20 1 8 11 20 1 11 11 9 20 2 6 11 20 2 7 11 20 2 8 11 20 2 9 11 11 20 3 6 11 11 20 3 7 11 11 20 3 8 11 11 20 3 9 11 20 4 6 11 20 4 7 11 20 4 8 11 20 4 9 11 11 20 5 6 11 20
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 5 7 11 11 20 5 8 11 20 5 9 11 20 1 6 12 20 11 7 12 20 1 8 12 20 1 12 9 20 2 6 12 20 2 7 12 20 2 8 12 20 2 9 12 20 3 6 12 12 20 3 7 12 20 3 8 12 20 3 9 12 20 4 6 12 20 4 7 12 20 4 8 12 20 4 9 12 20 5 6 12 20 5 7 12 20 5 8 12 20 5 9 12 20 1 6 13 20 1 7 13 20 1 8 13 20 1 13 9 20 2 6 13 20 2 7 13 20 2 8 13 20 2 9 13 20 3 6 13 20 3 7 13 20 3 8 13 20 3 9 13 20 4 6 13 20 4 7 13 20 4 8 13 13 20 4 9 13 13 20 5 6 13 20 5 7 13 20 5 8 13 20 5 9 13 20 1 6 10 21 21 1 7 10 21 1 8 10 10 21
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 1 9 10 21 2 6 10 21 2 7 10 21 2 8 10 21 2 9 10 21 3 6 10 21 3 7 10 21 3 8 10 21 3 9 10 21
4 6 10 21 4 4 7 10 21 4 8 10 21 4 9 10 21 5 6 10 21 5 7 10 21 5 8 10 21 5 9 10 21 1 1 11 6 11 21 1 7 11 21 1 8 11 21 11 11 9 21 2 6 11 21 2 7 11 21 2 8 11 21 2 9 11 11 21 3 6 11 21 3 7 11 21 3 8 11 21 3 9 11 21
4 6 11 21 4 7 11 21 4 8 11 21 4 9 11 21 5 6 11 21 5 7 11 21 5 8 11 21 5 9 11 21 1 6 12 21 1 7 12 21 1 8 12 21 1 12 21 9 2 6 12 21 2 7 12 21 2 8 12 21 2 9 12 21 3 6 12 21
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 3 7 12 21 3 8 12 12 21 3 9 12 12 21 4 6 12 21 4 7 12 21 21 4 8 12 21 4 9 12 21 5 6 12 21 21 5 7 12 21 5 8 12 21 21 5 9 12 21 1 13 21 6 1 7 13 21 1 8 13 21 1 9 13 21 2 6 13 21 2 7 13 21 2 8 13 21 2 9 13 21 3 6 13 21 21 3 7 13 21 3 8 13 21 3 9 13 21 4 6 13 21 21 4 7 13 21 4 8 13 21 4 9 13 21 21 5 6 13 21 21 5 7 13 21 21 5 8 13 13 21 5 9 13 21 1 6 14 18 1 7 14 18 1 8 14 18 1 14 18 9 2 6 14 18 2 7 14 18 2 8 14 18 2 9 14 18 3 6 14 18 3 7 14 18 3 8 14 18 3 9 14 18 4 6 14 18 4 7 14 18 4 8 14 18
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 4 9 14 18 5 6 14 18 5 7 14 18 5 8 14 18 5 9 14 18 1 6 15 18 1 15 18 7 1 8 15 18 1 15 18 9 2 6 15 18 2 7 15 18 2 8 15 18 2 9 15 18 3 6 15 18 3 7 15 18 3 8 15 15 18 3 9 15 18 4 6 15 18 4 7 15 18
4 8 15 18 4 9 15 18 5 6 15 18 5 7 15 18 5 8 15 18 5 9 15 18 18 1 6 16 18 1 16 18 7 1 8 18 16 1 16 18 9 2 6 16 18 2 7 16 18 2 8 16 18 2 9 16 18 3 6 16 18 3 7 16 18 18 3 8 16 16 18 3 9 16 18
4 6 16 18 4 7 16 18 4 4 8 16 18 4 9 16 18 5 6 16 18 5 7 16 18 5 8 16 18 18 5 9 16 16 18 1 6 17 17 18
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 1 18 7 17 1 8 17 17 18 1 9 17 18 2 6 17 18 2 7 17 18 2 8 17 18 2 9 17 18 3 6 17 18 3 7 17 18 3 8 17 18 3 9 17 18 4 6 17 17 18 4 7 17 18 4 8 17 17 18 4 9 17 18 5 6 17 18 5 7 17 18 5 8 17 18 5 9 17 18 1 14 19 6 1 19 7 14 1 8 14 19 1 14 19 9 14 2 6 14 19 2 7 14 19 2 8 14 19 2 9 14 19 3 6 14 19 3 7 14 19 3 8 14 19 3 9 14 19 4 6 14 19 4 7 14 19 4 8 14 19
4 9 14 19 5 6 14 19 5 7 14 19 5 8 14 19 5 9 14 19 1 15 19 6 1 15 19 7 1 8 15 19 1 15 19 9 2 6 15 19 2 7 15 19 2 8 15 19
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 2 9 15 19 3 6 15 19 3 7 15 19 3 8 15 19 3 9 15 19 4 6 15 19 4 7 15 19 4 8 15 19 4 9 15 19 5 6 15 19 5 7 15 19 5 8 15 19 5 9 15 19 1 16 16 19 6 1 16 19 7 16 1 19 8 16 1 16 19 9 2 6 16 19 2 7 16 16 19 2 8 16 19 2 9 16 19 3 6 16 16 19 3 7 16 16 19 3 8 16 19 3 9 16 19 4 6 16 19 4 7 16 16 19 4 8 16 19 4 9 16 16 19 5 6 16 19 5 7 16 19 5 8 16 16 19 5 9 16 19 1 6 17 19 1 17 19 7 1 8 17 19 17 1 17 19 9 2 6 17 19 2 7 17 19 2 8 17 19 2 9 17 17 19 3 6 17 19 3 7 17 19 3 8 17 17 19 3 9 17 19 4 6 17 17 19
First Parameter Second Second Third Parameter Fourth No. Parameter No. No. Parameter No. 4 7 17 19
4 8 17 19 4 9 17 19 5 6 17 19 5 7 17 19 5 8 17 19 5 9 17 19 1 6 14 20 1 7 14 20 11 8 14 20 1 1 9 14 20 2 6 14 20 2 7 14 20 2 8 14 20 2 9 14 20 3 6 14 20 3 7 14 20 3 8 14 20 3 9 14 20 4 6 14 20 4 7 14 20 4 8 14 20 4 9 14 20 5 6 14 20 5 7 14 20 5 8 14 20 5 9 14 20 1 15 6 20 11 15 7 20 11 8 15 20 1 9 15 20 2 6 15 20 2 7 15 20 2 8 15 20 2 9 15 20 3 6 15 20 3 7 15 20 3 8 15 20 3 9 15 20 4 6 15 20 4 7 15 20 4 8 15 20 4 9 15 20 5 6 15 20 5 7 15 20 5 8 15 20
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 5 9 15 20 1 6 16 16 20 11 7 16 20 11 8 16 20 11 16 9 20 2 6 16 20 2 7 16 20 2 8 16 20 2 9 16 20 3 6 16 20 3 7 16 20 3 8 16 20 3 9 16 20 4 6 16 20 4 7 16 20 4 8 16 20 4 4 9 16 20 5 6 16 20 5 7 16 20 5 8 16 20 5 9 16 20 1 17 6 20 1 7 17 20 11 8 17 20 1 1 17 9 17 20 2 6 17 20 2 7 17 20 2 8 17 20 2 9 17 20 3 6 17 20 3 7 17 20 3 8 17 20 3 9 17 20 4 6 17 20 4 7 17 20 4 8 17 20 4 9 17 20 5 6 17 20 5 7 17 20 5 8 17 20 5 9 17 20 1 6 14 21 1 14 7 21 1 8 14 21 11 21 9 14 2 6 14 21
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 2 7 14 21 2 8 14 21 2 9 14 21 3 6 14 21 3 7 14 21 3 8 14 21 3 9 14 21 4 6 14 21 4 7 14 21 4 8 14 21 4 4 9 14 21 5 6 14 21 5 7 14 21 5 8 14 21 5 9 14 21 11 15 6 21 1 15 7 15 21 1 8 15 21 1 15 9 21 2 6 15 21 2 7 15 21 2 8 15 21 2 9 15 21 21 3 6 15 21 3 7 15 21 3 8 15 21 3 9 15 21
4 6 15 21 21 4 7 15 21
4 8 15 21 4 9 15 21 5 6 15 21 5 7 15 21 5 8 15 21 5 9 15 21 1 16 6 21 1 16 21 7 1 8 16 21 1 1 16 21 9 2 6 16 21 2 7 16 21
2 8 16 21 2 9 16 21 3 6 16 21 3 7 16 21 3 8 16 21
151
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 3 9 16 21 4 6 16 21 4 7 16 21 4 8 16 21 4 9 16 21 5 6 16 21 5 7 16 21 5 8 16 21 21 5 9 16 21 11 6 17 21 1 7 17 21 1 8 17 21 1 9 17 21 2 6 17 21 2 7 17 21 21 2 8 17 21 2 9 17 21 3 6 17 21 21 3 7 17 21 3 8 17 21 3 9 17 21 4 6 17 21 4 7 17 21 4 8 17 21 4 4 9 17 21 5 6 17 17 21 5 7 17 21 5 8 17 21 5 9 17 21 11 10 14 18 1 11 14 18 1 12 14 18 1 13 14 18 2 10 14 18 2 11 14 18 2 12 14 18 2 13 14 18 3 10 14 18 3 11 14 18 3 12 14 18 3 13 14 18 4 10 14 18 4 11 14 18 4 12 14 18 4 13 14 18 5 10 14 18
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 5 11 11 14 18 5 12 14 18 5 13 14 18 1 1 10 15 18 11 11 15 18 1 12 15 18 1 13 15 18 2 10 15 18 2 11 15 18 2 12 15 18 2 13 15 18 3 10 15 18 3 11 15 18 3 12 15 18 3 13 15 18 4 10 15 18 4 11 11 15 18 4 12 15 18 4 13 15 18 5 10 15 18 5 11 15 18 5 12 15 18 5 13 15 18 11 10 16 18 1 11 16 18 1 12 16 16 18 1 13 16 18 2 10 16 16 18 2 11 16 18 2 12 16 18 2 13 16 18 3 10 16 16 18 3 11 16 18 3 12 16 18 3 13 16 18 4 10 16 16 18 4 11 16 18 4 12 16 18 4 13 16 18 5 10 16 18 5 11 16 16 18 5 12 16 18 5 13 16 18 1 10 17 18 1 11 11 17 18 1 12 17 18
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 1 13 17 18 2 10 17 18 2 11 11 17 18 2 12 17 18 2 13 17 17 18 3 10 17 18 3 11 17 18 3 12 17 18 3 13 17 18 4 10 17 18 4 11 17 17 18 4 12 17 17 18 4 13 17 18 5 10 17 18 5 11 11 17 17 18 5 12 17 18 5 13 17 18 1 10 14 19 1 11 19 14 1 12 14 19 1 13 14 19 2 10 14 19 2 11 14 19 2 12 14 19 2 13 14 19 3 10 14 19 3 11 11 14 19 3 12 14 19 3 13 14 19
4 10 14 19 4 11 11 14 19 4 12 14 19 4 13 14 19 5 10 14 19 5 11 14 19 5 12 14 19 5 13 14 19 1 10 15 19 1 11 15 19 1 12 15 19 1 13 15 19 2 10 15 19 2 11 15 19 2 12 15 19 2 13 15 19 3 10 15 19
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 3 11 11 15 19 3 12 15 19 3 13 15 19 4 10 15 19 4 11 15 19 4 12 15 19 4 13 15 19 5 10 15 19 5 11 15 19 5 12 15 19 5 13 15 19 1 10 16 19 1 11 11 16 19 1 12 16 19 1 13 16 19 2 10 16 19 2 11 11 16 19 2 12 16 19 2 13 16 19 3 10 16 16 19 3 11 16 19 3 12 16 19 3 13 16 16 19 4 10 16 19
4 11 16 19 4 12 16 16 19 4 13 16 16 19 5 10 16 16 19 5 11 11 16 19 5 12 16 19 5 13 16 16 19 1 1 10 17 19 1 11 17 19 1 12 17 19 1 13 17 19 2 10 17 17 19 2 11 17 17 19 2 12 17 19 2 13 17 17 19 3 10 17 19 3 11 11 17 17 19 3 12 17 19 3 13 17 17 19 4 10 17 19 4 11 17 19 4 12 17 19
155
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 4 13 17 19 5 10 17 17 19 5 11 11 17 19 5 12 17 19 5 13 13 17 19 1 10 14 20 1 11 11 14 20 1 12 14 14 20 1 13 14 20 2 10 14 20 2 11 14 20 2 12 14 20 2 13 14 20 3 10 14 20 3 11 14 20 3 12 14 20 3 13 13 14 20 4 10 14 20 4 11 14 20 4 12 14 20 4 13 14 20 5 10 14 20 5 11 11 14 20 5 12 14 20 5 13 14 20 1 10 15 20 1 11 11 15 20 1 12 15 20 1 13 15 20 2 10 15 20 2 11 11 15 20 2 12 15 20 2 13 15 20 3 10 15 20 3 11 15 20 3 12 15 20 3 13 15 20 4 10 15 20 4 11 15 20 4 12 15 20 4 13 15 20 5 10 15 20 5 11 15 20 5 12 15 20 5 13 15 20 1 10 16 20
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 1 11 16 20 1 12 16 16 20 1 13 16 20 2 10 16 20 2 11 16 20 2 12 16 20 2 13 16 20 3 10 16 20 3 11 16 20 3 12 16 20 3 13 16 20 4 10 16 20 4 11 11 16 20 4 12 16 20 4 13 16 20 5 10 16 20 5 11 16 20 5 12 16 20 5 13 16 20 1 10 17 20 1 11 17 20 1 12 17 20 1 13 17 17 20 2 10 17 20 2 11 17 20 2 12 17 17 20 2 13 17 20 3 10 17 17 20 3 11 17 20 3 12 17 20 3 13 17 20 4 10 17 17 20 4 11 17 20 4 12 17 20 4 13 17 17 20 5 10 17 20 5 11 17 20 5 12 17 20 5 13 17 20 1 10 14 21 1 11 14 21 1 12 14 21 1 13 14 21 2 10 14 21 2 11 14 21 2 12 14 21
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 2 13 14 21 3 10 14 21 3 11 14 21 3 12 14 21 3 13 14 21 4 4 10 14 21 4 11 14 21 4 12 14 21 4 13 14 21 5 10 14 21 5 11 14 21 5 12 14 21 5 13 14 21 1 10 15 21 1 11 11 15 21 21 1 12 15 21 1 13 15 21 2 10 15 21 2 11 15 21 2 12 15 21 2 13 15 21 3 10 15 21 3 11 15 21 3 12 15 21 3 13 15 21 4 10 15 21 4 11 11 15 21 4 12 15 21 21 4 13 13 15 21 5 10 15 21 5 11 15 21 5 12 15 21 5 13 15 21 1 10 16 21 1 11 16 21 1 12 16 21 1 13 16 21 2 10 16 21 2 11 16 21 2 12 16 21 2 13 16 21 3 10 16 21 3 11 16 21 3 12 16 21 3 13 13 16 21 4 10 16 21
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 4 11 11 16 16 21 4 12 16 21 4 13 16 21 5 10 16 21 5 11 16 21 5 12 16 21 5 13 16 21 1 10 17 21 1 11 11 17 21 1 12 17 21 1 13 17 21 2 10 17 21 2 11 11 17 17 21 2 12 17 21 21 2 13 17 21 21 3 10 17 21 3 11 11 17 21 3 12 17 17 21 3 13 13 17 17 21 4 4 10 17 17 21 4 11 11 17 21 4 12 17 21 4 13 17 17 21 21 5 10 17 21 5 11 17 17 21 5 12 17 17 21 5 13 17 21 6 10 14 18 6 11 14 18 6 12 14 18 6 13 14 18 7 10 14 18 7 11 14 18 7 12 14 18 7 13 14 18 8 10 14 18 8 11 11 14 18 8 12 14 18 8 13 14 18 9 10 14 18 9 11 11 14 18 9 12 14 18 9 13 14 18 6 10 15 18 6 11 11 15 18 6 12 15 18
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 6 13 15 18 7 10 15 18 7 11 11 15 18 7 12 15 18 7 13 15 18 8 10 15 18 8 11 15 18 8 12 15 18 8 13 15 18 9 10 15 18 9 11 11 15 18 9 12 15 18 9 13 15 18 6 10 16 18 6 11 16 18 6 12 16 18 6 13 16 18 7 10 16 18 7 11 16 18 7 12 16 18 7 13 16 18 8 10 16 18 8 11 16 18 8 12 16 18 8 13 16 18 9 10 16 18 9 11 16 18 9 12 16 16 18 9 13 16 18 6 10 17 17 18 6 11 17 18 6 12 17 17 18 6 13 17 18 7 10 17 18 7 11 11 17 18 7 12 17 18 7 13 17 17 18 8 10 17 17 18 8 11 17 18 8 12 17 18 8 13 17 18 9 10 17 18 9 11 17 17 18 9 12 17 18 9 13 17 18 6 10 14 19
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 6 11 11 14 19 6 12 14 19 6 13 14 19 7 10 14 19 7 11 11 14 14 19 7 12 14 19 7 13 14 19 8 10 14 19 8 11 11 14 19 8 12 14 19 8 13 14 19 9 10 14 14 19 9 11 14 14 19 9 12 14 14 19 9 13 14 19 6 10 15 19 6 11 15 19 6 12 15 19 6 13 15 19 7 10 15 19 7 11 11 15 19 7 12 15 19 7 13 15 19 8 10 15 19 8 11 15 19 8 12 15 19 8 13 15 19 9 10 15 19 9 11 11 15 19 9 12 15 19 9 13 15 19 6 10 16 16 19 6 11 16 19 6 12 16 19 6 13 16 19 7 10 16 19 7 11 16 19 7 12 16 19 7 13 16 19 8 10 16 19 8 11 16 16 19 8 12 16 19 8 13 16 19 9 10 16 16 19 9 11 11 16 19 9 12 16 16 19
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 9 13 16 16 19 6 10 17 19 6 11 11 17 17 19 6 12 17 19 6 13 17 19 7 10 17 19 7 11 17 17 19 7 12 17 19 7 13 17 19 8 10 17 17 19 8 11 17 17 19 8 12 17 19 8 13 17 19 9 10 17 19 9 11 11 17 19 9 12 17 19 9 13 17 19 6 10 14 20 6 11 14 20 6 12 14 20 6 13 14 20 7 10 14 20 7 11 14 20 7 12 14 20 7 13 14 20 8 10 14 20 8 11 14 14 20 8 12 14 14 20 8 13 13 14 20 9 10 14 20 9 11 11 14 20 9 12 14 14 20 9 13 14 20 6 10 15 20 6 11 11 15 20 6 12 15 20 6 13 15 20 7 10 15 20 7 11 11 15 20 7 12 15 20 7 13 15 20 8 10 15 20 8 11 15 20 8 12 15 20 8 13 15 20 9 10 15 20
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 9 11 15 20 9 12 15 20 9 13 15 20 6 10 16 20 6 11 16 20 6 12 16 20 6 13 16 20 7 10 16 20 7 11 16 20 7 12 16 20 7 13 16 20 8 10 16 20 8 11 11 16 20 8 12 16 20 8 13 16 20 9 10 16 20 9 11 11 16 20 9 12 16 16 20 9 13 16 20 6 10 17 20 6 11 17 20 6 12 17 20 6 13 17 17 20 7 10 17 20 7 11 17 17 20 7 12 17 17 20 7 13 17 20 8 10 17 17 20 8 11 17 20 8 12 17 20 8 13 17 20 9 10 17 20 9 11 17 20 9 12 17 20 9 13 17 20 6 10 14 21 6 11 14 21 6 12 14 21 6 13 14 21 7 10 14 21 7 11 11 14 21 7 12 14 21 7 13 14 21 8 10 14 21 8 11 14 21 8 12 14 21
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 8 13 14 21 9 10 14 21 9 11 11 14 21 9 12 14 21 9 13 14 21 6 10 15 21 6 11 11 15 21 6 12 15 21 6 13 15 21 7 10 15 21 7 11 15 21 7 12 15 21 7 13 15 21 8 10 15 21 8 11 15 21 21 8 12 15 21 8 13 13 15 21 9 10 15 21 21 9 11 15 21 9 12 15 21 9 13 15 21 6 10 16 16 21 6 11 11 16 21 21 6 12 16 21 21 6 13 16 21 7 10 16 21 7 11 16 21 7 12 16 16 21 21 7 13 16 21 8 10 16 21 8 11 16 21 21 8 12 16 16 21 8 13 16 21 9 10 16 21 9 11 16 21 9 12 16 21 9 13 16 21 6 10 17 21 21 6 11 17 21 6 12 17 21 6 13 17 21 7 10 17 21 7 11 11 17 21 7 12 17 17 21 7 13 17 21 8 10 17 21 21
WO wo 2019/113375 PCT/US2018/064335
First Parameter Second Third Parameter Fourth No. Parameter No. No. Parameter No. 8 11 17 21 8 12 17 21 8 13 17 21 21 9 10 17 21 9 11 17 21 9 12 17 21 21 9 13 17 21
TABLE 6. Five-way combinations of hematopoietic stem cell population parameters for
assessment
First Parameter Second Parameter Third Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 1 6 10 14 18 1 10 14 18 7 1 8 10 14 18 1 10 14 18 9 2 6 10 14 18 2 7 10 14 18 2 8 10 14 18 2 9 10 14 18 3 6 10 14 18 3 7 10 14 18 3 8 10 14 18 3 9 10 14 18 4 4 6 10 14 18
4 7 10 14 18 4 8 10 14 18 4 9 10 14 18 5 6 10 14 18 5 7 10 14 18 5 8 10 14 18 5 9 10 14 18 1 11 6 14 18 1 11 7 14 18 1 11 14 18 8 1 1 11 9 14 18 2 6 11 14 18 2 7 11 14 18 2 8 11 14 18 2 9 11 14 18 3 6 11 14 18 3 7 11 14 14 18 3 8 11 14 18 3 9 11 14 18 4 6 11 14 18
First Parameter Second Parameter Third Third Parameter Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 4 7 11 14 18
4 8 11 11 14 18
4 9 11 14 18 5 6 11 14 18 5 7 11 14 18 5 8 11 14 18 5 9 11 14 18 1 6 12 14 18 1 12 14 18 7 1 8 12 14 18 1 9 12 14 18 2 6 12 14 18 2 7 12 14 18 2 8 12 14 18 2 9 12 14 18 3 6 12 14 18 3 7 12 14 18 3 8 12 14 18 3 9 12 14 18
4 6 12 14 18 4 7 12 14 18 4 8 12 14 18 4 9 12 14 18 5 6 12 14 18 5 7 12 14 18 5 8 12 14 18 5 9 12 14 18 1 6 13 14 18 1 7 13 14 18 1 13 14 18 8 11 9 13 14 18 2 6 13 14 18 2 7 13 14 18 2 8 13 14 18 2 9 13 14 18 3 6 13 14 18 3 7 13 14 18 3 8 13 14 18 3 9 13 14 18
4 6 13 14 18 4 7 13 14 18 4 8 13 14 18 4 9 13 14 18 5 6 13 14 18 5 7 13 14 18 5 8 13 14 18
First Parameter Second Parameter Third Third Parameter Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 5 9 13 14 18 1 6 10 15 18 1 10 15 18 7 1 1 8 10 15 18 1 10 15 18 9 2 6 10 15 18 2 7 10 15 18 2 8 10 15 18 2 9 10 15 18 3 6 10 15 18 3 7 10 15 18 3 8 10 15 18 3 9 10 15 18
4 6 10 15 18
4 7 10 15 18 4 8 10 15 18
4 9 10 15 18 5 6 10 15 18 5 7 10 15 18 5 8 10 15 18 5 9 10 15 18 1 6 11 15 18 11 11 15 18 7 1 8 11 11 15 18 1 9 11 11 15 18 2 6 11 15 18 2 7 11 15 18 2 8 11 11 15 18 2 9 11 15 18 3 6 11 15 18 3 7 11 15 18 3 8 11 15 18 3 9 11 15 18 4 6 11 15 18
4 7 11 15 18 4 8 11 15 18 4 9 11 15 18 5 6 11 11 15 18 5 7 11 15 18 5 8 11 15 18 5 9 11 15 18 1 12 15 18 6 1 12 15 18 7 1 12 15 18 8 1 12 15 18 9 2 6 12 15 18
First Parameter Second Parameter Third Third Parameter Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 2 7 12 15 18 2 8 12 15 18 2 9 12 15 18 3 6 12 15 18 3 7 12 15 18 3 8 12 15 18 3 9 12 15 18
4 6 12 15 18 4 7 12 15 18
4 8 12 15 18 4 9 12 15 18 5 6 12 15 18 5 7 12 15 18 5 8 12 15 18 5 9 12 15 18 1 13 15 18 6 1 7 13 15 18 1 8 13 15 18 1 13 15 18 9 2 6 13 15 18 2 7 13 15 18 2 8 13 15 18 2 9 13 15 18 3 6 13 15 18 3 7 13 15 18 3 8 13 15 18 3 9 13 15 18
4 6 13 15 18 4 7 13 15 18 4 8 13 15 18 4 9 13 15 18 5 6 13 15 18 5 7 13 15 18 5 8 13 15 18 5 9 13 15 18 1 6 10 16 18 1 7 10 16 18 1 8 10 16 18 1 1 10 16 18 9 2 6 10 16 18 2 7 10 16 18 2 8 10 16 18 2 9 10 16 18 3 6 10 16 18 3 7 10 16 18 3 8 10 16 18
First Parameter Second Parameter Third Third Parameter Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 3 9 10 16 18
4 6 10 16 18 4 7 10 16 18 4 8 10 16 18 4 9 10 16 18 5 6 10 16 18 5 7 10 16 18 5 8 10 16 18 5 9 10 16 18 1 11 11 16 18 6 1 7 11 16 18 1 8 11 16 18 1 9 11 16 18 2 6 11 16 18 2 7 11 16 18 2 8 11 16 18 2 9 11 11 16 18 3 6 11 16 18 3 7 11 16 18 3 8 11 11 16 18 3 9 11 11 16 18 4 6 11 16 18 4 7 11 11 16 18 4 8 11 11 16 18 4 9 11 16 18 5 6 11 16 18 5 7 11 16 18 5 8 11 16 18 5 9 11 16 18 1 12 16 18 6 1 7 12 16 18 1 1 8 12 16 18 1 12 16 18 9 2 6 12 16 18 2 7 12 16 18 2 8 12 16 18 2 9 12 16 18 3 6 12 16 18 3 7 12 16 18 3 8 12 16 18 3 9 12 16 18 4 6 12 16 18
4 7 12 16 18 4 8 12 16 18 4 9 12 16 18 5 6 12 16 18
First Parameter Second Parameter Third Third Parameter Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 5 7 12 16 18 5 8 12 16 18 5 9 12 16 18 1 6 13 16 18 1 13 16 18 7 1 8 13 16 18 1 13 16 18 9 2 6 13 16 18 2 7 13 16 18 2 8 13 16 18 2 9 13 16 18 3 6 13 16 18 3 7 13 16 18 3 8 13 16 18 3 9 13 16 18
4 6 13 16 18
4 7 13 16 18 4 8 13 16 18 4 9 13 16 18 5 6 13 16 18 5 7 13 16 18 5 8 13 16 18 5 9 13 13 16 18 1 10 17 18 6 1 7 10 17 18 1 8 10 17 18 1 9 10 17 18 2 6 10 17 18 2 7 10 17 18 2 8 10 17 18 2 9 10 17 18 3 6 10 17 18 3 7 10 17 18 3 8 10 17 18 3 9 10 17 18 4 6 10 17 17 18 4 7 10 17 18 4 8 10 17 18 4 9 10 17 18 5 6 10 17 18 5 7 10 17 17 18 5 8 10 17 18 5 9 10 17 18 1 11 17 18 6 1 11 11 17 18 7 1 8 11 11 17 17 18
First Parameter Second Parameter Third Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 1 11 17 18 9 2 6 11 17 18 2 7 11 17 18 2 8 11 11 17 18 2 9 11 17 18 3 6 11 17 18 3 7 11 11 17 18 3 8 11 17 18 3 9 11 11 17 18
4 6 11 17 18 4 7 11 17 18
4 8 11 17 18 4 9 11 11 17 18 5 6 11 17 18 5 7 11 17 18 5 8 11 11 17 18 5 9 11 11 17 18 1 6 12 17 18 1 7 12 17 18 1 8 12 17 18 1 12 17 18 9 2 6 12 17 18 2 7 12 17 18 2 8 12 17 18 2 9 12 17 18 3 6 12 17 18 3 7 12 17 18 3 8 12 17 18 3 9 12 17 18
4 6 12 17 18
4 7 12 17 18 4 8 12 17 18 4 9 12 17 18 5 6 12 17 18 5 7 12 17 18 5 8 12 17 18 5 9 12 17 18 1 13 17 18 6 1 1 13 17 18 7 1 13 17 18 8 1 9 13 17 18 2 6 13 17 18 2 7 13 17 18 2 8 13 17 18 2 9 13 17 18 3 6 13 17 18
First Parameter Second Parameter Third Third Parameter Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 3 7 13 17 18 3 8 13 17 18 3 9 13 17 18 4 6 13 17 18
4 7 13 17 18
4 8 13 17 18
4 9 13 17 18 5 6 13 17 18 5 7 13 17 18 5 8 13 17 18 5 9 13 17 18 1 10 14 19 6 1 10 14 19 7 1 8 14 19 10 1 10 14 19 9 2 6 10 14 19 2 7 10 14 19 2 8 10 14 19 2 9 10 14 19 3 6 10 14 19 3 7 10 14 19 3 8 10 14 19 3 9 10 14 19 4 6 10 14 19
4 7 10 14 19
4 8 10 14 19
4 9 10 14 19 5 6 10 14 19 5 7 10 14 19 5 8 10 14 19 5 9 10 14 19 1 1 6 11 14 19 1 11 14 19 7 1 8 11 14 19 1 11 11 14 19 9 2 6 11 14 19 19 2 7 11 14 19 2 8 11 14 19 2 9 11 14 19 3 6 11 14 19 3 7 11 14 19 19 3 8 11 14 19 3 9 11 14 19 4 6 11 14 19
4 7 11 11 14 19 4 8 11 14 19
First Parameter Second Parameter Third Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 4 9 11 14 19 5 6 11 14 19 5 7 11 11 14 19 5 8 11 14 19 5 9 11 14 19 1 12 14 19 6 1 7 12 14 19 1 8 12 14 19 1 12 14 19 9 2 6 12 14 19 2 7 12 14 19 2 8 12 14 19 2 9 12 14 19 3 6 12 14 19 3 7 12 14 19 3 8 12 14 19 3 9 12 14 19 4 6 12 14 19 4 7 12 14 19
4 8 12 14 19 4 9 12 14 19 5 6 12 14 19 5 7 12 14 19 5 8 12 14 19 5 9 12 14 19 1 6 13 14 19 1 7 13 14 19 1 8 13 14 19 1 13 14 19 9 2 6 13 14 19 2 7 13 14 19 2 8 13 14 19 2 9 13 14 19 3 6 13 14 19 3 7 13 14 19 3 8 13 14 19 3 9 13 14 19 4 6 13 14 19 4 7 13 14 19 4 8 13 13 14 19 4 9 13 14 19 5 6 13 14 19 5 7 13 14 19 5 8 13 14 19 5 9 13 14 19 1 6 10 15 19
First Parameter Second Parameter Third Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 11 7 10 15 19 1 10 15 19 8 1 10 15 19 9 2 6 10 15 19 2 7 10 15 19 2 8 10 15 19 2 9 10 15 19 3 6 10 15 19 3 7 10 15 19 3 8 10 15 19 3 9 10 15 19
4 6 10 15 19
4 7 10 15 19
4 8 10 15 19 4 9 10 15 19 5 6 10 15 19 5 7 10 15 19 5 8 10 15 19 5 9 10 15 19 1 11 11 15 19 6 1 11 15 19 7 1 8 11 15 19 1 11 15 19 9 2 6 11 15 19 2 7 11 15 19 2 8 11 15 19 2 9 11 15 19 3 6 11 15 19 3 7 11 15 19 3 8 11 15 19 3 9 11 15 19
4 6 11 15 19
4 7 11 15 19 4 8 11 15 19 4 9 11 15 19 5 6 11 15 19 5 7 11 15 19 5 8 11 11 15 19 5 9 11 15 19 1 6 12 15 19 1 7 12 15 19 1 8 12 15 19 1 12 15 19 9 2 6 12 15 19 2 7 12 15 19 2 8 12 15 19
First Parameter Second Parameter Third Third Parameter Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 2 9 12 15 19 3 6 12 15 19 3 7 12 15 19 3 8 12 15 19 3 9 12 15 19 4 6 12 15 19 19
4 7 12 15 19 4 8 12 15 19 4 9 12 15 19 5 6 12 15 19 5 7 12 15 19 5 8 12 15 19 5 9 12 15 19 1 6 13 15 19 1 13 15 19 7 1 8 13 15 19 1 13 15 19 9 2 6 13 15 19 2 7 13 15 19 2 8 13 15 19 2 9 13 15 19 19 3 6 13 15 19 3 7 13 15 19 3 8 13 15 19 3 9 13 15 19
4 6 13 15 19
4 7 13 15 19
4 8 13 15 19 4 9 13 15 19 5 6 13 15 19 5 7 13 15 19 5 8 13 15 19 5 9 13 15 19 1 6 10 16 19 1 10 16 19 7 1 8 10 16 19 1 10 16 19 9 2 6 10 16 19 2 7 10 16 19 2 8 10 16 19 2 9 10 16 19 3 6 10 16 19 3 7 10 16 19 3 8 10 16 19 3 9 10 16 19 4 6 10 16 16 19
175
First Parameter Second Parameter Third Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 4 7 10 16 19
4 8 10 16 19
4 9 10 16 19 5 6 10 16 19 5 7 10 16 19 5 8 10 16 19 5 9 10 16 19 1 6 11 16 19 1 11 16 19 7 1 8 11 16 19 1 9 11 16 19 2 6 11 16 19 2 7 11 16 19 2 8 11 16 19 2 9 11 11 16 19 3 6 11 16 19 3 7 11 11 16 19 3 8 11 16 19 3 9 11 16 19
4 6 11 16 19
4 7 11 16 19 4 8 11 16 19 4 9 11 16 19 5 6 11 16 19 5 7 11 16 19 5 8 11 16 19 5 9 11 16 19 1 6 12 16 19 1 7 12 16 19 1 12 16 19 8 11 9 12 16 19 2 6 12 16 19 2 7 12 16 19 2 8 12 16 19 2 9 12 16 19 3 6 12 16 19 3 7 12 16 19 3 8 12 16 19 3 9 12 16 19
4 6 12 16 19 4 7 12 16 19 4 8 12 16 19
4 9 12 16 19 5 6 12 16 19 5 7 12 16 19 5 8 12 16 19
First Parameter Second Parameter Third Third Parameter Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 5 9 12 16 19 1 6 13 16 19 1 13 16 19 7 1 1 8 13 16 19 1 13 16 19 9 2 6 13 16 19 2 7 13 16 19 2 8 13 16 19 2 9 13 16 19 3 6 13 16 19 3 7 13 16 19 19 3 8 13 16 19 3 9 13 16 19
4 6 13 16 19
4 7 13 16 19 4 8 13 16 19
4 9 13 16 19 5 6 13 16 19 5 7 13 16 19 5 8 13 16 19 5 9 13 16 19 1 6 10 17 17 19 11 10 17 19 7 1 8 17 19 10 1 9 10 17 19 2 6 10 17 19 2 7 10 17 19 2 8 10 17 19 2 9 10 17 19 3 6 10 17 19 3 7 10 17 19 3 8 10 17 19 3 9 10 17 19 4 6 10 17 19
4 7 10 17 19 4 8 10 17 19 4 9 10 17 19 5 6 10 17 19 5 7 10 17 19 5 8 10 17 19 5 9 10 17 19 1 11 17 19 6 1 11 17 19 7 1 11 17 19 8 1 11 11 17 19 9 2 6 11 17 19
First Parameter Second Parameter Third Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 2 7 11 11 17 19 2 8 11 17 19 2 9 11 17 19 3 6 11 17 19 3 7 11 17 19 3 8 11 17 19 3 9 11 17 19
4 6 11 17 17 19 4 7 11 17 19
4 8 11 17 19 4 9 11 17 19 5 6 11 17 19 5 7 11 17 19 5 8 11 17 19 5 9 11 17 19 1 12 17 19 6 1 12 17 19 7 1 8 12 17 19 1 12 17 19 9 2 6 12 17 19 2 7 12 17 19 2 8 12 17 19 2 9 12 17 19 3 6 12 17 19 3 7 12 17 19 3 8 12 17 19 3 9 12 17 19
4 6 12 17 19
4 7 12 17 19 4 8 12 17 19 4 9 12 17 19 5 6 12 17 19 5 7 12 17 19 5 8 12 17 19 5 9 12 17 19 11 6 13 17 19 19 1 7 13 17 19 1 13 13 17 19 8 1 1 13 17 19 9 2 6 13 17 19 2 7 13 17 19 2 8 13 17 19 2 9 13 17 19 3 6 13 17 19 3 7 13 17 19 3 8 13 17 19
First Parameter Second Parameter Third Third Parameter Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 3 9 13 17 17 19
4 6 13 17 19 4 7 13 17 17 19 4 8 13 17 19 4 9 13 17 19 5 6 13 17 19 5 7 13 17 19 5 8 13 17 17 19 5 9 13 17 19 1 10 14 6 20 1 7 10 14 20 1 8 10 14 20 1 9 10 14 20 2 6 10 14 20 2 7 10 14 20 2 8 10 14 20 2 9 10 14 20 3 6 10 14 20 3 7 10 14 20 3 8 10 14 20 3 9 10 14 20 4 6 10 14 20 4 7 10 14 20 4 8 10 14 20 4 9 10 14 20 5 6 10 14 20 5 7 10 14 20 5 8 10 14 20 5 9 10 14 20 1 11 11 14 6 20 1 7 11 11 14 20 1 1 11 8 14 20 1 11 11 9 14 20 2 6 11 14 20 2 7 11 11 14 20 2 8 11 14 20 2 9 11 11 14 20 3 6 11 14 20 3 7 11 11 14 20 3 8 11 11 14 20 3 9 11 14 20 4 6 11 14 20 4 7 11 11 14 20 4 8 11 14 20 4 9 11 11 14 20 5 6 11 11 14 20
First Parameter Second Parameter Third Third Parameter Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 5 7 11 11 14 20 5 8 11 14 20 5 9 11 14 20 1 6 12 14 20 1 12 14 7 20 1 8 12 14 20 1 12 14 9 20 2 6 12 14 20 2 7 12 14 20 2 8 12 14 20 2 9 12 14 20 3 6 12 14 20 3 7 12 14 20 3 8 12 14 20 3 9 12 14 20 4 6 12 14 20 4 7 12 14 20 4 8 12 14 20 4 9 12 14 20 5 6 12 14 20 5 7 12 14 20 5 8 12 14 20 5 9 12 14 20 1 13 14 6 20 1 7 13 14 20 1 8 13 14 20 1 9 13 14 20 2 6 13 14 20 2 7 13 14 20 2 8 13 14 20 2 9 13 14 20 3 6 13 14 20 3 7 13 14 20 3 8 13 14 20 3 9 13 14 20 4 6 13 14 20 4 7 13 14 20 4 8 13 13 14 20 4 9 13 14 20 5 6 13 14 20 5 7 13 14 20 5 8 13 14 20 5 9 13 13 14 20 1 15 6 10 20 1 10 15 7 20 1 8 10 15 20
First Parameter Second Parameter Third Third Parameter Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 1 15 9 10 20 2 6 10 15 20 2 7 10 15 20 2 8 10 15 20 2 9 10 15 20 3 6 10 15 20 3 7 10 15 20 3 8 10 15 20 3 9 10 15 20 4 6 10 15 20 4 7 10 15 20 4 8 10 15 20 4 9 10 15 20 5 6 10 15 20 5 7 10 15 20 5 8 10 15 20 5 9 10 15 20 1 11 6 15 20 1 7 11 15 20 1 8 11 11 15 20 1 11 15 9 20 2 6 11 11 15 20 2 7 11 11 15 20 2 8 11 11 15 20 2 9 11 11 15 20 3 6 11 15 20 3 7 11 15 20 3 8 11 11 15 20 3 9 11 15 20 4 6 11 11 15 20 4 7 11 15 20 4 8 11 15 20 4 9 11 11 15 20 5 6 11 11 15 20 5 7 11 11 15 20 5 8 11 15 20 5 9 11 11 15 20 1 12 15 6 20 1 7 12 15 20 1 12 15 8 20 1 12 15 9 20 2 6 12 15 20 2 7 12 15 20 2 8 12 15 20 2 9 12 15 20 3 6 12 15 20
First Parameter Second Parameter Third Third Parameter Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 3 7 12 15 20 3 8 12 15 20 3 9 12 15 20 4 6 12 15 20 4 7 12 15 20 4 8 12 15 20 4 9 12 15 20 5 6 12 15 20 5 7 12 15 20 5 8 12 15 20 5 9 12 15 20 1 13 15 6 20 1 13 15 7 20 1 8 13 15 20 1 13 15 9 20 2 6 13 15 20 2 7 13 15 20 2 8 13 15 20 2 9 13 15 20 3 6 13 15 20 3 7 13 15 20 3 8 13 15 20 3 9 13 15 20 4 6 13 15 20 4 7 13 15 20 4 8 13 15 20 4 9 13 15 20 5 6 13 15 20 5 7 13 15 20 5 8 13 15 20 5 9 13 15 20 1 1 6 10 16 20 1 16 7 10 20 1 8 10 16 20 1 10 16 9 20 2 6 10 16 20 2 7 10 16 20 2 8 10 16 20 2 9 10 16 20 3 6 10 16 20 3 7 10 16 20 3 8 10 16 20 3 9 10 16 20 4 6 10 16 20 4 7 10 16 20 4 8 10 16 20
First Parameter Second Parameter Third Third Parameter Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 4 9 10 16 20 5 6 10 16 20 5 7 10 16 20 5 8 10 16 20 5 9 10 16 20 1 6 11 16 20 1 11 16 7 20 1 8 11 16 20 1 11 16 9 20 2 6 11 16 20 2 7 11 16 20 2 8 11 11 16 20 2 9 11 16 20 3 6 11 16 20 3 7 11 16 20 3 8 11 16 20 3 9 11 16 20 4 6 11 16 20 4 7 11 16 20 4 8 11 11 16 20 4 9 11 16 20 5 6 11 16 20 5 7 11 16 20 5 8 11 16 20 5 9 11 16 20 1 6 12 16 20 1 7 12 16 20 1 8 12 16 20 1 12 16 9 20 2 6 12 16 20 2 7 12 16 20 2 8 12 16 20 2 9 12 16 20 3 6 12 16 20 3 7 12 16 20 3 8 12 16 20 3 9 12 16 20 4 6 12 16 20 4 7 12 16 20 4 8 12 16 20 4 9 12 16 20 5 6 12 16 20 5 7 12 16 20 5 8 12 16 20 5 9 12 16 20 1 6 13 16 20
WO wo 2019/113375 PCT/US2018/064335
First Parameter Second Parameter Third Third Parameter Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 1 13 16 7 20 1 13 16 8 20 1 13 16 9 20 2 6 13 16 20 2 7 13 16 20 2 8 13 16 20 2 9 13 16 20 3 6 13 16 20 3 7 13 16 20 3 8 13 16 20 3 9 13 16 20 4 6 13 16 20 4 7 13 16 20 4 8 13 16 20 4 9 13 16 20 5 6 13 16 20 5 7 13 16 20 5 8 13 16 20 5 9 13 16 20 1 6 10 17 20 1 17 7 10 20 1 8 10 17 17 20 1 10 9 17 20 2 6 10 17 20 2 7 10 17 20 2 8 10 17 20 2 9 10 17 20 3 6 10 17 20 3 7 10 17 20 3 8 10 17 20 3 9 10 17 20 4 6 10 17 20 4 7 10 17 20 4 8 10 17 20 4 9 10 17 20 5 6 10 17 20 5 7 10 17 20 5 8 10 17 20 5 9 10 17 20 1 6 11 17 20 1 7 11 17 20 1 8 11 17 20 1 11 17 9 20 2 6 11 17 20 2 7 11 11 17 20 2 8 11 17 17 20
First Parameter Second Parameter Third Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 2 9 11 11 17 20 20 3 6 11 11 17 20 3 7 11 17 20 3 8 11 17 20 3 9 11 17 20 4 6 11 17 20 4 7 11 17 20 4 8 11 11 17 20 4 9 11 17 20 5 6 11 11 17 20 5 7 11 17 20 5 8 11 17 20 5 9 11 17 20 1 6 12 17 20 1 12 17 7 20 1 8 12 17 20 1 12 9 17 20 2 6 12 17 20 2 7 12 17 20 2 8 12 17 20 2 9 12 17 20 3 6 12 17 20 3 7 12 17 20 3 8 12 17 20 3 9 12 17 20 4 6 12 17 20 4 7 12 17 20 4 8 12 17 20 4 9 12 17 20 5 6 12 17 20 5 7 12 17 20 5 8 12 17 20 5 9 12 17 20 1 6 13 17 20 1 13 17 7 20 1 8 13 17 20 1 13 17 9 20 2 6 13 17 20 2 7 13 17 20 2 8 13 17 20 2 9 13 17 20 3 6 13 17 20 3 7 13 13 17 20 3 8 13 17 20 3 9 13 17 20 4 6 13 17 20
First Parameter Second Parameter Third Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 4 7 13 17 20 4 8 13 17 20 4 9 13 17 20 5 6 13 17 20 5 7 13 17 20 5 8 13 17 20 5 9 13 17 20 1 6 10 14 21 1 10 14 7 21 1 8 10 14 21 1 9 10 14 21 2 6 10 14 21 2 7 10 14 21 2 8 10 14 21 2 9 10 14 21 3 6 10 14 21 3 7 10 14 21 3 8 10 14 21 3 9 10 14 21 4 6 10 14 21
4 7 10 14 21 4 8 10 14 21 4 9 10 14 21 5 6 10 14 21 5 7 10 14 21 5 8 10 14 21 5 9 10 14 21 1 6 11 14 21 1 7 11 14 21 1 11 14 8 21 11 9 11 11 14 21 2 6 11 14 21 2 7 11 14 21 2 8 11 14 21 2 9 11 14 21 3 6 11 14 21 3 7 11 14 21 3 8 11 11 14 21 3 9 11 14 21 4 6 11 14 21 4 7 11 14 21 4 8 11 14 21 4 9 11 14 21 5 6 11 14 21 5 7 11 11 14 21 5 8 11 14 21
First Parameter Second Parameter Third Third Parameter Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 5 9 11 14 21 21 1 6 12 14 21 21 1 12 14 7 21 1 1 8 12 14 21 1 12 14 9 21 21 2 6 12 14 21 2 7 12 14 21 21 2 8 12 14 21 21 2 9 12 14 21 3 6 12 14 21 21 3 7 12 14 21 21 3 8 12 14 21 21 3 9 12 14 21 21 4 6 12 14 21 4 4 7 12 14 21 21 4 8 12 14 21 21 4 9 12 14 21 21 5 6 12 14 21 21 5 7 12 14 21 21 5 8 12 14 21 21 5 9 12 14 21 21 1 6 13 14 21 21 1 13 14 7 21 1 8 13 13 14 21 1 9 13 14 21 21 2 6 13 14 21 21 2 7 13 14 21 2 8 13 14 21 21 2 9 13 14 21 21 3 6 13 14 21 3 7 13 14 21 3 8 13 14 21 21 3 9 13 14 21 4 6 13 13 14 21 4 7 13 14 21
4 8 13 14 21 21 4 9 13 14 21 5 6 13 14 21 5 7 13 14 21 5 8 13 14 21 21 5 9 13 14 21 21 1 10 15 6 21 1 10 15 7 21 1 8 10 15 21 1 10 15 21 21 9 2 6 10 15 21 21
First Parameter Second Parameter Third Third Parameter Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 2 7 10 15 21 21 2 8 10 15 21 21 2 9 10 15 21 3 6 10 15 21 3 7 10 15 21 21 3 8 10 15 21 21 3 9 10 15 21 4 6 10 15 21 21 4 7 10 15 21 21 4 8 10 15 21 4 9 10 15 21 21 5 6 10 15 21 5 7 10 15 21 5 8 10 15 21 21 5 9 10 15 21 1 11 11 15 21 6 21 1 7 11 11 15 21 1 11 8 11 15 21 1 11 15 21 9 2 6 11 11 15 21 2 7 11 15 21 21 2 8 11 15 21 2 9 11 11 15 21 3 6 11 11 15 21 3 7 11 11 15 21 3 8 11 15 21 3 9 11 15 21 21 4 6 11 15 21 4 7 11 15 21 21 4 8 11 15 21 21 4 9 11 15 21 21 5 6 11 15 21 21 5 7 11 11 15 21 21 5 8 11 11 15 21 21 5 9 11 15 21 1 6 12 15 21 1 7 12 15 21 1 8 12 15 21 1 9 12 15 21 2 6 12 15 21 2 7 12 15 21 2 8 12 15 21 2 9 12 15 21 3 6 12 15 21 3 7 12 15 21 3 8 12 15 21 21
First Parameter Second Parameter Third Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 3 9 12 15 21 21 4 6 12 15 21 21 4 7 12 15 21 4 8 12 15 21 4 9 12 15 21 5 6 12 15 21 5 7 12 15 21 5 8 12 15 21 21 5 9 12 15 21 1 13 15 6 21 1 7 13 15 21 1 8 13 15 21 1 9 13 15 21 2 6 13 15 21 2 7 13 15 21 21 2 8 13 15 21 21 2 9 13 15 21 21 3 6 13 15 21 3 7 13 13 15 21 21 3 8 13 15 21 21 3 9 13 15 21 4 6 13 15 21 4 7 13 15 21 4 8 13 15 21 21 4 9 13 15 21 21 5 6 13 15 21 5 7 13 15 21 21 5 8 13 15 21 21 5 9 13 15 21 21 1 10 16 6 21 1 7 10 16 21 1 1 8 10 16 21 1 10 16 9 21 2 6 10 16 21 21 2 7 10 16 21 21 2 8 10 16 21 2 9 10 16 21 21 3 6 10 16 21 21 3 7 10 16 21 3 8 10 16 21 3 9 10 16 16 21 21 4 6 10 16 21
4 7 10 16 21 21 4 8 10 16 21 4 9 10 16 21 5 6 10 16 21
First Parameter Second Parameter Third Third Parameter Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 5 7 10 16 21 21 5 8 10 16 21 5 9 10 16 21 1 1 6 11 11 16 21 1 11 16 7 21 1 8 11 16 21 1 11 11 16 21 9 21 2 6 11 16 21 21 2 7 11 11 16 21 2 8 11 16 21 21 2 9 11 16 21 3 6 11 16 21 21 3 7 11 11 16 21 21 3 8 11 16 21 21 3 9 11 16 21 21 4 6 11 16 21 21
4 7 11 11 16 21 21 4 8 11 11 16 21 21 4 9 11 11 16 21 21 5 6 11 11 16 21 21 5 7 11 16 21 5 8 11 16 21 21 5 9 11 11 16 21 1 12 16 6 21 21 1 7 12 16 21 21 1 8 12 16 21 1 9 12 16 21 21 2 6 12 16 21 2 7 12 16 21 21 2 8 12 16 21 21 2 9 12 16 21 3 6 12 16 21 3 7 12 16 21 21 3 8 12 16 21 21 3 9 12 16 21 21 4 6 12 16 21 4 7 12 16 21 4 8 12 16 21 21 4 9 12 16 21 21 5 6 12 16 21 21 5 7 12 16 21 5 8 12 16 21 21 5 9 12 16 21 21 1 13 16 6 21 1 13 16 7 21 21 1 8 13 16 21 21
First Parameter Second Parameter Third Third Parameter Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 1 13 16 21 9 2 6 13 16 21 2 7 13 16 21 2 8 13 16 21 2 9 13 16 21 3 6 13 16 21 3 7 13 16 21 3 8 13 16 21 3 9 13 16 21 4 6 13 16 21 4 7 13 16 21 4 8 13 16 21 4 9 13 16 21 5 6 13 16 21 5 7 13 16 21 5 8 13 16 21 5 9 13 16 21 1 6 10 17 21 1 7 17 10 21 1 8 10 17 21 1 17 9 10 21 2 6 10 17 17 21 2 7 10 17 21 2 8 10 17 21 2 9 10 17 21 3 6 10 17 21 3 7 10 17 21 21 3 8 10 17 17 21 3 9 10 17 21 4 6 10 17 21 4 7 10 17 21 4 8 10 17 21 4 9 10 17 21 5 6 10 17 21 5 7 10 17 21 5 8 10 17 21 5 9 10 17 21 1 11 11 17 6 21 1 1 11 17 7 21 1 11 11 8 17 21 1 11 17 9 21 2 6 11 17 21 2 7 11 17 21 2 8 11 17 21 2 9 11 17 21 3 6 11 17 21
First Parameter Second Parameter Third Third Parameter Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 3 7 11 17 21 3 8 11 17 21 3 9 11 11 17 21 4 6 11 11 17 21
4 7 11 17 21 4 8 11 17 21 4 9 11 17 21 5 6 11 17 21 5 7 11 17 21 5 8 11 11 17 21 5 9 11 17 21 1 12 17 6 21 1 12 21 7 17 1 8 12 17 21 1 12 17 9 21 2 6 12 17 21 2 7 12 17 21 2 8 12 17 21 2 9 12 17 21 3 6 12 17 21 3 7 12 17 21 3 8 12 17 21 3 9 12 17 21 4 6 12 17 21 4 7 12 17 21 4 8 12 17 21 4 9 12 17 21 5 6 12 17 21 5 7 12 17 21 5 8 12 17 21 5 9 12 17 21 1 1 6 13 17 21 1 13 17 7 21 1 8 13 17 21 1 13 17 9 21 2 6 13 17 21 2 7 13 17 21 2 8 13 17 21 2 9 13 17 21 3 6 13 17 21 3 7 13 17 21 3 8 13 17 21 3 9 13 17 21 4 6 13 17 21 4 7 13 17 21 4 8 13 17 21
WO wo 2019/113375 PCT/US2018/064335
First Parameter Second Parameter Third Parameter Fourth Fifth Parameter No. No. No. Parameter No. No. 4 9 13 17 21 5 6 13 17 21 5 7 13 17 17 21 5 8 13 17 17 21 5 9 13 17 21
CXCR2 Agonists Gro-B, Gro-ß, Gro-B Gro-ß T, and variants thereof
[0190] Exemplary CXCR2 agonists that may be used in conjunction with the compositions
and methods described herein are Gro-B Gro-ß and variants thereof. Gro-B Gro-ß (also referred to as
growth-regulated protein B, ß, chemokine (C-X-C motif) ligand 2 (CXCL2), and macrophage
inflammatory protein 2-a (MIP2-a)) 2- (MIP2-)) isis a a cytokine cytokine capable capable ofof mobilizing mobilizing hematopoietic hematopoietic stem stem
and progenitor cells, for example, by stimulating the release of proteases, and particularly
MMP9, from peripheral neutrophils. Without being limited by mechanism, MMP9 may
induce mobilization of hematopoietic stem and progenitor cells from stem cell niches, such as
the bone marrow, to circulating peripheral blood by stimulating the degradation of proteins
such as stem cell factor, its corresponding receptor, CD117, and CXCL12, all of which
generally maintain hematopoietic stem and progenitor cells immobilized in bone marrow.
[0191] In addition to Gro-B, Gro-ß, exemplary CXCR2 agonists that may be used in conjunction
with the compositions and methods described herein are truncated forms of Gro-B, Gro-ß, such as
those that feature a deletion at the N-terminus of Gro-B Gro-ß of from 1 to 8 amino acids (e.g.,
peptides that feature an N-terminal deletion of 1 amino acids, 2 amino acids, 3 amino acids, 4
amino acids, 5 amino acids, 6 amino acids, 7 amino acids, or 8 amino acids). In some
embodiments, CXCR2 agonists that may be used in conjunction with the compositions and
methods described herein include Gro-ß T, which is characterized by a deletion of the first
four amino acids from the N-terminus of Gro-B. Gro-ß. Gro-B Gro-ß T exhibits particularly advantageous
biological properties, such as the ability to induce hematopoietic stem and progenitor cell
mobilization with a potency superior to that of Gro-B Gro-ß by multiple orders of magnitude. Gro-B Gro-ß
and Gro-ß T are described, for example, in US Patent No. 6,080,398, the disclosure of which
is incorporated herein by reference in its entirety.
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[0192] In addition, exemplary CXCR2 agonists that may be used in conjunction with the
compositions and methods described herein are variants of Gro-B Gro-ß containing an aspartic acid
residue in place of the asparagine residue at position 69 of SEQ ID NO: 1. This peptide,
referred to herein as Gro-B Gro-ß N69D, retains the hematopoietic stem and progenitor cell-
mobilizing functionality of Gro-ß, yet induces this effect with a superior potency. Similarly,
CXCR2 agonists that may be used with the compositions and methods described herein
include variants of Gro-B Gro-ß T containing an aspartic acid residue in place of the asparagine
residue at position 65 of SEQ ID NO: 2. This peptide, referred to herein as Gro-B Gro-ß T N65D,
not only retains hematopoietic stem and progenitor cell-mobilizing capacity, but exhibits a
potency that is substantially greater than that of Gro-B Gro-ß T. Gro-B Gro-ß N69D and Gro-B Gro-ß T N65D
are described, for example, in US Patent No. 6,447,766, the disclosure of which is
incorporated herein by reference in its entirety.
[0193] The amino acid sequences of Gro-B, Gro-ß, Gro-B Gro-ß T, Gro-B Gro-ß N69D, and Gro-ß T N65D are set
forth in TABLE 7, below.
TABLE 7. Amino acid sequences of Gro-B Gro-ß and select variants thereof
SEQ ID Description Amino Acid Sequence NO. APLATELRCQCLQTLQGIHLKN 1 Gro-B Gro-ß IQSVKVKSPGPHCAQTEVIATL KNGQKACLNPASPMVKKIIEK MLKNGKSN TELRCQCLQTLQGIHLKNIQSV TELRCQCLQTLQGIHLKNIQSV 2 Gro-B-T Gro-ß-T KVKSPGPHCAQTEVIATLKNGQ KVKSPGPHCAQTEVIATLKNGQ KACLNPASPMVKKIIEKMLKNG KACLNPASPMVKKIEKMLKNG KSN APLATELRCQCLQTLQGIHLKN 3 Gro-B Gro-ß N69D IQSVKVKSPGPHCAQTEVIATL KNGQKACLNPASPMVKKIIEK KNGQKACLNPASPMVKKIEK MLKDGKSN TELRCQCLQTLQGIHLKNIQSV TELRCQCLQTLQGIHLKNIQSV 4 Gro-B-T Gro-ß-T N65D N65D KVKSPGPHCAQTEVIATLKNGQ KACLNPASPMVKKIIEKMLKDG KACLNPASPMVKKIEKMLKDG KSN
[0194] Additional CXCR2 agonists that may be used in conjunction with the compositions
and methods described herein include other variants of Gro-B, Gro-ß, such as peptides that have one
or more amino acid substitutions, insertions, and/or deletions relative to Gro-B. Gro-ß. In some
embodiments, CXCR2 agonists that may be used in conjunction with the compositions and
methods described herein include peptides having at least 85% sequence identity to the amino
acid sequence of SEQ ID NO: 1 (e.g., a peptide having at least 85%, 90%, 95%, 96%, 97%,
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98%, 99%, 99.5%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1).
In some embodiments, the amino acid sequence of the CXCR2 agonist differs from that of
SEQ ID NO: 1 only by way of one or more conservative amino acid substitutions. In some
embodiments, in some embodiments, the amino acid sequence of the CXCR2 agonist differs
from that of SEQ ID NO: 1 by no more than 20, no more than 15, no more than 10, no more
than 5, or no more than 1 nonconservative amino acid substitutions.
[0195] Additional examples of CXCR2 agonists useful in conjunction with the compositions
and methods described herein are variants of Gro-B Gro-ß T, such as peptides that have one or more
amino acid substitutions, insertions, and/or deletions relative to Gro-B Gro-ß T. In some
embodiments, the CXCR2 agonist may be a peptide having at least 85% sequence identity to
the amino acid sequence of SEQ ID NO: 2 (e.g., a peptide having at least 85%, 90%, 95%,
96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 2). In some embodiments, the amino acid sequence of the CXCR2 agonist differs
from that of SEQ ID NO: 2 only by way of one or more conservative amino acid
substitutions. In some embodiments, in some embodiments, the amino acid sequence of the
CXCR2 agonist differs from that of SEQ ID NO: 2 by no more than 20, no more than 15, no
more than 10, no more than 5, or no more than 1 nonconservative amino acid substitutions.
[0196] Additional examples of CXCR2 agonists useful in conjunction with the compositions
and methods described herein are variants of Gro-B Gro-ß N69D, such as peptides that have one or
more amino acid substitutions, insertions, and/or deletions relative to Gro-B Gro-ß N69D. In some
embodiments, the CXCR2 agonist may be a peptide having at least 85% sequence identity to
the amino acid sequence of SEQ ID NO: 3 (e.g., a peptide having at least 85%, 90%, 95%,
96%, 97%, 98%, 99%, 99.%, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 3). In some embodiments, the amino acid sequence of the CXCR2 agonist differs
from that of SEQ ID NO: 3 only by way of one or more conservative amino acid
substitutions. In some embodiments, in some embodiments, the amino acid sequence of the
CXCR2 agonist differs from that of SEQ ID NO: 3 by no more than 20, no more than 15, no
more than 10, no more than 5, or no more than 1 nonconservative amino acid substitutions.
[0197] Additional examples of CXCR2 agonists useful in conjunction with the compositions
and methods described herein are variants of Gro-B Gro-ß T N65D, such as peptides that have one
or more amino acid substitutions, insertions, and/or deletions relative to Gro-B Gro-ß T N65D. In
some embodiments, the CXCR2 agonist may be a peptide having at least 85% sequence
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identity to the amino acid sequence of SEQ ID NO: 4 (e.g., a peptide having at least 85%,
90%, 95%, 96%, 97%, 98%, 99%, 99.%, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 4). In some embodiments, the amino acid sequence of the CXCR2
agonist differs from that of SEQ ID NO: 4 only by way of one or more conservative amino
acid substitutions. In some embodiments, in some embodiments, the amino acid sequence of
the CXCR2 agonist differs from that of SEQ ID NO: 4 by no more than 20, no more than 15,
no more than 10, no more than 5, or no more than 1 nonconservative amino acid
substitutions.
Agonistic anti-CXCR2 antibodies and antigen-binding fragments thereof
[0198] In some embodiments, the CXCR2 agonist is an antibody or antigen-binding fragment
thereof that binds CXCR2 and activates CXCR2 signal transduction. In some embodiments,
the CXCR2 agonist may be an antibody or antigen-binding fragment thereof that binds the
same epitope on CXCR2 as Gro-B Gro-ß or a variant or truncation thereof, such as Gro-B Gro-ß T, as
assessed, for example, by way of a competitive CXCR2 binding assay. In some
embodiments, the CXCR2 agonist is an antibody or an antigen-binding fragment thereof that
competes with Gro-B Gro-ß or a variant or truncation thereof, such as Gro-ß T, for binding to
CXCR2.
[0199] In some embodiments of any of the above aspects, the antibody or antigen-binding
fragment thereof is selected from the group consisting of a monoclonal antibody or antigen-
binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a
humanized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-
binding fragment thereof, a dual-variable immunoglobulin domain, a single-chain Fv
molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, a Fv
fragment, a Fab fragment, a F(ab')2 molecule,and F(ab') molecule, andaatandem tandemdi-scFv. di-scFv.In Insome someembodiments, embodiments,
the antibody has an isotype selected from the group consisting of IgG, IgA, IgM, IgD, and
IgE. IgE.
Synthetic CXCR2 Agonists
[0200] The peptidic CXCR2 agonists described herein, such as Gro-ß, Gro-B Gro-ß T, and variants
thereof, may be prepared synthetically, for instance, using solid phase peptide synthesis
techniques. Systems and processes for performing solid phase peptide synthesis include those
that are known in the art and have been described, for instance, in US Patent Nos. 9,169,287; 9, 169,287;
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9,388,212; 9,206,222; 6,028,172; and 5,233,044, among others, the disclosures of each of
which are incorporated herein by reference as they pertain to protocols and techniques for the
synthesis of peptides on solid support. Solid phase peptide synthesis is a process in which
amino acid residues are added to peptides that have been immobilized on a solid support, such
as a polymeric resin (e.g., a hydrophilic resin, such as a polyethylene-glycol-containing resin,
or hydrophobic resin, such as a polystyrene-based resin).
[0201] Peptides, such as those containing protecting groups at amino, hydroxy, thiol, and
carboxy substituents, among others, may be bound to a solid support such that the peptide is
effectively immobilized on the solid support. For example, the peptides may be bound to the
solid support via their C termini, thereby immobilizing the peptides for subsequent reaction in
at a resin-liquid interface.
[0202] The process of adding amino acid residues to immobilized peptides can include
exposing a deprotection reagent to the immobilized peptides to remove at least a portion of
the protection groups from at least a portion of the immobilized peptides. The deprotection
reagent exposure step can be configured, for instance, such that side-chain protection groups
are preserved, while N-terminal protection groups are removed. For instance, an exemplary
amino protecting contains a fluorenylmethyloxycarbonyl (Fmoc) substituent. A deprotection
reagent containing a strongly basic substance, such as piperidine (e.g., a piperidine solution in
an appropriate organic solvent, such as dimethyl formamide (DMF)) may be exposed to the
immobilized peptides such that the Fmoc protecting groups are removed from at least a
portion of the immobilized peptides. Other protecting groups suitable for the protection of
amino substituents include, for instance, the tert-butyloxycarbonyl (Boc) moiety. A
deprotection reagent comprising a strong acid, such as trifluoroacetic acid (TFA) may be
exposed to immobilized peptides containing a Boc-protected amino substituent SO so as to
remove the Boc protecting group by an ionization process. In this way, peptides can be
protected and deprotected at specific sites, such as at one or more side-chains or at the N- or
C-terminus of an immobilized peptide SO so as to append chemical functionality regioselectively
at one or more of these positions. This can be used, for instance, to derivatize a side-chain of
an immobilized peptide, or to synthesize a peptide, e.g., from the C-terminus to the N-
terminus.
[0203] The process of adding amino acid residues to immobilized peptides can include, for
instance, exposing protected, activated amino acids to the immobilized peptides such that at
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least a portion of the activated amino acids are bonded to the immobilized peptides to form
newly-bonded amino acid residues. For example, the peptides may be exposed to activated
amino acids that react with the deprotected N-termini of the peptides SO so as to elongate the
peptide chain by one amino acid. Amino acids can be activated for reaction with the
deprotected peptides by reaction of the amino acid with an agent that enhances the
electrophilicity of the backbone carbonyl carbon of the amino acid. For example,
phosphonium and uronium salts can, in the presence of a tertiary base (e.g.,
diisopropylethylamine (DIPEA) and triethylamine (TEA), among others), convert protected
amino acids into activated species (for example, BOP, PyBOP, HBTU, and TBTU all
generate HOBt esters). Other reagents can be used to help prevent racemization that may be
induced in the presence of a base. These reagents include carbodiimides (for example, DCC
or WSCDI) with an added auxiliary nucleophile (for example, 1-hydroxy-benzotriazole
(HOBt), 1-hydroxy-azabenzotriazole (HOAt), or HOSu) or derivatives thereof. Another
reagent that can be utilized to prevent racemization is TBTU. The mixed anhydride method,
using isobutyl chloroformate, with or without an added auxiliary nucleophile, can also be
used, as well as the azide method, due to the low racemization associated with this reagent.
These types of compounds can also increase the rate of carbodiimide-mediated couplings, as
well as prevent dehydration of Asn and Gln residues. Typical additional reagents include also
bases such as N,N-diisopropylethylamine (DIPEA), triethylamine (TEA) or N-
methylmorpholine (NMM). These reagents are described in detail, for instance, in US Patent
No. 8,546,350, the disclosure of which is incorporated herein in its entirety.
[0204] During the recombinant expression and folding of Gro-B Gro-ß and Gro-B Gro-ß T in aqueous
solution, a particular C-terminal asparagine residue (Asn69 within Gro-B Gro-ß and Asn65 within
Gro-ß T) is prone to deamidation. This process effectuates the conversion of the asparagine
residue to aspartic acid. Without wishing to be bound by any theory, the chemical synthesis
of Gro-B Gro-ß and Gro-B Gro-ß T may overcome this problem, for instance, by providing conditions that
reduce the exposure of this asparagine residue to nucleophilic solvent. When prepared
synthetically (i.e., chemically synthesized), for instance, using, e.g., the solid phase peptide
synthesis techniques described above, synthetic Gro-B, Gro-ß, Gro-B Gro-ß T, and variants thereof that may
be used in conjunction with the compositions and methods described herein may have a
purity of, e.g., at least about 95% relative to the deamidated versions of these peptides (i.e.,
contain less than 5% of the corresponding deamidated peptide). For instance, synthetic Gro-
B, ß, Gro-B Gro-ß T, and variants thereof that may be used in conjunction with the compositions and
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methods described herein may have a purity of about 95%, 95.5%, 96%, 96.5%, 97%, 97.5%,
98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or more, relative to the
deamidated versions of these peptides (e.g., the Asn69 deamidated version of SEQ ID NO: 1
or the Asn65 deamidated version of SEQ ID NO: 2). For instance, synthetic Gro-B, Gro-ß, Gro-B Gro-ß T,
and variants thereof may have, for instance, a purity of from about 95% to about 99.99%,
such as a purity of from about 95% to about 99.99%, about 96% to about 99.99%, about 97%
to about 99.99%, about 98% to about 99.99%, about 99% to about 99.99%, about 99.9% to
about 99.99%, about 95% to about 99.5%, about 96% to about 99.5%, about 95% to about
99%, or about 97% to about 99% relative to the deamidated versions of these peptides (e.g.,
the Asn69 deamidated version of SEQ ID NO: 1 or the Asn65 deamidated version of SEQ ID
NO: 2).
CXCR4 Antagonists
[0205] Exemplary CXCR4 antagonists for use in conjunction with the compositions
and methods described herein are compounds represented by formula (I)
Z - -linker linker -- Z' Z' (I) (I)
or a pharmaceutically acceptable salt thereof, wherein Z is:
(i) (i) a cyclic polyamine containing from 9 to 32 ring members, wherein
from 2 to 8 of the ring members are nitrogen atoms separated from one
another by 2 or more carbon atoms; or
(ii) (ii) an amine represented by formula (IA)
A N B (IA) (IA)
wherein A includes a monocyclic or bicyclic fused ring system including at
least one nitrogen atom and B is H or a substituent of from 1 to 20 atoms;
and wherein Z' is:
(i) (i) a cyclic polyamine containing from 9 to 32 ring members, wherein
from 2 to 8 of the ring members are nitrogen atoms separated from one
another by 2 or more carbon atoms;
(ii) (ii) an amine represented by formula (IB)
A' A' N B' B' (IB)
wherein A' includes a monocyclic or bicyclic fused ring system including at
least one nitrogen atom and B' is H or a substituent of from 1 to 20 atoms; or
(iii) a substituent represented by formula (IC)
--N(R)-(CR2)n-1 N(R) - (CR) - X-(IC) (IC)
wherein whereineach eachR R is is independently H or HC1-C6 independently alkyl, or C-C n is n alkyl, 1 or is 2, and 2, 1 or X is an Xaryl and is an aryl
or heteroaryl group or a mercaptan;
wherein the linker is a bond, optionally substituted alkylene (e.g., optionally substituted C1- C-
C6 alkylene), optionally C alkylene), optionallysubstituted heteroalkylene substituted (e.g., (e.g., heteroalkylene optionally substituted optionally C1-C6 substituted C-C
heteroalkylene), heteroalkylene), optionally substituted optionally alkenylene substituted (e.g., optionally alkenylene substituted (e.g., optionally C2-C6 substituted C-C
alkenylene), alkenylene),optionally substituted optionally heteroalkenylene substituted (e.g., optionally heteroalkenylene substituted (e.g., optionally C2-C6 substituted C-C
heteroalkenylene), heteroalkenylene), optionally substituted optionally alkynylene substituted (e.g., optionally alkynylene substitutedsubstituted (e.g., optionally C2-C6 C-C
alkynylene), alkynylene),optionally substituted optionally heteroalkynylene substituted (e.g., optionally heteroalkynylene substitutedsubstituted (e.g., optionally C2-C6 C-C
heteroalkynylene), optionally substituted cycloalkylene, optionally substituted
heterocycloalkylene, optionally substituted arylene, or optionally substituted heteroarylene.
[0206] In some embodiments, Z and Z' may each independently a cyclic polyamine
containing from 9 to 32 ring members, of which from 2 to 8 are nitrogen atoms separated
from one another by 2 or more carbon atoms. In some embodiments, Z and Z' are identical
substituents. As an example, Z may be a cyclic polyamine including from 10 to 24 ring
members. In some embodiments, Z may be a cyclic polyamine that contains 14 ring
members. In some embodiments, Z includes 4 nitrogen atoms. In some embodiments, Z is
8,11-tetraazocyclotetradecane 1,4,8,11-tetraazocyclotetradecane
[0207] In some embodiments, the linker is represented by formula (ID)
-xX D YT Y (ID) wherein ring D is an optionally substituted aryl group, an optionally substituted heteroaryl group, an optionally substituted cycloalkyl group, or an optionally substituted heterocycloalkyl group; and
X and Y are each independently optionally substituted alkylene (e.g., optionally substituted
C1-C6 alkylene), optionally C-C alkylene), optionallysubstituted heteroalkylene substituted (e.g.,(e.g., heteroalkylene optionally substituted optionally C1-C6 substituted C-C
heteroalkylene), heteroalkylene), optionally substituted optionally alkenylene substituted (e.g., optionally alkenylene substituted (e.g., optionally C2-C6 substituted C-C
alkenylene), alkenylene),optionally substituted optionally heteroalkenylene substituted (e.g., optionally heteroalkenylene substitutedsubstituted (e.g., optionally C2-C6 C-C
heteroalkenylene), heteroalkenylene), optionally substituted optionally alkynylene substituted (e.g., optionally alkynylene substitutedsubstituted (e.g., optionally C2-C6 C-C
alkynylene), alkynylene),oror optionally substituted optionally heteroalkynylene substituted (e.g., optionally heteroalkynylene substitutedsubstituted (e.g., optionally C2-C6 C-C
heteroalkynylene).
[0208] As an example, the linker may be represented by formula (IE)
X X
And Y x (IE) wherein ring D is an optionally substituted aryl group, an optionally substituted heteroaryl
group, an optionally substituted cycloalkyl group, or an optionally substituted
heterocycloalkyl group; and
X and Y are each independently optionally substituted alkylene (e.g., optionally substituted
C1-C6 alkylene), optionally C-C alkylene), optionallysubstituted heteroalkylene substituted (e.g.,(e.g., heteroalkylene optionally substituted optionally C1-C6 substituted C-C
heteroalkylene), heteroalkylene), optionally substituted optionally C2-C6 C-C substituted alkenylene (e.g.,(e.g., alkenylene optionally substituted optionally C2-C6 substituted C-C
alkenylene), alkenylene),optionally substituted optionally heteroalkenylene substituted (e.g., optionally heteroalkenylene substitutedsubstituted (e.g., optionally C2-C6 C-C
heteroalkenylene), heteroalkenylene), optionally substituted optionally alkynylene substituted (e.g., optionally alkynylene substitutedsubstituted (e.g., optionally C2-C6 C-C
alkynylene), alkynylene),oror optionally substituted optionally heteroalkynylene substituted (e.g., optionally heteroalkynylene substitutedsubstituted (e.g., optionally C2-C6 C-C
heteroalkynylene). In some embodiments, X and Y are each independently optionally
substituted C1-C6 alkylene. C-C alkylene. InIn some some embodiments, embodiments, X X and and Y Y are are identical identical substituents. substituents. InIn
some embodiments, X and Y may be each be methylene, ethylene, n-propylene, n-butylene,
n-pentylene, or n-hexylene groups. In some embodiments, X and Y are each methylene
groups.
[0209] The linker may be, for example, 1,3-phenylene, 2,6-pyridine, 3,5-pyridine, 2,5-
thiophene, 4,4'-(2,2'-bipyrimidine), 2,9-(1,10-phenanthroline), or the like. In some
embodiments, the linker is 1,4-phenylene-bis-(methylene)
[0210] CXCR4 antagonists useful in conjunction with the compositions and methods
described herein include plerixafor (also referred to herein as "AMD3100" and "Mozibil"), or
a pharmaceutically acceptable salt thereof, represented by formula (II), 1,1'-[1,4-
phenylenebis(methylene)]-bis-1,4,8,11-tetra-azacyclotetradecane phenylenebis(methylene)]-bis-1,4,8,11-tetra-azacyclotetradecane
HN HN N NH HN. HN N NH NH (II) (II)
[0211] Additional CXCR4 antagonists that may be used in conjunction with the compositions
and methods described herein include variants of plerixafor, such as a compound described in
US Patent No. 5,583,131, the disclosure of which is incorporated herein by reference as it
pertains to CXCR4 antagonists. In some embodiments, the CXCR4 antagonist may be a
compound selected from the group consisting of: 1,1'-[1,3-phenylenebis(methylene)]-bis-
1,4,8,11 stetra-azacyclotetradecane; 1,1'-[1,4-phenylene-bis-(methylene)]-bis-1,4,8,11- 1,4,8,11-tetra-azacyclotetradecane, 1,1'-[1,4-phenylene-bis-(methylene)]-bis-1,4,8,11-
tetraazacyclotetradecane; bis-zinc or bis-copper complex of 1,1'-[1,4-phenylene-bis-
(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane; (methylene)]-bis-1,4,8,11-tetraazacyclotetradecane,1,1'-[3,3'-biphenylene-bis-(methylene)]- 1,1'-[3,3'-biphenylene-bis-(methylene)]-
bis-1,4,8,11-tetraazacyclotetradecane; bis-1,4,8,11-tetraazacyclotetradecane; 11,11'-[1,4-phenylene-bis-(methylene)]-bis-1,4,7,11- 11,11'-[1,4-phenylene-bis-(methylene)]-bis-1,4,7,11-
1,11'-[1,4-phenylene-bis-(methylene)]-1,4,8,11 tetraazacyclotetradecane; 1,11'-[1,4-phenylene-bis-(methylene)]-1,4,8,11-
tetraazacyclotetradecane-1, 4,7,11-tetraazacyclotetradecane; 1,1'-[2,6-pyridine-bis-
(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane; 1,1-[3,5-pyridine-bis-(methylene)]-bis- (methylene)]-bis-1,4,8,11-tetraazacyclotetradecane, 1,1-[3,5-pyridine-bis-(methylene)]-bis-
1,4,8,11-tetraazacyclotetradecane; 1,1'-[2,5-thiophene-bis-(methylene)]-bis-1,4,8,11-
tetraazacyclotetradecane; 1,1'-[4,4'-(2,2'-bipyridine)-bis-(methylene)]-bis-1,4,8,11- (1,1'-[4,4'-(2,2'-bipyridine)-bis-(methylene)]-bis-1,4,8,11-
tetraazacyclotetradecane; 1,1'-[2,9-(1,10-phenanthroline)-bis-(methylene)]-bis-1,4,8,11- tetraazacyclotetradecane; 1,1'-[2,9-(1,10-phenanthroline)-bis-(methylene)]-bis-1,4,8,11-
tetraazacyclotetradecane; 1,1'-[1,3-phenylene-bis-(methylene)]-bis-1,4,7,10-
tetraazacyclotetradecane; 1,1'-[1,4-phenylene-bis-(methylene)]-bis-1,4,7,10 1,1'-[1,4-phenylene-bis-(methylene)]-bis-1,4,7,10-
tetraazacyclotetradecane; ('-[5-nitro-1,3-phenylenebis(methylene)]bis-1,4,8,11- 1'-[5-nitro-1,3-phenylenebis(methylene)]bis-1,4,8,11-
tetraazacyclotetradecane; 1',1'-[2,4,5,6-tetrachloro-1,3-phenyleneis(methylene)]bis-1,4,8,11-
tetraazacyclotetradecane; 1,1'-[2,3,5,6-tetra-fluoro-1,4-phenylenebis(methylene)]bis-1,4,8,11-
tetraazacyclotetradecane; tetraazacyclotetradecane; 1,1'-[1,4-naphthylene-bis-(methylene)]bis-1,4,8,11- - 1,1'-[1,4-naphthylene-bis-(methylene)]bis-1,4,8,11-
tetraazacyclotetradecane; 1,1'-[1,3-phenylenebis-(methylene)]bis-1,5,9-triazacyclododecane 1,1'-[1,3-phenylenebis-(methylene)]bis-1,5,9-triazacyclododecane;
1,1'-[1,4-phenylene-bis-(methylene)]-1,5,9-triazacyclododecane 1,1'-[2,5-dimethyl-1,4-
phenylenebis-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane 1,1'-[2,5-dichloro-1,4-
phenylenebis-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane; 1,1'-[2-bromo-1,4- phenylenebis-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane 1,1'-[2-bromo-1,4-
202 wo 2019/113375 WO PCT/US2018/064335 phenylenebis-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane phenylenebis-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecaneand and1,1'-[6-phenyl-2,4- 1,1'-[6-phenyl-2,4- pyridinebis-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecar pyridinebis-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane.
[0212] In some embodiments, the CXCR4 antagonist is a compound described in US
2006/0035829, the disclosure of which is incorporated herein by reference as it pertains to
CXCR4 antagonists. In some embodiments, the CXCR4 antagonist may be a compound
selected from the group consisting of: 3,7,11,17-tetraazabicyclo(13.3.1)heptadeca-
1(17),13,15-triene;
4,7,10,17-tetraazabicyclo(13.3.1)heptadeca-1(17),13,15-triene; 1,4,7,10- 4,7,10,17-tetraazabicyclo(13.3.1)heptadeca-1(17),13,15-triene,1,4,7,10-
tetraazacyclotetradecane; 1,4,7-triazacyclotetradecane; and 4,7,10-
riazabicyclo(13.3.1)heptadeca-1(17),13,15-triene triazabicyclo(13.3.1)heptadeca-1(17),13,15-triene.
[0213] The CXCR4 antagonist may be a compound described in WO 2001/044229, the
disclosure of which is incorporated herein by reference as it pertains to CXCR4 antagonists.
In some embodiments, the CXCR4 antagonist may be a compound selected from the group
consisting of: N-[4-(11-fluoro-1,4,7-triazacyclotetradecanyl)-1,4-phenylenebis(methylene)]-
2-(aminomethyl)pyridine; N-[4-(11,11-difluoro-1,4,7-triazacyclotetradecanyl)-1, 2-(aminomethyl)pyridine;N-[4-(11,11-difluoro-1,4,7-triazacyclotetradecanyl)-1,4-
nenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[4-(1,4,7-triazacyclotetradecan-2- phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[4-(1,4,7-triazacyclotetradecan-2-
ony1)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[12-(5-oxa-1,9- onyl)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine N-[12-(5-oxa-1,9-
diazacyclotetradecanyl)-1,4-phenylenebis(methylene)]-2-(aminomethy1)pyridine N-[4-(11- diazacyclotetradecanyl)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine, N-[4-(11-
oxa-1,4,7-triazacyclotetradecanyl)-1,4-phenylenebis(methylene)]-2-(aminomethy) oxa-1,4,7-triazacyclotetradecanyl)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine;
N-[4-(11-thia-1,4,7-triazacyclotetradecanyl)-1,4-phenylenebis(methylene)]-2-
;N-[4-(11-sulfoxo-1,4,7-triazacyclotetradecanyl)-1,4- (aminomethyl)pyridine; N-[4-(11-sulfoxo-1,4,7-triazacyclotetradecanyl)-1,4-
phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[4-(11-sulfono-1,4,7- phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[4-(11-sulfono-1,4,7-
iazacyclotetradecanyl)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine; and triazacyclotetradecanyl)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine; and N-[4- N-[4-
(3-carboxo-1,4,7-triazacyclotetradecanyl)-1,4-phenylenebis(methylene)]-2-
(aminomethyl)pyridine.
[0214] Additional CXCR4 antagonists useful in conjunction with the compositions and
methods described herein include compounds described in WO 2000/002870, the disclosure
of which is incorporated herein by reference as it pertains to CXCR4 antagonists. In some
embodiments, the CXCR4 antagonist may be a compound selected from the group consisting
of: N-[1,4,8,11-tetraazacyclotetra-decanyl-1,4-phenylenebis-(methylene)] N-[1,4,8,11-tetraazacyclotetra-decanyl-1,4-phenylenebis-(methylene)]-2-
N-[1,4,8,11-tetraazacyclotetra-decanyl-1,4 (aminomethyl)pyridine; N-[1,4,8,11-tetraazacyclotetra-decanyl-1,4-
phenylenebis(methylene)]-N-methy1-2-(aminomethyl)pyridine; N-[1,4,8,11- phenylenebis(methylene)]-N-methyl-2-(aminomethyl)pyridine: wo 2019/113375 WO PCT/US2018/064335 etraazacyclotetra-decanyl-1,4-phenylenebis(methylene)]-4-(aminomethyl)pyridine N- tetraazacyclotetra-decanyl-1,4-phenylenebis(methylene)]-4-(aminomethyl)pyridine;N-
(1,4,8,11-tetraazacyclotetra-decanyl-1,4-phenylenebis(methylene)]-3-(aminomethyl)pyridine 1,4,8,11-tetraazacyclotetra-decanyl-1,4-phenylenebis(methylene)]-3-(aminomethyl)pyridine,
8,11-tetraazacyclotetra-decanyl-1,4-phenylenebis(methylene)]-(2-aminomethyl-5- J-[1,4,8,11-tetraazacyclotetra-decanyl-1,4-phenylenebis(methylene)]-(2-aminomethy1-5-
methyl)pyrazine; N-[1,4,8,11-tetraazacyclotetra-decanyl-1,4-phenylenebis(methylene)] N-[1,4,8,11-tetraazacyclotetra-decanyl-1,4-phenylenebis(methylene)]-2-
(aminoethyl) pyridine; N-[1,4,8,11-tetraazacyclotetra-decanyl-1,4-phenylenebis(methylene)]-
2-(aminomethyl)thiophene; N-[1,4,8,11-tetraazacyclotetra-decany1-1,4- 2-(aminomethyl)thiophene; N-[1,4,8,11-tetraazacyclotetra-decanyl-1,4-
phenylenebis(methylene)]-2-(aminomethyl)mercaptan; phenylenebis(methylene)]-2-(aminomethyl)mercaptan; N-[1,4,8,11-tetraazacyclotetra- N-[1,4,8,11-tetraazacyclotetra-
decanyl-1,4-phenylenebis(methylene)]-2-aminotbenzylamine; decanyl-1,4-phenylenebis(methylene)]-2-amino benzylamine;N-[1,4,8,11-tetraazacyclotetra- N-[1,4,8,11-tetraazacyclotetra-
decanyl-1,4-phenylenebis(methylene)]-4-amino benzylamine, decanyl-1,4-phenylenebis(methylene)]-4-amino benzylamine; N-[1,4,8,11-tetraazacyclotetra- N-[1,4,8,11-tetraazacyclotetra-
decanyl-1,4-phenylenebis(methylene)]-4-(aminoethyl)imidazole; decanyl-1,4-phenylenebis(methylene)]4-(aminoethyl)imidazole, 1N-[1,4,8,11- N-[1,4,8,11-
tetraazacyclotetra-decanyl-1,4-phenylenebis(methylene)]-benzylamine; N-[4-(1,4,7- tetraazacyclotetra-decanyl-1,4-phenylenebis(methylene)]-benzylamine, N-[4-(1,4,7-
triazacyclotetra-decany1)-1,4-phenylenebis(methylene)]-2-(aminomethy1)pyridine;N-[7- triazacyclotetra-decanyl)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine;,N-[7-
(4,7,10,17-tetraazabicyclo[13.3.1]heptadeca-1(17),13,15-trieny1)-1,4- (4,7,10,17-tetraazabicyclo[13.3.1]heptadeca-1(17),13,15-trienyl)-1,4-
henylenebis(methylene)]-2-(aminomethyl)pyridine; N-[7-(4,7,10- phenylenebis(methylene)]-2-(aminomethyl)pyridine, N-[7-(4,7,10-
riazabicyclo[13.3.1]heptadeca-1(17),13,15-trieny1)-1,4-phenylenebis(methylene)]-2- triazabicyclo[133.1]heptadeca-1(17),13,15-trienyl)-1,4-phenylenebis(methylene)]-2-
;N-[1-(1,4,7-triazacyclotetra-decanyl)-1,4-phenylenebis(methylene)]- (aminomethyl)pyridine; N-[1-(1,4,7-triazacyclotetra-decanyl)-1,4-phenylenebis(methylene)]-
2-(aminomethyl)pyridine; ;N-[4-[4,7,10,17-tetraazabicyclo[13.3.1]heptadeca-1(17),13,15- N-[4-[4,7,10,17-tetraazabicyclo[13.3.1]heptadeca-1(17),13,15-
trieny1]-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine, N-[4-[4,7,10 trienyl]-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[4-[4,7,10-
triazabicyclo[13.3.1]heptadeca-1(17),13,15-trieny1]-1,4-phenylenebis(methylene)]-2- triazabicyclo[133.1]heptadeca-1(17),13,15-trienyl]-1,4-phenylenebis(methylene)]-2-
(aminomethyl)pyridine;N-[1,4,8,11-tetraazacyclotetradecanyl-1,4- (aminomethyl)pyridine; N-[1,4,8,11-tetraazacyclotetradecanyl-1,4-
phenylenebis(methylene)]-purine,1-[1,4,8,11-tetraazacyclotetradecanyl-1,4 phenylenebis(methylene)]-purine -[1,4,8,11-tetraazacyclotetradecanyl-1,4-
phenylenebix(methylene)]-4-phenylpiperazine N-[4-(1,7-diazacyclotetradecanyl)-1,4- phenylenebix(methylene)]-4-phenylpiperazine; N-[4-(1,7-diazacyclotetradecanyl)-1,4-
nenylenebis(methylene)]-2-(aminomethyl)pyridine; and phenylenebis(methylene)]-2-(aminomethyl)pyridine; and N-[7-(4,10- N-[7-(4,10-
diazabicyclo[13.3.1]heptadeca-1(17),13,15-trieny1)-1,4-phenylenebis(methylene)]-2- diazabicyclo[13.3 1]heptadeca-1(17),13,15-trienyl)-1,4-phenylenebis(methylene)]-2-
(aminomethyl)pyridine.
[0215] In some embodiments, the CXCR4 antagonist is a compound selected from the group
consisting of: consisting :1-[2,6-dimethoxypyrid-4-yl(methylene)]-1,4,8,11-tetraazacyclotetradecane; of: -[2,6-dimethoxypyrid-4-yl(methylene)]-1,4,8,1l-tetraazacyclotetradecane, 1- - 1-
-chloropyrid-4-yl(methylene)]-1,4,8,11-tetraazacyclotetradecane; 1-[2,6-dimethylpyrid-4-
[2-chloropyrid-4-yl(methylene)]-1,4,8,11-tetraazacyclotetradecane;
y1(methylene)]-1,4,8,11-tetraazacyclotetradecane; 1-[2-methylpyrid-4-yl(methylene)]- yl(methylene)]-1,4,8,11-tetraazacyclotetradecane; 1-[2-methylpyrid-4-yl(methylene)]-
1,4,8,11-tetraazacyclotetradecane; 1,4,8,11-tetraazacyclotetradecane; 1-[2,6-dichloropyrid-4-yl(methylene)]-1,4,8,11- 1-[2,6-dichloropyrid-4-yl(methylene)]-1,4,8,11-
tetraazacyclotetradecane; 1-[2-chloropyrid-5-yl(methylene)]-1,4,8,11-
tetraazacyclotetradecane; and 7-[4-methylphenyl (methylene)]-4,7,10,17-
etraazabicyclo[13.3.1]heptadeca-1(17),13,15-triene tetraazabicyclo[13.3.1]heptadeca-1(17),13,15-triene.
WO wo 2019/113375 PCT/US2018/064335
[0216] In some embodiments, the CXCR4 antagonist is a compound described in US Patent
No. 5,698,546, the disclosure of which is incorporated herein by reference as it pertains to
CXCR4 antagonists. In some embodiments, the CXCR4 antagonist may be a compound
selected from the group consisting of: 7,7'-[1,4-phenylene-bis(methylene)]bis-3,7,11,17-
tetraazabicyclo[13.3.1]heptadeca-1(17),13,15-triene; 7,7'-[1,4-phenylene- 7,7-[1,4-phenylene-
bis(methylene)]bis[15-chloro-3,7,11,17-tetraazabicyclo[13.3.1]heptadeca-1 ois(methylene)]bis[15-chloro-3,7,11,17-tetraazabicyclo[13.3.1]heptadeca-1 (17),13,15- (17),13,15-
triene]; 7,7'-[1,4-phenylene-bis(methylene)]bis[15-methoxy-3,7,11,17 7,7'-[1,4-phenylene-bis(methylene)]bis[15-methoxy-3,7,11,17-
tetraazabicyclo[13.3.1]heptadeca-1(17),13,15-triene]; 7,7'-[1,4-phenylene- tetraazabicyclo[13.3.1]heptadeca-1(17),13,15-triene],7,7-[1,4-phenylene-
bis ene)]bis-3,7,11,17-tetraazabicyclo[13.3.1]-heptadeca-13,16-triene-15-one; 7,7'- - bis(methylene)]bis-3,7,11,17-tetraazabicyclo[13.3.1]-heptadeca-13,16-triene-15-one,7,7'-
4-phenylene-bis(methylene)]bis-4,7,10,17-tetraazabicyclo[13.3.1]-heptadeca-1(17),13,15
[1,4-phenylene-bis(methylene)]bis-4,7,10,17-tetraazabicyclo[13.3.1]-heptadeca-1(17),13,15-
triene; triene, 8,8'-[1,4-phenylene-bis(methylene)]bis-4,8,12,19-tetraazabicyclo[15.3.1]nonade 8,8-[1,4-phenylene-bis(methylene)]bis-4,8,12,19-tetraazabicyclo[15.3.1]nonadeca-
1(19),15,17-triene; 1(19),15,17-triene; 6,6'-[1,4-phenylene-bis(methylene)]bis-3,6,9,15- 6,6'-[1,4-phenylene-bis(methylene)]bis-3,6,9,15-
tetraazabicyclo[11.3.1]pentadeca-1 (15),11,13-triene; tetraazabicyclo[11.3.1|pentadeca-1 (15),11,13-triene; 6,6'-[1,3-phenylene-bis(methylene)]bis- 6,6'-[1,3-phenylene-bis(methylene)]bis-
3,6,9,15-tetraazabicyclo[11.3.1]pentadeca-1 (15),11,13-triene;and 3,6,9,15-tetraazabicyclo[113.1|pentadeca-1(15),11,13-triene; and17,17-I1,4-phenylene- 17,17'-[1,4-phenylene-
bis(methylene)]bis-3,6,14,17,23,24-hexazatricyclo[173.1.1°¹²]tetacosa- bis(methylene)]bis-3,6,14,17,23,24-hexaazatricyclo[17.3.1.18,12]tetracosa-
(23),8,10,12(24),19,21-hexaene. 1(23),8,10,12(24),19,21-hexaene.
[0217] In some embodiments, the CXCR4 antagonist is a compound described in US Patent
No. 5,021,409, the disclosure of which is incorporated herein by reference as it pertains to
CXCR4 antagonists. In some embodiments, the CXCR4 antagonist may be a compound
selected from the group consisting of: 2,2'-bicyclam, 6,6'-bicyclam; 3,3'-(bis-1,5,9,13-
3,3'-(bis-1,5,8,11,14-pentaazacyclohexadecane); methylene (or tetraaza cyclohexadecane); 3,3'-(bis-1,5,8,11,14-pentaazacyclohexadecane),
polymethylene) di-1-N-1,4,8,11-tetraaza polymethylene) cyclotetradecane; cyclotetradecane; 3,3'-bis-1,5,9,13- 3,3'-bis-1,5,9,13- tetraazacyclohexadecane; 3,3'-bis-1,5,8,11,14-pentaazacyclohexadecane; 5,5'-bis-1,4,8,11-
tetraazacyclotetradecane; 2,5'-bis-1,4,8,11-tetraazacyclotetradecane; 2,6'-bis-1,4,8,11-
11,11'-(1,2-ethanediyl)bis-1,4,8,11-tetraazacyclotetradecane tetraazacyclotetradecane; 11,11'-(1,2-ethanediyl)bis-1,4,8,ll-tetraazacyclotetradecane,
11,11'-(1,2-propanediyl)bis-1,4,8,11-tetraazacyclotetradecane; 11,11'-(1,2-propanediyl)bis-1,4,8,11-tetraazacyclotetradecane, 11,11'-(1,2-butanediyl)bis- 11,11'- (1,2-butanediyl)bis-
1,4,8,11-tetraazacyclotetradecane; 11,11'-(1,2-pentanediyl)bis-1,4,8,11-
tetraazacyclotetradecane; and and tetraazacyclotetradecane; 111,11'-(1,2-hexanediyl)bis-1,4,8,11-tetraazacyclotetradecane 11,11'-(1,2-hexanediyl)bis-1,4,8,11-tetraazacyclotetradecane
[0218] In some embodiments, the CXCR4 antagonist is a compound described in WO
2000/056729, the disclosure of which is incorporated herein by reference as it pertains to
CXCR4 antagonists. In some embodiments, the CXCR4 antagonist may be a compound
selected from the group consisting of: N-(2-pyridinylmethyl)-N'-(6,7,8,9-tetrahydro-5H-
cyclohepta[b]pyridin-9-y1)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(5,6,7,8- cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine;
WO wo 2019/113375 PCT/US2018/064335
etrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethy1)-N'-(6,7-dihydro- tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine, N-(2-pyridinylmethyl)-N'-(6,7-dihydro-
5H-cyclopenta[b]pyridin-7-yl)-1,4-benzenedimethanamine N-(2-pyridinylmethyl)-N'- 5H-cyclopenta[b]pyridin-7-yl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-
(1,2,3,4-tetrahydro-1-naphthalenyl)-1,4-benzenedimethanamine; (1,2,3,4-tetrahydro-1-naphthalenyl)-1,4-benzenedimethanamine; N-(2-pyridinyImethyl)-N' N-(2-pyridinylmethyl)-N'-
(1-naphthalenyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(8-quinolinyl)-1,4- (1-naphthalenyl)-1,4-benzenedimethanamine, N-(2-pyridinylmethy1)-N'-(8-quinolinyl)-1,4-
benzenedimethanamine; benzenedimethanamine; N-(2-pyridinylmethy1)-N'-[2-[(2-pyridinylmethy1)amino]ethy1]-N'- N-(2-pyridinylmethyl)-N-[2-[(2-pyridinylmethyl)amino]ethyl]-N-
(1-methyl-1,2,3,4-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; (1-methyl-1,2,3,4-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine,N-(2- N-(2-
pyridinylmethy1)-N'-[2-[(1H-imidazol-2-ylmethy1)amino]ethy1]-N'-(1-methyl-1,2,3,4- pyridinylmethyl)-N'-[2-[(1H-imidazol-2-ylmethyl)amino]ethyl]-N-(1-methyl-1,2,3,4-
etrahydro-8-quinolinyl)-1,4-benzenedimethanamine, N-(2-pyridinylmethyl)-N'-(1,2,3,4- tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine,
tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine, N-(2-pyridinylmethyl)-N'-I2-[(1H- tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethy1)-N'-[2-[(1H-
imidazol-2-ylmethy1)amino]ethy1]-N'-(1,2,3,4-tetrahydro-1-naphthalenyl)-1,4- imidazol-2-ylmethyl)amino]ethyl]-N'-(1,2,3,4-tetrahydro-1-naphthalenyl)-1,4-
benzenedimethanamine; N-(2-pyridinylmethy1)-N'-(2-phenyl-5,6,7,8-tetrahydro-8 N-(2-pyridinylmethyl)-N'-(2-phenyl-5,6,7,8-tetahydro-8-
quinolinyl)-1,4-benzenedimethanamine;N,N'-bis(2-pyridinylmethyl)-N'-(2-phenyl-5,6,7,8- quinolinyl)-1,4-benzenedimethanamine; N,N'-bis(2-pyridinylmethy1)-N'-(2-phenyl-5,6,7,8
etrahydro-8-quinolinyl)-1,4-benzenedimethanamine; tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine;
[0219]N-(2-pyridinylmethy1)-N'-(5,6,7,8-tetrahydro-5-quinolinyl)-1,4
[0219] N-(2-pyridinylmethyl)-N'-(5,6,7,8-tetrahydro-5-quinolinyl)-1,4-
benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(1H-imidazol-2-ylmethyl)-N'-(5,6,7,8- N-(2-pyridinylmethyl)-N'-(1H-imidazol-2-ylmethyl)-N-(5,6,7,8-
etrahydro-5-quinolinyl)-1,4-benzenedimethanamine N-(2-pyridinylmethyl)-N'-(1H- tetrahydro-5-quinolinyl)-1,4-benzenedimethanamine, N-(2-pyridinylmethyl)-N'-(1H-
imidazol-2-ylmethy1)-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2- imidazol-2-ylmethyl)-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine;N-(2-
yridinylmethyl)-N'-[(2-amino-3-pheny1)propyl]-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4 pyridinylmethyl)-N'-[(2-amino-3-phenyl)propyl]-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-
benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(1H-imidazol-4-ylmethyl)-N'-(5,6,7,8- benzenedimethanamine, N-(2-pyridinylmethy1)-N'-(1H-imidazol-4-ylmethyl)-N'-(5,6,7,8-
etrahydro-8-quinolinyl)-1,4-benzenedimethanamine N-(2-pyridinylmethyl)-N'-(2- tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine;1 N-(2-pyridinylmethyl)-N'-(2-
quinolinylmethy1)-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine;N-(2- quinolinylmethyl)-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine,N-(2-
byridinylmethyl)-N'-(2-(2-naphthoy1)aminoethy1)-N'-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4 pyridinylmethyl)-N'-(2-(2-naphthoyl)aminoethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-
benzenedimethanamine; N-(2-pyridinylmethy1)-N'-[(S)-(2-acetylamino-3-phenyl)propyl]-N'- N-(2-pyridinylmethyl)-N'-[(S)-(2-acetylamino-3-phenyl)propyl]-N'-
(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-[(S)- (5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine, N-(2-pyridinylmethyl)-N'-[(S)-
(2-acetylamino-3-pheny1)propyl]-N'-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4- (2-acetylamino-3-phenyl)propyl]-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-
benzenedimethanamine; benzenedimethanamine, N-(2-pyridinylmethyl)-N'-[3-((2- N-(2-pyridinylmethyl)-N'-[3-(2-
naphthalenylmethyl)amino)propyl]-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1, naphthalenylmethyl)amino)propyl]-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-
N-(2-pyridinylmethy1)-N'-[2-(S)-pyrollidinylmethy1]-N'-(5,6,7,8 benzenedimethanamine; N-(2-pyridinylmethyl)-N'-[2-(S)-pyrollidinylmethyl]-N'-(5,6,7,8-
etrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-[2-(R)- tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-[2-(R)-
pyrollidinylmethy1]-N'-(5,6,78-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2- pyrollidinylmethyl]-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine;,N-(2-
pyridinylmethyl)-N'-[3-pyrazolylmethy1]-N'-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4- pyridinylmethyl)-N'-[3-pyrazolylmethyl]-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-
;N-(2-pyridinylmethyl)-N'-[2-pyrrolylmethy1]-N'-(5,6,7,8-tetrahydro- benzenedimethanamine; N-(2-pyridinylmethyl)-N'-[2-pyrrolylmethyl]-N-(5,6,7,8-tetrahydro-
8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N-I2-thiopheneylmethyl]- 8-quinolinyl)-1,4-benzenedimethanamine, N-(2-pyridinylmethyl)-N'-[2-thiopheneylmethyl]-
206
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N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine;N-(2-pyridinylmethyl)-N'- N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine N-(2-pyridinylmethy1)-N'
2-thiazolylmethyl]-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-
[2-thiazolylmethyl]-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine,N-(2-
pyridinylmethyl)-N'-[2-furanylmethy1]-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4- pyridinylmethyl)-N'-[2-furanylmethyl]-N'-(S,6,7,8-tetrahydro-8-quinolinyl)-1,4-
benzenedimethanamine; N-(2-pyridinylmethyl)-N'-[2-[(phenylmethyl)amino]ethy1]-N' N-(2-pyridinylmethyl)-N'-[2-[(phenylmethyl)amino]ethyl]-N'-
5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethy1)-N'-(2- (5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(2-
aminoethyl)-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine;N-(2- aminoethyl)-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine;1 N-(2-
pyridinylmethyl)-N'-3-pyrrolidinyl-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4- pyridinylmethyl)-N'-3-pyrrolidinyl-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-
benzenedimethanamine; N-(2-pyridinylmethyl)-N'-4-piperidinyl-N'-(5,6,7,8-tetrahydro-8 N-(2-pyridinylmethyl)-N'-4-piperidinyl-N'-(5,6,7,8-tetahydro-8-
quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-[2-[(phenylamino]ethyl]- quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethy1)-N'-[2-[(phenyl)amino]ethyl]-
N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine;N-(2-pyridinylmethyl)-N'- N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine N-(2-pyridinylmethyl)-N'-
(7-methoxy-1,2,3,4-tetrahydro-2-naphthalenyl)-1,4-benzenedimethanamine; (7-methoxy-1,2,3,4-tetrahydro-2-naphthalenyl)-1,4-benzenedimethanamine, N-(2- N-(2-
pyridinylmethy1)-N'-(6-methoxy-1,2,3,4-tetrahydro-2-naphthalenyl)-1,4 pyridinylmethyl)-N'-(6-methoxy-1,2,3,4-tetrahydro-2-naphthalenyl)-1,4-
benzenedimethanamine; benzenedimethanamine, N-(2-pyridinylmethy1)-N'-(1-methyl-1,2,3,4-tetrahydro-2- N-(2-pyridinylmethyl)-N'-(1-methyl-1,2,3,4-tetrahydro-2-
haphthaleny1)-1,4-benzenedimethanamine; N-(2-pyridinylmethy1)-N'-(7-methoxy-3,4- naphthalenyl)-1,4-benzenedimethanamine;, N-(2-pyridinylmethyl)-N'-(7-methoxy-3,4-
dihydronaphthalenyl)-1-(aminomethy1)-4-benzamide; N-(2-pyridinylmethyl)-N'-(6-methoxy- dihydronaphthalenyl)-1-(aminomethyl)-4-benzamide, N-(2-pyridinylmethyl)-N'-(6-methoxy-
3,4-dihydronaphthaleny1)-1-(aminomethy1)-4-benzamide N-(2-pyridinylmethyl)-N'-(1H- 3,4-dihydronaphthalenyl)-1-(aminomethyl)-4-benzamide; N-(2-pyridinylmethyl)-N'-(1H-
imidazol-2-ylmethy1)-N'-(7-methoxy-1,2,3,4-tetrahydro-2-naphthaleny1)-1,4- imidazol-2-ylmethyl)-N'-(7-methoxy-1,2,3,4-tetrahydro-2-naphthalenyl)-1,4-
benzenedimethanamine; benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(8-hydroxy-1,2,3,4-tetrahydro-2- N-(2-pyridinylmethyl)-N'-(8-hydroxy-1,2,3,4-tetrahydro-2-
haphthaleny1)-1,4-benzenedimethanamine; N-(2-pyridinylmethy1)-N'-(1H-imidazol-2- naphthalenyl)-1,4-benzenedimethanamine;, N-(2-pyridinylmethyl)-N'-(1H-imidazol-2-
ylmethy1)-N'-(8-hydroxy-1,2,3,4-tetrahydro-2-naphthaleny1)-1,4-benzenedimethanamine;] ylmethyl)-N'-(8-hydroxy-1,2,3,4-tetrahydro-2-naphthalenyl)-1,4-benzenedimethanamine,N-N-
(2-pyridinylmethyl)-N'-(8-Fluoro-1,2,3,4-tetrahydro-2-naphthalenyl)-1,4- (2-pyridinylmethyl)-N'-(8-Fluoro-1,2,3,4-tetrahydro-2-naphthalenyl)-1,4-
;N-(2-pyridinylmethy1)-N'-(1H-imidazol-2-ylmethyl)-N'-(8-Fluoro- benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(1H-imidazol-2-ylmethyl)-N'-(8-Fluoro-
,2,3,4-tetrahydro-2-naphthalenyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'- 1,2,3,4-tetrahydro-2-naphthalenyl)-1,4-benzenedimethanamine, N-(2-pyridinylmethyl)-N'-
(5,6,7,8-tetrahydro-7-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(1H- (5,6,7,8-tetrahydro-7-quinolinyl)-1,4-benzenedimethanamine:
amidazol-2-ylmethy1)-N'-(5,6,7,8-tetrahydro-7-quinoliny1)-1,4-benzenedimethanamine N-(2- imidazol-2-ylmethyl)-N'-(5,6,7,8-tetrahydro-7-quinolinyl)-1,4-benzenedimethanamine,N-(2-
pyridinylmethy1)-N'-[2-[(2-naphthalenylmethy1)amino]ethy1]-N'-(5,6,7,8-tetrahydro-8- pyridinylmethyl)-N'-[2-[(2-naphthalenylmethyl)amino]ethyl]-N'-(5,6,7,8-tetrahydro-8-
uinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-[2-(isobutylamino)ethyl]- quinolinyl)-1,4-benzenedimethanamine, N-(2-pyridinylmethy1)-N'-[2-(isobutylamino)ethy1]-
N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine;I N-(2-pyridinylmethyl)-N'- N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine N-(2-pyridinylmethyl)-N'-
[2-[(2-pyridinylmethyl)amino]ethyl]-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,
[2-[(2-pyridinylmethyl)amino]ethyl]-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-
benzenedimethanamine; N-(2-pyridinylmethy1)-N'-[2-[(2-furanylmethyl)amino]ethyl]-N' N-(2-pyridinylmethyl)-N'-[2-[(2-furanylmethyl)amino]ethyI]-N-
17,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(2- 5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine;
guanidinoethy1)-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine;N-(2- guanidinoethyl)-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine;1 N-(2-
yridinylmethyl)-N'-[2-[bis-[(2-methoxy)phenylmethyl]amino]ethy1]-N'-(5,6,7,8-tetrahydro pyridinylmethyl)-N'-[2-[bis-[(2-methoxy)phenylmethylJamino]ethyl]-N'-(5,6,7,8-tetrahydo- wo 2019/113375 WO PCT/US2018/064335
8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-[2-[(1H-imidazol-4- 8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethy1)-N'-[2-[(1H-imidazol-4-
ylmethyl)amino]ethyl]-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N- ylmethyl)amino]ethyl]-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine,N-
(2-pyridinylmethy1)-N'-[2-[(1H-imidazol-2-ylmethyl)amino]ethyl]-N'-(5,6,7,8-tetrahydro- (2-pyridinylmethyl)-N'-[2-[(IH-imidazol-2-ylmethyl)amino]ethyl]-N-(5,6,7,8-tetrahydro-8-
quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-I2-(phenylureido)ethyl]- quinolinyl)-1,4-benzenedimethanamine, N-(2-pyridinylmethyl)-N'-[2-(phenylureido)ethyl]-
N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethy1)-N'- N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine N-(2-pyridinylmethyl)-N'.
[[N"-(n-butyl)carboxamido]methy1]-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4
[[N"-(n-butyl)carboxamido]methyl]-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-
benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(carboxamidomethy1)-N'-(5,6,7,8- N-(2-pyridinylmethyl)-N'-(carboxamidomethyl)-N'-(5,6,7,8-
letrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-[(N"- tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine, N-(2-pyridinylmethyl)-N'-[(N"-
phenyl)carboxamidomethy1]-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4- phenyl)carboxamidomethyl]-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-
N-(2-pyridinylmethy1)-N'-(carboxymethy1)-N'-(5,6,7,8-tetrahydro-8- benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(carboxymethyl)-N-(5,6,7,8-tetrahydro-8-
quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(phenylmethyl)-N': quinolinyl)-1,4-benzenedimethanamine, N-(2-pyridinylmethyl)-N'-(phenylmethyl)-N'
,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethy1)-N'-(1H- (5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine, N-(2-pyridinylmethyl)-N'-(1H-
benzimidazol-2-ylmethy1)-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; benzimidazol-2-ylmethyl)-N'-(5,6,7,8-tetrahydro-&-quinolinyl)-1,4-benzenedimethanamine;
N-(2-pyridinylmethy1)-N'-(5,6-dimethyl-1H-benzimidazol-2-ylmethy1)-N'-(5,6,7, N-(2-pyridinylmethyl)-N'-(5,6-dimethyl-1H-benzimidazol-2-ylmethyl)-N'-(5,6,7,8-
tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine (hydrobromide salt); N-(2-
pyridinylmethyl)-N'-(5-nitro-1H-benzimidazol-2-ylmethyl)-N'-(5,6,7,8-tetrahydro-8 pyridinylmethyl)-N'-(5-nitto-1H-benzimidazol-2-ylmethyl)-N-(5,6,7,8-tetrahydro-8-
uinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-[(1H)-5-azabenzimidazol- quinolinyl)-1,4-benzenedimethanamine;
Imethyl]-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; 1N-(2- 2-ylmethyl]-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine, N-(2-
pyridinylmethyl)-N-(4-phenyl-1H-imidazol-2-ylmethy1)-N'-(5,6,7,8-tetrahydro-8-quinolinyl)- pyridinylmethyl)-N-(4-phenyl-1H-imidazol-2-ylmethyl)-N'-(S,6,7,8-tetrahydro-8-quinolinyl)-
14-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-[2-(2-pyridinyl)ethy1]-N'-(5,6,7,8 1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-[2-(2-pyridinyl)ethyl]-N'-(5,6,7,8-
etrahydro-8-quinolinyl)-1,4-benzenedimethanamine N-(2-pyridinylmethyl)-N'-(2- tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(2-
benzoxazolyl)-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine;N-(2- benzoxazolyl)-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine,1 N-(2-
hy1)-N'-(trans-2-aminocyclohexyl)-N'-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4- pyridinylmethyl)-N'-(trans-2-aminocyclohexyl)-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-
benzenedimethanamine;N-(2-pyridinylmethyl)-N'-(2-phenylethy1)-N'-(5,6,7,8-tetrahydro-8- benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(2-phenylethyl)-N'-(5,6,7,8-tetrahydro-8-
quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethy1)-N'-(3-phenylpropyl)-N'- quinolinyl)-1,4-benzenedimethanamine;, N-(2-pyridinylmethyl)-N'-(3-phenylpropyl)-N'
5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine N-(2-pyridinylmethyl)-N'- (5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine, N-(2-pyridinylmethyl)-N'-
(trans-2-aminocyclopentyl)-N'-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine (trans-2-aminocyclopentyl)-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine)
N-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl1]-N-(5,6,7,8-tetrahydro- N-[[4-[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-N-(5,6,7,8-tetrahydro-8-
quinolinyl)-glycinamide; N-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-N-
(5,6,7,8-tetrahydro-8-quinolinyl)-(L)-alaninamide; N-[[4-[[(2- (5,6,7,8-tetrahydro-8-quinolinyl)-(L)-alaninanmide; N-[[4-[[(2-
inylmethyl)amino]methyl]phenyl]methyl]-N-(5,6,7,8-tetrahydro-8-quinolinyl)-(I pyridinylmethyl)amino]methyl|phenyl]methyl]-N-(5,6,7,8-tetrahydro-8-quinolinyl)-(L)-
aspartamide; e;N-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-N-(5,6,7,8- N-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-N-(5,6,7,8-
trahydro-8-quinolinyl)-pyrazinamide; N-[[4-[[(2- tetrahydro-8-quinolinyl)-pyrazinamide; N-[[4-[[(2-
WO wo 2019/113375 PCT/US2018/064335
pyridinylmethyl)amino]methy1]phenyl]methy1]-N-(5,6,7,8-tetrahydro-8-quinolinyl)-(L)- pyridinylmethyl)amino]methyl]phenyl]methyl]-N-(5,6,7,8-tetrahydro-8-quinolinyl)-(L)-
prolinamide;N-[[4-[[(2-pyridinylmethy1)amino]methyl]phenyl]methyl]-N-(5,6,7,8- prolinamide; N-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-N-(5,6,7,8-
strahydro-8-quinoliny1)-(L)-lysinamide; N-[[4-[[(2- tetrahydro-8-quinolinyl)-(L)-lysinamide; N-[[4-[[(2-
pyridinylmethyl)amino]methy1]phenyl]methy1]-N-(5,6,7,8-tetrahydro-8-quinolinyl)- pyridinylmethyl)amino]methyl]phenyl]methyl]-N-(5,6,7,&-tetrahydro-8-quinolinyl)-
;N-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methy1]-N-(5,6,7,8-tetrahydro- benzamide; ; N-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-N-(5,6,7,8-tetrahydro-
8-quinolinyl)-picolinamide; N'-Benzyl-N-[[4-[[(2-
pyridinylmethyl)amino]methy1]phenyl]methy1]-N-(5,6,7,8-tetrahydro-8-quinolinyl)-urea;N'- pyridinylmethyl)amino]methyl|phenyl]methyl]-N-(5,6,7,8-tetrahydro-8-quinolinyl)-urea;N'-
phenyl-N-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-N-(5,6,7,8-tetrahydro-8- phenyl-N-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-N-(5,6,7,8-tetrahydro-8-
quinolinyl)-urea; N-(6,7,8,9-tetrahydro-5H-cyclohepta[bacteriapyridin-9-yl)-4-[(2- ;N-(6,78,9-tetrahydro-5H-cycloheptabacteriapyridin-9-y1)-4-[[(2-
byridinylmethyl)amino]methyl]benzamide,N-(5,6,7,8-tetrahydro-8-quinol pyridinylmethyl)amino]methyl|benzamide; N-(5,6,7,8-tetrahydro-8-quinolinyl)-4-[[(2- inyl)-4-[[(2-
pyridinylmethyl)amino]methyl]benzamide; N,N'-bis(2-pyridinylmethyl)-N'-(5,6,7,8- pyridinylmethyl)amino]methyl]benzamide; N,N'-bis(2-pyridinylmethyl)-N'-(5,6,7,8-
etrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N,N'-bis(2-pyridinylmethyl)-N'- tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine N,N'-bis(2-pyridinylmethyl)-N'-
6,7,8,9-tetrahydro-5H-cyclohepta[bacteriapyridin-9-y1)-1,4-benzenedimethanamine;I N,N'- 6,7,8,9-tetrahydro-5H-cycloheptalbacteriapyridin-9-yl)-1,4-benzenedimethanamine;1 N,N'-
bis(2-pyridinylmethyl)-N'-(6,7-dihydro-5H-cyclopenta[bacteriapyridin-7-y1)-1,4- bis(2-pyridinylmethyl)-N'-(6,7-dihydro-5H-cyclopenta[bacteriapyridin-7-yl)-1,4-
benzenedimethanamine; benzenedimethanamine, ;N,N'-bis(2-pyridinylmethy1)-N'-(1,2,3,4-tetrahydro-1-naphthalenyl) N,N'-bis(2-pyridinylmethyl)-N'-(1,2,3,4-tetrahydro-1-naphthalenyl)
1,4-benzenedimethanamine; N,N'-bis(2-pyridinylmethyl)-N'-[(5,6,7,8-tetrahydro-8- 1,4-benzenedimethanamine; N,N'-bis(2-pyridinylmethy1)-N'-[(5,6,7,8-tetrahydro-8-
uinolinyl)methy1]-1,4-benzenedimethanamine; N,N'-bis(2-pyridinylmethyl)-N'[(6,7- quinolinyl)methyl]-1,4-benzenedimethanamine;
dihydro-5H-cyclopenta[bacteriapyridin-7-y1)methy1]-1,4-benzenedimethanamine N-(2- dihydro-5H-cyclopenta[bacteriapyridin-7-yl)methyl]-1,4-benzenedimethanamine;,N-(2-
pyridinylmethyl)-N-(2-methoxyethyl)-N'-(5,6,78-tetrahydro-8-quinolinyl)-1,4- pyridinylmethyl)-N-(2-methoxyethyl)-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-
benzenedimethanamine; benzenedimethanamine, N-(2-pyridinylmethy1)-N-[2-(4-methoxyphenyl)ethy1]-N'-(5,6,7,8 N-(2-pyridinylmethyl)-N-[2-(4-methoxyphenyl)ethyl]-N-(5,6,7,8-
tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine, N,N'-bis(2-pyridinylmethyl)-1,4- tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine, (N,N'-bis(2-pyridinylmethy1)-1,4-
5,6,7,8-tetrahydro-8-quinolinyl)benzenedimethanamine N-[(2,3-dimethoxyphenyl)methyl]- (5,6,7,8-tetrahydro-8-quinolinyl)benzenedimethanamine; N-[(2,3-dimethoxypheny1)methyl]-
N'-(2-pyridinylmethy1)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine,
N,N'-bis(2-pyridinylmethyl)-N-[1-(N"-phenyl-N"-methylureido)-4-piperidinyl]-1, N,N'-bis(2-pyridinylmethyl)-N-[1-(N"-phenyl-N"-methylureido)-4-piperidinyl]-1,3-
benzenedimethanamine; N,N'-bis(2-pyridinylmethyl)-N-[N'-p-toluenesulfonylphenylalanyl)- benzenedimethanamine, N,N'-bis(2-pyridinylmethyl)-N-[N"-p-toluenesulfonylphenylalanyl)-
4-piperidinyl]-1,3-benzenedimethanamine; N,N'-bis(2-pyridinylmethyl)-N-[1-[3-(2- 4-piperidinyl]-1,3-benzenedimethanamine; N,N'-bis(2-pyridinylmethyl)-N-[1-[3-(2-
loropheny1)-5-methyl-isoxazol-4-oy1]-4-piperidinyl]-1,3-benzenedimethanamin N-[(2- chlorophenyl)-5-methyl-isoxazol-4-oyl]-4-piperidinyl]-1,3-benzenedimethanamine;,N-[(2-
hydroxypheny1)methy1]-N'-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H- hydroxyphenyl)methyl]-N'-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-
yclohepta[bacteriapyridin-9-y1)-1,4-benzenedimethanamine N-[(4-cyanophenyl)methyl]-N'- cyclohepta[bacteriapyridin-9-yl)-1,4-benzenedimethanamine N-[(4-cyanophenyl)methy1]-N'-
30 (2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[bacteriapyridin-9-y1)-1,4- (2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[bacteriapyridin-9-yl)-1,4-
benzenedimethanamine; T-[(4-cyanophenyl)methy1]-N'-(2-pyridinylmethy1)-N-(5,67,8- N-[(4-cyanophenyl)methyl]-N'-(2-pyridinylmethyl)-N-(5,6,7,8-
etrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[(4-acetamidopheny1)methy1]-N'-(2- tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[(4-acetamidophenyl)methyl]-N'-(2-
yridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine;N-[(4- pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine;N-[(4-
WO wo 2019/113375 PCT/US2018/064335
phenoxyphenyl)methy1]-N'-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H- phenoxyphenyl)methyl]-N-(2-pyridinylmethyl)-N-(6,7,8,9-tetahydro-5H-
clohepta[bacteriapyridin-9-yl)-1,4-benzenedimethanamine N-[(1-methyl-2- cyclohepta[bacteriapyridin-9-yl)-1,4-benzenedimethanamine; N-[(1-methyl-2-
carboxamido)ethy1]-N,N'-bis(2-pyridinylmethy1)-1,3-benzenedimethanamine; N-[(4- carboxamido)ethyl]-N,N'-bis(2-pyridinylmethyl)-1,3-benzenedimethanamine;N-](4-
benzyloxyphenyl)methyl]-N'-(2-pyridinylmethy1)-N-(6,7,8,9-tetrahydro-5H- benzyloxyphenyl)methyl]-N'-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-
yclohepta[bacteriapyridin-9-y1)-1,4-benzenedimethanamine; N-[(thiophene-2-yl)methy1]-N'- cyclohepta[bacteriapyridin-9-yl)-1,4-benzenedimethanamine,N-[(thiophene-2-y)methyl]-N;
(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[bacteriapyridin-9-yl)-1,4- (2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[bacteriapyridin-9-yl)-1,4-
benzenedimethanamine; ;N-[1-(benzy1)-3-pyrrolidinyl]-N,N'-bis(2-pyridinylmethyl)-1,3- N-[1-(benzyl)-3-pyrrolidinyl]-N,N'-bis(2-pyridinylmethyl)-1,3-
benzenedimethanamine; N-[[1-methyl-3-(pyrazol-3-y1)]propyl]-N,N'-bis(2-pyridinylmethyl)- N-[[1-methyl-3-(pyrazol-3-yl)]propyl]-N,N'-bis(2-pyridinylmethyl)-
1,3-benzenedimethanamine; N-[1-(phenyl)ethyl1]-N,N'-bis(2-pyridinylmethyl)-1,3- N-[1-(phenyl)ethyl]-N,N'-bis(2-pyridinylmethyl)-1,3-
N-[(3,4-methylenedioxyphenyl)methy1]-N'-(2-pyridinylmethyl)-N benzenedimethanamine; N-[(3,4-methylenedioxyphenyl)methyl]-N'-(2-pyridinylmethyl)-N-
6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine;N-[1-benzyl- 6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N-[1-benzyl-
B-carboxymethyl-4-piperidinyl]-N,N'-bis(2-pyridinylmethy1)-1,3-benzenedimethanamine;n 3-carboxymethyl-4-piperidinyl]-N,N-bis(2-pyridinylmethyl)-1,3-benzenedimethanamine; N- N-
[(3,4-methylenedioxyphenyl)methyl]-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-
quinolinyl)-1,4-benzenedimethanamine; N-(3-pyridinylmethyl)-N'-(2-pyridinylmethyl)-N- quinolinyl)-1,4-benzenedimethananine; N-(3-pyridinylmethy1)-N'-(2-pyridinylmethyl)-N-
(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine;N-[[1- (6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine, N-[[1-
methyl-2-(2-toly1)carboxamido]ethy1]-N,N'-bis(2-pyridinylmethy1)-1,3- methyl-2-(2-tolyl)carboxamido]ethyl]-N,N'-bis(2-pyridinylmethyl)-1,3-
benzenedimethanamine; ;N-[(1,5-dimethy1-2-phenyl-3-pyrazolinone-4-yl)methy1]-N'-(2- N-[(1,5-dimethyl-2-phenyl-3-pyrazolinone-4-yl)methyl]-N-(2-
pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine;N-[(4- pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine,1 N-[(4-
propoxyphenyl)methy1]-N'-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H- propoxyphenyl)methyl]N'-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-
yclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine N-(1-phenyl-3,5-dimethylpyrazolin- cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine, N-(1-phenyl-3,5-dimethylpyrazolin-
4-ylmethy1)-N'-(2-pyridinylmethy1)-N-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4- 4-ylmethyl)-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-
benzenedimethanamine; N-[H-imidazol-4-ylmethy1]-N,N'-bis(2-pyridinylmethy1)-1,3- N-[H-imidazol-4-ylmethyl]-N,N'-bis(2-pyridinylmethyl)-1,3-
benzenedimethanamine; benzenedimethanamine; N-[(3-methoxy-4,5-methylenedioxyphenyl)methy1]-N'-(2- N-[(3-methoxy-4,5-methylenedioxyphenyl)methyl]-N-(2-
pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4- pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-
benzenedimethanamine; N-[(3-cyanophenyl)methyl]-N'-(2-pyridinylmethyl)-N-(6,7,8,9- benzenedimethanamine, N-[(3-cyanophenyl)methy1]-N'-(2-pyridinylmethy1)-N-(6,7,8,9-
etrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine N-[(3- tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine, N-[(3-
cyanophenyl)methy1]-N'-(2-pyridinylmethy1)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4- cyanophenyl)methyl]-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-
benzenedimethanamine;N-(5-ethylthiophene-2-ylmethy1)-N'-(2-pyridinylmethy1)-N-(6,7,8,94 benzenedimethanamine; N-(5-ethylthiophene-2-ylmethyl)-N'-(2-pyridinylmethyl)-N-(6,7,8,9-
etrahydro-5H-cyclohepta[b]pyridin-9-y1)-1,4-benzenedimethanamine; N-(5-ethylthiophene- tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N-(5-ethylthiophene-
2-ylmethy1)-N'-(2-pyridinylmethy1)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4- 2-ylmethyl)-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-
benzenedimethanamine; benzenedimethanamine; ;N-[(2,6-difluorophenyl)methyl]-N'-(2-pyridinylmethyl)-N-(6,7,8,9- N-[(2,6-difluorophenyl)methyl]-N'-(2-pyridinylmethyl)-N-(6,7,8,9-
tetrahydro-5H-cyclohepta[b]pyridin-9-y1)-1,4-benzenedimethanamine;N-[(2,6- tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine, N-[(2,6-
difluorophenyl)methyl]-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-&-quinolinyl)-1,4- difluorophenyl)methyl]-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4- wo 2019/113375 WO PCT/US2018/064335 benzenedimethanamine; N-[(2-difluoromethoxyphenyl)methy1]-N'-(2-pyridinylmethy1)-N- N-[(2-difluoromethoxyphenyl)methyl]-N'-(2-pyridinylmethyl)-N-
(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-y1)-1,4-benzenedimethanamine; N-(2- (6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-vl)-1,4-benzenedimethanamine,N-(2-
fluoromethoxyphenylmethy1)-N'-(2-pyridinylmethy1)-N-(5,6,7,8-tetrahydro-8-quinoling difluoromethoxyphenylmethyl)-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-
1,4-benzenedimethanamine; N-(1,4-benzodioxan-6-ylmethyl)-N'-(2-pyridinylmethyl)-N- 1,4-benzenedimethanamine; N-(1,4-benzodioxan-6-ylmethyl)-N'-(2-pyridinylmethyl)-N
7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-y1)-1,4-benzenedimethanamine;] N,N'-bis(2- (6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine;
pyridinylmethy1)-N-[1-(N"-phenyl-N"-methylureido)-4-piperidinyl]-1,4- pyridinylmethyl)-N-[1-(N"-phenyl-N"-methylureido)-4-piperidinyl]-1,4-
benzenedimethanamine;N,N'-bis(2-pyridinylmethyl)-N-[N"-p-toluenesulfonylphenylalanyl). benzenedimethanamine, N,N'-bis(2-pyridinylmethyl)-N-[N'-p-toluenesulfonylphenylalanyl)-
piperidinyl]-1,4-benzenedimethanamine; N-[1-(3-pyridinecarboxamido)-4-piperidinyl]- 4-piperidinyl]-1,4-benzenedimethanamine; N-[1-(3-pyridinecarboxamido)-4-piperidinyl]-
N,N'-bis(2-pyridinylmethy1)-1,4-benzenedimethanamine N-[1-(cyclopropylcarboxamido)-4- N,N'-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine N-[1-(cyclopropylcarboxamido)-4-
piperidinyl]-N,N'-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; piperidinyl]-N,N'-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine,N-[1-(1- N-[1-(1-
phenylcyclopropylcarboxamido)-4-piperidinyl]-N,N'-bis(2-pyridinylmethy1)-1,4 phenylcyclopropylcarboxamido)-4-piperidinyl]-N,N'-bis(2-pyridinylmethyl)-1,4-
N-(1,4-benzodioxan-6-ylmethy1)-N'-(2-pyridinylmethy1)-N-(5,6,7,8- benzenedimethanamine; N-(1,4-benzodioxan-6-ylmethyl)-N'-(2-pyridinylmethyl)-N-(5,6,7,8-
etrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[1-[3-(2-chloropheny1)-5-methyl- tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[1-[3-(2-chlorophenyl)-5-methyl-
isoxazol-4-carboxamido]-4-piperidinyl]-N,N'-bis(2-pyridinylmethyl)-1,4-
benzenedimethanamine; ;N-[1-(2-thiomethylpyridine-3-carboxamido)-4-piperidinyl]-N,NI N-[1-(2-thiomethylpyridine-3-carboxamido)-4-piperidinyl]-N,N'-
bis(2-pyridinylmethy1)-1,4-benzenedimethanamine, N-[(2,4-difluorophenyl)methyl]-N'-(2- bis(2-pyridinylmethyl)-1,4-benzenedimethanamine, N-[(2,4-difluorophenyl)methy1]-N'-(2-
pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine N-(1- pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine;1 N-(1-
methylpyrrol-2-ylmethyl)-N'-(2-pyridinylmethy1)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4 methylpyrrol-2-ylmethyl)-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-
benzenedimethanamine; N-[(2-hydroxyphenyl)methy1]-N'-(2-pyridinylmethy1)-N-(5,6,78 N-[(2-hydroxyphenyl)methyl]-N'-(2-pyridinylmethyl)-N-(5,6,7,8-
etrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[(3-methoxy-4,5- tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[(3-methoxy-4,5-
methylenedioxyphenyl)methy1]-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl) methylenedioxyphenyl)methyl]-N-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydto-8-quinolinyl).
1,4-benzenedimethanamine; N-(3-pyridinylmethyl)-N'-(2-pyridinylmethyl)-N-(5,6,7,8- 1,4-benzenedimethanamine,
tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[2-(N"-morpholinomethyl)-1- tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[2-(N"-morpholinomethy1)-1-
cyclopentyl]-N,N'-bis(2-pyridinylmethy1)-1,4-benzenedimethanamine;N-[(1-methyl-3- cyclopentyl]-N,N'-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine: N-[(1-methyl-3-
piperidinyl)propyl]-N,N'-bis(2-pyridinylmethy1)-1,4-benzenedimethanamine; N-(1- piperidinyl)propyl]-N,N'-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine, N-(1-
methylbenzimidazol-2-ylmethy1)-N'-(2-pyridinylmethy1)-N-(5,6,7,8-tetrahydro-8- methylbenzimidazol-2-ylmethyl)-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8
quinoliny1)-1,4-benzenedimethanamine; N-[1-(benzyl)-3-pyrrol quinolinyl)-1,4-benzenedimethanamine, N-[1-(benzyl)-3-pyrrolidinyl]-N,N'-bis(2- idinyl]-N,N'-bis(2-
pyridinylmethyl)-1,4-benzenedimethanamine; pyridinylmethyl)-1,4-benzenedimethanamine; N-[[(1-phenyl-3-(N"-morpholino)]propyl]- N-[[(1-phenyl-3-(N"-morpholino)]propyl]-
N,N'-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N,N'-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine;N-[1-(iso-propyl)-4-piperidinyl]- N-[1-(iso-propyl)-4-piperidinyl]-
,N'-bis(2-pyridinylmethy1)-1,4-benzenedimethanamine; N-[1-(ethoxycarbonyl)-4- N,N'-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-[1-(ethoxycarbonyl)-4-
piperidinyl]-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4- piperidinyl]-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolnyl)-1,4
benzenedimethanamine; N-[(1-methyl-3-pyrazolyl)propyl]-N'-(2-pyridinylmethyl)-N- benzenedimethanamine, N-[(1-methyl-3-pyrazoly1)propyl]-N'-(2-pyridinylmethyl)-N
(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine;]N-[1-methyl-2-(N",N"- (5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine, N-[1-methyl-2-(N",N"-
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diethylcarboxamido)ethy1]-N,N'-bis(2-pyridinylmethy1)-1,4-benzenedimethanamine;N-[(1- diethylcarboxamido)ethyl]-N,N'-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine,N-[(1-
methyl-2-phenylsulfonyl)ethy1]-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinol; methyl-2-phenylsulfonyl)ethyl]-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyi]).
1,4-benzenedimethanamine; N-[(2-chloro-4,5-methylenedioxyphenyl)methyI]-N'-(2- 1,4-benzenedimethanamine; N-[(2-chloro-4,5-methylenedioxyphenyl)methy1]-N'-(2-
byridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine;N-[1- pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[1-
methyl-2-[N"-(4-chloropheny1)carboxamido]ethy1]-N'-(2-pyridinylmethy1)-N-(5,6,7 methyl-2-[N"-(4-chlorophenyl)carboxamidolethyl]-N'-(2-pyridinylmethyl)-N-(5,6,7,8-
tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(1-acetoxyindol-3-ylmethyl)-N'-(2- tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(1-acetoxyindol-3-ylmethyl)-N'-(2-
pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-y1)-1,4- pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cycloheptalb]pyridin-9-yl)-1,4-
benzenedimethanamine; N-[(3-benzyloxy-4-methoxyphenyl)methyl]-N'-(2-pyridinylmethyl)- benzenedimethanamine, N-[(3-benzyloxy-4-methoxyphenyl)methy1]-N'-(2-pyridinylmethyl)
N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-y1)-1,4-benzenedimethanamine; N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N-(3- N-(3-
quinolylmethyl)-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-
benzenedimethanamine; N-[(8-hydroxy)-2-quinolylmethy1]-N'-(2-pyridinylmethy1)-N- N-[(8-hydroxy)-2-quinolylmethyl]-N-(2-pyridinylmethyl)-N-
(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-y1)-1,4-benzenedimethanamine; (6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine, N-(2- N-(2-
quinolylmethyl)-N'-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9- quinolylmethyl)-N'-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-
y1)-1,4-benzenedimethanamine; N-[(4-acetamidophenyl)methyl]-N'-(2-pyridinylmethyl)-N- yl)-1,4-benzenedimethanamine; N-[(4-acetamidophenyl)methy1]-N'-(2-pyridinylmethyl)-N-
(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-y1)-1,4-benzenedimethanamine;N-[1H- (6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine,1 N-[1H-
imidazol-2-ylmethy1]-N,N'-bis(2-pyridinylmethy1)-1,4-benzenedimethanamine; N-(3- imidazol-2-ylmethyl]-N,N'-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine;1 N-(3-
quinolylmethyl)-N'-(2-pyridinylmethy1)-N-(6,7,8,9-tetrahydro-5H-cycloheptab]pyridin-9- quinolylmethyl)-N'-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[bpyridin-9-
y1)-1,4-benzenedimethanamine;N-(2-thiazolylmethyl)-N'-(2-pyridinylmethyl)-N-(6,7,8,9- yl)-1,4-benzenedimethanamine; N-(2-thiazolylmethyl)-N'-(2-pyridinylmethyl)-N-(6,7,8,9-
tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine;N-(4- tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine,N-(4-
pyridinylmethyl)-N'-(2-pyridinylmethy1)-N-(6,7,8,9-tetrahydro-5H-cycloheptab]pyridin-9 pyridinylmethyl)-N'-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyidin-9-
y1)-1,4-benzenedimethanamine; N-[(5-benzyloxy)benzo[b]pyrrol-3-ylmethyI]-N,N'-bis(2- yl)-1,4-benzenedimethanamine, N-[(5-benzyloxy)benzo[b]pyrrol-3-ylmethy1]-N,N'-bis(2-
pyridinylmethy1)-1,4-benzenedimethanamine N-(1-methylpyrazol-2-ylmethy1)-N'-(2- pyridinylmethyl)-1,4-benzenedimethanamine, N-(1-methylpyrazol-2-ylmethyl)-N'-(2-
pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4- pyridinylmethy1)-N-(6,78,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)-1,4
benzenedimethanamine; benzenedimethanamine, N-[(4-methyl)-1H-imidazol-5-ylmethy1]-N,N'-bis(2- N-[(4-methyl)-1H-imidazol-5-ylmethyl]-N,N'-bis(2-
pyridinylmethyl)-1,4-benzenedimethanamine; N-[[(4-dimethylamino)-1-napthalenyl]methyl]- pyridinylmethyl)-1,4-benzenedimethanamine, N-[[(4-dimethylamino)-1-napthalenyl]methyl]
N,N'-bis(2-pyridinylmethy1)-1,4-benzenedimethanamine; N,N'-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-[1,5-dimethyl-2-phenyl-3- N-[1,5-dimethyl-2-phenyl-3-
yrazolinone-4-ylmethy1]-N,N'-bis(2-pyridinylmethy1)-1,4-benzenedimethanamine; N-[1-[(1- pyrazolinone-4-ylmethyl]-N,N'-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine,1
acetyl-2-(R)-prolinyl]-4-piperidinyl]-N-[2-(2-pyridinyl)ethyl]-N'-(2-pyridinylmethy1)-1,3- acetyl-2-(R)-prolinyl]-4-piperidinyl]-N-[2-(2-pyridinyl)ethyl]-N-(2-pyridinylmethyl)-1,3-
benzenedimethanamine; benzenedimethanamine; ;N-[1-[2-acetamidobenzoyl-4-piperidinyl]-4-piperidinyl]-N-[2-(2- N-[1-[2-acetamidobenzoyl-4-piperidinyl]-4-piperidinyl]-N-[2-(2-
pyridinyl)ethy1]-N'-(2-pyridinylmethy1)-1,3-benzenedimethanamine; pyridinyl)ethyl]-N'-(2-pyridinylmethyl)-1,3-benzenedimethanamineN-[(2-cyano-2- N-[(2-cyano-2-
1)ethy1]-N'-(2-pyridinylmethy1)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl)- phenyl)ethyl]-N'-(2-pyridinylmethyl)-N-(6,7,8,9-tetahydro-5H-cycloheptalb]pyridin-9-yl)-
N-[(N"-acetyltryptophanyl)-4-piperidinyl]-N-[2-(2 1,4-benzenedimethanamine; N-[(N"-acetyltryptophanyl)-4-piperidinyl]-N-[2-(2-
pyridinyl)ethy1]-N'-(2-pyridinylmethy1)-1,3-benzenedimethanamine; N-[(N"-benzoylvalinyl)- pyridinyl)ethyl]-N'-(2-pyridinylmethyl)-1,3-benzenedimethanamine;,N-[(N'-benzoylvalnyl)-
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--piperidinyl]-N-[2-(2-pyridinyl)ethyl]-N'-(2-pyridinylmethy1)-1,3-benzenedimethanamine; 4-piperidinyl]-N-[2-(2-pyridinyl)ethyl]-N'-(2-pyridinylmethyl)-1,3-benzenedimethanamne,
N-[(4-dimethylaminophenyl)methy1]-N'-(2-pyridinylmethy1)-N-(6,7,8,9-tetrahydro-5H- N-[(4-dimethylaminophenyl)methyl]-N'4(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-
cyclohepta[b]pyridin-9-y1)-1,4-benzenedimethanamine;N-(4-pyridinylmethyl)-N'-(2- cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N-(4-pyridinylmethyl)-N'-(2-
yridinylmethy1)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine N-(1- pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine;N-(1-
methylbenzimadazol-2-ylmethy1)-N'-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H- methylbenzimadazol-2-ylmethyl)-N'-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-
yclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine N-[1-butyl-4-piperidinyl]-N-[2-(2- cyclohepta[b]pyridin-9-yl)-1,4-benzenedimethanamine; N-[1-butyl-4-piperidinyl]-N-[2-(2-
pyridinyl)ethy1]-N'-(2-pyridinylmethy1)-1,3-benzenedimethanamine;N-[1-benzoyl-4- pyridinyl)ethyl]-N'-(2-pyridinylmethyl)-1,3-benzenedimethanamine: N-[1-benzoyl-4-
Diperidinyl]-N-[2-(2-pyridinyl)ethy1]-N'-(2-pyridinylmethy1)-1,3-benzenedimethanamine; piperidinyl]-N-[2-(2-pyridinyl)ethyl]N'-(2-pyridinylmethyl)-1,3-benzenedimethanamine; N-N-
[1-(benzyl)-3-pyrrolidinyl]-N-[2-(2-pyridinyl)ethy1]-N'-(2-pyridinylmethy1)-1,3-
[1-(benzyl)-3-pyrrolidinyl]-N-[2-(2-pyridinyl)ethyl]-N'-(2-pyridinylmethyl)-1,3-
; N-[(1-methyl)benzo[b]pyrrol-3-ylmethy1]-N-[2-(2-pyridinyl)ethyl]- benzenedimethanamine; N-[(1-methyl)benzo[b]pyrrol-3-ylmethyl]-N-[2-(2-pyridinyl)ethyl]-
N'-(2-pyridinylmethyl)-1,3-benzenedimethanamine; N'-(2-pyridinylmethyl)-1,3-benzenedimethanamine, N-[1H-imidazol-4-ylmethy1]-N-[2-(2- N-[1H-imidazol-4-ylmethyl]-N-[2-(2-
pyridinyl)ethyl]-N'-(2-pyridinylmethy1)-1,3-benzenedimethanamine; N-[1-(benzyl)-4- pyridinyl)ethyl]-N'-(2-pyridinylmethyl)-1,3-benzenedimethanamine N-[1-(benzyl)-4-
piperidinyl]-N-[2-(2-pyridinyl)ethyl]-N'-(2-pyridinylmethyl)-1,4-benzenedimethanamine; N- piperidinyl]-N-[2-(2-pyridinyl)ethyl]-N'-(2-pyridinylmethyl)-1,4-benzenedimethanamine,N-
1-methylbenzimidazol-2-ylmethy1]-N-[2-(2-pyridinyl)ethy1]-N'-(2-pyridinylmethy1)-1,4
[1-methylbenzimidazol-2-ylmethyl]-N-[2-(2-pyridinyl)ethyl]-N'-(2-pyridinylmethyl)-1,4-
benzenedimethanamine; ;N-[(2-phenyl)benzo[b]pyrrol-3-ylmethy1]-N-[2-(2-pyridinyl)ethyl] N-[(2-phenyl)benzo[b]pyrrol-3-ylmethyl]-N-[2-(2-pyridinyl)ethyl]-
N'-(2-pyridinylmethyl)-1,4-benzenedimethanamine N-[(6-methylpyridin-2-yl)methy1]-N'-(2- N'-(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-[(6-methylpyridin-2-yl)methyl]-N'-(2-
ridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(3- pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine,1 N-(3-
methyl-1H-pyrazol-5-ylmethy1)-N'-(2-pyridinylmethy1)-N-(5,6,7,8-tetrahydro-8-quinolinyl)- methyl-1H-pyrazol-5-ylmethyl)-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-&-quinolinyl)-
1,3-benzenedimethanamine; N-[(2-methoxyphenyl)methyl]-N'-(2-pyridinylmethyl)-N- 1,3-benzenedimethanamine; N-[(2-methoxyphenyl)methy1]-N'-(2-pyridinylmethyl)-N
(5,6,7,8-tetrahydro-8-quinolinyl)-1,3-benzenedimethanamine; N-[(2-ethoxyphenyl)methyl]-
N'-(2-pyridinylmethy1)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9 N'-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5FH-cyclohepta[b]pyridin-9-yl)-1,3-
benzenedimethanamine; N-(benzyloxyethyl)-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-
quinolinyl)-1,3-benzenedimethanamine; N-[(2-ethoxy-1-naphthalenyl)methyl]-N'-(2- quinolinyl)-1,3-benzenedimethanamine; N-[(2-ethoxy-1-naphthalenyl)methyl]-N'-(2
pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinoliny1)-1,3-benzenedimethanamine;N-[(6- pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,3-benzenedimethanamine,N-[(6-
mnethylpyridin-2-y1)methy1]-N'-(2-pyridinylmethy1)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1 methylpyridin-2-yl)methyl]-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,3-
benzenedimethanamine; 1-[[4-[[(2-pyridinylmethy1)amino]methyl]phenyl]methyl]guanidine; -[[-[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]guanidine;
N-(2-pyridinylmethyl)-N-(8-methyl-8-azabicyclo[3.2.1]octan-3-y1)-1,4- N-(2-pyridinylmethyl)-N-(8-methyl-8-azabicyclo[3.2.1]octan-3-yl)-1,4-
benzenedimethanamine; 1-[[4-[[(2-
pyridinylmethyl)amino]methyl]phenyl]methyl]homopiperazine; 1-[[3-[[(2- pyridinylmethyl)amino]methyl]phenyl]methyl]homopiperazine,1-[[3-[[(2-
pyridinylmethyl)amino]methyl]phenyl]methyl]homopiperazine; pyridinylmethyl)amino]methyl]phenyl]methyllhomopiperazine; trans and cis-1-[[4-[[(2-
pyridinylmethyl)amino]methyl]phenyl]methy1]-3,5-piperidinediamine; pyridinylmethyl)amino]methyl]phenylJmethyl]-3,5-piperidinediamine, N,N'-[1,4- N,N'-[1,4-
Phenylenebis(methylene)]bis-4-(2-pyrimidyl)piperazine;1-[[4-[[(2- Phenylenebis(methylene)]bis-4-(2-pyrimidyl)piperazine;, 1-[[4-[(2-
yridinylmethyl)amino]methyl]phenyl]methyl]-1-(2-pyridinyl)methylamine; 2-(2-pyridinyl) pyridinylmethyl)amino]methyl]phenyl]methyl]-1-(2-pyridinyl)methylamine;2-(2-pyridinyl)-
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5-[[(2-pyridinylmethyl)amino]methy1]-1,2,3,4-tetrahydroisoquinoline;1 1-[[4-[[(2- 5-[[(2-pyridinylmethyl)amino]methyl]-1,2,3,4-tetrahydroisoquinoline 1-[[4-[[(2-
pyridinylmethyl)amino]methyl]phenyl]methy1]-3,4-diaminopyrrolidine; 1-[[4-[[(2- pyridinylmethyl)amino]methyl]phenyl]methyl]-3,4-diaminopyrrolidine,
pyridinylmethy1)amino]methyl]phenyl]methy1]-3,4-diacetylaminopyrrolidine, 8-[4-[(2- pyridinylmethyl)amino]methyl|phenyl]methyl]-3,4-diacetylaminopyrrolidine; 8-[[4-[[(2-
pyridinylmethyl)amino]methyl]phenyl]methy1]-2,5,8-triaza-3-oxabicyclo[4.3.0]nonane; and pyridinylmethyl)amino]methyl]phenyl]methyl]2,5,8-tiaza-3-oxabicyclo[4.3.0]nonane, and
8-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methy1]-2,5,8-triazabicyclo[4.3.0]nonane. 8-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-2,5,8-triazabicyclo[4.3.0)nonane.
[0220] Additional CXCR4 antagonists that may be used to in conjunction with the
compositions and methods described herein include those described in WO 2001/085196,
WO 1999/050461, WO 2001/094420, and WO 2003/090512, the disclosures of each of
which are incorporated herein by reference as they pertain to compounds that inhibit CXCR4
activity or expression.
Expansion of Hematopoietic Stem and Progenitor Cells
[0221] Prior to infusion into a patient, hematopoietic and progenitor cells may be expanded
ex vivo, for example, by contacting the cells with an aryl hydrocarbon receptor antagonist.
Aryl hydrocarbon receptor antagonists useful in conjunction with the compositions and
methods described herein include those described in US Patent No. 9,580,426, the disclosure
of which is incorporated herein by reference in its entirety.
[0222] In some embodiments, aryl hydrocarbon receptor antagonists include those
represented by formula (III)
R2 L R N N R3 R1 N N R R R4 (III) R in which:
L L is isselected selected from - -NR5a(CH2)2-3 ,-NR5a(CH2)2NR5b-,-NR5a(CH2)2S-, -
NR53CH2CH(OH)- and -NR5aCH(CH3)CH- wherein R5a and R5b are independently NRCHCH(OH)- and wherein R and R are independently selected selectedfrom fromhydrogen andand hydrogen C1-4alkyl; alkyl;
R1 isselected R is selectedfrom fromthiophenyl, thiophenyl,IH-benzoimidazolyl, 1H-benzoimidazolyl,isoquinolinyl, isoquinolinyl,1H-imidazopyridinyl, 1H-imidazopyridinyl,
benzothiophenyl, pyrimidinyl, pyridinyl, pyrazinyl, pyridazinyl, and thiazolyl; In some
embodiments, wherein the thiophenyl, 1H-benzoimidazolyl, isoquinolinyl, 1H-
214 wo 2019/113375 WO PCT/US2018/064335 imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyridinyl, pyrazinyl, pyridazinyl, or thiazolyl of R1 can be R can be optionally optionally substituted substituted by by 11 to to 33 radicals radicals independently independently selected selected from from cyano, cyano, hydroxy, hydroxy,C1-4 C- alkyl, alkyl,C1-4 C- alkoxy, alkoxy,halo, halo,halo-substituted-C14 halo-substituted-C alkyl, halo-substituted-C1. alkyl, halo-substituted-C.
4alkoxy, alkoxy, amino, amino,-C(O)R8a -C(0)R,-S(O)0-2R8a: -S(O)-R,-C(O)OR8a -C(0)OR and and-C(O)NR82R8b; -C(O)NRR; wherein whereinR8a R and and R8b are R are independently independently selected selected from from hydrogen hydrogen and and C1-4alkyl; C-alkyl;
R2 is selected ffrom-S(O)2NR6aR6b,-NR6aC(O)R6b,-NR6aC(O)NR6bR6c,phenyl, 1H- R is selected from -S(O)NRR, -NRC(O)Rb-, phenyl, 1H- pyrrolopyridin-3-yl, 1H-pyrrolopyridin-5-yl, 1H-indolyl thiophenyl, pyridinyl, 1H-1,2,4-
triazolyl, 2-oxoimidazolidinyl, 1H-pyrazolyl, 2-oxo-2,3-dihydro-1H-benzoimidazolyl and
1H-indazolyl; wherein R6a, R, R R6b and and R6cindependently Rc are are independently selected selected from from hydrogen hydrogen and C1- and C-
4alkyl; and the phenyl, 1H-pyrrolopyridin-3-yl, 1H-pyrrolo[2,3-b]pyridin-5-yl, 1H-indolyl,
thiophenyl, pyridinyl, 1H-1,2,4-triazolyl, 2-oxoimidazolidinyl, 1H-pyrazolyl, 2-oxo-2,3-
dihydro-1H-benzoimidazoly or dihydro-1H-benzoimidazolyl or 1H-indazolyl 1H-indazolyl of of R2 R is optionally substituted with 1 to 3
radicals independently selected from hydroxy, halo, methyl, methoxy, amino, -
-OS(O)2NR7aR7 O(CH)NRR, -S(O)NRR, -OS(O)NRRb and and -NR7aS(O)2R76; -NRS(O)R; wherein wherein R R7a andand R7b areindependently Rb are independentlyselected selectedfrom fromhydrogen hydrogenand andC- C1-4 alkyl; alkyl;
R3 is R is selected selected from fromhydrogen, C1-4 hydrogen, C-alkyl andand alkyl biphenyl; and and biphenyl;
R4 is selected R is selected from fromC1-10 alkyl, alkyl, prop-1-en-2-yl,cyclohexyl, prop-1-en-2-yl, cyclohexyl, cyclopropyl, cyclopropyl,2-(2-oxopyrrolidin-1- 2-(2-oxopyrrolidin-1-
yl)ethyl, oxetan-2-yl, oxetan-3-yl, benzhydryl, tetrahydro-2H-pyran-2-yl, tetrahydro-2H-
pyran-3-yl, phenyl, tetrahydrofuran-3-yl, and benzyl, (4-pentylphenyl)(phenyl)methyl and 1- -
-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-y1)-1H-1,2,3-triazol-4-yl)ethyl wherein (1-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-1H-1,2,3-triazol-4-yl)ethyl wherein said said alkyl, alkyl,
cyclopropyl, cyclohexyl, 2-(2-oxopyrrolidin-1-yl)ethyl, oxetan-3-yl, oxetan-2-yl, benzhydryl,
tetrahydro-2H-pyran-2-yl, tetrahydro-2H-pyran-3-yl, phenyl, tetrahydrofuran-3-yl, benzyl,
(4-pentylpheny1)(phenyl)methyl or 1-(1-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-1H- (4-pentylphenyl)(phenyl)methyl 1-(1-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-y1)-1H-
1,2,3-triazol-4-y1)ethyl can be optionally substituted with 1 to 3 radicals independently 1,2,3-triazol-4-yl)ethyl
selected selectedfrom fromhydroxy, C1-4alkyl hydroxy, C.alkylandand halo-substituted-C1-4alkyl; or a salt halo-substituted-C.4alkyl; or athereof. salt thereof.
[0223] In some embodiments, aryl hydrocarbon receptor antagonists useful in conjunction
with the compositions and methods described herein include SR-1, represented by formula
(1), below.
OH HN N N
N N S (1)
Methods Methods for forthe theRecombinant Expression Recombinant of Peptides Expression and Proteins of Peptides and Proteins
[0224] Peptides and proteins described herein (e.g., CXCR2 agonists, such as Gro-ß, Gro-B Gro-ß
T, Gro-B Gro-ß N69D, Gro-B Gro-ß T N65D, and variants thereof) can be expressed in host cells, for
example, by delivering to the host cell a nucleic acid encoding the corresponding peptide or
protein. The sections that follow describe a variety of techniques that can be used for the
purposes of introducing nucleic acids encoding peptides and proteins described herein to a
host cell for the purposes of recombinant expression.
Transfection techniques
[0225] Techniques that can be used to introduce a polynucleotide, such as nucleic acid
encoding a CXCR2 agonist, such as Gro-B, Gro-ß, Gro-B Gro-ß T, Gro-B Gro-ß N69D, Gro-B Gro-ß T N65D, or a
variant thereof, into a cell (e.g., a mammalian cell, such as a human cell) are known in the art.
In some embodiments, electroporation can be used to permeabilize mammalian cells (e.g.,
human cells) by the application of an electrostatic potential to the cell of interest.
Mammalian cells, such as human cells, subjected to an external electric field in this manner
are subsequently predisposed to the uptake of exogenous nucleic acids. Electroporation of
mammalian cells is described in detail, e.g., in Chu et al. (1987) Nucleic Acids Research
15:1311, 15:1311,the thedisclosure of which disclosure is incorporated of which herein by is incorporated reference. herein A similar technique, by reference. A similar technique,
, utilizes Nucleofection TM utilizes an an applied applied electric electric field field in in order order to to stimulate stimulate the the uptake uptake of of
TM and exogenous polynucleotides into the nucleus of a eukaryotic cell. Nucleofection and
protocols useful for performing this technique are described in detail, e.g., in Distler et al.
(2005) Experimental Dermatology 14:315, as well as in US 2010/0317114, the disclosures of
each of which are incorporated herein by reference.
[0226] Additional techniques useful for the transfection of host cells for the purposes of
recombinant peptide and protein expression include the squeeze-poration methodology. This
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technique induces the rapid mechanical deformation of cells in order to stimulate the uptake
of exogenous DNA through membranous pores that form in response to the applied stress.
This technology is advantageous in that a vector is not required for delivery of nucleic acids
into a cell, such as a human cell. Squeeze-poration is described in detail, e.g., in Sharei et al.
(2013) Journal of Visualized Experiments 81:e50980, the disclosure of which is incorporated
herein by reference.
[0227] Lipofection represents another technique useful for transfection of cells. This method
involves the loading of nucleic acids into a liposome, which often presents cationic functional
groups, such as quaternary or protonated amines, towards the liposome exterior. This
promotes electrostatic interactions between the liposome and a cell due to the anionic nature
of the cell membrane, which ultimately leads to uptake of the exogenous nucleic acids, for
example, by direct fusion of the liposome with the cell membrane or by endocytosis of the
complex. Lipofection is described in detail, for example, in US Patent No. 7,442,386, the
disclosure of which is incorporated herein by reference. Similar techniques that exploit ionic
interactions with the cell membrane to provoke the uptake of foreign nucleic acids include
contacting a cell with a cationic polymer-nucleic acid complex. Exemplary cationic
molecules that associate with polynucleotides SO so as to impart a positive charge favorable for
interaction with the cell membrane are activated dendrimers (described, e.g., in Dennig
(2003) Topics in Current Chemistry 228:227, the disclosure of which is incorporated herein
by reference) and diethylaminoethyl (DEAE)-dextran, the use of which as a transfection
agent is described in detail, for example, in Gulick et al. (1997) Current Protocols in
Molecular Biology 40:1:9.2:9.2.1, 40:I:9.2:9.2.1, the disclosure of which is incorporated herein by reference.
Magnetic beads are another tool that can be used to transfect cells in a mild and efficient
manner, as this methodology utilizes an applied magnetic field in order to direct the uptake of
nucleic acids. This technology is described in detail, for example, in US 2010/0227406, the
disclosure of which is incorporated herein by reference.
[0228] Another useful tool for inducing the uptake of exogenous nucleic acids by cells is
laserfection, a technique that involves exposing a cell to electromagnetic radiation of a
particular wavelength in order to gently permeabilize the cells and allow polynucleotides to
penetrate the cell membrane. This technique is described in detail, e.g., in Rhodes et al.
(2007) Methods in Cell Biology 82:309, the disclosure of which is incorporated herein by
reference.
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[0229] Microvesicles represent another potential vehicle that can be used to introduce a
nucleic acid encoding a peptide or protein described herein into a host cell for the purpose of
recombinant expression. In some embodiments, microvesicles that have been induced by the
co-overexpression of the glycoprotein VSV-G with, e.g., a genome-modifying protein, such
as a nuclease, can be used to efficiently deliver proteins into a cell that subsequently catalyze
the site-specific cleavage of an endogenous polynucleotide sequence SO so as to prepare the
genome of the cell for the covalent incorporation of a polynucleotide of interest, such as a
gene or regulatory sequence. The use of such vesicles, also referred to as Gesicles, for the
genetic modification of eukaryotic cells is described in detail, e.g., in Quinn et al., Genetic
Modification of Target Cells by Direct Delivery of Active Protein [abstract]. In: Methylation
changes in early embryonic genes in cancer [abstract], in: Proceedings of the 18th Annual
Meeting of the American Society of Gene and Cell Therapy; 2015 May 13, Abstract No. 122.
Viral vectors for nucleic acid delivery
[0230] Viral genomes provide a rich source of vectors that can be used for the efficient
delivery of exogenous nucleic acids encoding peptides and proteins described herein, such as
CXCR2 agonists, including Gro-B, Gro-ß, Gro-B Gro-ß T, Gro-B Gro-ß N69D, Gro-B Gro-ß T N65D, and variants
thereof, into host cells for the purpose of recombinant expression. Viral genomes are
particularly useful vectors for gene delivery because the polynucleotides contained within
such genomes may be incorporated into the genome of a cell, for example, by way of
generalized or specialized transduction. These processes may occur as part of the natural
replication cycle of a viral vector, and may not require added proteins or reagents in order to
induce gene integration. Examples of viral vectors that may be used to introduce a nucleic
acid molecule encoding a peptide or protein described herein into a host cell for recombinant
expression include parvovirus, such as adeno-associated virus (AAV), retrovirus, adenovirus
(e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), coronavirus, negative strand RNA viruses such as
orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis
virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses, such as
picornavirus and alphavirus, and double stranded DNA viruses including adenovirus,
herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus),
and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox).
Other viruses useful for delivering polynucleotides encoding peptides and proteins described
herein to host cells for recombinant expression purposes include Norwalk virus, togavirus,
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flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples
of retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type
viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses
and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds.,
Lippincott-Raven Publishers, Philadelphia, 1996). Other examples include murine leukemia
viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline
leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus,
baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian
immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses. Other
examples of vectors are described, for example, in US Patent No. 5,801,030, the disclosure of
which is incorporated herein by reference as it pertains to viral vectors for use in gene
delivery and recombinant protein and peptide expression.
Methods of Treatment
[0231] As described herein, hematopoietic stem cell transplant therapy can be administered
to a subject in need of treatment SO so as to populate or repopulate one or more blood cell types,
such as a blood cell lineage that is deficient or defective in a patient suffering from a stem
cell disorder. Hematopoietic stem and progenitor cells exhibit multi-potency, and can thus
differentiate into multiple different blood lineages including, but not limited to, granulocytes
(e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes,
erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producing megakaryocytes,
platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts,
and lymphocytes (e.g., NK cells, B-cells and T-cells). Hematopoietic stem cells are
additionally capable of self-renewal, and can thus give rise to daughter cells that have
equivalent potential as the mother cell, and also feature the capacity to be reintroduced into a
transplant recipient whereupon they home to the hematopoietic stem cell niche and re-
establish productive and sustained hematopoiesis. Thus, hematopoietic stem and progenitor
cells represent a useful therapeutic modality for the treatment of a wide array of disorders in
which a patient has a deficiency or defect in a cell type of the hematopoietic lineage. The
deficiency or defect may be caused, for example, by depletion of a population of endogenous
cells of the hematopoietic system due to administration of a chemotherapeutic agent (e.g., in
the case of a patient suffering from a cancer, such as a hematologic cancer described herein).
The deficiency or defect may be caused, for example, by depletion of a population of
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endogenous hematopoietic cells due to the activity of self-reactive immune cells, such as T
lymphocytes or B lymphocytes that cross-react with self antigens (e.g., in the case of a patient
suffering from an autoimmune disorder, such as an autoimmune disorder described herein).
Additionally or alternatively, the deficiency or defect in cellular activity may be caused by
aberrant expression of an enzyme (e.g., in the case of a patient suffering from various
metabolic disorders, such as a metabolic disorder described herein).
[0232] Thus, hematopoietic stem cells can be administered to a patient defective or deficient
in one or more cell types of the hematopoietic lineage in order to re-constitute the defective
or deficient population of cells in vivo, thereby treating the pathology associated with the
defect or depletion in the endogenous blood cell population. Hematopoietic stem and
progenitor cells can be used to treat, e.g., a non-malignant hemoglobinopathy (e.g., a
hemoglobinopathy selected from the group consisting of sickle cell anemia, thalassemia,
Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome). In these cases, for
example, a CXCR4 antagonist and/or a CXCR2 agonist may be administered to a donor, such
as a donor identified as likely to exhibit release of a population of hematopoietic stem and
progenitor cells from a stem cell niche, such as the bone marrow, into circulating peripheral
blood in response to such treatment. The hematopoietic stem and progenitor cells thus
mobilized may then be withdrawn from the donor and administered to a patient, where the
cells may home to a hematopoietic stem cell niche and re-constitute a population of cells that
are damaged or deficient in the patient.
[0233] Additionally or alternatively, hematopoietic stem and progenitor cells can be used to
treat an immunodeficiency, such as a congenital immunodeficiency. Additionally or
alternatively, the compositions and methods described herein can be used to treat an acquired
immunodeficiency (e.g., an acquired immunodeficiency selected from the group consisting of
HIV and AIDS). In these cases, for example, a CXCR4 antagonist and/or a CXCR2 agonist
may be administered to a donor, such as a donor identified as likely to exhibit release of a
population of hematopoietic stem and progenitor cells from a stem cell niche, such as the
bone marrow, into circulating peripheral blood in response to such treatment. The
hematopoietic stem and progenitor cells thus mobilized may then be withdrawn from the
donor and administered to a patient, where the cells may home to a hematopoietic stem cell
niche and re-constitute a population of immune cells (e.g., T lymphocytes, B lymphocytes,
NK cells, or other immune cells) that are damaged or deficient in the patient.
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[0234] Hematopoietic stem and progenitor cells can also be used to treat a metabolic disorder
(e.g., a metabolic disorder selected from the group consisting of glycogen storage diseases,
mucopolysaccharidoses, Gaucher's Disease, Hurlers Disease, sphingolipidoses, and
metachromatic leukodystrophy). In these cases, for example, a CXCR4 antagonist and/or a
CXCR2 agonist may be administered to a donor, such as a donor identified as likely to
exhibit release of a population of hematopoietic stem and progenitor cells from a stem cell
niche, such as the bone marrow, into circulating peripheral blood in response to such
treatment. The hematopoietic stem and progenitor cells thus mobilized may then be
withdrawn from the donor and administered to a patient, where the cells may home to a
hematopoietic stem cell niche and re-constitute a population of hematopoietic cells that are
damaged or deficient in the patient.
[0235] Additionally or alternatively, hematopoietic stem or progenitor cells can be used to
treat a malignancy or proliferative disorder, such as a hematologic cancer or
myeloproliferative disease. In the case of cancer treatment, for example, a CXCR4
antagonist and/or a CXCR2 agonist may be administered to a donor, such as a donor
identified as likely to exhibit release of a population of hematopoietic stem and progenitor
cells from a stem cell niche, such as the bone marrow, into circulating peripheral blood in
response to such treatment. The hematopoietic stem and progenitor cells thus mobilized may
then be withdrawn from the donor and administered to a patient, where the cells may home to
a hematopoietic stem cell niche and re-constitute a population of cells that are damaged or
deficient in the patient, such as a population of hematopoietic cells that is damaged or
deficient due to the administration of one or more chemotherapeutic agents to the patient. In
some embodiments, hematopoietic stem or progenitor cells may be infused into a patient in
order to repopulate a population of cells depleted during cancer cell eradication, such as
during systemic chemotherapy. Exemplary hematological cancers that can be treated by way
of administration of hematopoietic stem and progenitor cells in accordance with the
compositions and methods described herein are acute myeloid leukemia, acute lymphoid
leukemia, chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloma, diffuse
large B-cell lymphoma, and non-Hodgkin's lymphoma, as well as other cancerous conditions,
including neuroblastoma.
[0236] Hematopoietic stem or progenitor cells mobilized to the peripheral blood of a subject
may be withdrawn (e.g., harvested or collected) from the subject by any suitable technique.
For example, the hematopoietic stem or progenitor cells may be withdrawn by a blood draw.
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In some embodiments, hematopoietic stem or progenitor cells mobilized to a subject's
peripheral blood as contemplated herein may be harvested (i.e., collected) using apheresis. In
some embodiments, apheresis may be used to enrich a donor's blood with mobilized
hematopoietic stem or progenitor cells.
[0237] Additional diseases that can be treated by the administration of hematopoietic stem
and progenitor cells to a patient include, without limitation, adenosine deaminase deficiency
and severe combined immunodeficiency, hyper immunoglobulin M syndrome, Chediak-
Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta,
storage diseases, thalassemia major, systemic sclerosis, systemic lupus erythematosus,
multiple sclerosis, and juvenile rheumatoid arthritis.
[0238] In addition, administration of hematopoietic stem and progenitor cells can be used to
treat autoimmune disorders. In some embodiments, upon infusion into a patient, transplanted
hematopoietic stem and progenitor cells may home to a stem cell niche, such as the bone
marrow, and establish productive hematopoiesis. This, in turn, can re-constitute a population
of cells depleted during autoimmune cell eradication, which may occur due to the activity of
self-reactive lymphocytes (e.g., self-reactive T lymphocytes and/or self-reactive B
lymphocytes). Autoimmune diseases that can be treated by way of administering
hematopoietic stem and progenitor cells to a patient include, without limitation, psoriasis,
psoriatic arthritis, Type 1 diabetes mellitus (Type 1 diabetes), rheumatoid arthritis (RA),
human systemic lupus (SLE), multiple sclerosis (MS), inflammatory bowel disease (IBD),
lymphocytic colitis, acute disseminated encephalomyelitis (ADEM), Addison's disease,
alopecia universalis, ankylosing spondylitis, antiphospholipid antibody syndrome (APS),
aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner
ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune
oophoritis, Balo disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Chagas'
disease, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory
demyelinating polyneuropathy, Crohn's disease, cicatricial pemphigoid, coeliac sprue-
dermatitis herpetiformis, cold agglutinin disease, CREST syndrome, Degos disease, discoid
lupus, dysautonomia, endometriosis, essential mixed cryoglobulinemia, fibromyalgia-
fibromyositis, Goodpasture' S syndrome, Grave's disease, Guillain-Barre syndrome (GBS),
Hashimoto' S thyroiditis, Hidradenitis suppurativa, idiopathic and/or acute thrombocytopenic
purpura, idiopathic pulmonary fibrosis, IgA neuropathy, interstitial cystitis, juvenile arthritis,
Kawasaki's disease, lichen planus, Lyme disease, Meniere disease, mixed connective tissue
WO wo 2019/113375 PCT/US2018/064335
disease (MCTD), myasthenia gravis, neuromyotonia, opsoclonus myoclonus syndrome
(OMS), optic neuritis, Ord's thyroiditis, pemphigus vulgaris, pernicious anemia,
polychondritis, polymyositis and dermatomyositis, primary biliary cirrhosis, polyarteritis
nodosa, polyglandular syndromes, polymyalgia rheumatica, primary agammaglobulinemia,
Raynaud phenomenon, Reiter' syndrome, rheumatic S syndrome, fever, rheumatic sarcoidosis, fever, scleroderma, sarcoidosis, scleroderma,
Sjögren's syndrome, stiff person syndrome, Takayasu's arteritis, temporal arteritis (also
known as "giant cell arteritis"), ulcerative colitis, collagenous colitis, uveitis, vasculitis,
Wegener' Sgranulomatosis. vitiligo, vulvodynia ("vulvar vestibulitis"), and Wegener's granulomatosis.
[0239] In some embodiments, a method of harvesting hematopoietic stem cells from a human
subject is provided. The method comprises administering a CXCR2 agonist and a CXCR4
antagonist to the human subject and harvesting the hematopoietic stem cells from peripheral
blood of the human subject.
[0240] In some embodiments, a method of transplanting hematopoietic stem cells into a
human patient in need thereof is provided. The method comprises administering a CXCR2
agonist and a CXCR4 antagonist to a hematopoietic stem cell donor, harvesting the
hematopoietic stem cells from peripheral blood of the donor, and transplanting the harvested
hematopoietic stem cells into the patient.
Selection of donors and patients
[0241] In some embodiments, the patient is the donor. In such cases, withdrawn
hematopoietic stem or progenitor cells may be re-infused into the patient, such that the cells
may subsequently home hematopoietic tissue and establish productive hematopoiesis, thereby
populating or repopulating a line of cells that is defective or deficient in the patient (e.g., a
population of megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeoblasts,
basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-
presenting cells, macrophages, dendritic cells, natural killer cells, T-lymphocytes, and B-
lymphocytes). In this scenario, the transplanted hematopoietic stem or progenitor cells are
least likely to undergo graft rejection, as the infused cells are derived from the patient and
express the same HLA class I and class II antigens as expressed by the patient.
[0242] Alternatively, the patient and the donor may be distinct. In some embodiments, the
patient and the donor are related, and may, for example, be HLA-matched. As described
herein, HLA-matched donor-recipient pairs have a decreased risk of graft rejection, as
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endogenous T cells and NK cells within the transplant recipient are less likely to recognize
the incoming hematopoietic stem or progenitor cell graft as foreign, and are thus less likely to
mount an immune response against the transplant. Exemplary HLA-matched donor-recipient
pairs are donors and recipients that are genetically related, such as familial donor-recipient
pairs (e.g., sibling donor-recipient pairs).
[0243] In some embodiments, the patient and the donor are HLA-mismatched, which occurs
when at least one HLA antigen, in particular with respect to HLA-A, HLA-B and HLA-DR,
is mismatched between the donor and recipient. To reduce the likelihood of graft rejection,
for example, one haplotype may be matched between the donor and recipient, and the other
may be mismatched.
CD34im CD34dim cells cells
[0244] When the donor and patient are distinct, methods of treatment using hematopoietic
stem stem cells cellscomprising CD34dim comprising cells CD34im are particularly cells useful,useful, are particularly in part in because part CD34dim becausecells arecells are CD34i
capable of suppressing alloreactive T lymphocyte proliferation when administered to a
recipient, thereby reducing the risk of graft VS. host disease (GVHD). (D'Aveni (D' Aveniet etal. al.(2015), (2015),
supra.) In certain embodiments, administration of a CXCR2 agonist and a CXCR4
antagonist according to the methods disclosed herein mobilizes hematopoietic stem cells
comprising CD34dim cells CD34i cells from from the the bone bone marrow marrow ofof a a donor donor into into peripheral peripheral blood. blood. The The
CD34dim CD34i cells cells are are present present inin a higher a higher amount amount inin the the peripheral peripheral blood blood asas compared compared toto
peripheral blood from an unmobilized mammal. In certain embodiments, the CD34i cells CD34dir cells
are present in a higher amount in the peripheral blood than if the hematopoietic stem cells
were mobilized using the CXCR4 antagonist alone.
[0245] Accordingly, the methods disclosed herein are useful in performing an allogeneic
hematopoietic stem cell transplant in a patient in need thereof. For example, the method can
include infusing into the patient a therapeutically effective amount of allogeneic
hematopoietic stem cells, wherein the hematopoietic stem cells were mobilized from bone
marrow of a human donor into peripheral blood of the human donor using the methods
herein. In certain embodiments, the method includes administering to the donor (i) a CXCR2
agonist selected from the group consisting of Gro-B, Gro-ß, Gro-B Gro-ß T, and variants thereof at a dose
of from about 50 ug/kg µg/kg to about 1,000 ug/kg µg/kg and (ii) a CXCR4 antagonist.
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[0246] In addition, CD34dim CD34di¹ cells have been shown to increase overall survival (OS),
decrease non-relapse mortality (NRM, i.e., the time to death without relapse/recurrence), and
lower the risk of infection (e.g., cytomegalovirus (CMV) infection) in a patient having a
standard risk disease receiving an allogenic hematopoietic cell transplant. (Nakasone et al.
"CD34 "CD34+monocytes monocytesmobilized mobilizedby byG-CSF G-CSFin indonor donorPB PBand andclinical clinicaloutcomes outcomesafter afterall-HCT all-HCT
from related donors," Poster presented at 44th Annual Meeting of the European Society for
Blood and Marrow Transplantation, March 18-21, 2018, Lisbon, Portugal.)
[0247] Accordingly, in certain embodiments, the methods of treating a stem cell disorder in a
human patient disclosed herein can include infusing into the patient a therapeutically
effective amount of the hematopoietic stem cells mobilized by any one of the methods
disclosed herein, wherein the mobilized hematopoietic stem cells comprise CD34 CD34idim cells, cells,
and wherein the treatment results in increased OS, decreased NRM, and/or lowered risk of
infection (e.g., CMV infection).
[0248] In addition, the methods described herein can be used in preventing, reducing the risk
of developing, or reducing the severity of a post-transplant infection in a patient in need
thereof. The method can include infusing into the patient a therapeutically effective amount
of hematopoietic stem cells, wherein the hematopoietic stem cells were mobilized from bone
marrow of a human donor into peripheral blood of the human donor according to the methods
described herein, for example, administering to the human donor (i) a CXCR2 agonist
selected from the group consisting of Gro-B, Gro-ß, Gro-B Gro-ß T, and variants thereof at a dose of from
about 50 ug/kg µg/kg to about 1,000 ug/kg µg/kg and (ii) a CXCR4 antagonist. In certain embodments,
the infection is a CMV infection.
[0249] In addition, the disclosure relates to a method of preventing, reducing the risk of
developing, or reducing the severity of graft versus host disease (GVHD) in a patient in need
thereof, wherein the method includes infusing into the patient a therapeutically effective
amount of hematopoietic stem cells, wherein the hematopoietic stem cells were mobilized
from bone marrow of a mammalian donor into peripheral blood by the methods described
herein, e.g., including administering to the mammalian donor a CXCR2 agonist and a
CXCR4 antagonist.
[0250] In certain embodiments, the hematopoietic stem and progenitor cells mobilized from
the bone marrow of a donor into peripheral blood comprise at least 1%, at least 2% at least
5% 5% at at least least10%, at at 10%, least 15%,15%, least at least 20% or20% at least at least or at20% or more least 20% CD34 dim cells or more CD34iascells as
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compared to peripheral blood from an unmobilized mammal. In certain embodiments, the
hematopoietic stem and progenitor cells mobilized from the bone marrow of a donor into
peripheral blood comprise at from about 1% to about 5%, from about 1% to about 10%, from
about 1% to about 15%, from about 1% to about 20%, from about 2% to about 25%, 2% to
about 5%, from about 2% to about 10%, from about 2% to about 15%, from about 2% to
about 20%, from about 2% to about 25%, from about 5% to about 10%, from about 5% to
about 15%, from about 5% to about 20%, from about 5% to about 25%, from about 10% to
about 15%, from about 10% to about 20%, from about 10% to about 25%, from about 15% to
about 20%, from about 15% to about 25% CD34dim cells CD34i cells asas compared compared toto peripheral peripheral blood blood
from an unmobilized mammal.
[0251] In certain embodiments, the hematopoietic stem and progenitor cells mobilized from
the bone marrow of a donor into peripheral blood comprise at least 1.5-fold, at least 2-fold, at
least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at
least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold,
at least 15-fold, at least 20-fold, at least 30-fold, at least 50-fold more CD34dim cells CD34i cells asas
compared to peripheral blood from an unmobilized mammal. In certain embodiments, the
hematopoietic stem and progenitor cells mobilized from the bone marrow of a donor into
peripheral blood comprise between about 1.5-fold and 30-fold, between about 5-fold and
about 25-fold, between about 10-fold and about 20-fold, or between about 12-fold and about
CD34i cells 17-fold more CD34dim asas cells compared toto compared peripheral blood peripheral from blood anan from unmobilized mammal. unmobilized mammal.
[0252] In certain embodiments, the hematopoietic stem and progenitor cells mobilized from
the bone marrow of a donor into peripheral blood comprise at least 1%, at least 2% at least
5% at least 10%, at least 15%, at least 20% or at least 20% or more CD34 CD34icells cellsthan thanif ifthe the
hematopoietic stem cells were mobilized using the CXCR4 antagonist alone. In certain
embodiments, the hematopoietic stem and progenitor cells mobilized from the bone marrow
of a donor into peripheral blood comprise at from about 1% to about 5%, from about 1% to
about 10%, from about 1% to about 15%, from about 1% to about 20%, from about 2% to
about 25%, 2% to about 5%, from about 2% to about 10%, from about 2% to about 15%,
from about 2% to about 20%, from about 2% to about 25%, from about 5% to about 10%,
from about 5% to about 15%, from about 5% to about 20%, from about 5% to about 25%,
from about 10% to about 15%, from about 10% to about 20%, from about 10% to about 25%,
CD34i cells from about 15% to about 20%, from about 15% to about 25% CD34din than cells ifif than the the
hematopoietic stem cells were mobilized using the CXCR4 antagonist alone.
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[0253] In certain embodiments, the hematopoietic stem and progenitor cells mobilized from
the bone marrow of a donor into peripheral blood comprise at least 1.5-fold, at least 2-fold, at
least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at
least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold,
CD34im cells at least 15-fold, at least 20-fold, at least 30-fold, at least 50-fold more CD34dim cells than than if if
the hematopoietic stem cells were mobilized using the CXCR4 antagonist alone. In certain
embodiments, the hematopoietic stem and progenitor cells mobilized from the bone marrow
of a donor into peripheral blood comprise between about 1.5-fold and 30-fold, between about
5-fold and about 25-fold, between about 10-fold and about 20-fold, or between about 12-fold
and about 17-fold more CD34dim cells CD34i cells than than ifif the the hematopoietic hematopoietic stem stem cells cells were were mobilized mobilized
using the CXCR4 antagonist alone.
Methods of Genetic Modification of Hematopoietic Stem and Progenitor Cells
[0254] Prior to infusion into a patient, such as a patient having one or more stem cell
disorders described herein, hematopoietic stem cells obtained from a donor (or progeny
thereof) may be genetically modified, for example, by disrupting an endogenous gene. This
strategy can be used, for example, to silence the expression of one or more major
histocompatibility complex genes in a hematopoietic stem cell that is allogeneic with respect
to the patient, thereby reducing the likelihood of graft rejection upon transplantation.
[0255] A wide array of methods has been established for the disruption of target genes in a
population of cells. In some embodiments, one such method is through the use of a clustered
regularly interspaced short palindromic repeats (CRISPR)/Cas system, a system that
originally evolved as an adaptive defense mechanism in bacteria and archaea against viral
infection. The CRISPR/Cas system includes palindromic repeat sequences within plasmid
DNA and an associated Cas9 nuclease. This ensemble of DNA and protein directs site
specific DNA cleavage of a target sequence by first incorporating foreign DNA into CRISPR
loci. Polynucleotides containing these foreign sequences and the repeat-spacer elements of
the CRISPR locus are in turn transcribed in a host cell to create a guide RNA, which can
subsequently anneal to a target sequence and localize the Cas9 nuclease to this site. In this
manner, highly site-specific cas9-mediated DNA cleavage can be engendered in a foreign
polynucleotide because the interaction that brings cas9 within close proximity of the target
DNA molecule is governed by RNA:DNA hybridization. As a result, one can theoretically
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design a CRISPR/Cas system to cleave any target DNA molecule of interest. This technique
has been exploited in order to edit eukaryotic genomes (Hwang et al. (2013) Nature
Biotechnology 31:227, the disclosure of which is incorporated herein by reference) and can
be used as an efficient means of site-specifically editing hematopoietic stem cell genomes in
order to cleave DNA, for example, prior to the incorporation of a gene encoding a target
protein. The use of CRISPR/Cas to modulate gene expression has been described in, e.g., US
8,697,359, the disclosure of which is incorporated herein by reference. Alternative methods
for site-specifically cleaving genomic DNA prior to the incorporation of a gene of interest in
a hematopoietic stem cell include the use of zinc finger nucleases (ZFNs) and transcription
activator-like effector nucleases (TALENs). Unlike the CRISPR/Cas system, these enzymes
do not contain a guiding polynucleotide to localize to a specific target sequence. Target
specificity is instead controlled by DNA binding domains within these enzymes. The use of
ZFNs and TALENs in genome editing applications is described, e.g., in Urnov et al. (2010)
Nature Reviews Genetics 11:636; and in Joung et al. (2013) Nature Reviews Molecular Cell
Biology 14:49, the disclosure of both of which are incorporated herein by reference.
[0256] Additional genome editing techniques that can be used to incorporate polynucleotides
encoding target genes into the genome of a hematopoietic stem cell include the use of
ARCUS TM meganucleases meganucleases that that cancan be be rationally rationally designed designed SO so as as to to site-specifically site-specifically cleave cleave
genomic DNA. The use of these enzymes for the incorporation of genes encoding target
genes into the genome of a mammalian cell is advantageous in view of the defined structure-
activity relationships that have been established for such enzymes. Single chain
meganucleases can be modified at certain amino acid positions in order to create nucleases
that selectively cleave DNA at desired locations, enabling the site-specific incorporation of a
target gene into the nuclear DNA of a hematopoietic stem cell. These single-chain nucleases
have been described extensively in, e.g., US 8,021,867 and US 8,445,251, the disclosures of
each of which are incorporated herein by reference.
Kinetics of CXCR2 Agonist and CXCR4 Antagonist Dosing
[0257] For cases in which the donor is administered both a CXCR4 antagonist and a CXCR2
agonist, the two agents may be administered to the donor concurrently. In some
embodiments, the CXCR4 antagonist and the CXCR2 agonist may be co-formulated with one
another and administered in the same pharmaceutical composition. Alternatively, the
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CXCR4 antagonist and the CXCR2 agonist may be formulated in distinct pharmaceutical
compositions and administered separately but simultaneously to the donor.
[0258] In some embodiments, the CXCR4 antagonist is administered to the donor prior to
administration of the CXCR2 agonist. In some embodiments, the CXCR4 antagonist may be
administered to the donor from about 30 minutes to about 180 minutes prior to administration
of the CXCR2 agonist, such as from about 40 minutes to about 160 minutes, about 50
minutes to about 150 minutes, about 60 minutes to about 140 minutes, about 70 minutes to
about 130 minutes, about 60 minutes to about 120 minutes, about 70 minutes to about 110
minutes, or about 80 minutes to about 100 minutes (e.g., about 30 minutes, about 35 minutes,
about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes,
about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes,
about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about 110
minutes, about 115 minutes, about 120 minutes, about 125 minutes, about 130 minutes, about
135 minutes, about 140 minutes, about 145 minutes, about 150 minutes, about 155 minutes,
about 160 minutes, about 165 minutes, about 170 minutes, about 175 minutes, or about 180
minutes prior to administration of the CXCR2 agonist). In some embodiments, the CXCR4
antagonist is administered to the donor from about 30 minutes to about 60 minutes prior to
administration of the CXCR2 agonist (e.g., about 30 minutes, about 35 minutes, about 40
minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes prior to
administration of the CXCR2 agonist). In some embodiments, the CXCR4 antagonist may be
administered to the donor about 45 minutes prior to administration of the CXCR2 agonist.
[0259] Isolation of the population of hematopoietic stem or progenitor cells may commence
from about 10 minutes to about 60 minutes following completion of the administration of the
CXCR4 antagonist and the CXCR2 agonist (e.g., about 10 minutes to about 1.9 hours, about
20 minutes to about 1.8 hours, about 25 minutes to about 1.7 hours, about 30 minutes to
about 1.6hours, about 1.6 hours, about about 40 minutes 40 minutes to about to about 1.5 (e.g., 1.5 hours hours about (e.g.,10 about 10 about minutes, minutes, 15 about 15
minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40
minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, or about
120 minutes following completion of the administration of the CXCR4 antagonist and the
CXCR2 agonist). In some embodiments, isolation of the population of hematopoietic stem or
progenitor cells may commence from about 10 minutes to about 20 minutes following
completion of the administration of the CXCR4 antagonist and the CXCR2 agonist (e.g.,
about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes,
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about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes,
or about 20 minutes following completion of the administration of the CXCR4 antagonist and
the CXCR2 agonist). In some embodiments, isolation of the population of hematopoietic
stem or progenitor cells commences about 15 minutes following completion of the
administration of the CXCR4 antagonist and the CXCR2 agonist.
[0260] In some embodiments, the population of hematopoietic stem or progenitor cells is
isolated from the donor over a period of from about 15 minutes to about 6 hours, such as
from about 20 minutes to about 4.5 hours, about 30 minutes to about 4 hours, about 40
minutes toabout minutes to about3.53.5 hours, hours, about about 50 minutes 50 minutes to 3about to about hours,3 or hours, about or about 1 hour 1 hour to about 2 to about 2
hours (e.g., over a period of about 15 minutes, about 20 minutes, about 30 minutes, about 35
minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60
minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85
minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about
110 minutes, about 115 minutes, about 120 minutes, about 180 minutes, about 240 minutes,
about 300 minutes, or about 360 minutes). In some embodiments, the population of
hematopoietic stem and progenitor cells may be isolated from the donor over a period of from
about 30 minutes to about 1 hour (e.g., over a period of about 30 minutes, about 35 minutes,
about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60
minutes).
[0261] In some embodiments, the hematopoietic stem or progenitor cells may be harvested
by apheresis. In some embodiments, the hematopoietic stem or progenitor cells may be
harvested by drawing peripheral blood from the donor (i.e., subject).
Routes of Administration of CXCR2 Agonists and CXCR4 Antagonists
[0262] The CXCR4 antagonists and CXCR2 agonists described herein may be administered
to a patient by a variety of routes, such as intravenously, subcutaneously, intramuscularly, or
parenterally. The most suitable route for administration in any given case will depend on the
particular agent administered, the patient, pharmaceutical formulation methods,
administration methods (e.g., administration time and administration route), the patient's age,
body weight, sex, severity of the diseases being treated, the patient's diet, and the patient's
excretion rate. Preferably, the CXCR2 agonist (e.g., Gro-B, Gro-ß, Gro-B Gro-ß T, or a variant thereof)
may be administered to a donor intravenously. Under these conditions, CXCR2 agonists,
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such as those described herein, rapidly give rise to populations of cells that are enriched in
CD34 CD90 CD34+ CD45RA CD90+ cells CD45RA (hematopoietic cells stem (hematopoietic cells), stem and cells), reduce and the reduce mobilization the ofof mobilization
other cell types, such as leukocytes, neutrophils, lymphocytes, and monocytes. This property
is described in further detail in Example 1, below.
Pharmaceutical Compositions
[0263] The CXCR2 agonists and CXCR4 antagonists contemplated herein may each be
formulated into a pharmaceutical composition for administration to a subject, such as a
mammalian subject (e.g., a human subject). For instance, contemplated herein are
pharmaceutical compositions comprising a CXCR2 agonist and/or a CXCR4 antagonist, in
admixture with one or more suitable diluents, carriers, and/or excipients. Pharmaceutical
compositions may include sterile aqueous suspensions. Conventional procedures and
ingredients for the selection and preparation of suitable formulations are described, for
example, in Remington: The Science and Practice of Pharmacy (2012, 22nd ed.) 22 ed.) and and inin The The
United States Pharmacopeia: The National Formulary (2015, USP 38 NF 33), the disclosure
of which is incorporated herein by reference in its entirety.
[0264] A pharmaceutical composition may be administered to a subject, such as a human
subject, alone or in combination with pharmaceutically acceptable carriers, the proportion of
which may be determined by the quantity of active pharmaceutical ingredient (i.e., CXCR2
agonist and/or a CXCR4 antagonist), chosen route of administration, and standard
pharmaceutical practice.
Administration and Dosing of CXCR2 Agonists and/or CXCR4 Antagonists
[0265] Contemplated CXCR2 agonists and CXCR4 antagonists, may be administered to a
subject, such as a mammalian subject (e.g., a human subject), by one or more routes of
administration. For instance, contemplated CXCR2 agonists and CXCR4 antagonists may be
administered to a subject by intravenous, intraperitoneal, intramuscular, intraarterial, or
subcutaneous infusion, among others.
[0266] Contemplated CXCR2 agonists and CXCR4 antagonists may be administered to a
subject in one or more doses. For example, a CXCR2 agonist and/or CXCR4 antagonist may
be administered as a single dose or in two, three, four, five, or more doses. When multiple
doses are administered, subsequent doses may be provided during the same day or one or more days, weeks, months, or years following the initial dose. For instance, the contemplated
CXCR2 agonists and CXCR4 antagonists described herein may be administered to a subject,
such as a human subject one or more times daily, weekly, monthly, or yearly, depending on
such factors as, for instance, the subject's age, body weight, sex, the subject's diet, and the
subject's excretion rate. In certain embodiments, the contemplated CXCR2 agonists and
CXCR4 antagonists are each administered in a single dose once per day.
[0267] Hematopoietic stem or progenitor cells and pharmaceutical compositions described
herein may be administered to a subject in one or more doses. When multiple doses are
administered, subsequent doses may be provided one or more days, weeks, months, or years
following the initial dose. For instance, the hematopoietic stem cells and pharmaceutical
compositions described herein may be administered to a subject, such as a human subject
suffering from one or more diseases, conditions, or disorders described herein, one or more
times daily, weekly, monthly, or yearly, depending on such factors as, for instance, the
subject's age, body weight, sex, severity of the diseases being treated, the subject's diet, and
the subject's excretion rate.
Examples
[0268] The following examples are put forth SO so as to provide those of ordinary skill in the art
with a description of how the compositions and methods described herein may be used, made,
and evaluated, and are intended to be purely exemplary of the invention and are not intended
to limit the scope of what the inventors regard as their invention.
Example 1. The effects of Gro-B Gro-ß T on the mobilization of hematopoietic stem cells in
mice and Rhesus monkeys
[0269] Mobilized peripheral blood grafts are currently the predominant source of
hematopoietic stem and progenitor cells (HSPC) for both autologous and allogeneic
transplantation. The most common clinical hematopoietic stem cell mobilization protocol is
five days of Filgrastim (G-CSF). This regimen requires daily injections, has been associated
with bone pain and often results in unpredictably low yields. A rapid mobilization method
that ideally only required a single treatment and had robust and predictable kinetics would be
a significant improvement over the current standard of care. In mice, a unique CXCR2
agonist, Gro-B Gro-ß T, induces rapid mobilization of stem and progenitor cells 15 minutes after a
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single injection. When co-administered with plerixafor (AMD3100), an inhibitor of CXCR4,
a synergistic increase in mobilization results, and grafts are enriched in highly engraftable,
long-term hematopoietic stem cells (see, e.g., FIG. 1A; LT-HSC = Lin- c-kit Sca-1+ CD150+ Sca-1 CD150
CD48). Grafts CD48*). Graftscontaining containingcells cellsmobilized mobilizedby byGro-ß Gro-BTTand andplerixaflor plerixaflorled ledto tohigher higherrelative relative
numbers of competitive repopulating units (CRU) at week 16 than did grafts containing cells
mobilized by G-CSF alone (FIG. 1B).
[0270] In this example, data are presented to demonstrate that combination treatment with
Gro-ß T and AMD3100 results in significantly enhanced mobilization of CD34 cells and Gro-B
colony forming units (CFU) compared to that achieved with AMD3100 alone in nonhuman
primates (NHP).
[0271] Mobilization of hematopoietic stem cells was investigated in rhesus macaques using
Gro-B Gro-ß T and plerixafor (also referred to as AMD3100) as described below.
Methods Methods
[0272] Male rhesus macaques were treated with AMD3100 alone or in combination with
Gro-B Gro-ß T. Blood was collected immediately prior to and 0.5, 1, 2, 4 and 24 hours after
treatment and analyzed by multicolor flow cytometry to quantitate HSPC numbers.
Additional aliquots of mobilized blood were plated in methylcellulose and CFU were
enumerated seven days later.
Results
[0273] FIG. 2A shows the pharmacokinetic profile of various dosages of Gro-B Gro-ß T when
administered intravenously to Rhesus monkeys. FIG. 2B shows the pharmacokinetic profile
of various dosages of Gro-B Gro-ß T when administered subcutaneously to Rhesus monkeys. In all
experiments, Gro-B Gro-ß T was administered to subjects concurrently with plerixafor.
[0274] As shown in FIGS. 3-6, Gro-B Gro-ß T and AMD3100 mobilizes white blood cells into the
peripheral blood. Animals were mobilized with AMD3100 alone or in combination with
Gro-B Gro-ß T. Peripheral blood was collected at the time points shown and white blood cells
enumerated on a HESKA Hematology Analyzer. Total number of white blood cells,
neutrophils, lymphocytes and monocytes per uL µL of peripheral blood were determined. Data
shown in FIGS. 2-5 are expressed as mean SEM and ± SEM represent and 5 animals represent per 5 animals group. per group.
Statistical significance was determined based on 2-way ANOVA with post-hoc Dunnett's
multiple comparisons test (** p < 0.01).
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[0275] Importantly, as shown in FIGS. 7 and 8, Gro-B Gro-ß T and AMD3100 induces robust
mobilization of CD34+ cells into CD34 cells into the the peripheral peripheral blood. blood. The The data data shown shown in in FIGS. FIGS. 77 and and
8include the percentage of whole blood accounted for by CD34+ hematopoietic stem CD34 hematopoietic stem and and
progenitor cells for each treatment group. Absolute numbers and fold change in CD34+ cells CD34 cells
per uL µL of peripheral blood were quantified with a single platform quantitative method. Data
shown in FIGS. 7 and 8 are expressed as mean SEM and ± SEM represent and 5 animals represent per 5 animals group. per group.
Statistical significance was determined based on 2-way ANOVA with post-hoc Dunnett's
multiple comparisons multiple comparisonstest (* p(* test < 0.05, < ** p**< 'p<0.01, p < 0.05, 0.01, **** **** p < 0.0001). p <0.0001).
[0276] Additionally, as shown in FIGS. 9 and 10, CD34+ cells mobilized CD34 cells mobilized in in response response to to Gro- Gro-
BT + AMD3100 are enriched for primitive CD34+ ß T+ CD90+CD45RA- CD34 CD90+ CD45RA-stem stemand andprogenitor progenitor
cells. The data shown in FIGS. 9 and 10 include the percentage of whole blood accounted
for by CD34+ CD90+ CD34 CD90 CD45RA CD45RA hematopoietic hematopoietic stem stem and and progenitor progenitor cells cells for for each each treatment treatment
group. group. Absolute Absolutenumbers and and numbers foldfold change in CD34+ change CD90+ CD90 in CD34 CD45RA cells cells CD45RA per uL per of µL of
peripheral blood are shown. Data shown in FIGS. 9 and 10 are expressed as mean SEM ± SEM
and represent 5 animals per group. Statistical significance was determined based on 2-way
ANOVA withpost-hoc ANOVA with post-hoc Dunnett's Dunnett's multiple multiple comparisons comparisons test (*test p < (* p <**p 0.05. 0.05. 0.01, < 0.01, *** <p<
0.001, **** pp << 0.0001). 0.001,**** 0.0001).
[0277] Further, as shown in FIG. 11, Gro-B Gro-ß T and AMD3100 mobilizes hematopoietic stem
and progenitor cells with colony forming potential. The number of CFU per mL of peripheral
blood was enumerated after seven days of culture in methylcellulose. Data shown in FIG. 11
are expressed as mean SEM and ± SEM represent and 3-5 represent animals 3-5 per animals group. per Statistical group. significance Statistical significance
was determined based on 2-way ANOVA with post-hoc Dunnett's multiple comparisons test
(* p < 0.05). The ratio < The of of ratio MMP-9 to to MMP-9 TIMP-1 is is TIMP-1 additionally elevated additionally following elevated treatment following treatment
with Gro-B Gro-ß T and AMD3100 (FIGS. 12-14).
[0278] Additional data summarizing the mobilization of CD34+ cells(e.g., CD34 cells (e.g.,CD34 CD34+ CD90 CD90
CD45RA cells) in Rhesus monkeys using various doses of Gro-beta T and AMD3100 are
reported in TABLES 8-11, below. Quantities are reported in TABLES 8-11 using the
following notation: "Median value (Minimum value observed - maximum value observed)."
234 wo 2019/113375 WO PCT/US2018/064335
TABLE 8. Mobilization response observed in Rhesus monkeys upon administration of
plerixafor (1 mg/kg, subcutaneously)
Ratio of Ratio of quantity of fold Quantity of Ratio of Fold Fold increase increase Ratio of fold increase of cells observed 4 CD34 quantity of CD90+ VS. baseline vs. increase of hours following CD90 CD34 Cell type CD34 cells following SC sc CD34 cells to CD90 SC sc CD45RA to other cell cells to administration other cell administration CD45RA populations other cell populations cells to of AMD3100: of AMD3100: other cell population S populations 0.352 41054 (23012 - 1.4 (0.6 - (0.259 - 9.1 (7.8 - 16.5) CD34 CD34 58496) -- -- -- 2.6) 0.551)
CD34 CD34 14820 (6123 - 2.838 (1.816 - 14.9 (5.3 - CD90+ CD90 -- 0.7 (0.4 - 1.7) -- 17893) 3.864) 23.3) CD54RA CD54RA 0.0009 0.0003 3.90E+07 (3.16 - 3.7 (1.0 - (0.0006 - (0.0002 - 2.3 (1.6 - 4.6) 2.3 (1.6 - 4.6) WBCs 4.97E+07) 7.9) 0.0012) 0.0004) 0.0004) 0.0015 0.0006 2.10E+07 (1.61 - 3.0 (0.4 - Neutrophils (0.0011 - (0.0004 - 5.0 (3.0 - 14.0) 2.1 (0.6 - 3.6) 3.31E+07) 6.2) 0.0021) 0.0007) 0.0025 0.0011 1.35E+07 (1.12 - 5.7 (2.4 - (0.0020 - (0.0005 - 2.5 (1.3 - 3.1) 4.0 (2.9 - 8.2) Lymphocytes 14.0) 1.62E+07) 0.0043) 0.0013) 0.0111 0.0039 3.10E+06 (1.55 - 1.6 (0.7 - (0.0047 - (0.0020 - 7.9 (5.4 - 14.1) 1.2 (0.6 - 2.0) Monocytes 3.5) 4.93E+06) 0.0377) 0.0115)
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TABLE 9. Mobilization response observed in Rhesus monkeys upon administration of Gro-
ß T (450 ug/kg, µg/kg, intravenously) and plerixafor (1 mg/kg, subcutaneously)
Ratio of fold Quantity of Ratio of Fold increase increase cells observed Ratio of quantity of VS. vs. baseline Ratio of fold quantity of following iv of CD34 4 hours CD34 CD90 increase of Cell type following iv CD34+ cells CD34 cells administratio CD34 cells CD90 CD45RA administration to other cell cells to other n of Gro-beta to other cell CD45RA cells to of Gro-beta T populations cell T at 450 populations other cell at 450 ug/kg: µg/kg: populations ug/kg: µg/kg: populatio ns 47194 (38004 - 0.676 (0.393 - 21.7 (11.2 - 1.2 (1.1 - CD34 CD34 103119) -- 0.745) 27.2) -- 4.8)
CD34 0.8 (0.2 - CD34 CD90+ CD90 25701 (16770 - 1.479 (1.341 - -- 30.7 (18.3 - -- 2.545) -- 73.9) 0.9) 0.9) CD54RA 76870) CD54RA 0.0014 0.0009 3.84E+07 (2.72 5.1 (3.4 - 6.0 (5.5 - (0.0008 - (0.0003 - 3.8 (2.7 3.8 (2.7- 5.4) 5.4) WBCs - 5.27E+07) 6.9) 26.9) 0.0021) 0.0016) 0.0016) 0.0036 0.0024 1.79E+07 (1.03 6.4 (2.1 - 8.2 (3.5 - Neutrophils (0.0018 - (0.0007 - 3.4 (3.1 3.4 (3.1- 5.2) 5.2) - 2.44E+07) 8.1) 22.0) 0.0058) 0.0043) 0.0043) 0.0031 0.0021 1.76E+07 (0.8 - 5.7 (4.8 - 9.3 (5.6 - Lymphocytes (0.0021 - (0.0008 - 3.2 (2.0 3.2 (2.0- 5.4) 5.4) 2.39E+07) 8.4) 37.0) 0.0094) 0.0069) 0.0118 0.0073 5.48E+06 (3.54 11.7 (8.7 - 1.6 (1.1 - 1.9 (1.5 - Monocytes (0.0071 - (0.0028 - - 5.99E+06) 21.1) 2.3) 8.5) 0.0174) 0.0130)
236 wo 2019/113375 WO PCT/US2018/064335 PCT/US2018/064335
TABLE 10. Mobilization response observed in Rhesus monkeys upon administration of
Gro-ß T (450 ug/kg, µg/kg, subcutaneously and plerixafor (1 mg/kg, subcutaneously)
Ratio of Quantity of Ratio of fold quantity of Fold increase Ratio of cells observed Ratio of increase of vs. baseline fold 6 hours quantity of CD34 CD34+ CD90 CD34 CD90 following SC sc increase of Cell type following SC sc CD34+ cells CD34 cells CD90 administration CD34 cells CD45RA administration to other cell CD45RA cells to other cells to of Gro-beta T to other cell of Gro-beta T populations cell other cell at 450 ug/kg: µg/kg: populations at 450 ug/kg: µg/kg: populations populations 41178 (19413 - 0.359 (0.318 CD34+ 6.3 (4.8 - 13.2) 1.0 (1.0 - 1.3) CD34 72140) -- -- - 0.441) 0.441) -- --
14782 (6177 - 2.786 (2.266 1.0 (0.8 - CD34+ CD90 CD34 CD90 6.6 (6.1 6.6 (6.1- 13.6) 13.6) -- -- -- -- 31841) - 3.143) 1.0) CD54RA 0.0007 0.0003 6.31E+07 (5.83 1.3 (0.9 - (0.0003 - (0.0001 - 5.4 5.4 (4.8 (4.8- 5.8) 5.8) 1.4 (1.1 2.3) WBCs - 6.88E+07) 6.88E+07) 2.3) 0.0011) 0.0005) 0.0011 0.0004 4.00E+07 (3.64 1.3 (0.8 - Neutrophils (0.0004 - (0.0001 - 5.6 (4.8 5.6 (4.8- 6.1) 6.1) 1.4 (1.0 2.4) - 4.98E+07) 2.4) 0.0018) 0.0008) 0.0008) 0.0022 0.0008 1.4 (1.2 - 1.85E+07 (1.57 (0.0012 - (0.0004 - 4.5 (3.9 - 5.7) 1.6 (1.5 1.6 (1.5- 2.4) 2.4) Lymphocytes - 1.97E+07) 1.97E+07) 2.3) 0.0037) 0.0016) 0.0016) 0.0185 0.0066 2.23E+06 (2.05 0.7 0.7 (0.6 (0.6- Monocytes (0.0075 - (0.0024 - 9.7 (7.4 9.7 (7.4- 17.1) 17.1) 0.8 (0.7 0.8 (0.7- 0.8) 0.8) - 2.60E+06) 0.8) 0.0352) 0.0155)
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TABLE 11. Mobilization response observed in Rhesus monkeys upon administration of
Gro-B Gro-ß T (1.2 mg/kg, subcutaneously) and plerixafor (1 mg/kg, subcutaneously)
Ratio of Ratio of fold Quantity of quantity of Fold increase increase of cells observed Ratio of Ratio of fold quantity of CD34 VS. baseline increase of CD34+ 4 hours Cell type following SC sc CD34 cells CD90+ CD90 following SC sc CD34 cells CD90+ CD90 CD45RA administration CD45RA administration to other cell to other cell cells to of Gro-beta T cells to other of Gro-beta T populations populations other cell at 1.2 mg/kg: cell at 1.2 mg/kg: populations populations 36219 (26331 - 0.449 (0.278 12.0 (7.2 - CD34+ CD34 -- -- -- -- 1.3 (0.8 - 1.9) 71704) - 0.548) 15.0)
CD34+ CD34 17471 (7645 - 2.232 (1.826 13.1 (10.3 13.1 (10.3- - 0.8 (0.5 - 1.3) CD90 25964) - 3.594) -- 23.9) -- --
CD54RA CD54RA 0.0008 0.0003 4.94E+07 (4.07 5.7 (4.5 - 9.7) 1.8 (1.4 - 2.7) 2.5 (1.1 - 4.2) (0.0005 - (0.0001 - WBCs -6.66E+07) 6.66E+07) 0.0011) 0.0005) 0.0013 0.0006 3.23E+07 (2.31 9.7 (5.5 - 15.1) 1.2 (0.7 - 1.9) Neutrophils (0.0008 - (0.0002 - 1.6 (0.7 - 2.6) - 3.52E+07) 3.52E+07) 0.0023) 0.0008) 0.0024 0.0010 4.9 (2.0 - 1.36E+07 (0.66 (0.0019 - (0.0005 - 3.3 (1.7 - 5.2) 3.6 (2.4 - 8.9) Lymphocytes - 2.91E+07) 12.0) 0.0068) 0.0037) 0.0130 0.0052 4.08E+06 (1.16 13.7 (7.2 - (0.0068 - (0.0029 - 0.8 (0.5 - 2.1) 1.1 (0.6 - 2.8) Monocytes 23.2) - 5.37E+06) 5.37E+06) 0.0237) 0.0078) 0.0078)
Conclusions Conclusions
5 [0279] A single treatment of Gro-ß T, in combination with AMD3100, induces robust
mobilization of stem and progenitor cells within four hours of administration in nonhuman
primates. Additionally, Gro-B Gro-ß T, in combination with AMD3100, results in 2-3 fold more
CD34+ CD90+ CD45RA CD34 CD90 CD45RA stem stem and andprogenitor progenitorcells relative cells to AMD3100 relative alone,alone, to AMD3100 suggesting a suggesting a
significant graft quality improvement.
10 [0280] Further, as evidenced by these data, Gro-B Gro-ß T, in combination with AMD3100,
may offer a more robust and safer alternative to G-CSF in autologous and allogeneic
transplant, including diseases such as sickle cell disease (SCD) and multiple sclerosis (MS)
where G-CSF is contraindicated or associated with adverse events.
WO wo 2019/113375 PCT/US2018/064335
Example 2. Determining whether a population of hematopoietic stem cells mobilized
with a CXCR2 agonist and/or a CXCR4 antagonist is suitable for ex vivo expansion
and/or therapeutic use
[0281] Using the compositions and methods described herein, a practitioner of skill in the art
may mobilize a population of hematopoietic stem or progenitor cells in a mammalian donor,
such as a human donor. In some embodiments, the practitioner may administer a CXCR4
antagonist and a CXCR2 agonist in amounts sufficient to engender the release of a population
of a population of hematopoietic stem cells into circulating peripheral blood while reducing
the mobilization of other cells of the hematopoietic lineage, such as leukocytes, neutrophils,
lymphocytes, and monocytes.
[0282] When administering a CXCR4 antagonist in combination with a CXCR2 agonist, the
physician may administer the two agents to the donor simultaneously or at different times. In
some embodiments, the CXCR4 antagonist may be administered to the donor from about 30
minutes to about 180 minutes prior to administration of the CXCR2 agonist agonist,, such such as as from from
about 40 minutes to about 160 minutes, about 50 minutes to about 150 minutes, about 60
minutes to about 140 minutes, about 70 minutes to about 130 minutes, about 60 minutes to
about 120 minutes, about 70 minutes to about 110 minutes, or about 80 minutes to about 100
minutes (e.g., about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about
50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about
75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about
100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, about 120 minutes,
about 125 minutes, about 130 minutes, about 135 minutes, about 140 minutes, about 145
minutes, about 150 minutes, about 155 minutes, about 160 minutes, about 165 minutes, about
170 minutes, about 175 minutes, or about 180 minutes prior to administration of the CXCR2
agonist).
[0283] To assess the efficacy of the mobilization regimen, a peripheral blood sample may be
isolated from the subject following administration of the CXCR2 agonist and/or CXCR4
antagonist. The sample may then be characterized, for example, by acquiring an input value
for each of one or more parameters of the sample, such as a parameter listed in TABLE 2.
Exemplary parameters that may be used to assess the efficacy of the hematopoietic stem cell
mobilization regimen are ratios of hematopoietic stem cells to cells of other types, such as
leukocytes, neutrophils, lymphocytes, and monocytes, as well as the relative frequency of
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WO wo 2019/113375 PCT/US2018/064335
hematopoietic stem cells in the sample. Input values for these parameters may be acquired,
for example, using immunophenotyping methods known in the art, such as flow cytometry
and fluorescence activated cell sorting (FACS) techniques.
[0284] When acquiring and analyzing input values for more than one parameter listed in
TABLE 2, one may analyze a combination of parameters. In some embodiments, one may
analyze a ratio of hematopoietic stem cells to leukocytes, a ratio of hematopoietic stem cells
to neutrophils, a ratio of hematopoietic stem cells to lymphocytes, a ratio of hematopoietic
stem cells to monocytes, and/or the relative frequency of hematopoietic stem cells in a
sample obtained from the peripheral blood of a donor following administration of a CXCR2
agonist and/or a CXCR4 antagonist. One may analyze, for example, a combination of
parameters set forth in any one of TABLES 3-6.
[0285] Upon acquiring an input value for each of the one or more parameters, one may then
compare the input value(s) to the reference criterion for each parameter. If the reference
criterion is satisfied (e.g., if the ratio of hematopoietic stem cells to another hematopoietic
cell type is sufficiently high, or if the relative frequency of hematopoietic stem cells in the
sample obtained from the peripheral blood of the donor is sufficiently high), then the cells
may be released for ex vivo expansion and/or for therapeutic use.
Example 3. Treatment of a hematologic disorder by administration of a hematopoietic
stem or progenitor cell graft
[0286] Using the compositions and methods described herein, a practitioner of skill in the art
may treat a stem cell disorder, such as a hematologic pathology described herein, by
administering to a patient a hematopoietic stem or progenitor cell graft. For example, a
practitioner may identify a human donor of hematopoietic stem or progenitor cells as likely to
respond to a mobilization regimen, as outlined in Example 1, and may subsequently mobilize
a population of hematopoietic stem or progenitor cells accordingly, for example, as set forth
in Example 2. Following mobilization, the physician may isolate a population of
hematopoietic stem or progenitor cells from the donor. Isolation of the cells may commence,
for example, from about 10 minutes to about 60 minutes following completion of the
administration of a CXCR4 antagonist and/or a CXCR2 agonist, such as from about 15
minutes to about 55 minutes, about 20 minutes to about 50 minutes, about 25 minutes to
about 45 minutes, or about 30 minutes to about 40 minutes following completion of the
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WO wo 2019/113375 PCT/US2018/064335
administration of these agents (e.g., about 10 minutes, about 15 minutes, about 20 minutes,
about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes,
about 50 minutes, about 55 minutes, or about 60 minutes following completion of the
administration of the CXCR4 antagonist and the CXCR2 agonist).
[0287] The isolation procedure may be carried out over a period of from about 15 minutes to
about 6 hours, such as from about 20 minutes to about 4.5 hours, about 30 minutes to about 4
hours, about 40 minutes to about 3.5 hours, about 50 minutes to about 3 hours, or about 1
hour to about 2 hours (e.g., over a period of about 15 minutes, about 20 minutes, about 30
minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55
minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80
minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about
105 minutes, about 110 minutes, about 115 minutes, or about 120 minutes). In some
embodiments, the population of hematopoietic stem and progenitor cells may be isolated
from the donor over a period of from about 30 minutes to about 1 hour (e.g., over a period of
about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes,
about 55 minutes, or about 60 minutes).
[0288] Following the isolation process, a patient may then receive an infusion (e.g., an
intravenous infusion) of the mobilized and isolated hematopoietic stem or progenitor cells.
The patient may be the donor, or may be a patient that is HLA-matched with respect to the
donor, thereby reducing the likelihood of graft rejection. The patient may be one that is
suffering, for example, from a cancer, such as a hematologic cancer described herein.
Additionally or alternatively, the patient may be one that is suffering from an autoimmune
disease or metabolic disorder described herein. The physician may administer the patient the
mobilized and isolated hematopoietic stem or progenitor cells, for example, at a dosage of
from from 11 x X1010³ 3 to to1 1x X10' 10hematopoietic hematopoieticstemstem cells/kg (e.g.,(e.g., cells/kg about 1about X 105 1CD34+ X 10cells/kg to CD34 cells/kg to
about 1 x X 107 CD34+ 10 CD34 cells/kg, cells/kg, about about 2 105 x 10CD34+ CD34 cells/kg to about 9 X x 106 CD34+ 10 CD34 cells/kg, cells/kg,
about X 3 105 x 10CD34+ CD34 cells/kg to about 8 X 106 CD34+ 10 CD34 cells/kg, cells/kg, about about 4 4 X X 10105 CD34+ CD34 cells/kg cells/kg
to about to about7 7x x106 10CD34+ CD34 cells/kg, cells/kg,depending on various depending factors, on various such assuch factors, the patient's age, as the patient's age,
weight, and the severity of the disease being treated.
[0289] The physician may monitor the engraftment of the hematopoietic stem cell transplant,
for example, by withdrawing a blood sample from the patient and determining the increase in in
concentration of hematopoietic stem cells or cells of the hematopoietic lineage (such as
WO wo 2019/113375 PCT/US2018/064335
megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeoblasts, basophils,
neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting
cells, macrophages, dendritic cells, natural killer cells, T-lymphocytes, and B-lymphocytes)
following administration of the transplant. This analysis may be conducted, for example,
from about 1 hour to about 6 months, from about 2 hours to about 5 months, from about 3
hours to about 4 months, from about 4 hours to about 3 months, from about 10 hours to about
7 days, from about 24 hours to about 96 hours, or more following hematopoietic stem cell
transplant therapy (e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9
hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18
hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5
days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9
weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks,
18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, or more). A
finding that the concentration of hematopoietic stem cells or cells of the hematopoietic
lineage has increased (e.g., by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, or more) following the transplant
therapy relative to the concentration of the corresponding cell type prior to transplant therapy
provides one indication that the hematopoietic stem or progenitor cell transplant therapy is
efficacious in treating the stem cell disorder.
CD34im cells Example 4: Mobilization of CD34dim cellsusing usingaaCXCR2 CXCR2agonist agonistand/or and/oraaCXCR4 CXCR4
antagonist
[0290] In this example, data are presented to demonstrate that combination treatment with
Gro-B Gro-ß T and AMD3100 (plerixafor) results in significantly enhanced mobilization of
CD34dim CD34i cells cells compared compared toto that that achieved achieved with with AMD3100 AMD3100 alone alone inin nonhuman nonhuman primates primates
(NHP).
[0291] Cells were mobilized according to the method of Example 1. A sample of peripheral
blood was tested using a flow cytometer for the markers CD34, CD11b, CD14, CD16, CD45,
and SSC.
[0292] As shown in FIG. 15, intravenous administration of 450 ug/kg µg/kg Gro-ß T and
subcutaneous administration of 1 mg/kg AMD3100 surprisingly leads to the mobilization of a
CD34i CD11b. population of cells that are CD34dim Subpopulations CD11b+. within Subpopulations this within population this areare population
CD14 CD14 CD16*, CD16, CD14 CD14 CD16`, CD16; and andcombinations combinationsthereof. FIG.FIG. thereof. 16 shows that mobilization 16 shows of that mobilization of
WO wo 2019/113375 PCT/US2018/064335
the CD34du the CD34i cells cells is is significantly significantly enhanced enhanced upon upon administration administration of of the the combination combination of of Gro-ß Gro-B
T and AMD3100 as compared to AMD3100 alone.
Gro-B T in combination with AMD3100 mobilizes immunosuppressive Example 5: Gro-ß
VS. host disease monocytes that inhibit graft vs.
[0293] This example demonstrates that combination treatment with Gro-B Gro-ß T and AMD3100
(plerixafor) results in significantly enhanced mobilization of CD34dim CD34 dimcells cellshaving having
immunosuppressive properties and that transplantation of this population of cells leads to a
significantly reduced incidence of acute graft versus host disease (aGVHD) in a xenograft
GVHD mouse model, as compared to transplantation of peripheral blood mononuclear cells
CD34i cells. having a lower concentration of CD34dim cells.
[0294] Rhesus macaques were treated with Gro-B Gro-ß T, AMD3100, Gro-ß T and AMD3100, or
G-CSF alone. A sample of peripheral blood was tested using a flow cytometry. Animals
mobilized with Gro-B Gro-ß T and AMD3100 showed a more than fifteen-fold increase over
baseline in the number of CD34dim cells CD34i cells atat 4 4 hours hours post-treatment post-treatment (p<0.0001, (p<0.0001, n=13, n=13, FIG. FIG. 17). 17).
By comparison, animals mobilized with G-CSF alone showed only about a nine-fold increase
over over baseline baselineinin thethe number of CD34dim number cells of CD34i at 4 at cells hours after after 4 hours the last dose the of dose last G-CSF of G-CSF
(p<0.0001, n=3, FIG. 17).
[0295] Further, the composition of unmobilized cells and grafts mobilized by G-CSF, Gro-B Gro-ß
T and AMD3100 and AMD3100 alone is provided in FIG 18. As shown, grafts mobilized
Gro-ß T and AMD3100 show a 3 fold increase in CD34dim using Gro-B CD34i cells and cells a a and 3 3 fold increase fold inin increase
T-cells as compared to grafts mobilized using G-CSF.
[0296] To determine if CD34dim cells CD34i cells had had immunosuppressive immunosuppressive properties, properties, they they were were sorted sorted
from the peripheral blood of Rhesus macaques treated with Gro-B Gro-ß T and AMD3100. The
CD34dim CD34 dimcells cellswere wereco-cultured co-culturedwith withcarboxyfluorescein carboxyfluoresceinsuccinimidyl succinimidylester ester(CFSE)-labeled (CFSE)-labeled
autologous T cells stimulated with anti-CD2/CD3/CD28-coated beads in vitro. As shown in
FIG. 19, Gro-B Gro-ß T and AMD3100 mobilized CD34dim cellssuppressed CD34im cells suppressedT-cell T-cellproliferation proliferationas as
measured by CFSE staining after four days.
[0297] To assess whether these immunosuppressive cells may prevent GVHD, we developed
a xenograft GVHD model in NOD scid gamma (NSG) mice, which are immunodeficient
mice. Gro-B Gro-ß T and AMD3100 mobilized peripheral blood (6 X 106 PBMCs) containing 10 PBMCs) containing aa
high percentage of CD34dim cellswere CD34im cells wereinjected injectedinto intosublethally sublethallyirradiated irradiatedNSG NSGmice. mice.This This
was compared to unmobilized primate PBMCs (6 X x 106 PBMCs) containing 10 PBMCs) containing aa relatively relatively low low
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numbers of numbers ofCD34dim CD34i cells cells and andG-CSF G-CSF mobilized mobilized primate primate PBMCsPBMCs (6 XPBMCs). (6 X 106 10 PBMCs). As As
shown in the survival curve in FIG. 20, at day 24, all mice (13/13) transplanted with
unmobilized PBMCs had died of aGVHD compared to 5/16 mice transplanted with
AMD3100 mobilized PBMCs, 3/16 mice transplanted with G-CSF-mobilized PBMCs and
none of the mice transplanted with Gro-B Gro-ß T and AMD3100-mobilized PBMCs. At day 60
post-transplant, 15/16 mice transplanted with Gro-B Gro-ß T and AMD3100 remained alive,
whereas only 10/16 mice transplanted with AMD3100 mobilized peripheral blood and 11/16
mice transplanted with G-CSF mobilized peripheral blood remained alive (see FIG. 20). As
shown in FIG. 21A, mice transplanted with unmobilized PBMCs ("Unmobilized") also
demonstrated higher numbers of donor T-cells (approximately 160-fold higher, see FIG.
21A) compared to mice transplanted with Gro-B Gro-ß T and AMD3100 mobilized PBMCs ("Gro-B ("Gro-ß
T + Plerixafor") at day 14 post-transplant (*p<0.01, n=6-8). Additionally, also as shown in
FIG. 21A, mice transplanted with PBMCs mobilized with AMD3100 (i.e., plerixafor) alone
("Plerixafor") or PBMCs mobilized with G-CSF alone ("G-CSF") demonstrated higher
numbers of T-cells (approximately 60-fold higher (p<0.05) and approximately 10-fold higher
(p<0.01), respectively) as compared to mice transplanted with Gro-B Gro-ß T and AMD3100-
mobilized PBMCs.
CD34i cells
[0298] To confirm that the CD34dim inin cells the Gro-ß the T T Gro-B and AMD3100-mobilized and graft AMD3100-mobilized were graft were
responsible for the protection against aGvHD in the NSG mouse xenotransplantation model
shown in FIG. 20, additional cohorts of mice were transplanted with 6x106 6x 10 PBMCs from
Gro-ß T and AMD3100-mobilized rhesus monkeys, where CD34dim Gro-B CD34i cells were cells first were depleted first depleted
by fluorescence activated cell sorting and T cell numbers and survival were compared to
cohorts transplanted with Gro-ß T and AMD3100-mobilized PBMCs and cohorts
transplanted with unmobilized PBMCs. As shown in FIG. 21B, at day 14 post-transplant,
mice transplanted with Gro-ß T and AMD3100-mobilized PBMCs ("Gro-B ("Gro-ß T T++ Plerixafor") Plerixafor")
had significantly reduced numbers of rhesus CD45+ CD3+ T cells in peripheral blood as
compared to mice transplanted with unmobilized PBMCs ("Unmobilized") (p < 0.001,
n=10/group). By contrast, mice transplanted with Gro-ß T and AMD3100 mobilized PBMCs
that were depleted of CD34dim cells showed no significant difference in T-cell numbers by
day 14 post-transplant (n=10) as compared to mice transplanted with unmobilized PBMCs
("Unmobilized") (see FIG. 21B). As shown in FIG. 21C, by day 44 post transplant, 0/10
mice transplanted with unmobilized PBMCs ("Unmobilized") survived versus 8/10 mice
transplanted with Gro-B Gro-ß T and AMD3100-mobilized PBMCs ("Gro-B ("Gro-ß T T++plerixafor") plerixafor")
244
WO wo 2019/113375 PCT/US2018/064335
(p<0.05) and only 4/10 mice transplanted with Gro-B Gro-ß T and AMD3100 mobilized PBMCs
("Gro-ß T that were depleted of CD34dim cells ("Gro-B T++ plerixafor plerixafor CD34i -depleted") (no(no CD34dim-depleted``)
significant difference versus Unmobilized).
[0299] Accordingly, these data demonstrate that co-administration of Gro-B Gro-ß T and AMD3100
results not only in rapid and efficacious mobilization of highly enriched HSCs but that these
CD34i cell HSCs are enriched in a CD34dim population cell with population potent with immunosuppressive potent activity immunosuppressive activity
compared to AMD3100 alone or the current standard of care, G-CSF, and that transplant of
this CD34dim cell CD34i cell population population can can result result inin a a reduced reduced incidence incidence ofof oror risk risk for for aGVHD. aGVHD.
Other Embodiments
[0300] All publications, patents, and patent applications mentioned in this specification are
incorporated herein by reference to the same extent as if each independent publication or
patent application was specifically and individually indicated to be incorporated by reference.
[0301] While the invention has been described in connection with specific embodiments
thereof, it will be understood that it is capable of further modifications and this application is
intended to cover any variations, uses, or adaptations of the invention following, in general,
the principles of the invention and including such departures from the invention that come
within known or customary practice within the art to which the invention pertains and may be
applied to the essential features hereinbefore set forth, and follows in the scope of the claims.
[0302] Other embodiments are within the claims.

Claims (22)

CLAIMS 18 Sep 2025 What is claimed is:
1. A method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian subject into peripheral blood, the method comprising administering to the subject (i) a C-X-C chemokine receptor type 2 (CXCR2) agonist selected from the group consisting of growth regulated oncogene beta (Gro-β), truncated Gro-β (Gro-β T), a 2018378804
variant of Gro-β having at least 95% sequence identity to SEQ ID NO: 1, and a variant of Gro-β T having at least 95% sequence identity to SEQ ID NO: 2, at a dose of from 50 μg/kg to 150 μg/kg and (ii) a C-X-C chemokine receptor type 4 (CXCR4) antagonist.
2. The method of claim 1, wherein the CXCR2 agonist is administered to the subject at a dose of 50 μg/kg.
3. The method of claim 1, wherein the CXCR2 agonist is administered to the subject at a dose of 70 μg/kg.
4. The method of claim 1, wherein the CXCR2 agonist is administered to the subject at a dose of 75 μg/kg.
5. The method of any one of claims 1-4, wherein the CXCR2 agonist is administered intravenously to the subject.
6. The method of any one of claims 1-4, wherein the CXCR4 antagonist is administered subcutaneously to the subject.
7. The method of claim 6, wherein the CXCR4 antagonist is plerixafor or a pharmaceutically acceptable salt thereof.
8. The method of claim 7, wherein the CXCR4 antagonist or pharmaceutically acceptable salt thereof is administered to the subject at a dose of from 50 µg/kg to 500 µg/kg.
9. The method of claim 8, wherein the CXCR4 antagonist or pharmaceutically acceptable salt thereof is administered to the subject at a dose of from 100 µg/kg to 300 µg/kg.
10. The method of claim 9, wherein the CXCR4 or pharmaceutically acceptable salt 18 Sep 2025
thereof is administered to the subject at a dose of 240 µg/kg.
11. A method of mobilizing a population of hematopoietic stem cells from the bone marrow of a human subject into peripheral blood, the method comprising administering to the subject (i) a CXCR2 agonist selected from the group consisting of Gro-β, Gro-β T, a variant of Gro-β having at least 95% sequence identity to SEQ ID NO: 1, and a variant of Gro-β T having at least 95% sequence identity to SEQ ID NO: 2, and (ii) a CXCR4 antagonist, 2018378804
wherein the a CXCR4 antagonist is administered prior to the CXCR2 agonist.
12. The method of claim 11, wherein the CXCR4 antagonist is plerixafor or pharmaceutically acceptable salt thereof.
13. The method of claim 11 or claim 12, wherein the CXCR4 antagonist is administered between 30-180 minutes before the CXCR2 agonist.
14. The method of claim 13, wherein the CXCR4 antagonist is administered between 70-130 minutes before the CXCR2 agonist.
15. The method of claim 14, wherein the CXCR4 antagonist is administered 120 minutes before the CXCR2 agonist.
16. The method of claim 11, wherein the dose of CXCR2 agonist is from 50 μg/kg to 150 μg/kg.
17. The method of claim 16, wherein the CXCR2 agonist is administered to the subject at a dose of 50 μg/kg.
18. The method of claim 16, wherein the CXCR2 agonist is administered to the subject at a dose of 70 μg/kg.
19. The method of claim 16, wherein the CXCR2 agonist is administered to the subject at a dose of 75 μg/kg.
20. The method of claim 16, wherein the CXCR2 agonist is administered to the subject at a dose of 150 μg/kg.
21. The method of any one of claims 11-20, wherein the CXCR2 agonist is 18 Sep 2025
administered intravenously to the subject.
22. The method of any one of claims 11-20, wherein the CXCR4 antagonist is administered subcutaneously to the subject. 2018378804
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