DOSING REGIMENS FOR THE MOBILIZATION OF HEMATOPOIETIC STEM AND PROGENITOR CELLS

REGIMES POSOLOGIQUES POUR LA MOBILISATION DE CELLULES SOUCHES ET PROGENITRICES HEMATOPOIETIQUES

English Abstract

The invention provides compositions and methods useful for mobilizing populations of hematopoietic stem and progenitor cells within a donor, as well as for determining whether samples of mobilized cells are suitable for release for ex vivo expansion and/or therapeutic use. In accordance with the compositions and methods described herein, mobilized hematopoietic stem and progenitor cells can be withdrawn from a donor and administered to a patient for the treatment of various stem cell disorders, including hematopoietic diseases, metabolic disorders, cancers, and autoimmune diseases, among others. In certain embodiments, the compositions and methods described herein lead to the mobilization of a population of CD34dim cells that have immunosuppressive effects and that can reduce the incidence of graft vs. host disease.

French Abstract

L'invention concerne des compositions et des procédés utiles pour mobiliser des populations de cellules souches et progénitrices hématopoïétiques chez un donneur, ainsi que pour déterminer si les échantillons de cellules mobilisées sont appropriés à une libération pour une expansion ex vivo et/ou une utilisation thérapeutique. Grâce aux compositions et aux procédés l'invention, des cellules souches et progénitrices hématopoïétiques mobilisées peuvent être prélevées sur un donneur et administrées à un patient pour le traitement de divers troubles des cellules souches, notamment des maladies hématopoïétiques, des troubles métaboliques, des cancers et des maladies auto-immunes, entre autres. Dans certains modes de réalisation, les compositions et les procédés décrits ici permettent la mobilisation d'une population de cellules CD34dim qui ont des effets immunosuppresseurs et qui peuvent réduire l'incidence de la maladie du greffon contre l'hôte.

Claims

CLAIMS
What is claimed is:
1. 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 (i) a CXCR2 agonist selected from the group consisting of Gro-
.beta., Gro-.beta. T, and
variants thereof at a dose of from about 50 µg/kg to about 300 mg/kg and
(ii) a CXCR4
antagonist.
2. The method of claim 1, wherein the CXCR2 agonist is Gro-.beta. T.
3. The method of claim 1 or 2, wherein the CXCR2 agonist is administered to
the
donor at a dose of from about 100 µg/kg to about 250 µg/kg.
4. The method of claim 3, wherein the CXCR2 agonist is administered to the
donor
at a dose of from about 125 µg/kg to about 225 µg/kg.
5. The method of claim 4, wherein the CXCR2 agonist is administered to the
donor
at a dose of about 150 µg/kg.
6. The method of any one of claims 1-5, wherein the CXCR2 agonist is
administered
intravenously to the donor.
7. The method of any one of claims 1-6, wherein the CXCR4 antagonist is
administered subcutaneously to the donor.
8. The method of any one of claims 1-7, wherein the CXCR4 antagonist is
plerixafor
or a pharmaceutically acceptable salt thereof.
9. The method of claim 8, wherein the plerixafor or pharmaceutically
acceptable salt
thereof is administered to the donor at a dose of from about 50 µg/kg to
about 500 µg/kg.
10. The method of claim 9, wherein the plerixafor or pharmaceutically
acceptable salt
thereof is administered to the donor at a dose of from about 200 µg/kg to
about 300 µg/kg.
11. The method of claim 10, wherein the plerixafor or pharmaceutically
acceptable
salt thereof is administered to the donor at a dose of about 240 µg/kg.
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12. 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 a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to
produce a
population of cells having a ratio of CD34+ cells to leukocytes of from about
0.0008 to about
0.0021 in a sample of peripheral blood of the donor following administration
of the CXCR2
agonist and CXCR4 antagonist.
13. The method of claim 12, wherein the ratio of CD34+ stem cells to
leukocytes in
the sample is from about 0.0010 to about 0.0018.
14. 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 a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to
enrich the
peripheral blood of the donor with CD34+ cells relative to leukocytes by a
ratio of from about
3.4:1 to about 6.9:1 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.
15. The method of claim 14, wherein the peripheral blood of the donor is
enriched
with CD34+ cells relative to leukocytes by a ratio of from about 4.0:1 to
about 6.0:1.
16. 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 a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to
produce a
population of cells having a ratio of CD34+ cells to neutrophils of from about
0.0018 to about
0.0058 in a sample of peripheral blood of the donor following administration
of the CXCR2
agonist and CXCR4 antagonist.
17. The method of claim 16, wherein the ratio of CD34+ cells to neutrophils
in the
sample is from about 0.0026 to about 0.0046.
18. 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 a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to
enrich the
peripheral blood of the donor with CD34+ cells relative to neutrophils by a
ratio of from
247

about 2.1:1 to about 8.1:1 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.
19. The method of claim 18, wherein the peripheral blood of the donor is
enriched
with CD34+ cells relative to neutrophils by a ratio of from about 5.4:1 to
about 7.4:1.
20. 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 a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to
produce a
population of cells having a ratio of CD34+ cells to lymphocytes of from about
0.0021 to
about 0.0094 in a sample of peripheral blood of the donor following
administration of the
CXCR2 agonist and CXCR4 antagonist.
21. The method of claim 20, wherein the ratio of CD34+ cells to lymphocytes
in the
sample is from about 0.0025 to about 0.0035.
22. 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 a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to
enrich the
peripheral blood of the donor with CD34+ cells relative to lymphocytes by a
ratio of from
about 4.8:1 to about 8.4 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.
23. The method of claim 22, wherein the peripheral blood of the donor is
enriched
with CD34+ cells relative to lymphocytes by a ratio of from about 5.0:1 to
about 6.5:1.
24. 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 a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to
produce a
population of cells having a ratio of CD34+ cells to monocytes of from about
0.0071 to about
0.0174 in a sample of peripheral blood of the donor following administration
of the CXCR2
agonist.
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25. The method of claim 24, wherein the ratio of CD34+ cells to monocytes
in the
sample is from about 0.0100 to about 0.0140.
26. 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 a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to
enrich the
peripheral blood of the donor with CD34+ cells relative to monocytes by a
ratio of from
about 1.1:1 to about 2.3:1 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.
27. The method of claim 26, wherein the peripheral blood of the donor is
enriched
with CD34+ cells relative to monocytes by a ratio of from about 1.3:1 to about
1.9:1.
28. 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 a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to
produce a
population of cells having a frequency of CD34+ cells of from about 0.051% to
about
0.140% in a sample of peripheral blood of the donor following administration
of the CXCR2
agonist and CXCR4 antagonist.
29. The method of claim 28, wherein the frequency of CD34+ cells in the
sample is
from about 0.080% to about 0.120%.
30. 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 a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to
induce an
increase in the frequency of CD34+ cells in the peripheral blood of the donor
by from about
3.4-fold to about 7.1-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.
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31. The method of claim 30, wherein the frequency of CD34+ cells in the
peripheral
blood of the donor is increased by from about 4.0-fold to about 6.0-fold
following
administration of the CXCR2 agonist and CXCR4 antagonist.
32. 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 a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to
produce a
population of cells having a ratio of CD34+ CD90+ CD45RA- cells to leukocytes
of from
about 0.0003 to about 0.0016 in a sample of peripheral blood of the donor
following
administration of the CXCR2 agonist and CXCR4 antagonist.
33. The method of claim 32, wherein the ratio of CD34+ CD90+ CD45RA- cells
to
leukocytes in the sample is from about 0.0006 to about 0.0012.
34. 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 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.
35. The method of claim 34, wherein the peripheral blood of the donor is
enriched
with CD34+ CD90+ CD45RA- cells relative to leukocytes by a ratio of from about
5.5:1 to
about 6.5:1.
36. 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 a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to
produce a
population of cells having a ratio of CD34+ CD90+ CD45RA- cells to neutrophils
of from
about 0.0007 to about 0.0043 in a sample of peripheral blood of the donor
following
administration of the CXCR2 agonist and CXCR4 antagonist.
37. The method of claim 36, wherein the ratio of CD34+ CD90+ CD45RA- cells
to
neutrophils in the sample is from about 0.0014 to about 0.0034.
250

38. 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 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
neutrophils by a
ratio of from about 3.5:1 to 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.
39. The method of claim 38, wherein the peripheral blood of the donor is
enriched
with CD34+ CD90+ CD45RA- cells relative to neutrophils by a ratio of from
about 7.0:1 to
about 9.0:1.
40. 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 a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to
produce a
population of cells having a ratio of CD34+ CD90+ CD45RA- cells to lymphocytes
of from
about 0.0008 to about 0.0069 in a sample of peripheral blood of the donor
following
administration of the CXCR2 agonist and CXCR4 antagonist.
41. The method of claim 40, wherein the ratio of CD34+ CD90+ CD45RA- cells
to
lymphocytes in the sample is from about 0.0011 to about 0.0031.
42. 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 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
lymphocytes by
a ratio of from about 5.6:1 to 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.
43. The method of claim 42, wherein the peripheral blood of the donor is
enriched
with CD34+ CD90+ CD45RA- cells relative to lymphocytes by a ratio of from
about 8.0:1 to
about 10.0:1.
251

44. 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 a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to
produce a
population of cells having a ratio of CD34+ CD90+ CD45RA- cells to monocytes
of from
about 0.0028 to about 0.0130 in a sample of peripheral blood of the donor
following
administration of the CXCR2 agonist.
45. The method of claim 44, wherein the ratio of CD34+ CD90+ CD45RA- cells
to
monocytes in the sample is from about 0.0063 to about 0.0083.
46. 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 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
monocytes by a
ratio of from about 1.5:1 to about 8.5:1 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.
47. The method of claim 46, wherein the peripheral blood of the donor is
enriched
with CD34+ CD90+ CD45RA- cells relative to monocytes by a ratio of from about
1.3:1 to
about 2.5:1.
48. 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 a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to
produce a
population of cells having a ratio of CD34+ CD90+ CD45RA- cells to CD34+ cells
of from
about 0.393 to about 0.745 in a sample of peripheral blood of the donor
following
administration of the CXCR2 agonist and CXCR4 antagonist.
49. The method of claim 48, wherein the ratio of CD34+ CD90+ CD45RA- cells
to
CD34+ cells in the sample is from about 0.625 to about 0.725.
50. 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 a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to
enrich the
252

peripheral blood of the donor with CD34+ CD90+ CD45RA- cells relative to CD34+
cells by
a ratio of from about 1.1:1 to 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.
51. The method of claim 50, wherein the peripheral blood of the donor is
enriched
with CD34+ CD90+ CD45RA- cells relative to CD34+ cells by a ratio of from
about 1.1:1 to
about 1.5:1.
52. 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 a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to
produce a
population of cells having a frequency of CD34+ CD90+ CD45RA- cells of from
about
0.020% to about 0.110% in a sample of peripheral blood of the donor following
administration of the CXCR2 agonist and CXCR4 antagonist.
53. The method of claim 52, wherein the frequency of CD34+ CD90+ CD45RA-
cells
in the sample is from about 0.046% to about 0.086%.
54. 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 a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to
induce an
increase in the frequency of CD34+ CD90+ CD45RA- cells in the peripheral blood
of the
donor by from about 5.1-fold to about 25.7-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.
55. The method of claim 54, wherein the frequency of CD34+ CD90+ CD45RA-
cells
in the peripheral blood of the donor is increased by from about 5.1-fold to
about 7.1-fold
following administration of the CXCR2 agonist and CXCR4 antagonist.
56. A method of mobilizing a population of hematopoietic stem cells from
the bone
marrow of a mammalian donor into peripheral blood, the method comprising:
253

a. administering to the donor mobilizing amounts of a CXCR2 agonist and a
CXCR4 antagonist;
b. acquiring an input value for each of one or more parameters listed in TABLE
2 characterizing a sample of peripheral blood of the donor; and
c. 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.
57. The method of claim 56, wherein the one or more reference parameters
are a set of
parameters listed in any one of TABLES 3-6.
58. The method of claim 56 or 57, further comprising expanding the
hematopoietic
stem cells ex vivo if the sample is released for ex vivo expansion.
59. The method of any one of claims 56-58, further comprising infusing the
hematopoietic stem cells, or progeny thereof, into a mammalian patient
suffering from one or
more stem cell disorders if the sample is released for use in the treatment
thereof.
60. The method of any one of claims 12-59, wherein 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.
61. The method of claim 60, wherein the sample is isolated from the donor
at about 4
hours following administration of the CXCR2 agonist and CXCR4 antagonist.
62. The method of any one of claims 12-61, wherein the CXCR2 agonist is Gro-
.beta. T
or a variant thereof.
63. The method of claim 62, wherein the CXCR2 agonist is a peptide having
at least
85% sequence identity to the amino acid sequence of SEQ ID NO: 2.
64. The method of claim 63, wherein 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.
65. The method of claim 63, wherein the CXCR2 agonist is Gro-.beta. T.
254

66. The method of any one of claims 12-61, wherein the CXCR2 agonist is Gro-
.beta. or a
variant thereof.
67. The method of claim 66, wherein the CXCR2 agonist is a peptide having
at least
85% sequence identity to the amino acid sequence of SEQ ID NO: 1.
68. The method of claim 67, wherein 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.
9. The method of claim 67, wherein the CXCR2 agonist is Gro-.beta..
70. The method of any one of claims 12-69, wherein the CXCR2 agonist is
administered to the donor at a dose of from about 50 µg/kg to about 1
mg/kg.
71. The method of claim 70, wherein the CXCR2 agonist is administered to
the donor
at a dose of from about 50 µg/kg to about 300 µg/kg.
72. The method of claim 71, wherein the CXCR2 agonist is administered to
the donor
at a dose of from about 100 µg/kg to about 250 µg/kg.
73. The method of claim 72, wherein the CXCR2 agonist is administered to
the donor
at a dose of about 150 µg/kg.
74. The method of any one of claims 1-73, wherein the CXCR2 agonist is
administered intravenously to the donor.
75. The method of any one of claims 12-74, wherein the CXCR2 agonist and
the
CXCR4 antagonist are administered to the donor concurrently.
76. The method of any one of claims 12-75, wherein 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) an amine represented by formula (IA)
255

<IMG>
wherein A comprises a monocyclic or bicyclic fused ring system comprising
at least one nitrogen atom and B is H or a substituent of from 1 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)
<IMG>
wherein A' comprises a monocyclic or bicyclic fused ring system comprising
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) ¨ (CR2n ¨ X (IC)
wherein each R is independently H or C1-C6 alkyl, n is 1 or 2, and X is an
aryl
or heteroaryl group or a mercaptan;
wherein the linker is a bond, optionally substituted C1-C6 alkylene,
optionally
substituted C1-C6 heteroalkylene, optionally substituted C2-C6 alkenylene,
optionally
substituted C2-C6 heteroalkenylene, optionally substituted C2-C6 alkynylene,
optionally
substituted C2-C6 heteroalkynylene, optionally substituted cycloalkylene,
optionally
substituted heterocycloalkylene, optionally substituted arylene, or optionally
substituted
heteroarylene.
77. The method of claim 76, wherein 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.
78. The method of claim 76 or 77, wherein Z and Z' are identical
substituents.
79. The method of any one of claims 76-78, wherein Z is a cyclic polyamine
comprising from 10 to 24 ring members.
256

80. The method of claim 79, wherein Z is a cyclic polyamine comprising 14
ring
members.
81. The method of any one of claims 76-80, wherein Z comprises 4 nitrogen
atoms.
82. The method of any one of claims 76-81, wherein Z is 1,4,8,11-
tetraazocyclotetradecane.
83. The method of any one of claims 76-82, wherein the linker is
represented by
formula (ID)
<IMG>
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,
optionally
substituted C1-C6 heteroalkylene, optionally substituted C2-C6 alkenylene,
optionally
substituted C2-C6 heteroalkenylene, optionally substituted C2-C6 alkynylene,
or optionally
substituted C2-C6 heteroalkynylene.
84. The method of claim 83, wherein the linker is represented by formula
(IE)
<IMG>
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,
optionally
substituted C1-C6 heteroalkylene, optionally substituted C2-C6 alkenylene,
optionally
substituted C2-C6 heteroalkenylene, optionally substituted C2-C6 alkynylene,
or optionally
substituted C2-C6 heteroalkynylene.
85. The method of claim 83 or 84, wherein X and Y are each independently
optionally
substituted C1-C6 alkylene.
86. The method of any one of claim 83-85, wherein X and Y are identical
substituents.
257

87. The method of claim 86, wherein X and Y are each methylene groups.
88. The method of any one of claims 76-87, wherein the CXCR4 antagonist is
plerixafor or a pharmaceutically acceptable salt thereof.
89. The method of claim 88, wherein the plerixafor or pharmaceutically
acceptable
salt thereof is administered subcutaneously to the donor.
90. The method of claim 88 or 89, wherein the plerixafor or
pharmaceutically
acceptable salt thereof is administered to the donor at a dose of from about
50 µg/kg to about
500 µg/kg.
91. The method of claim 90, wherein the plerixafor or pharmaceutically
acceptable
salt thereof is administered to the donor at a dose of from about 200 µg/kg
to about 300
µg/kg.
92. The method of claim 91, wherein the plerixafor or pharmaceutically
acceptable
salt thereof is administered to the donor at a dose of about 240 µg/kg.
93. A pharmaceutical composition comprising a population of hematopoietic
stem
cells or progeny thereof isolated from a mammalian donor, wherein the ratio of
CD34+ cells
to leukocytes in the population is from about 0.0008 to about 0.0021.
94. The pharmaceutical composition of claim 93, wherein the ratio of CD34+
cells to
leukocytes in the population is from about 0.0010 to about 0.0018.
95. A pharmaceutical composition comprising a population of hematopoietic
stem
cells or progeny thereof isolated from a mammalian donor, wherein the ratio of
CD34+ cells
to neutrophils in the population is from about 0.0018 to about 0.0058.
96. The pharmaceutical composition of claim 95, wherein the ratio of CD34+
cells to
neutrophils in the population is from about 0.0026 to about 0.0046.
97. A pharmaceutical composition comprising a population of hematopoietic
stem
cells or progeny thereof isolated from a mammalian donor, wherein the ratio of
CD34+ cells
to lymphocytes in the population is from about 0.0021 to about 0.0094.
98. The pharmaceutical composition of claim 97, wherein the ratio of CD34+
cells to
lymphocytes in the population is from about 0.0025 to about 0.0035.
258

99. A pharmaceutical composition comprising a population of hematopoietic
stem
cells or progeny thereof isolated from a mammalian donor, wherein the ratio of
CD34+ cells
to monocytes in the population is from about 0.0071 to about 0.0174.
100. The pharmaceutical composition of claim 99, wherein the ratio of CD34+
cells to
monocytes in the population is from about 0.0100 to about 0.0140.
101. A pharmaceutical composition comprising a population of hematopoietic
stem
cells or progeny thereof isolated from a mammalian donor, wherein the
frequency of CD34+
cells in the population is from about 0.051% to about 0.140%.
102. The pharmaceutical composition of claim 101, wherein the frequency of
CD34+
cells in the population is from about 0.080% to about 0.120%.
103. A pharmaceutical composition comprising a population of hematopoietic
stem
cells or progeny thereof isolated from a mammalian donor, wherein the ratio of
CD34+ cells
to leukocytes in the population is from about 0.0003 to about 0.0016.
104. The pharmaceutical composition of claim 103, wherein the ratio of CD34+
CD90+ CD45RA- cells to leukocytes in the population is from about 0.0006 to
about 0.0012.
105. A pharmaceutical composition comprising a population of hematopoietic
stem
cells or progeny thereof isolated from a mammalian donor, wherein the ratio of
CD34+
CD90+ CD45RA- cells to neutrophils in the population is from about 0.0007 to
about 0.0043.
106. The pharmaceutical composition of claim 105, wherein the ratio of CD34+
CD90+ CD45RA- cells to neutrophils in the population is from about 0.0014 to
about 0.0034.
107. A pharmaceutical composition comprising a population of hematopoietic
stem
cells or progeny thereof isolated from a mammalian donor, wherein the ratio of
CD34+
CD90+ CD45RA- cells to lymphocytes in the population is from about 0.0008 to
about
0.0069.
108. The pharmaceutical composition of claim 107, wherein the ratio of CD34+
CD90+ CD45RA- cells to lymphocytes in the population is from about 0.0011 to
about
0.0031.
259

109. A pharmaceutical composition comprising a population of hematopoietic
stem
cells or progeny thereof isolated from a mammalian donor, wherein the ratio of
CD34+
CD90+ CD45RA- cells to monocytes in the population is from about 0.0028 to
about 0.0130.
110. The pharmaceutical composition of claim 109, wherein the ratio of CD34+
CD90+ CD45RA- cells to monocytes in the population is from about 0.0063 to
about 0.0083.
111. A pharmaceutical composition comprising a population of hematopoietic
stem
cells or progeny thereof isolated from a mammalian donor, wherein the ratio of
CD34+
CD90+ CD45RA- cells to CD34+ cells in the population is from about 0.393 to
about 0.745.
112. The pharmaceutical composition of claim 111, wherein the ratio of CD34+
CD90+ CD45RA- cells to CD34+ cells in the population is from about 0.625 to
about 0.725.
113. A pharmaceutical composition comprising a population of hematopoietic
stem
cells or progeny thereof isolated from a mammalian donor, wherein the
frequency of CD34+
CD90+ CD45RA- cells in the population is from about 0.020% to about 0.110%.
114. The pharmaceutical composition of claim 113, wherein the frequency of
CD34+
CD90+ CD45RA- cells in the population is from about 0.046% to about 0.086%.
115. A method of treating a stem cell disorder in a mammalian patient, the
method
comprising:
a. mobilizing a population of hematopoietic stem cells in a mammalian donor
in
accordance with the method of any one of claims 1-92; and
b. infusing a therapeutically effective amount of the hematopoietic stem
cells, or
progeny thereof, into the patient.
116. A method of treating a stem cell disorder in a mammalian patient, the
method
comprising infusing into the patient a therapeutically effective amount of the
hematopoietic
stem cells mobilized by the method of any one of claims 1-92, or progeny
thereof
117. A method of treating a stem cell disorder in a mammalian patient, the
method
comprising administering to the patient the pharmaceutical composition of any
one of claims
93-114.
118. The method of any one of claims 115-117, wherein the stem cell disorder
is a
hemoglobinopathy disorder.
260

119. The method of claim 118, wherein the hemoglobinopathy disorder is
selected
from the group consisting of sickle cell anemia, thalassemia, Fanconi anemia,
aplastic
anemia, and Wiskott-Aldrich syndrome.
120. The method of any one of claims 115-117, wherein the stem cell disorder
is a
myelodysplastic disorder.
121. The method of any one of claims 115-117, wherein the stem cell disorder
is an
immunodeficiency disorder.
122. The method of claim 121, wherein the immunodeficiency disorder is a
congenital
immunodeficiency.
123. The method of claim 121, wherein the immunodeficiency disorder is an
acquired
immunodeficiency.
124. The method of claim 123, wherein the acquired immunodeficiency is human
immunodeficiency virus or acquired immune deficiency syndrome.
125. The method of any one of claims 115-117, wherein the stem cell disorder
is a
metabolic disorder.
126. The method of claim 125, wherein the metabolic disorder is selected from
the
group consisting of glycogen storage diseases, mucopolysaccharidoses,
Gaucher's Disease,
Hurlers Disease, sphingolipidoses, and metachromatic leukodystrophy.
127. The method of any one of claims 115-117, wherein the stem cell disorder
is
cancer.
128. The method of claim 127, wherein the cancer is selected from the group
consisting
of leukemia, lymphoma, multiple myeloma, and neuroblastoma.
129. The method of claim 123, wherein the cancer is a hematological cancer.
130. The method of claim 127, wherein 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.
261

131. The method of any one of claims 115-117, wherein 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, osteopetrosis, osteogenesis imperfecta,
storage diseases,
thalassemia major, systemic sclerosis, systemic lupus erythematosus, multiple
sclerosis, and
juvenile rheumatoid arthritis.
132. The method of any one of claims 115-117, wherein the stem cell disorder
is an
autoimmune disorder.
133. The method of claim 132, wherein the autoimmune disorder is selected from
the
group consisting of 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 syndrome, Grave's disease, Guillain-
Barre
syndrome, 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 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.
134. The method of any one of claims 115-133, wherein the hematopoietic stem
cells
are autologous with respect to the patient.
262

135. The method of any one of claims 115-133, wherein the hematopoietic stem
cells
are allogeneic with respect to the patient.
136. The method of claim 135, wherein the hematopoietic stem cells are HLA-
matched
with respect to the patient.
137. The method of any one of claims 115-136, wherein the hematopoietic stem
cells
have been genetically modified to disrupt an endogenous gene.
138. The method of claim 137, wherein the endogenous gene encodes a major
histocompatibility complex protein.
139. The method of any one of claims 115-138, wherein 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.
140. The method of any one of claims 115-139, wherein 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.
141. The method of any one of claims 115-140, wherein 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.
142. The method of any one of claims 1-92, wherein the mammalian donor is a
human
donor.
143. The pharmaceutical composition of any one of claims 93-114, wherein the
mammalian donor is a human donor.
144. The method of any one of claims 115-141, wherein the mammalian donor is a
human donor and the mammalian patient is a human patient.
145. The method of any one of the above claims, further comprising isolating
the
hematopoietic stem cells or progeny thereof by drawing peripheral blood from
the donor.
263

146. The method of any one of the above claims, further comprising using
apheresis to
collect the hematopoietic stem cells or progeny thereof from the donor.
147. The method of any one of the above claims, wherein the Gro-.beta., Gro-
.beta. T, and
variants thereof have a purity of at least about 95% relative to the
deamidated versions of
these peptides.
148. An enriched preparation of human blood cells comprising a population of
hematopoietic stem cells or progeny thereof, wherein the ratio of CD34+ cells
to leukocytes
in the preparation is from about 0.0008 to about 0.0021.
149. A enriched preparation of human blood cells comprising a population of
hematopoietic stem cells or progeny thereof, wherein the ratio of CD34+ CD90+
CD45RA-
cells to neutrophils in the population is from about 0.0007 to about 0.0043.
150. A human blood cell preparation comprising hematopoietic stem cells or
progeny
thereof prepared using the method of any one of claims 1-92.
151. A method of mobilizing CD34dim 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-.beta., Gro-.beta. T, and variants
thereof at a dose of from
about 50 m/kg to about 1,000 m/kg and (ii) a CXCR4 antagonist.
152. 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-.beta., Gro-.beta. T, and variants thereof at a
dose of from about 50 m/kg
to about 1,000 m/kg and (ii) a CXCR4 antagonist.
153. 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
264

CXCR2 agonist selected from the group consisting of Gro-.beta., Gro-.beta. T,
and variants thereof at
a dose of from about 50 µg/kg to about 1,000 µg/kg and (ii) a CXCR4
antagonist.
154. 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-
.beta., Gro-.beta. T, and
variants thereof at a dose of from about 50 µg/kg to about 1,000 µg/kg
and (ii) a CXCR4
antagonist.
155. The method of any one of claims 151-154, wherein the CD34dim cells are
present
in the peripheral blood in a amount that is at least two- to ten-fold higher
than if the
hematopoietic stem cells were mobilized using the CXCR4 antagonist alone.
156. The method of any one of claims 151-155, wherein the CD34dim cells are
capable
of suppressing alloreactive T lymphocyte proliferation when administered to a
recipient.
157. The method of any one of claims 151-156, wherein the CXCR2 agonist is Gro-
.beta.
T.
158. The method of any one of claims 151-157, wherein the CXCR2 agonist is
administered to the donor at a dose of from about 100 µg/kg to about 250
µg/kg.
159. The method of claim 158, wherein the CXCR2 agonist is administered to the
donor at a dose of from about 125 µg/kg to about 225 µg/kg.
160. The method of claim 159, wherein the CXCR2 agonist is administered to the
donor at a dose of about 150 µg/kg.
161. The method of any one of claims 151-160, wherein the CXCR2 agonist is
administered intravenously to the donor.
162. The method of any one of claims 151-161, wherein the CXCR4 antagonist is
administered subcutaneously to the donor.
265

163. The method of any one of claims 151-162, wherein the CXCR4 antagonist is
plerixafor or a pharmaceutically acceptable salt thereof.
164. The method of claim 163, wherein the plerixafor or pharmaceutically
acceptable
salt thereof is administered to the donor at a dose of from about 50 µg/kg
to about 500 µg/kg.
165. The method of claim 164, wherein the plerixafor or pharmaceutically
acceptable
salt thereof is administered to the donor at a dose of from about 200 µg/kg
to about 300
µg/kg.
166. The method of claim 165, wherein the plerixafor or pharmaceutically
acceptable
salt thereof is administered to the donor at a dose of about 240 µg/kg.
167. The population of CD34dim cells derived from the method of any one of
claims
151-165.
266

Description

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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
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numbers of MDSCs may have better outcomes including lower rates of acute GVHD
(Vendramin et at., (2014) BBMT 20(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-f3 or a variant thereof, such as a
truncated form of Gro-
(e.g., Gro-f3 T), as described herein, optionally in combination with a C-X-C
chemokine
receptor type 4 (CXCR4) antagonist, such as 1,1'41,4-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
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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 at. (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, metachromaticleukodystrophy, 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.
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[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+ cells to leukocytes of from about 0.0008 to about 0.0021 in a sample of
peripheral
blood of the donor following administration of the CXCR2 agonist and CXCR4
antagonist.
In some embodiments, the ratio of CD34+ cells to leukocytes 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, or 0.00225. In some embodiments, the ratio
of 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,
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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 leukocytes in the sample is from about 0.0012 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+ cells to leukocytes in the sample is
about 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 to leukocytes by a ratio of from about 3.40:1 to about
6.90:1 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+ cells relative to
leukocytes by a
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 with CD34+ cells relative to leukocytes by a ratio of about from
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,
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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+ cells
relative 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, 4.65:1, 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, or 5.50. In some
embodiments, the
peripheral blood of the donor is enriched with CD34+ cells relative to
leukocytes by a ratio 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 CD34+ cells of at least about 38,000 cells/ml, such as a density of CD34+
cells 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 leukocytes of no more than about 5.3 x 107 cells/ml, such as a
density of
leukocytes of about 2.3 x 107 cells/ml to about 5.3 x 107 cells/ml, about 2.5
x 107 cells/ml to
about 5.1 x 107 cells/ml, 2.9 x 107 cells/ml to about 4.5 x 107 cells/ml,
about 3 x 107 cells/ml
to about 4 x 107 cells/ml (e.g., 5.3 x 107 cells/ml, 5.2 x 107 cells/ml, 5.1 x
107 cells/ml, 5 x 107
cells/ml, 4.9 x 107 cells/ml, 4.8 x 107 cells/ml, 4.7 x 107 cells/ml, 4.6 x
107 cells/ml, 4.5 x 107
cells/ml, 4.4 x 107 cells/ml, 4.3 x 107 cells/ml 4.2 x 107 cells/ml, 4.1 x 107
cells/ml 4 x 107
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cells/ml, 3.9 x 107 cells/ml, 3.8 x 107 cells/ml, 3.7 x 107 cells/ml, 3.6 x
107 cells/ml, 3.5 x 107
cells/ml, 3.4 x 107 cells/ml, 3.3 x 107 cells/ml, 3.2 x 107 cells/ml, 3.1 x
107 cells/ml, 3 x 107
cells/ml, 2.9 x 107 cells/ml, 2.8 x 107 cells/ml, 2.7 x 107 cells/ml, 2.6 x
107 cells/ml, 2.5 x 107
cells/ml, 2.4 x 107 cells/ml, 2.3 x 107 cells/ml, or less). 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+ cells 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 107 cells/ml to about 5.3 x 107 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+ cells of from about
40,000
cells/ml to about 80,000 cells/ml, and having a density of leukocytes of from
about 2.5 x 107
cells/ml to about 5 x 107 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+ cells of from about 50,000
cells/ml to about
90,000 cells/ml, and having a density of leukocytes of from about 3 x 107
cells/ml to about 4
x 107 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 neutrophils of from about 0.0018 to about 0.0058 in a sample of
peripheral
blood of the donor following administration of the CXCR2 agonist and CXCR4
antagonist.
In some embodiments, the ratio of CD34+ cells to neutrophils in the sample 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,
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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, 0.00520, 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, or 0.00580. In some embodiments, the ratio of CD34+ cells to
neutrophils in 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
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about 0.0046. In some embodiments, the ratio of CD34+ cells to neutrophils in
the sample is
from about 0.0026 to about 0.0046, such as a ratio of CD34+ cells 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
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 to neutrophils by a ratio of from about 2.1:1 to about
8.1:1 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+ cells relative to
neutrophils by a
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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
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 with CD34+ cells relative to neutrophils by a ratio of from about
from about 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
embodiments, the peripheral blood of the donor is enriched with CD34+ cells
relative to
neutrophils by a ratio of about 6.4:1.
[0015] In yet 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 lymphocytes of from about 0.0021 to about 0.0094 in a sample of
peripheral
blood of the donor following administration of the CXCR2 agonist and CXCR4
antagonist.
In some embodiments, the ratio of CD34+ cells to lymphocytes in the sample 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,

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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, 0.00520, 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,
11

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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, 0.00690, 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,
12

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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+ cells to
lymphocytes in 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,
0.00347, 0.00348, 0.00349, or 0.00350. In some embodiments, the ratio of CD34+
cells 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 to lymphocytes by a ratio of from about 4.8:1 to 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+ cells relative to
lymphocytes by a
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,
13

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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 CD34+ cells relative to lymphocytes by a ratio of from 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 to lymphocytes by a
ratio of 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 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 107 cells/ml, such as a
density of
lymphocytes of about 1 x 107 cells/ml to about 2.3 x 107 cells/ml, about 1.3 x
107 cells/ml to
about 2.1 x 107ce11s/ml, or about 1.5 x 107 cells/ml to about 1.9 x 107
cells/ml (e.g., about 2.4
14

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x 107 cells/ml, 2.3 x 107 cells/ml, 2.2 x 07 cells/ml, 2.1 x 107 cells/ml, 2 x
107 cells/ml, 1.9 x
107 cells/ml, 1.8 x 107 cells/ml, 1.7 x 107 cells/ml, 1.6 x 107 cells/ml, 1.5
x 107 cells/ml 1.4 x
107 cells/ml, 1.3 x 107 cells/ml, 1.2 x 107 cells/ml, 1.1 x 107 cells/ml, 1 x
107 cells/ml, or less,
0.9 x 107 cells/ml, 0.8 x 107 cells/ml, or less). 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+ cells of from about
38,000
cells/ml to about 100,000 cells/ml, and having a density of lymphocytes of
from about 1 x 107
cells/ml to about 2.3 x 107 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+ cells of from about
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 about 2.3 x 107 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+ cells of from about
50,000
cells/ml to about 90,000 cells/ml, and having a density of lymphocytes of from
about 1.5 x
107 cells/ml to about 2 x 107 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 monocytes of from about 0.0071 to about 0.0174 in a sample of
peripheral
blood of the donor following administration of the CXCR2 agonist. In some
embodiments,
the ratio of CD34+ cells to monocytes in the sample 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,

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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
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+
cells to monocytes in the sample is from about 0.0100 to about 0.0140, such as
a ratio of
CD34+ cells to monocytes in the sample of about 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,
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0.0139, or 0.0140. In some embodiments, the ratio of CD34+ cells to monocytes
in 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 CD34+ cells of at least about 38,000 cells/ml, such as a density of CD34+
cells 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 as a density
of monocytes
of from 3.4 x 106 cells/ml to about 5.9 x 106 cells/ml, about 3.5 x 106
cells/ml to about 5.7 x
106 cells/ml, or about 4 x 106 cells/ml to about 5 x 106 cells/ml (e.g., 5.9 x
106 cells/ml, 5.8 x
.. 106 cells/ml, 5.7 x 106 cells/ml, 5.6 x 106 cells/ml, 5.5 x 106 cells/ml,
5.4 x 106 cells/ml, 5.3 x
106 cells/ml, 5.2 x 106 cells/ml, 5.1 x 106 cells/ml, 5 x 106 cells/ml, 4.9 x
106 cells/ml, 4.8 x
106 cells/ml, 4.7 x 106 cells/ml, 4.6 x 106 cells/ml, 4.5 x 106 cells/ml, 4.4
x 106 cells/ml, 4.3 x
106 cells/ml, 4.2 x 106 cells/ml, 4.1 x 106 cells/ml, 4 x 106 cells/ml, 3.9 x
106 cells/ml, 3.8 x
106 cells/ml, 3.7 x 106 cells/ml, 3.6 x 106 cells/ml, 3.5 x 106 cells/ml, 3.4
x 106 cells/ml, or
less). 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+ cells of from about 38,000 cells/ml to about 100,000
cells/ml, and
having a density of monocytes of from about 3.4 x 106 cells/ml to about 6 x
106 cells/ml. In
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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 from about 40,000 cells/ml to about 80,000 cells/ml, and having
a density of
monocytes of from about 4 x 106 cells/ml to about 5.5 x 106 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+
cells of from
about 50,000 cells/ml to about 90,000 cells/ml, and having a density of
monocytes of from
about 4 x 106 cells/ml to about 5 x 106 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 to monocytes by a ratio of from about 1.1:1 to about
2.3:1 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+ 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, or 2.30:1. In some embodiments, the peripheral blood of the
donor is enriched
with CD34+ cells relative to monocytes by a ratio of about from about 1.3:1 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+ cells of from about 0.051% to about 0.140% 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+
cells of 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%, 0.064%, 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%,
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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%,
0.10000,
0.101%, 0.102%, 0.103%, 0.104%, 0.105%, 0.106%, 0.107%, 0.108%, 0.109%,
0.1100o,
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
from about
0.050 A to about 0.120%, about 0.060 A to about 0.1100o, or about 0.080 A to
about 0.1000o.
In some embodiments, the population of cells has a frequency of CD34+ cells of
from 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
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+
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 the peripheral blood of the donor
is 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+ cells in the peripheral blood of the
donor is
increased by from about 4.0-fold to about 6.0-fold following administration of
the CXCR2
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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 of CD34+ cells in the peripheral blood of the donor 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+ CD45RA- cells to leukocytes of from about 0.0003 to about 0.0016
in 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- 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+ CD45RA- cells to leukocytes in the sample is from about 0.0008 to
about
0.0010. In some embodiments, the ratio of CD34+ CD90+ CD45RA- cells to
leukocytes in
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,

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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 to leukocytes in the sample is about 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+
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: , 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: , 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,
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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+
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+
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+ CD45RA- cells of at least about 16,000 cells/ml, such as a
density of from
about 20,000 cells/ml to about 75,000 cells/ml, about 25,000 cells/ml to about
70,000
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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 x 107 cells/ml, such as a density of
leukocytes of about
2.3 x 107 cells/ml to about 5.3 x 107 cells/ml, about 2.5 x 107 cells/ml to
about 5.1 x 107
cells/ml, 2.9 x 107 cells/ml to about 4.5 x 107 cells/ml, about 3 x 107
cells/ml to about 4 x 107
cells/ml (e.g., 5.3 x 107 cells/ml, 5.2 x 107 cells/ml, 5.1 x 107 cells/ml, 5
x 107 cells/ml, 4.9 x
107 cells/ml, 4.8 x 107 cells/ml, 4.7 x 107 cells/ml, 4.6 x 107 cells/ml, 4.5
x 107 cells/ml, 4.4 x
107 cells/ml, 4.3 x 107 cells/ml 4.2 x 107 cells/ml, 4.1 x 107 cells/ml 4 x
107 cells/ml, 3.9 x 107
cells/ml, 3.8 x 107 cells/ml, 3.7 x 107 cells/ml, 3.6 x 107 cells/ml, 3.5 x
107 cells/ml, 3.4 x 107
cells/ml, 3.3 x 107 cells/ml, 3.2 x 107 cells/ml, 3.1 x 107 cells/ml, 3 x 107
cells/ml, 2.9 x 107
cells/ml, 2.8 x 107 cells/ml, 2.7 x 107 cells/ml, 2.6 x 107 cells/ml, 2.5 x
107 cells/ml, 2.4 x 107
cells/ml, 2.3 x 107 cells/ml, or less). 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+ CD45RAT cells
of from
about 20,000 cells/ml to about 75,000 cells/ml, and having a density of
leukocytes of from
about 2.3 x 107 cells/ml to about 5.3 x 107 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+
CD45RAT 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/ml to about 5 x 107 cells/ml. In some embodiments,
the method
includes administering to the donor a CXCR2 agonist and a CXCR4 antagonist in
amounts
23

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sufficient to produce a population of cells having a density of CD34+ CD90+
CD45RAT cells
of from about 40,000 cells/ml to about 50,000 cells/ml, and having a density
of leukocytes of
from about 3 x 107 cells/ml to about 4 x 107 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+ CD45RAT cells to neutrophils of from about 0.0007 to about 0.0043
in 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+ CD45RAT cells
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,
24

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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+ CD45RA- cells to neutrophils in the sample is from
about 0.002 to
about 0.003. In some embodiments, the ratio of CD34+ CD90+ CD45RA- cells to
neutrophils
in the sample is from about 0.0014 to about 0.0034, such as a ratio of 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,

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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- cells to neutrophils in the
sample is 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+ CD45RA- cells relative to neutrophils by a ratio of from about
3.5:1 to 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,
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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+ CD45RA- cells relative to
neutrophils by 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+ CD45RA- cells relative to neutrophils
by a ratio of
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+
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+ CD45RA- cells of at least about 16,000 cells/ml, such as a
density of from
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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 of no more than about 2.5 x 107 cells/ml, such as a density of
neutrophils of about
1 x 107 cells/ml to about 2.5 x 107 cells/ml, about 1.3 x 107 cells/ml to
about 2 x 107ce11s/ml,
or about 1.5 x 107ce11s/m1 to about 1.9 x 107 cells/ml (e.g., about 2.5 x 107
cells/ml, 2.4 x 107
cells/ml, 2.3 x 107 cells/ml, 2.2 x 07 cells/ml, 2.1 x 107 cells/ml, 2 x 107
cells/ml, 1.9 x 107
cells/ml, 1.8 x 107 cells/ml, 1.7 x 107 cells/ml, 1.6 x 107 cells/ml, 1.5 x
107 cells/ml 1.4 x 107
cells/ml, 1.3 x 107 cells/ml, 1.2 x 107 cells/ml, 1.1 x 107 cells/ml, 1 x 107
cells/ml, or less). 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+ CD45RAT cells of from about 20,000 cells/ml to about 75,000
cells/ml, and
having a density of neutrophils of from about 1 x 107 cells/ml to about 2.5 x
107 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+ CD45RAT cells of from about 30,000 cells/ml to about 60,000
cells/ml, and
having a density of neutrophils of from about 1.3 x 107 cells/ml to about 2.3
x 107 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+ CD45RAT cells of from about 40,000 cells/ml to about 50,000
cells/ml, and
having a density of neutrophils of from about 1.5 x 107 cells/ml to about 2 x
107 cells/ml.
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[0029] In yet 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- cells to lymphocytes of from about 0.0008 to about 0.0069
in 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- 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,
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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, 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,

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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- cells to lymphocytes in the
sample
is from about 0.0011 to about 0.0031, such as a ratio of CD34+ CD90+ CD45RA-
cells to
lymphocytes in the sample 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+
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+ CD90+ CD45RA- cells relative to lymphocytes by a ratio of from about
5.6:1 to 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
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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+
CD45RAT 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, 13.05:1, 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,
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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,
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,
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+
CD90+
CD45RA- cells relative to lymphocytes by a ratio of about from about 8.0:1 to
about 10.0:1,
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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+ CD45RA- cells
relative to
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- cells of at least about 16,000 cells/ml, such as a
density of 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 2.4 x 107 cells/ml, such as a density of
lymphocytes of
about 1 x 107 cells/ml to about 2.3 x 107 cells/ml, about 1.3 x 107 cells/ml
to about 2.1 x
107ce11s/ml, or about 1.5 x 107 cells/ml to about 1.9 x 107 cells/ml (e.g.,
about 2.4 x 107
cells/ml, 2.3 x 107 cells/ml, 2.2 x 07 cells/ml, 2.1 x 107 cells/ml, 2 x 107
cells/ml, 1.9 x 107
cells/ml, 1.8 x 107 cells/ml, 1.7 x 107 cells/ml, 1.6 x 107 cells/ml, 1.5 x
107 cells/ml 1.4 x 107
cells/ml, 1.3 x 107 cells/ml, 1.2 x 107 cells/ml, 1.1 x 107 cells/ml, 1 x 107
cells/ml, or less, 0.9
x 107 cells/ml, 0.8 x 107 cells/ml, or less). In some embodiments, the method
includes
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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
of from
about 20,000 cells/ml to about 75,000 cells/ml, and having a density of
lymphocytes of from
about 1 x 107 cells/ml to about 2.3 x 107 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
of 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 about 2.3 x 107 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
of from about 40,000 cells/ml to about 50,000 cells/ml, and having a density
of lymphocytes
of from about 1.5 x 107 cells/ml to about 2 x 107 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- cells to monocytes of from about 0.0028 to about 0.0130 in
a sample
of peripheral blood of the donor following administration of the CXCR2
agonist. In some
embodiments, the ratio of CD34+ CD90+ CD45RA- cells to monocytes in the 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,

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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, 0.00674, 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,
36

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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, 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, or 0.0130. In some embodiments, the ratio of 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 to monocytes in the sample of 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,
37

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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+ CD45RA- cells to monocytes in the sample
is 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+ CD45RA- cells relative to monocytes by a ratio of from about 1.5:1
to about
8.5:1 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 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,
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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+ CD45RA- cells relative to monocytes by a
ratio of
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- cells of at least about 16,000 cells/ml, such as a
density of 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 106 cells/ml, such as a density of
monocytes of from 3.4
x 106 cells/ml to about 5.9 x 106 cells/ml, about 3.5 x 106 cells/ml to about
5.7 x 106 cells/ml,
or about 4 x 106 cells/ml to about 5 x 106 cells/ml (e.g., 5.9 x 106 cells/ml,
5.8 x 106 cells/ml,
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5.7 x 106 cells/ml, 5.6 x 106 cells/ml, 5.5 x 106 cells/ml, 5.4 x 106
cells/ml, 5.3 x 106 cells/ml,
5.2 x 106 cells/ml, 5.1 x 106 cells/ml, 5 x 106 cells/ml, 4.9 x 106 cells/ml,
4.8 x 106 cells/ml,
4.7 x 106 cells/ml, 4.6 x 106 cells/ml, 4.5 x 106 cells/ml, 4.4 x 106
cells/ml, 4.3 x 106 cells/ml,
4.2 x 106 cells/ml, 4.1 x 106 cells/ml, 4 x 106 cells/ml, 3.9 x 106 cells/ml,
3.8 x 106 cells/ml,
3.7 x 106 cells/ml, 3.6 x 106 cells/ml, 3.5 x 106 cells/ml, 3.4 x 106
cells/ml, or less). 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 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 106 cells/ml to about 6 x
106 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 of from about 30,000 cells/ml to about 60,000
cells/ml, and
having a density of monocytes of from about 4 x 106 cells/ml to about 5.5 x
106 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 of from about 40,000 cells/ml to about 50,000
cells/ml, and
having a density of monocytes of from about 4 x 106 cells/ml to about 5 x 106
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+ CD45RA- cells to CD34+ cells of from about 0.393 to about 0.745 in
a sample
of peripheral blood of the donor following administration of the CXCR2
agonist. In some
embodiments, the ratio of CD34+ CD90+ CD45RA- cells to CD34+ cells in the
sample may be
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,

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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, 0.741, 0.742, 0.743, 0.744,
or 0.745. In some
embodiments, the ratio of CD34+ CD90+ CD45RAT cells to CD34+ cells in the
sample is from
about 0.625 to about 0.725, such as a ratio of CD34+ CD90+ CD45RAT cells to
CD34+ cells 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+ CD45RAT cells to CD34+ cells in the
sample is
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
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a CXCR4 antagonist in amounts sufficient to enrich the peripheral blood of the
donor with
CD34+ CD90+ CD45RA- cells relative to CD34+ cells by a ratio of from about
1.1:1 to 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+
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
some embodiments, the peripheral blood of the donor is enriched with CD34+
CD90+
CD45RA- cells relative to CD34+ cells by a ratio of about 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 of CD34+ CD90+ CD45RA- cells of from about 0.020% to about 0.110% 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- cells of about 0.020%, 0.021%, 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%,
0.045%,
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 a frequency of CD34+ CD90+ CD45RA- cells of from about 0.046% to about
0.086%,
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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+ CD45RA- cells of about
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+
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, 8.8-fold, 8.9-fold, 9.0-fold, 9.1-fold, 9.2-fold, 9.3-fold, 9.4-fold,
9.5-fold, 9.6-fold, 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, 11.6-fold, 11.7-fold, 11.8-fold, 11.9-fold, 12.0-fold, 12.1-fold,
12.2-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, 15.2-fold, 15.3-fold, 15.4-fold, 15.5-fold, 15.6-fold, 15.7-fold,
15.8-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, 17.0-fold, 17.1-fold, 17.2-fold, 17.3-fold, 17.4-fold, 17.5-fold,
17.6-fold, 17.7-fold,
17.8-fold, 17.9-fold, 18.0-fold, 18.1-fold, 18.2-fold, 18.3-fold, 18.4-fold,
18.5-fold, 18.6-fold,
18.7-fold, 18.8-fold, 18.9-fold, 19.0-fold, 19.1-fold, 19.2-fold, 19.3-fold,
19.4-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,
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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+ CD90+ CD45RAT cells in the peripheral
blood of
the donor is increased by from about 5.1-fold to about 7.1-fold following
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+ CD45RAT cells in the peripheral
blood of 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-f3 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%,
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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-f3
T. In some embodiments, the Gro-f3 T is not covalently modified. In some
embodiments, the
Gro-f3 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-f3 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-f3.
In some embodiments, the Gro-f3 T is not covalently modified. In some
embodiments, the
Gro-f3 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-f3 or Gro-f3 T, such
as
unmodified Gro-f3 or Gro-f3 T) is administered to the donor at a dose of from
about 501.tg/kg

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to about 1 mg/kg, such as a dose of about 501.tg/kg, 551.tg/kg, 601.tg/kg,
651.tg/kg, 701.tg/kg,
751.tg/kg, 801.tg/kg, 851.tg/kg, 901.tg/kg, 95 jig/kg, 1001.tg/kg, 105 jig/kg,
1101.tg/kg, 115
jig/kg, 1201.tg/kg, 1251.tg/kg, 1301.tg/kg, 1351.tg/kg, 1401.tg/kg,
1451.tg/kg, 1501.tg/kg, 155
jig/kg, 1601.tg/kg, 1651.tg/kg, 1701.tg/kg, 1751.tg/kg, 1801.tg/kg, 185
jig/kg, 1901.tg/kg, 195
jig/kg, 2001.tg/kg, 205 jig/kg, 2101.tg/kg, 2151.tg/kg, 2201.tg/kg,
2251.tg/kg, 2301.tg/kg, 235
jig/kg, 2401.tg/kg, 2451.tg/kg, 2501.tg/kg, 2551.tg/kg, 2601.tg/kg,
2651.tg/kg, 2701.tg/kg, 275
jig/kg, 2801.tg/kg, 2851.tg/kg, 2901.tg/kg, 2951.tg/kg, 3001.tg/kg,
3051.tg/kg, 3101.tg/kg, 315
jig/kg, 3201.tg/kg, 3251.tg/kg, 3301.tg/kg, 3351.tg/kg, 3401.tg/kg,
3451.tg/kg, 3501.tg/kg, 355
jig/kg, 3601.tg/kg, 3651.tg/kg, 3701.tg/kg, 3751.tg/kg, 3801.tg/kg,
4001.tg/kg, 405 jig/kg, 410
jig/kg, 4151.tg/kg, 4251.tg/kg, 4301.tg/kg, 4351.tg/kg, 4401.tg/kg,
4451.tg/kg, 4501.tg/kg, 210
jig/kg, 3001.tg/kg, 4001.tg/kg, 4051.tg/kg, 4101.tg/kg, 4151.tg/kg,
4201.tg/kg, 425 jig/kg, 430
jig/kg, 4351.tg/kg, 4401.tg/kg, 4451.tg/kg, 4501.tg/kg, 4551.tg/kg,
4601.tg/kg, 465 jig/kg, 470
jig/kg, 4751.tg/kg, 4801.tg/kg, 4851.tg/kg, 4901.tg/kg, 4951.tg/kg,
5001.tg/kg, 505 jig/kg, 510
jig/kg, 5051.tg/kg, 5151.tg/kg, 5201.tg/kg, 5251.tg/kg, 5301.tg/kg,
5451.tg/kg, 5501.tg/kg, 555
jig/kg, 5601.tg/kg, 5651.tg/kg, 5701.tg/kg, 5751.tg/kg, 5801.tg/kg,
5851.tg/kg, 5901.tg/kg, 595
jig/kg, 6001.tg/kg, 605 jig/kg, 6101.tg/kg, 6151.tg/kg, 6201.tg/kg,
6251.tg/kg, 6301.tg/kg, 635
jig/kg, 6401.tg/kg, 6451.tg/kg, 6501.tg/kg, 6551.tg/kg, 6601.tg/kg,
6651.tg/kg, 6701.tg/kg, 675
jig/kg, 6801.tg/kg, 6851.tg/kg, 6901.tg/kg, 6951.tg/kg, 7001.tg/kg,
7051.tg/kg, 7101.tg/kg, 715
jig/kg, 7201.tg/kg, 7251.tg/kg, 7301.tg/kg, 7351.tg/kg, 7401.tg/kg,
7451.tg/kg, 7501.tg/kg, 755
jig/kg, 7601.tg/kg, 7651.tg/kg, 7701.tg/kg, 7751.tg/kg, 7801.tg/kg,
7851.tg/kg, 7901.tg/kg, 795
jig/kg, 8001.tg/kg, 8051.tg/kg, 8101.tg/kg, 8151.tg/kg, 8201.tg/kg,
8251.tg/kg, 8301.tg/kg, 835
jig/kg, 8401.tg/kg, 8451.tg/kg, 8501.tg/kg, 8551.tg/kg, 8601.tg/kg,
8651.tg/kg, 8701.tg/Kg, 875
jig/kg, 8801.tg/kg, 8851.tg/kg, 8901.tg/kg, 8951.tg/kg, 9001.tg/kg,
9051.tg/kg, 9101.tg/kg, 915
jig/kg, 9201.tg/kg, 9251.tg/kg, 9301.tg/kg, 9351.tg/kg, 9401.tg/kg,
9451.tg/kg, 9501.tg/kg, 955
jig/kg, 9601.tg/kg, 9651.tg/kg, 9701.tg/kg, 9751.tg/kg, 9801.tg/kg,
9851.tg/kg, 9901.tg/kg, 995
jig/kg, or 1,0001.tg/kg. In some embodiments, the CXCR2 agonist (e.g., Gro-f3
or Gro-f3 T,
such as unmodified Gro-f3 or Gro-f3 T) is administered to the donor at a dose
of from about 50
jig/kg to about 3001.tg/kg, such as a dose of from about 1001.tg/kg to about
2501.tg/kg, or
from about 1251.tg/kg to about 225 jig/kg. In some embodiments, the CXCR2
agonist (e.g.,
Gro-f3 or Gro-f3 T, such as unmodified Gro-f3 or Gro-f3 T) is administered to
the donor at a
dose of about 1501.tg/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)
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into peripheral blood, the method including administering to the donor a CXCR2
agonist
selected from the group consisting of Gro-f3, Gro-f3 T, and variants thereof
at a dose of from
about 50 pg/kg to about 1 mg/kg (e.g., a dose of 501.tg/kg, 551.tg/kg,
601.tg/kg, 651.tg/kg, 70
jig/kg, 751.tg/kg, 801.tg/kg, 851.tg/kg, 901.tg/kg, 95 jig/kg, 10011g/kg,
10511g/kg, 11011g/kg,
11511g/kg, 12011g/kg, 12511g/kg, 13011g/kg, 135 jig/kg, 14011g/kg, 145 jig/kg,
15011g/kg,
15511g/kg, 16011g/kg, 16511g/kg, 17011g/kg, 175 jig/kg, 18011g/kg, 185 jig/kg,
19011g/kg,
19511g/kg, 20011g/kg, 20511g/kg, 21011g/kg, 21511g/kg, 22011g/kg, 22511g/kg,
23011g/kg,
23511g/kg, 24011g/kg, 24511g/kg, 25011g/kg, 255 jig/kg, 26011g/kg, 265 jig/kg,
27011g/kg,
27511g/kg, 28011g/kg, 28511g/kg, 29011g/kg, 295 jig/kg, 30011g/kg, 305 jig/kg,
31011g/kg,
31511g/kg, 32011g/kg, 32511g/kg, 33011g/kg, 335 jig/kg, 34011g/kg, 345 jig/kg,
35011g/kg,
35511g/kg, 36011g/kg, 36511g/kg, 37011g/kg, 375 jig/kg, 38011g/kg, 40011g/kg,
405 jig/kg,
41011g/kg, 415 jig/kg, 42511g/kg, 43011g/kg, 435 jig/kg, 44011g/kg, 445
jig/kg, 45011g/kg,
21011g/kg, 30011g/kg, 40011g/kg, 405 jig/kg, 41011g/kg, 415 jig/kg, 42011g/kg,
425 jig/kg,
43011g/kg, 435 jig/kg, 44011g/kg, 445 jig/kg, 45011g/kg, 455 jig/kg,
46011g/kg, 465 jig/kg,
47011g/kg, 475 jig/kg, 48011g/kg, 485 jig/kg, 49011g/kg, 495 jig/kg,
50011g/kg, 50511g/kg,
51011g/kg, 505 jig/kg, 51511g/kg, 52011g/kg, 525 jig/kg, 53011g/kg, 545
jig/kg, 55011g/kg,
55511g/kg, 56011g/kg, 56511g/kg, 57011g/kg, 575 jig/kg, 58011g/kg, 585 jig/kg,
59011g/kg,
59511g/kg, 60011g/kg, 60511g/kg, 61011g/kg, 615 jig/kg, 62011g/kg, 625 jig/kg,
63011g/kg,
63511g/kg, 64011g/kg, 64511g/kg, 65011g/kg, 655 jig/kg, 66011g/kg, 665 jig/kg,
67011g/kg,
67511g/kg, 68011g/kg, 68511g/kg, 69011g/kg, 695 jig/kg, 70011g/kg, 705 jig/kg,
71011g/kg,
71511g/kg, 72011g/kg, 72511g/kg, 73011g/kg, 735 jig/kg, 74011g/kg, 745 jig/kg,
75011g/kg,
75511g/kg, 76011g/kg, 76511g/kg, 77011g/kg, 775 jig/kg, 78011g/kg, 785 jig/kg,
79011g/kg,
79511g/kg, 80011g/kg, 80511g/kg, 81011g/kg, 815 jig/kg, 82011g/kg, 825 jig/kg,
83011g/kg,
83511g/kg, 84011g/kg, 84511g/kg, 85011g/kg, 855 jig/kg, 86011g/kg, 86511g/kg,
87011g/Kg,
87511g/kg, 88011g/kg, 88511g/kg, 89011g/kg, 895 jig/kg, 90011g/kg, 905 jig/kg,
91011g/kg,
91511g/kg, 92011g/kg, 92511g/kg, 93011g/kg, 935 jig/kg, 94011g/kg, 945 jig/kg,
95011g/kg,
95511g/kg, 96011g/kg, 96511g/kg, 97011g/kg, 975 jig/kg, 98011g/kg, 98511g/kg,
99011g/kg,
99511g/kg, or 1,00011g/kg). In some embodiments, the method further includes
administering a CXCR4 antagonist to the donor.
[0045] In some embodiments of any of the above aspects of the invention, the
CXCR2
agonist (e.g., Gro-f3 or Gro-f3 T, such as unmodified Gro-f3 or Gro-f3 T) is
administered to the
donor at a dose of from about 50 jig/kg to about 300 jig/kg, such as a dose of
about 50 jig/kg,
551.tg/kg, 601.tg/kg, 651.tg/kg, 701.tg/kg, 75 jig/kg, 801.tg/kg, 85 jig/kg,
901.tg/kg, 95 jig/kg,
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100 pg/kg, 105 pg/kg, 110 pg/kg, 115 pg/kg, 120 pg/kg, 125 pg/kg, 130 pg/kg,
135 ug/kg,
140 pg/kg, 145 pg/kg, 150 pg/kg, 155 pg/kg, 160 pg/kg, 165 pg/kg, 170 pg/kg,
175 ug/kg,
180 pg/kg, 185 pg/kg, 190 pg/kg, 195 pg/kg, 200 pg/kg, 205 pg/kg, 210 pg/kg,
215 ug/kg,
220 pg/kg, 225 pg/kg, 230 pg/kg, 235 pg/kg, 240 pg/kg, 245 pg/kg, 250 pg/kg,
255 ug/kg,
260 pg/kg, 265 pg/kg, 270 pg/kg, 275 pg/kg, 280 pg/kg, 285 ug/kg, 290 pg/kg,
295 pg/kg, or
300 jig/kg.
[0046] In some embodiments of any of the above aspects of the invention, the
CXCR2
agonist (e.g., Gro-f3 or Gro-f3 T, such as unmodified Gro-f3 or Gro-f3 T) is
administered to the
donor at a dose of from about 100 jig/kg to about 250 pg/kg, such as a dose of
about 100
.. jig/kg, 105 jig/kg, 110 jig/kg, 115 jig/kg, 120 jig/kg, 125 jig/kg, 130
jig/kg, 135 jig/kg, 140
jig/kg, 145 jig/kg, 150 jig/kg, 155 jig/kg, 160 jig/kg, 165 jig/kg, 170
jig/kg, 175 jig/kg, 180
jig/kg, 185 jig/kg, 190 jig/kg, 195 jig/kg, 200 jig/kg, 205 jig/kg, 210
jig/kg, 215 jig/kg, 220
jig/kg, 225 jig/kg, 230 jig/kg, 235 jig/kg, 240 jig/kg, 245 jig/kg, or 250
jig/kg.
[0047] In some embodiments of any of the above aspects of the invention, the
CXCR2
agonist (e.g., Gro-f3 or Gro-f3 T, such as unmodified Gro-f3 or Gro-f3 T) is
administered to the
donor at a dose of about 150 jig/kg. For example, in some embodiments, the
CXCR2 agonist
(e.g., Gro-f3 or Gro-f3 T, such as unmodified Gro-f3 or Gro-f3 T) is
administered to the donor at
a dose of from about 50 jig/kg per day to about 1 mg/kg per day, such as a
dose of about 50
jig/kg per day, 55 jig/kg per day, 60 jig/kg per day, 65 jig/kg per day, 70
jig/kg per day, 75
jig/kg per day, 80 jig/kg per day, 85 jig/kg per day, 90 jig/kg per day, 95
jig/kg per day, 100
jig/kg per day, 105 jig/kg per day, 110 jig/kg per day, 115 jig/kg per day,
120 jig/kg per day,
125 jig/kg per day, 130 jig/kg per day, 135 jig/kg per day, 140 jig/kg per
day, 145 jig/kg per
day, 150 jig/kg per day, 155 jig/kg per day, 160 jig/kg per day, 165 jig/kg
per day, 170 jig/kg
per day, 175 jig/kg per day, 180 jig/kg per day, 185 jig/kg per day, 190
jig/kg per day, 195
jig/kg per day, 200 jig/kg per day, 205 jig/kg per day, 210 jig/kg per day,
215 jig/kg per day,
220 jig/kg per day, 225 jig/kg per day, 230 jig/kg per day, 235 jig/kg per
day, 240 jig/kg per
day, 245 jig/kg per day, 250 jig/kg per day, 255 jig/kg per day, 260 jig/kg
per day, 265 jig/kg
per day, 270 jig/kg per day, 275 jig/kg per day, 280 jig/kg per day, 285
jig/kg per day, 290
jig/kg per day, 295 jig/kg per day, 300 jig/kg per day, 305 jig/kg per day,
310 jig/kg per day,
315 jig/kg per day, 320 jig/kg per day, 325 jig/kg per day, 330 jig/kg per
day, 335 jig/kg per
day, 340 jig/kg per day, 345 jig/kg per day, 350 jig/kg per day, 355 jig/kg
per day, 360 jig/kg
per day, 365 jig/kg per day, 370 jig/kg per day, 375 jig/kg per day, 380
jig/kg per day, 400
jig/kg per day, 405 jig/kg per day, 410 jig/kg per day, 415 jig/kg per day,
425 jig/kg per day,
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43011g/kg per day, 43511g/kg per day, 44011g/kg per day, 445 1.tg/kg per day,
45011g/kg per
day, 21011g/kg per day, 30011g/kg per day, 40011g/kg per day, 40511g/kg per
day, 41011g/kg
per day, 41511g/kg per day, 42011g/kg per day, 425 1.tg/kg per day, 43011g/kg
per day, 435
1.tg/kg per day, 44011g/kg per day, 445 1.tg/kg per day, 45011g/kg per day,
45511g/kg per day,
.. 46011g/kg per day, 46511g/kg per day, 47011g/kg per day, 475 1.tg/kg per
day, 48011g/kg per
day, 485 1.tg/kg per day, 49011g/kg per day, 49511g/kg per day, 50011g/kg per
day, 50511g/kg
per day, 51011g/kg per day, 50511g/kg per day, 515 jig/kg per day, 52011g/kg
per day, 525
jig/kg per day, 53011g/kg per day, 545 jig/kg per day, 55011g/kg per day,
55511g/kg per day,
56011g/kg per day, 56511g/kg per day, 57011g/kg per day, 575 jig/kg per day,
58011g/kg per
day, 585 jig/kg per day, 59011g/kg per day, 59511g/kg per day, 60011g/kg per
day, 60511g/kg
per day, 61011g/kg per day, 61511g/kg per day, 62011g/kg per day, 625 jig/kg
per day, 630
jig/kg per day, 635 jig/kg per day, 64011g/kg per day, 64511g/kg per day,
65011g/kg per day,
65511g/kg per day, 66011g/kg per day, 66511g/kg per day, 67011g/kg per day,
675 jig/kg per
day, 68011g/kg per day, 685 jig/kg per day, 69011g/kg per day, 69511g/kg per
day, 70011g/kg
per day, 70511g/kg per day, 71011g/kg per day, 715 jig/kg per day, 72011g/kg
per day, 725
jig/kg per day, 73011g/kg per day, 735 jig/kg per day, 74011g/kg per day,
74511g/kg per day,
75011g/kg per day, 75511g/kg per day, 76011g/kg per day, 765 jig/kg per day,
77011g/kg per
day, 775 jig/kg per day, 78011g/kg per day, 78511g/kg per day, 79011g/kg per
day, 79511g/kg
per day, 80011g/kg per day, 80511g/kg per day, 81011g/kg per day, 815 jig/kg
per day, 820
jig/kg per day, 825 jig/kg per day, 83011g/kg per day, 83511g/kg per day,
84011g/kg per day,
84511g/kg per day, 85011g/kg per day, 85511g/kg per day, 86011g/kg per day,
865 jig/kg per
day, 87011g/kg per day, 875 jig/kg per day, 88011g/kg per day, 88511g/kg per
day, 89011g/kg
per day, 89511g/kg per day, 90011g/kg per day, 905 jig/kg per day, 91011g/kg
per day, 915
jig/kg per day, 92011g/kg per day, 925 jig/kg per day, 93011g/kg per day,
93511g/kg per day,
94011g/kg per day, 94511g/kg per day, 95011g/kg per day, 955 jig/kg per day,
96011g/kg per
day, 965 jig/kg per day, 97011g/kg per day, 97511g/kg per day, 98011g/kg per
day, 98511g/kg
per day, 99011g/kg per day, 99511g/kg per day, or 1,00011g/kg per day. In some
embodiments, the CXCR2 agonist (e.g., Gro-f3 or Gro-f3 T, such as unmodified
Gro-f3 or Gro-
0 T) is administered to the donor at a dose of from about 501.tg/kg per day to
about 30011g/kg
per day, such as a dose of from about 10011g/kg per day to about 25011g/kg per
day, or from
about 125 jig/kg per day to about 225 jig/kg per day, or from about 125 jig/kg
per day to
about 175 jig/kg per day. In some embodiments, the CXCR2 agonist (e.g., Gro-f3
or Gro-f3 T,
such as unmodified Gro-f3 or Gro-f3 T) is administered to the donor at a dose
of about 150
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1.tg/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, I Basic
Cl/n. 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-f3 or Gro-f3 T, such as unmodified Gro-f3 or Gro-f3 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) an amine represented by formula (IA)
A
N
(IA)
wherein A includes a monocyclic or bicyclic fused ring system including at
least one nitrogen
atom and B is H or a sub stituent of from 1 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)

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N I
B'i (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)õ ¨ X (IC)
wherein each R is independently H or C1-C6 alkyl, n is 1 or 2, and X is an
aryl or heteroaryl
group or a mercaptan;
wherein the linker is a bond, optionally substituted C1-C6 alkylene,
optionally
substituted Ci-C6 heteroalkylene, optionally substituted C2-C6 alkenylene,
optionally
substituted C2-C6 heteroalkenylene, optionally substituted C2-C6 alkynylene,
optionally
substituted C2-C6 heteroalkynylene, optionally substituted cycloalkylene,
optionally
substituted heterocycloalkylene, optionally substituted arylene, or optionally
substituted
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 sub stituents. 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.
[0052] In some embodiments, the linker is represented by formula (ID)
1-X 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,
optionally
substituted C1-C6 heteroalkylene, optionally substituted C2-C6 alkenylene,
optionally
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substituted C2-C6 heteroalkenylene, optionally substituted C2-C6 alkynylene,
or optionally
substituted C2-C6 heteroalkynylene.
In some embodiments, the linker is represented by formula (IE)
Nvx
YA" (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 Ci-C6 alkylene,
optionally
substituted Ci-C6 heteroalkylene, optionally substituted C2-C6 alkenylene,
optionally
substituted C2-C6 heteroalkenylene, optionally substituted C2-C6 alkynylene,
or optionally
substituted C2-C6 heteroalkynylene.
[0053] In some embodiments, X and Y are each independently optionally
substituted Ci-C6
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 [tg/kg
to about 500
[tg/kg, such as a dose of about 50 g/kg, 55 [tg/kg, 60 g/kg, 65 g/kg, 70
g/kg, 75 g/kg,
80 g/kg, 85 g/kg, 90 [tg/kg, 95 [tg/kg, 100 [tg/kg, 105 g/kg, 110 [tg/kg,
115 g/kg, 120
[tg/kg, 125 [tg/kg, 130 g/kg, 135 g/kg, 140 g/kg, 145 [tg/kg, 150 g/kg,
155 g/kg, 160
[tg/kg, 165 [tg/kg, 170 g/kg, 175 g/kg, 180 g/kg, 185 [tg/kg, 190 g/kg,
195 [tg/kg, 200
[tg/kg, 205 [tg/kg, 210 g/kg, 215 g/kg, 220 g/kg, 225 [tg/kg, 230 g/kg,
235 [tg/kg, 240
[tg/kg, 245 [tg/kg, 250 g/kg, 255 g/kg, 260 g/kg, 265 g/kg, 270 g/kg, 275
g/kg, 280
[tg/kg, 285 [tg/kg, 290 g/kg, 295 g/kg, 300 g/kg, 305 [tg/kg, 310 g/kg,
315 [tg/kg, 320
[tg/kg, 325 [tg/kg, 330 [tg/kg, 335 g/kg, 340 [tg/kg, 345 [tg/kg, 350 [tg/kg,
355 [tg/kg, 360
[tg/kg, 365 [tg/kg, 370 g/kg, 375 g/kg, 380 [tg/kg, 385 g/kg, 390 [tg/kg,
395 g/kg, 400
[tg/kg, 405 [tg/kg, 410 g/kg, 415 g/kg, 420 g/kg, 425 [tg/kg, 430 g/kg,
435 [tg/kg, 440
[tg/kg, 445 [tg/kg, 450 g/kg, 455 g/kg, 460 g/kg, 465 [tg/kg, 470 g/kg,
475 [tg/kg, 480
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ug/kg, 485 ug/kg, 490 ug/kg, 495 ug/kg, or 500 ug/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 to about 300 ug/kg, such as a dose
of about 240
pig/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 per day to about 500 ug/kg per day, such as a dose of about 50 ug/kg
per day, 55
ug/kg per day, 60 ug/kg per day, 65 ug/kg per day, 70 ug/kg per day, 75 ug/kg
per day, 80
ug/kg per day, 85 ug/kg per day, 90 ug/kg per day, 95 ug/kg per day, 100 ug/kg
per day, 105
ug/kg per day, 110 ug/kg per day, 115 ug/kg per day, 120 ug/kg per day, 125
ug/kg per day,
130 ug/kg per day, 135 ug/kg per day, 140 ug/kg per day, 145 ug/kg per day,
150 ug/kg per
day, 155 ug/kg per day, 160 ug/kg per day, 165 ug/kg per day, 170 ug/kg per
day, 175 ug/kg
per day, 180 ug/kg per day, 185 ug/kg per day, 190 ug/kg per day, 195 ug/kg
per day, 200
ug/kg per day, 205 ug/kg per day, 210 ug/kg per day, 215 ug/kg per day, 220
ug/kg per day,
225 ug/kg per day, 230 ug/kg per day, 235 ug/kg per day, 240 ug/kg per day,
245 ug/kg per
day, 250 ug/kg per day, 255 ug/kg per day, 260 ug/kg per day, 265 ug/kg per
day, 270 ug/kg
per day, 275 ug/kg per day, 280 ug/kg per day, 285 ug/kg per day, 290 ug/kg
per day, 295
ug/kg per day, 300 ug/kg per day, 305 ug/kg per day, 310 ug/kg per day, 315
ug/kg per day,
320 ug/kg per day, 325 ug/kg per day, 330 ug/kg per day, 335 ug/kg per day,
340 ug/kg per
day, 345 ug/kg per day, 350 ug/kg per day, 355 ug/kg per day, 360 ug/kg per
day, 365 ug/kg
per day, 370 ug/kg per day, 375 ug/kg per day, 380 ug/kg per day, 385 ug/kg
per day, 390
ug/kg per day, 395 ug/kg per day, 400 ug/kg per day, 405 ug/kg per day, 410
ug/kg per day,
415 ug/kg per day, 420 ug/kg per day, 425 ug/kg per day, 430 ug/kg per day,
435 ug/kg per
day, 440 ug/kg per day, 445 ug/kg per day, 450 ug/kg per day, 455 ug/kg per
day, 460 ug/kg
per day, 465 ug/kg per day, 470 ug/kg per day, 475 ug/kg per day, 480 ug/kg
per day, 485
ug/kg per day, 490 ug/kg per day, 495 ug/kg per day, or 500 ug/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 per
day to about 300
ug/kg per day, such as a dose of about 240 ug/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
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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 leukocytes in the
population is
from about 0.0008 to about 0.0021. In some embodiments, the ratio of 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,
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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 leukocytes is from about 0.0010 to
about 0.0018,
such as a ratio of CD34+ cells to leukocytes 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, 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 leukocytes is about 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 (e.g., a human donor), wherein the ratio of CD34+ cells to neutrophils
in the population
is from about 0.0018 to about 0.0058. In some embodiments, the ratio of 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,

CA 03083783 2020-05-27
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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, 0.00520, 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, or 0.00580. In some embodiments, the ratio
of CD34+
cells to neutrophils is from about 0.0026 to about 0.0046, such as a ratio of
CD34+ cells to
neutrophils 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,
56

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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 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., a human donor), wherein the ratio of CD34+ cells to lymphocytes in the
population is
from about 0.0021 to about 0.0094. In some embodiments, the ratio of 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,
57

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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, 0.00520, 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,
58

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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+
cells to lymphocytes is from about 0.0025 to about 0.0035, such as a ratio of
CD34+ cells to
lymphocytes of about 0.00250, 0.00251, 0.00252, 0.00253, 0.00254, 0.00255,
0.00256,
59

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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+
cells to lymphocytes is 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 monocytes in the
population is
from about 0.0071 to about 0.0174. In some embodiments, the ratio of 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,

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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 monocytes is from about
0.0100 to about
0.0140, such as a ratio of CD34+ cells to monocytes of about 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, or 0.0140. In some embodiments, the ratio of CD34+ cells to
monocytes 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+ cells in the population
is from about
0.051% to about 0.140%. In some embodiments, the population of cells may have
a
frequency of CD34+ cells of 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%, 0.064%, 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%,
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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%, 0.1100o, 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 from about 0.080% to about 0.120%, such as a
frequency of
CD34+ 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 about 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+ CD45RA- cells to
leukocytes in the
population is from about 0.0003 to about 0.0016. In some embodiments, the
ratio of 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+ CD45RA- cells to leukocytes is from about 0.0006 to
about
0.0012, such as a ratio of CD34+ CD90+ CD45RA- cells to leukocytes of about
0.00060,
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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+ CD4SRA- cells to
leukocytes 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+ CD4SRA- cells to
neutrophils in the population is from about 0.0007 to about 0.0043. In some
embodiments,
the ratio of CD34+ CD90+ CD4SRA- cells to neutrophils 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,
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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+ CD45RA- cells to
neutrophils is
from about 0.0014 to about 0.0034, such as a ratio of CD34+ CD90+ CD45RA-
cells 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,
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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- 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+ CD45RA- cells to
lymphocytes in
the population is from about 0.0008 to about 0.0069. In some embodiments, the
ratio of
CD34+ CD90+ CD45RA- cells to lymphocytes 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,

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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, 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,
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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- cells to
lymphocytes
is from about 0.0011 to about 0.0031, such as a ratio of CD34+ CD90+ CD45RA-
cells to
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+ 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+ CD45RA- cells to
monocytes in the
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population is from about 0.0028 to about 0.0130. In some embodiments, the
ratio of CD34+
CD90+ CD45RAT 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,
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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, 0.00730, 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,
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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, or 0.0130. In
some
embodiments, the ratio of CD34+ CD90+ CD45RA- cells to monocytes is from about
0.0063
to about 0.0083, such as a ratio of CD34+ CD90+ CD45RA- cells to monocytes of
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

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0.00830. In some embodiments, the ratio of CD34+ CD90+ CD45RA- cells 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- cells to CD34+
cells in
the population is from about 0.393 to about 0.745. In some embodiments, the
ratio of CD34+
CD90+ CD45RA- cells to CD34+ cells may be 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, 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,
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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 from about 0.625 to about 0.725, such as a
ratio of CD34+
CD90+ CD45RA- cells to CD34+ cells 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 to
CD34+ cells is about 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- cells in
the
population is from about 0.020% to about 0.110%. In some embodiments, the
population of
cells may have a frequency of CD34+ CD90+ CD45RA- cells of about 0.020%,
0.021%,
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%, 0.045%, 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 a frequency of CD34+ CD90+ CD45RA--
cells 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%,
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0.085%, or 0.086%. In some embodiments, the population of cells has a
frequency of CD34+
CD90+ CD45RAT cells of about 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 (e.g., a human patient), the method including infusing 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,
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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 syndrome, Grave's
disease,
Guillain-Barre syndrome, 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 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, Sjogren'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
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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
CD34d1m 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-f3,
Gro-f3 T, and variants thereof at a dose of from about 501.tg/kg to about
1,00011g/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-f3, Gro-f3
T, and
variants thereof at a dose of from about 501.tg/kg to about 1,00011g/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

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Gro-f3, Gro-f3 T, and variants thereof at a dose of from about 501.tg/kg to
about 1,0001.tg/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-f3, Gro-f3 T, and variants thereof at a dose of from about 501.tg/kg to
about 1,0001.tg/kg
and (ii) a CXCR4 antagonist.
[0082] In certain embodiments, the CD34dim cells are present in a higher
amount in the
peripheral blood than if the hematopoietic stem cells were mobilized using the
CXCR4
antagonist alone. In certain embodiments, the CD34d1m cells are capable of
suppressing
alloreactive T lymphocyte proliferation when administered to a recipient.
.. [0083] In certain embodiments, the CXCR2 agonist is Gro-f3 T. In certain
embodiments, the
CXCR2 agonist is administered to the donor at a dose of from about 1001.tg/kg
to about 250
i.tg/kg. In certain embodiments, the CXCR2 agonist is administered to the
donor at a dose of
from about 1251.tg/kg to about 225 tg/kg. In certain embodiments, the CXCR2
agonist is
administered to the donor at a dose of about 1501.tg/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 salt thereof In certain embodiments, the plerixafor or
pharmaceutically
acceptable salt thereof is administered to the donor at a dose of from about
50 i.tg/kg to about
500 i.tg/kg. In certain embodiments, the plerixafor or pharmaceutically
acceptable salt
thereof is administered to the donor at a dose of from about 200 tg/kg to
about 300 tg/kg. In
certain embodiments, the plerixafor or pharmaceutically acceptable salt
thereof is
administered to the donor at a dose of about 240 tg/kg.
[0085] In certain embodiments, the method further includes testing a sample of
peripheral
blood for the presence of CD34d1m cells and releasing the sample for ex vivo
expansion of the
CD34d1m cells.
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[0086] In certain embodiments, the disclosure relates to the population of
CD34d1m cells
derived from any of the above methods, or a composition comprising the same.
Brief Description of the Figures
[0087] FIG. 1A provides a graph showing that, when Gro-0 T is co-administered
with
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+
Sca-1+ CD150+ CD48+). FIG. 1B provides a graph showing that grafts containing
cells
mobilized by Gro-f3 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-0 T when administered intravenously to Rhesus monkeys. FIG. 2B is a graph
demonstrating the pharmacokinetic profile of various dosages of Gro-0 T when
administered
subcutaneously to Rhesus monkeys. In all experiments, Gro-0 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-0 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-0 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-0 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-0 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
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response of neutrophils to various dosages of Gro-f3 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-f3 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-f3 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-f3 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-f3 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-f3 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-f3 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-f3 T was
administered to
subjects concurrently with plerixafor.
[0093] FIG. 7A shows a series of graphs demonstrating the mobilization
response of CD34+
cells to various dosages of Gro-f3 T upon intravenous administration to Rhesus
monkeys.
CD34+ cell response is shown both in terms of the frequency of CD34+ cells in
the sample
obtained from peripheral blood of the subjects (top) and the fold change in
CD34+ cell
frequency relative to baseline CD34+ cell frequency prior to administration
(bottom). FIG.
7B shows a series of graphs demonstrating the mobilization response of CD34+
cells to
various dosages of Gro-f3 T upon subcutaneous administration to Rhesus
monkeys. CD34+
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cell response is shown both in terms of the frequency of CD34+ cells in the
sample obtained
from peripheral blood of the subjects (top) and the fold change in CD34+ cell
frequency
relative to baseline CD34+ cell frequency prior to administration (bottom). In
all
experiments, Gro-f3 T was administered to subjects concurrently with
plerixafor.
[0094] FIG. 8A shows a series of graphs demonstrating the mobilization
response of CD34+
cells to various dosages of Gro-f3 T upon intravenous administration to Rhesus
monkeys.
CD34+ cell response is shown both in terms of the quantity of cells mobilized
(top) and the
fold change in CD34+ cell density relative to baseline CD34+ cell density
prior to
administration (bottom). FIG. 8B shows a series of graphs demonstrating the
mobilization
response of CD34+ cells to various dosages of Gro-f3 T upon subcutaneous
administration to
Rhesus monkeys. CD34+ cell response is shown both in terms of the quantity of
cells
mobilized (top) and the fold change in CD34+ cell density relative to baseline
CD34+ cell
density prior to administration (bottom). In all experiments, Gro-f3 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+ CD90+ CD45RA- cells) to various dosages of Gro-
f3 T upon
intravenous administration to Rhesus monkeys. CD34+ CD90+ CD45RA- cell
response is
shown both in terms of the frequency of CD34+ CD90+ CD45RA- cells in the
sample
obtained from peripheral blood of the subjects (top) and the fold change in
CD34+ CD90+
CD45RA- cell frequency relative to baseline CD34+ CD90+ CD45RA- cell frequency
prior to
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-f3 T upon subcutaneous administration to Rhesus monkeys. CD34+ CD90+
CD45RA-
cell response is shown both in terms of the frequency of CD34+ CD90+ CD45RA-
cells in the
sample obtained from peripheral blood of the subjects (top) and the fold
change in CD34+
CD90+ CD45RA- cell frequency relative to baseline CD34+ CD90+ CD45RA- cell
frequency
prior to administration (bottom). In all experiments, Gro-f3 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 various dosages of Gro-
f3 T upon
intravenous administration to Rhesus monkeys. CD34+ CD90+ CD45RA- cell
response is
shown both in terms of the quantity of cells mobilized (top) and the fold
change in CD34+
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CD90+ CD45RA-cell density relative to baseline CD34+ CD90+ CD45RA-cell density
prior to
administration (bottom). FIG. 10B shows a series of graphs demonstrating the
mobilization
response of hematopoietic stem cells (CD34+ CD90+ CD45RA- cells) to various
dosages of
Gro-f3 T upon subcutaneous administration to Rhesus monkeys. CD34+ cell
response is
shown both in terms of the quantity of cells mobilized (top) and the fold
change in CD34+
CD90+ CD45RA- cell density relative to baseline CD34+ CD90+ CD45RA- cell
density prior
to administration (bottom). In all experiments, Gro-f3 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-f3 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-f3 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-f3 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-f3 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-f3 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-f3
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-f3
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
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concentration relative to baseline TIMP-1 concentration prior to
administration (bottom). In
all experiments, Gro-f3 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-f3 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-f3 T upon subcutaneous administration
to Rhesus
monkeys. In all experiments, Gro-f3 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 [tg/kg Gro-
f3 T and
subcutaneous administration of 1 mg/kg plerixafor (AMD3100) leads to the
mobilization of a
population of CD34d1m 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 g/kg Gro-f3 T and subcutaneous
administration of 1
mg/kg plerixafor (AMD3100) or (2) subcutaneous administration of 1 mg/kg
plerixafor
(AMD3100). The combination of Gro-f3 T and plerixafor (as compared to
plerixafor alone)
leads to enhanced mobilization of CD34dim cells.
[0103] FIG. 17 is a graph quantifying the concentration of CD34d1m cells in
peripheral blood
from untreated Rhesus monkeys ("Unmobilized"), Rhesus monkeys that have been
treated
with intravenous administration of 450 [tg/kg Gro-f3 T and subcutaneous
administration of 1
mg/kg plerixafor ("Gro-f3 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 [tg/kg (q.d. x 5) G-
CSF ("G-CSF").
CD34d1m cells were present at a significantly higher concentration in blood
mobilized using
Gro-f3 T plus plerixafor.
[0104] FIG. 18 is a graph depicting the composition of unmobilized cells and
grafts
mobilized by G-CSF, Gro-f3 T and AMD3100 and AMD3100 alone. As shown, grafts
mobilized using Gro-f3 T and AMD3100 show a 3 fold increase in CD34d1m cells
and a 3 fold
increase in T-cells as compared to grafts mobilized using G-CSF.
[0105] FIG. 19 provides graphs showing that Gro-f3 T and AMD3100 mobilized
CD34dim
cells suppressed T-cell proliferation as measured by carboxyfluorescein
succinimidyl ester
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(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-f3 T and AMD3100
mobilized
PBMCs. At day 60 post-transplant, 15/16 mice transplanted with Gro-f3 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
(comparing Gro- I T and AMD3100 v. unmobilized) and p<0.05 (comparing Gro- I 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-f3 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
PBMCs, Gro-f3 T and plerixafor (i.e., AMD3100) mobilized PBMCs, and Gro-f3 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-f3 T and
AMD3100
mobilized PBMCs, and Gro-f3 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-f3 or a variant thereof, such as a
truncated form of Gro-
0 (e.g., Gro-f3 T), as described herein, optionally in combination with a C-X-
C chemokine
receptor type 4 (CXCR4) antagonist, such as 1,1'41,4-phenylenebis(methylene)]-
bis-
1,4,8,11-tetra-azacyclotetradecane or a variant thereof, may be administered
to a donor, as
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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-f3, Gro-f3 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 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
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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, metachromaticleukodystrophy, 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 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
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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
(Ka) or, in
cases in which one of the binding partners is an enzyme, as an inhibition
constant (K1).
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,
r1gG, 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 are capable of specifically
binding to a target
protein. As used herein, the Fab and F(ab')2 fragments refer to antibody
fragments 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, a
triabody, 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
domains; (ii) a F(ab')2 fragment, a bivalent fragment containing two Fab
fragments linked by
a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the
VH and CHI
domains; (iv) a Fv fragment consisting of the VL and VH domains of a single
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antibody, (v) a dAb including VH and VL domains; (vi) a dAb fragment that
consists of a 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 at. (1988) Science 242:423-426 and Huston et at. (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 et at. (2015) Science Translational Medicine 7(281):1-12. In some
embodiments, a
population of CD34d1m cells may be CD14+.
[0120] The person of skill can readily recognize a CD34d1m cell population
when viewing a
flow cytometry plot as a population of cells that is CD34 + but has a
brightness between the
base level of fluorescence (e.g., autofluorescence) in the cell population
being examined and
the brightness of a CD34 + bright cell (e.g., a hematopoietic stem cell and/or
a CD34 + CD90+
cell) population. For example, in certain embodiments, a CD34d1m cell
population exhibits
between 5% and 95% of the brightness of a CD34 + bright cell (e.g., a
hematopoietic stem
cell) population, but is brighter than a CD34- cell population.
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[0121] In certain embodiments, CD34d1m cells exhibit between 1000 and 900 0,
1000 and 800 o,
10% and 70%, 10% and 6000, 1000 and 5000, 1000 and 4000, 1000 and 3000, 1000
and 20%,
2000 and 90%, 2000 and 80%, 2000 and 70%, 2000 and 60%, 2000 and 50%, 2000 and
40%,
20% and 30%, 30% and 90%, 30% and 80%, 30% and 70%, 30% and 60%, 30% and 5000,
3000 and 4000, 4000 and 9000, 4000 and 8000, 4000 and 7000, 4000 and 6000,
4000 and 5000,
500 0 and 900 0, 500 0 and 800 0, 500 0 and 700 0, 500 0 and 600 0, 600 0 and
900 0, 600 0 and 800 0,
60% and 70%, 70% and 90%, 70% and 80%, or 80% and 90%, of the brightness of
CD34+
bright cells (e.g., hematopoietic stem cells), but is brighter than CD34-
cells. In certain
embodiments, the CD34d1m cells are at least 5 A brighter, at least 10%
brighter, at least 20%
brighter or at least 30 A brighter than CD34- cells, but is less bright than
CD34 + bright cells
(e.g., a hematopoietic stem cells).
[0122] In certain embodiments, CD34d1m cells are identified in a cell sample
by using flow
cytometry with magnetic beads instead of fluorescence. The magnetic beads will
selectively
pull down CD34 + bright cells, leaving CD34d1m cells in the cell sample.
[0123] In certain embodiments, CD34d1m cells are identified by measuring the
number of
copies of CD34 expressed by the cells. For example, CD34d1m cells can exhibit
between 5 A
and 95% of the number of copies of CD34 as compared to CD34 + bright cells
(e.g.,
hematopoietic stem cells), or between 10% and 90%, 10% and 80%, 10% and 70%,
10% and
60%, 100o and 500o, 100o and 40%, 100o and 30%, 100o 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%, 500o and 90%, 500o
and
80%, 500o and 70%, 500o 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 (e.g., hematopoietic stem 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
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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 13-sheet configuration, connected by
three CDRs,
which form loops that connect, and in some cases form part of, the 13-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 at.,
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-(3 or a variant or truncation thereof, such
as Gro-(3 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-(3 or a
variant or truncation
thereof, such as Gro-(3 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
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Amino Acid 3 Letter 1 Letter Side-chain Electrostatic Steric
Code Code Polarity character at Volumel.
physiological
pH (7.4)
Alanine Ala A nonpolar neutral small
Arginine Arg R polar cationic large
Asparagine Asn N polar neutral intermediate
Aspartic acid Asp D polar anionic intermediate
Cysteine Cys C nonpolar neutral intermediate
Glutamic acid Glu E polar anionic intermediate
Glutamine Gln Q polar neutral intermediate
Glycine Gly G nonpolar neutral small
Histidine His H polar Both neutral large
and cationic
forms in
equilibrium
at pH 7.4
Isoleucine Ile I nonpolar neutral large
Leucine Leu L nonpolar neutral large
Lysine Lys K polar cationic large
Methionine Met M nonpolar neutral large
Phenylalanine Phe F nonpolar neutral large
Proline Pro P non-polar neutral intermediate
Serine Ser S polar neutral small
Threonine Thr T polar neutral intermediate
Tryptophan Trp W nonpolar neutral bulky
Tyrosine Tyr Y polar neutral large
Valine Val V nonpolar neutral intermediate
tbased on volume in A3: 50-100 is small, 100-150 is intermediate, 150-200 is
large, and >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
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one amino acid for a member of the same amino acid family (e.g., a
substitution of Ser for
Thr or Lys 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 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 as to position the VH and VL
domains of
neighboring peptide chains spatially proximal to one another (see, for
example, Holliger et at.
(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; US
2010/0251395; and US 2012/0222143, the disclosures of each of which are
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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 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
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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,
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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+. 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 are immature cells that express the CD34 cell surface marker. In
humans,
CD34 + cells are believed to include a subpopulation of cells with the stem
cell 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-, CD45RA-, CD90+, CD49F+,
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 Ten 19, CD11b, Grl, CD3, CD4, CD8, B220, IL7ra), whereas ST-HSCs are
CD34, SCA-1+, C-kit, CD135-, Slamfl/CD150+, and lin- (negative for mature
lineage
markers including Ten 19, CD11b, Grl, 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 MEW class II (DP, DQ and
DR)
exists in different alleles. For example, two unrelated individuals may carry
class I HLA-B,
genes B5, and Bw41, respectively. Allelic gene products differ in one or more
amino acids in
the a and/or 0 domain(s). Large panels of specific antibodies or nucleic acid
reagents 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 MEW 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
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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 at. (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,
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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,
metachromaticleukodystrophy, 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
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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-f3 or a variant thereof, such
as a truncation of
Gro-f3, for example, Gro-f3 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 are CD34+, CD38-, CD45RA-, CD90+, CD49F+, and lin- (negative 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
(In some embodiments, a quantity of plerixafor, or a variant thereof, and/or
Gro-f3, or a
variant thereof, such as a truncation of Gro-f3, for example, Gro-f3 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/0_, of peripheral blood, such as from about
21 to about 39
CD34+ cells/0_, of peripheral blood, about 22 to about 38 CD34+ cells/0_, of
peripheral
blood, about 23 to about 37 CD34+ cells/0_, of peripheral blood, about 24 to
about 36 CD34+
cells/0_, of peripheral blood, about 25 to about 35 CD34+ cells/0_, of
peripheral blood, about
26 to about 34 CD34+ cells/0_, of peripheral blood, about 27 to about 33 CD34+
cells/0_, of
peripheral blood, about 28 to about 32 CD34+ cells/0_, of peripheral blood, or
about 29 to
about 31 CD34+ cells/0_, of peripheral blood (e.g., about 20 CD34+ cells/0_,
of peripheral
blood, 21 CD34+ cells/0_, of peripheral blood, 22 CD34+ cells/0_, of
peripheral blood, 23
CD34+ cells/0_, of peripheral blood, 24, CD34+ cells/0_, of peripheral blood,
25 CD34+
cells/0_, of peripheral blood, 26 CD34+ cells/0_, of peripheral blood, 27
CD34+ cells/0_, of
peripheral blood, 28 CD34+ cells/0_, of peripheral blood, 29 CD34+ cells/0_,
of peripheral
blood, 30 CD34+ cells/0_, of peripheral blood, 31 CD34+ cells/0_, of
peripheral blood, 32
CD34+ cells/0_, of peripheral blood 33 CD34+ cells/0_, of peripheral blood, 34
CD34+
cells/0_, of peripheral blood, 35 CD34+ cells/0_, of peripheral blood, 36
CD34+ cells/0_, of
peripheral blood, 37 CD34+ cells/0_, of peripheral blood, 38 CD34+ cells/0_,
of peripheral
blood, 39 CD34+ cells/0_, of peripheral blood, 40 CD34+ cells/0_, of
peripheral blood, or
more. For instance, mobilizing amounts of a CXCR2 agonist, such as Gro-f3 T,
include from
about 50 tg/kg of recipient to about 1 mg/kg of recipient, such as from about
50 pg/kg to
about 300 tg/kg, 100 pg/kg to about 250 pg/kg, or about 150 tg/kg. Mobilizing
amounts of
a CXCR4 antagonist, such as plerixafor or a pharmaceutically acceptable salt
thereof, include
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from about 50 g/kg of recipient to about 500 g/kg of recipient, such as from
about 200
[tg/kg to about 300 g/kg, or about 240 [tg/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¨
peripheral 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 by a linker. The linker that joins the VL and VH regions of a scFv
fragment can be a
peptide linker composed of proteinogenic amino acids. Alternative linkers can
be used to 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 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 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 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
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 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, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-
butyl, 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. The divalent positions may be on the same or different atoms
within the alkyl
chain. Examples of alkylene include methylene, ethylene, propylene,
isopropylene, and the
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.
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[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 or more heteroatoms (e.g., oxygen, nitrogen, or sulfur, among
others) 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 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[4,3-b]pyridyl, quinolyl, isoquinolyl, tetrazolyl,
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-f3, Gro-f3 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-f3, Gro-f3 T, or
a variant thereof, when administered intravenously at a dose of from about 50
[tg/kg to about
1 mg/kg, preferably from about 100 [tg/kg to about 250 [tg/kg, and even more
preferably at a
dose of about 150 [tg/kg, exhibit the ability to rapidly mobilize
hematopoietic stem and
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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-f3,
Gro-f3 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-f3, Gro-f3 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+
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
Ratio of CD34+ cells neutrophils
Ratio of CD34+ CD90+
(e.g., CD34+ CD90+ 8 At least
0.0006
CD45RA- cells to neutrophils
CD45RA- cells) to
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
+
sample isolated from 11 Ratio of
CD34 cells toAt least 0.0025
lymphocytes
mammalian donor
Ratio of CD34+ 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
At least 0.0111
monocytes
Ratio of CD34+ CD90+
16 At least 0.0020
CD45RA- cells to monocytes
Ratio of CD34+ CD90+
17 At least 0.0039
CD45RA- cells to monocytes
Frequency of CD34+ cells
Proportion of CD34+ relative to total quantity of At least
0.051%
18
cells (e.g., 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 of CD34+ CD90+
CD45RA- cells relative to
20 At least 0.020%
total quantity of cells in
sample isolated from donor
Frequency of CD34+ CD90+
CD45RAT 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+ cell ratio parameter (e.g., one or more of parameter numbers 1-17 in
TABLE 2) 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
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First Parameter Second
No. Parameter No.
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
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 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
112

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First Parameter 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
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
113

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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
14
10 15
10 16
10 17
11 14
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 18
11 19
11 20
11 21
12 18
12 19
12 20
114

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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 18
17 19
17 20
17 21
TABLE 4. Three-way combinations of hematopoietic stem cell population
parameters for
assessment
First Parameter Second Parameter Third 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
6 10
115

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First Parameter Second Parameter Third Parameter
No. No. No.
7 10
5 8 10
5 9 10
1 6 11
1 7 11
1 8 11
1 9 11
2 6 11
2 7 11
2 8 11
2 9 11
3 6 11
3 7 11
3 8 11
3 9 11
4 6 11
4 7 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 12
2 8 12
2 9 12
3 6 12
3 7 12
3 8 12
3 9 12
4 6 12
4 7 12
4 8 12
4 9 12
5 6 12
5 7 12
5 8 12
5 9 12
1 6 13
1 7 13
1 8 13
116

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First Parameter Second Parameter Third Parameter
No. No. No.
1 9 13
2 6 13
2 7 13
2 8 13
2 9 13
3 6 13
3 7 13
3 8 13
3 9 13
4 6 13
4 7 13
4 8 13
4 9 13
6 13
5 7 13
5 8 13
5 9 13
1 6 14
1 7 14
1 8 14
1 9 14
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
117

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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
6 15
5 7 15
5 8 15
5 9 15
1 6 16
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 9 17
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
118

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First Parameter Second Parameter Third Parameter
No. No. No.
4 9 17
6 17
5 7 17
5 8 17
5 9 17
1 6 18
1 7 18
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 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 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
119

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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
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
2 9 21
3 6 21
3 7 21
3 8 21
3 9 21
4 6 21
4 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
120

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First Parameter Second Parameter Third 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
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 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
121

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First Parameter Second Parameter Third Parameter
No. No. No.
4 11 16
4 12 16
4 13 16
10 16
5 11 16
5 12 16
5 13 16
1 10 17
1 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 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 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 18
4 12 18
4 13 18
5 10 18
5 11 18
5 12 18
122

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First Parameter Second Parameter Third Parameter
No. No. No.
13 18
1 10 19
1 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 10 21
1 11 21
1 12 21
1 13 21
2 10 21
123

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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
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 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
124

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First Parameter Second Parameter Third Parameter
No. No. No.
3 17 19
4 14 19
4 15 19
4 16 19
4 17 19
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
125

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First Parameter Second Parameter Third Parameter
No. No. No.
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 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 15
6 12 15
6 13 15
7 10 15
7 11 15
7 12 15
7 13 15
8 10 15
8 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 12 16
7 13 16
8 10 16
8 11 16
8 12 16
126

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First Parameter Second Parameter Third 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 17
6 13 17
7 10 17
7 11 17
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 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 10 19
7 11 19
7 12 19
7 13 19
8 10 19
127

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First Parameter Second Parameter Third Parameter
No. No. No.
8 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 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 20
9 12 20
9 13 20
6 10 21
6 11 21
6 12 21
6 13 21
7 10 21
7 11 21
7 12 21
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
128

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First Parameter Second Parameter Third 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 14 19
7 15 19
7 16 19
7 17 19
8 14 19
8 15 19
8 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 20
6 17 20
7 14 20
7 15 20
7 16 20
7 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
129

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First Parameter Second Parameter Third Parameter
No. No. No.
7 15 21
7 16 21
7 17 21
8 14 21
8 15 21
8 16 21
8 17 21
9 14 21
9 15 21
9 16 21
9 17 21
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 19
13 14 19
13 15 19
13 16 19
13 17 19
10 14 20
10 15 20
10 16 20
130

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First Parameter Second Parameter Third Parameter
No. No. No.
17 20
11 14 20
11 15 20
11 16 20
11 17 20
12 14 20
12 15 20
12 16 20
12 17 20
13 14 20
13 15 20
13 16 20
13 17 20
10 14 21
10 15 21
10 16 21
10 17 21
11 14 21
11 15 21
11 16 21
11 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 14
3 6 10 14
3 7 10 14
3 8 10 14
131

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First Parameter Second Third Parameter Fourth
No. Parameter No. No. Parameter No.
3 9 10 14
4 6 10 14
4 7 10 14
4 8 10 14
4 9 10 14
6 10 14
5 7 10 14
5 8 10 14
5 9 10 14
1 6 11 14
1 7 11 14
1 8 11 14
1 9 11 14
2 6 11 14
2 7 11 14
2 8 11 14
2 9 11 14
3 6 11 14
3 7 11 14
3 8 11 14
3 9 11 14
4 6 11 14
4 7 11 14
4 8 11 14
4 9 11 14
5 6 11 14
5 7 11 14
5 8 11 14
5 9 11 14
1 6 12 14
1 7 12 14
1 8 12 14
1 9 12 14
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
132

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First Parameter Second Third Parameter Fourth
No. Parameter No. No. Parameter No.
7 12 14
5 8 12 14
5 9 12 14
1 6 13 14
1 7 13 14
1 8 13 14
1 9 13 14
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 9 10 15
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 6 11 15
1 7 11 15
1 8 11 15
133

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First Parameter Second Third Parameter Fourth
No. Parameter No. No. Parameter No.
1 9 11 15
2 6 11 15
2 7 11 15
2 8 11 15
2 9 11 15
3 6 11 15
3 7 11 15
3 8 11 15
3 9 11 15
4 6 11 15
4 7 11 15
4 8 11 15
4 9 11 15
6 11 15
5 7 11 15
5 8 11 15
5 9 11 15
1 6 12 15
1 7 12 15
1 8 12 15
1 9 12 15
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 9 13 15
2 6 13 15
2 7 13 15
2 8 13 15
2 9 13 15
3 6 13 15
134

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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
6 13 15
5 7 13 15
5 8 13 15
5 9 13 15
1 6 10 16
1 7 10 16
1 8 10 16
1 9 10 16
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 6 11 16
1 7 11 16
1 8 11 16
1 9 11 16
2 6 11 16
2 7 11 16
2 8 11 16
2 9 11 16
3 6 11 16
3 7 11 16
3 8 11 16
3 9 11 16
4 6 11 16
4 7 11 16
4 8 11 16
135

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First Parameter Second Third Parameter Fourth
No. Parameter No. No. Parameter No.
4 9 11 16
6 11 16
5 7 11 16
5 8 11 16
5 9 11 16
1 6 12 16
1 7 12 16
1 8 12 16
1 9 12 16
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 7 13 16
1 8 13 16
1 9 13 16
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 16
1 6 10 17
136

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First Parameter Second Third Parameter Fourth
No. Parameter No. No. Parameter No.
1 7 10 17
1 8 10 17
1 9 10 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 17
4 7 10 17
4 8 10 17
4 9 10 17
6 10 17
5 7 10 17
5 8 10 17
5 9 10 17
1 6 11 17
1 7 11 17
1 8 11 17
1 9 11 17
2 6 11 17
2 7 11 17
2 8 11 17
2 9 11 17
3 6 11 17
3 7 11 17
3 8 11 17
3 9 11 17
4 6 11 17
4 7 11 17
4 8 11 17
4 9 11 17
5 6 11 17
5 7 11 17
5 8 11 17
5 9 11 17
1 6 12 17
1 7 12 17
1 8 12 17
1 9 12 17
2 6 12 17
2 7 12 17
2 8 12 17
137

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
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
6 12 17
5 7 12 17
5 8 12 17
5 9 12 17
1 6 13 17
1 7 13 17
1 8 13 17
1 9 13 17
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 7 10 18
1 8 10 18
1 9 10 18
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 18
138

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter Second Third Parameter Fourth
No. Parameter No. No. Parameter No.
4 7 10 18
4 8 10 18
4 9 10 18
6 10 18
5 7 10 18
5 8 10 18
5 9 10 18
1 6 11 18
1 7 11 18
1 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
3 9 11 18
4 6 11 18
4 7 11 18
4 8 11 18
4 9 11 18
5 6 11 18
5 7 11 18
5 8 11 18
5 9 11 18
1 6 12 18
1 7 12 18
1 8 12 18
1 9 12 18
2 6 12 18
2 7 12 18
2 8 12 18
2 9 12 18
3 6 12 18
3 7 12 18
3 8 12 18
3 9 12 18
4 6 12 18
4 7 12 18
4 8 12 18
4 9 12 18
5 6 12 18
5 7 12 18
5 8 12 18
139

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter Second Third Parameter Fourth
No. Parameter No. No. Parameter No.
9 12 18
1 6 13 18
1 7 13 18
1 8 13 18
1 9 13 18
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 6 10 19
1 7 10 19
1 8 10 19
1 9 10 19
2 6 10 19
2 7 10 19
2 8 10 19
2 9 10 19
3 6 10 19
3 7 10 19
3 8 10 19
3 9 10 19
4 6 10 19
4 7 10 19
4 8 10 19
4 9 10 19
5 6 10 19
5 7 10 19
5 8 10 19
5 9 10 19
1 6 11 19
1 7 11 19
1 8 11 19
1 9 11 19
2 6 11 19
140

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter Second Third Parameter Fourth
No. Parameter No. No. Parameter No.
2 7 11 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 9 11 19
6 11 19
5 7 11 19
5 8 11 19
5 9 11 19
1 6 12 19
1 7 12 19
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 19
5 8 12 19
5 9 12 19
1 6 13 19
1 7 13 19
1 8 13 19
1 9 13 19
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
141

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
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
6 13 19
5 7 13 19
5 8 13 19
5 9 13 19
1 6 10 20
1 7 10 20
1 8 10 20
1 9 10 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 6 11 20
1 7 11 20
1 8 11 20
1 9 11 20
2 6 11 20
2 7 11 20
2 8 11 20
2 9 11 20
3 6 11 20
3 7 11 20
3 8 11 20
3 9 11 20
4 6 11 20
4 7 11 20
4 8 11 20
4 9 11 20
5 6 11 20
142

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter Second Third Parameter Fourth
No. Parameter No. No. Parameter No.
7 11 20
5 8 11 20
5 9 11 20
1 6 12 20
1 7 12 20
1 8 12 20
1 9 12 20
2 6 12 20
2 7 12 20
2 8 12 20
2 9 12 20
3 6 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 9 13 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 20
4 9 13 20
5 6 13 20
5 7 13 20
5 8 13 20
5 9 13 20
1 6 10 21
1 7 10 21
1 8 10 21
143

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
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 7 10 21
4 8 10 21
4 9 10 21
6 10 21
5 7 10 21
5 8 10 21
5 9 10 21
1 6 11 21
1 7 11 21
1 8 11 21
1 9 11 21
2 6 11 21
2 7 11 21
2 8 11 21
2 9 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 9 12 21
2 6 12 21
2 7 12 21
2 8 12 21
2 9 12 21
3 6 12 21
144

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter Second Third Parameter Fourth
No. Parameter No. No. Parameter No.
3 7 12 21
3 8 12 21
3 9 12 21
4 6 12 21
4 7 12 21
4 8 12 21
4 9 12 21
6 12 21
5 7 12 21
5 8 12 21
5 9 12 21
1 6 13 21
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
3 7 13 21
3 8 13 21
3 9 13 21
4 6 13 21
4 7 13 21
4 8 13 21
4 9 13 21
5 6 13 21
5 7 13 21
5 8 13 21
5 9 13 21
1 6 14 18
1 7 14 18
1 8 14 18
1 9 14 18
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
145

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter Second Third Parameter Fourth
No. Parameter No. No. Parameter No.
4 9 14 18
6 14 18
5 7 14 18
5 8 14 18
5 9 14 18
1 6 15 18
1 7 15 18
1 8 15 18
1 9 15 18
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 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
1 6 16 18
1 7 16 18
1 8 16 18
1 9 16 18
2 6 16 18
2 7 16 18
2 8 16 18
2 9 16 18
3 6 16 18
3 7 16 18
3 8 16 18
3 9 16 18
4 6 16 18
4 7 16 18
4 8 16 18
4 9 16 18
5 6 16 18
5 7 16 18
5 8 16 18
5 9 16 18
1 6 17 18
146

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter Second Third Parameter Fourth
No. Parameter No. No. Parameter No.
1 7 17 18
1 8 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 18
4 7 17 18
4 8 17 18
4 9 17 18
6 17 18
5 7 17 18
5 8 17 18
5 9 17 18
1 6 14 19
1 7 14 19
1 8 14 19
1 9 14 19
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 6 15 19
1 7 15 19
1 8 15 19
1 9 15 19
2 6 15 19
2 7 15 19
2 8 15 19
147

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
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
6 15 19
5 7 15 19
5 8 15 19
5 9 15 19
1 6 16 19
1 7 16 19
1 8 16 19
1 9 16 19
2 6 16 19
2 7 16 19
2 8 16 19
2 9 16 19
3 6 16 19
3 7 16 19
3 8 16 19
3 9 16 19
4 6 16 19
4 7 16 19
4 8 16 19
4 9 16 19
5 6 16 19
5 7 16 19
5 8 16 19
5 9 16 19
1 6 17 19
1 7 17 19
1 8 17 19
1 9 17 19
2 6 17 19
2 7 17 19
2 8 17 19
2 9 17 19
3 6 17 19
3 7 17 19
3 8 17 19
3 9 17 19
4 6 17 19
148

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter Second Third Parameter Fourth
No. Parameter No. No. Parameter No.
4 7 17 19
4 8 17 19
4 9 17 19
6 17 19
5 7 17 19
5 8 17 19
5 9 17 19
1 6 14 20
1 7 14 20
1 8 14 20
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 6 15 20
1 7 15 20
1 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
149

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter Second Third Parameter Fourth
No. Parameter No. No. Parameter No.
9 15 20
1 6 16 20
1 7 16 20
1 8 16 20
1 9 16 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 9 16 20
5 6 16 20
5 7 16 20
5 8 16 20
5 9 16 20
1 6 17 20
1 7 17 20
1 8 17 20
1 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 7 14 21
1 8 14 21
1 9 14 21
2 6 14 21
150

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
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 9 14 21
6 14 21
5 7 14 21
5 8 14 21
5 9 14 21
1 6 15 21
1 7 15 21
1 8 15 21
1 9 15 21
2 6 15 21
2 7 15 21
2 8 15 21
2 9 15 21
3 6 15 21
3 7 15 21
3 8 15 21
3 9 15 21
4 6 15 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 6 16 21
1 7 16 21
1 8 16 21
1 9 16 21
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

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
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
6 16 21
5 7 16 21
5 8 16 21
5 9 16 21
1 6 17 21
1 7 17 21
1 8 17 21
1 9 17 21
2 6 17 21
2 7 17 21
2 8 17 21
2 9 17 21
3 6 17 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 9 17 21
5 6 17 21
5 7 17 21
5 8 17 21
5 9 17 21
1 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
152

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter Second Third Parameter Fourth
No. Parameter No. No. Parameter No.
11 14 18
5 12 14 18
5 13 14 18
1 10 15 18
1 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 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
1 10 16 18
1 11 16 18
1 12 16 18
1 13 16 18
2 10 16 18
2 11 16 18
2 12 16 18
2 13 16 18
3 10 16 18
3 11 16 18
3 12 16 18
3 13 16 18
4 10 16 18
4 11 16 18
4 12 16 18
4 13 16 18
5 10 16 18
5 11 16 18
5 12 16 18
5 13 16 18
1 10 17 18
1 11 17 18
1 12 17 18
153

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter Second Third Parameter Fourth
No. Parameter No. No. Parameter No.
1 13 17 18
2 10 17 18
2 11 17 18
2 12 17 18
2 13 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 18
4 12 17 18
4 13 17 18
10 17 18
5 11 17 18
5 12 17 18
5 13 17 18
1 10 14 19
1 11 14 19
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 14 19
3 12 14 19
3 13 14 19
4 10 14 19
4 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
154

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter Second Third Parameter Fourth
No. Parameter No. No. Parameter No.
3 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
10 15 19
5 11 15 19
5 12 15 19
5 13 15 19
1 10 16 19
1 11 16 19
1 12 16 19
1 13 16 19
2 10 16 19
2 11 16 19
2 12 16 19
2 13 16 19
3 10 16 19
3 11 16 19
3 12 16 19
3 13 16 19
4 10 16 19
4 11 16 19
4 12 16 19
4 13 16 19
5 10 16 19
5 11 16 19
5 12 16 19
5 13 16 19
1 10 17 19
1 11 17 19
1 12 17 19
1 13 17 19
2 10 17 19
2 11 17 19
2 12 17 19
2 13 17 19
3 10 17 19
3 11 17 19
3 12 17 19
3 13 17 19
4 10 17 19
4 11 17 19
4 12 17 19
155

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter Second Third Parameter Fourth
No. Parameter No. No. Parameter No.
4 13 17 19
10 17 19
5 11 17 19
5 12 17 19
5 13 17 19
1 10 14 20
1 11 14 20
1 12 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 14 20
4 10 14 20
4 11 14 20
4 12 14 20
4 13 14 20
5 10 14 20
5 11 14 20
5 12 14 20
5 13 14 20
1 10 15 20
1 11 15 20
1 12 15 20
1 13 15 20
2 10 15 20
2 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
156

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter Second Third Parameter Fourth
No. Parameter No. No. Parameter No.
1 11 16 20
1 12 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 16 20
4 12 16 20
4 13 16 20
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 20
2 10 17 20
2 11 17 20
2 12 17 20
2 13 17 20
3 10 17 20
3 11 17 20
3 12 17 20
3 13 17 20
4 10 17 20
4 11 17 20
4 12 17 20
4 13 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
157

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
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 10 14 21
4 11 14 21
4 12 14 21
4 13 14 21
10 14 21
5 11 14 21
5 12 14 21
5 13 14 21
1 10 15 21
1 11 15 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 15 21
4 12 15 21
4 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 16 21
4 10 16 21
158

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter Second Third Parameter Fourth
No. Parameter No. No. Parameter No.
4 11 16 21
4 12 16 21
4 13 16 21
10 16 21
5 11 16 21
5 12 16 21
5 13 16 21
1 10 17 21
1 11 17 21
1 12 17 21
1 13 17 21
2 10 17 21
2 11 17 21
2 12 17 21
2 13 17 21
3 10 17 21
3 11 17 21
3 12 17 21
3 13 17 21
4 10 17 21
4 11 17 21
4 12 17 21
4 13 17 21
5 10 17 21
5 11 17 21
5 12 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 14 18
8 12 14 18
8 13 14 18
9 10 14 18
9 11 14 18
9 12 14 18
9 13 14 18
6 10 15 18
6 11 15 18
6 12 15 18
159

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter Second Third Parameter Fourth
No. Parameter No. No. Parameter No.
6 13 15 18
7 10 15 18
7 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 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 18
9 13 16 18
6 10 17 18
6 11 17 18
6 12 17 18
6 13 17 18
7 10 17 18
7 11 17 18
7 12 17 18
7 13 17 18
8 10 17 18
8 11 17 18
8 12 17 18
8 13 17 18
9 10 17 18
9 11 17 18
9 12 17 18
9 13 17 18
6 10 14 19
160

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter Second Third Parameter Fourth
No. Parameter No. No. Parameter No.
6 11 14 19
6 12 14 19
6 13 14 19
7 10 14 19
7 11 14 19
7 12 14 19
7 13 14 19
8 10 14 19
8 11 14 19
8 12 14 19
8 13 14 19
9 10 14 19
9 11 14 19
9 12 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 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 15 19
9 12 15 19
9 13 15 19
6 10 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 19
8 12 16 19
8 13 16 19
9 10 16 19
9 11 16 19
9 12 16 19
161

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter Second Third Parameter Fourth
No. Parameter No. No. Parameter No.
9 13 16 19
6 10 17 19
6 11 17 19
6 12 17 19
6 13 17 19
7 10 17 19
7 11 17 19
7 12 17 19
7 13 17 19
8 10 17 19
8 11 17 19
8 12 17 19
8 13 17 19
9 10 17 19
9 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 20
8 12 14 20
8 13 14 20
9 10 14 20
9 11 14 20
9 12 14 20
9 13 14 20
6 10 15 20
6 11 15 20
6 12 15 20
6 13 15 20
7 10 15 20
7 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
162

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
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 16 20
8 12 16 20
8 13 16 20
9 10 16 20
9 11 16 20
9 12 16 20
9 13 16 20
6 10 17 20
6 11 17 20
6 12 17 20
6 13 17 20
7 10 17 20
7 11 17 20
7 12 17 20
7 13 17 20
8 10 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 14 21
7 12 14 21
7 13 14 21
8 10 14 21
8 11 14 21
8 12 14 21
163

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter Second Third Parameter Fourth
No. Parameter No. No. Parameter No.
8 13 14 21
9 10 14 21
9 11 14 21
9 12 14 21
9 13 14 21
6 10 15 21
6 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
8 12 15 21
8 13 15 21
9 10 15 21
9 11 15 21
9 12 15 21
9 13 15 21
6 10 16 21
6 11 16 21
6 12 16 21
6 13 16 21
7 10 16 21
7 11 16 21
7 12 16 21
7 13 16 21
8 10 16 21
8 11 16 21
8 12 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
6 11 17 21
6 12 17 21
6 13 17 21
7 10 17 21
7 11 17 21
7 12 17 21
7 13 17 21
8 10 17 21
164

CA 03083783 2020-05-27
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
9 10 17 21
9 11 17 21
9 12 17 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 7 10 14 18
1 8 10 14 18
1 9 10 14 18
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 6 10 14 18
4 7 10 14 18
4 8 10 14 18
4 9 10 14 18
6 10 14 18
5 7 10 14 18
5 8 10 14 18
5 9 10 14 18
1 6 11 14 18
1 7 11 14 18
1 8 11 14 18
1 9 11 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 18
3 8 11 14 18
3 9 11 14 18
4 6 11 14 18
165

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter second Parameter Third Parameter Fourth
Fifth Parameter
No. No. No. Parameter No. No.
4 7 11 14 18
4 8 11 14 18
4 9 11 14 18
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 7 12 14 18
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 8 13 14 18
1 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
166

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter second Parameter Third Parameter Fourth
Fifth Parameter
No. No. No. Parameter No. No.
9 13 14 18
1 6 10 15 18
1 7 10 15 18
1 8 10 15 18
1 9 10 15 18
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
1 7 11 15 18
1 8 11 15 18
1 9 11 15 18
2 6 11 15 18
2 7 11 15 18
2 8 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 15 18
5 7 11 15 18
5 8 11 15 18
5 9 11 15 18
1 6 12 15 18
1 7 12 15 18
1 8 12 15 18
1 9 12 15 18
2 6 12 15 18
167

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter second Parameter Third 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
6 12 15 18
5 7 12 15 18
5 8 12 15 18
5 9 12 15 18
1 6 13 15 18
1 7 13 15 18
1 8 13 15 18
1 9 13 15 18
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 9 10 16 18
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
168

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter second Parameter Third 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
6 10 16 18
5 7 10 16 18
5 8 10 16 18
5 9 10 16 18
1 6 11 16 18
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 16 18
3 6 11 16 18
3 7 11 16 18
3 8 11 16 18
3 9 11 16 18
4 6 11 16 18
4 7 11 16 18
4 8 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 6 12 16 18
1 7 12 16 18
1 8 12 16 18
1 9 12 16 18
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
169

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter second Parameter Third Parameter Fourth
Fifth Parameter
No. No. No. Parameter No. No.
7 12 16 18
5 8 12 16 18
5 9 12 16 18
1 6 13 16 18
1 7 13 16 18
1 8 13 16 18
1 9 13 16 18
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 16 18
1 6 10 17 18
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 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 18
5 8 10 17 18
5 9 10 17 18
1 6 11 17 18
1 7 11 17 18
1 8 11 17 18
170

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter second Parameter Third Parameter Fourth
Fifth Parameter
No. No. No. Parameter No. No.
1 9 11 17 18
2 6 11 17 18
2 7 11 17 18
2 8 11 17 18
2 9 11 17 18
3 6 11 17 18
3 7 11 17 18
3 8 11 17 18
3 9 11 17 18
4 6 11 17 18
4 7 11 17 18
4 8 11 17 18
4 9 11 17 18
6 11 17 18
5 7 11 17 18
5 8 11 17 18
5 9 11 17 18
1 6 12 17 18
1 7 12 17 18
1 8 12 17 18
1 9 12 17 18
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 6 13 17 18
1 7 13 17 18
1 8 13 17 18
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
171

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter second Parameter Third 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
6 13 17 18
5 7 13 17 18
5 8 13 17 18
5 9 13 17 18
1 6 10 14 19
1 7 10 14 19
1 8 10 14 19
1 9 10 14 19
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 6 11 14 19
1 7 11 14 19
1 8 11 14 19
1 9 11 14 19
2 6 11 14 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
3 8 11 14 19
3 9 11 14 19
4 6 11 14 19
4 7 11 14 19
4 8 11 14 19
172

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter second Parameter Third Parameter Fourth
Fifth Parameter
No. No. No. Parameter No. No.
4 9 11 14 19
6 11 14 19
5 7 11 14 19
5 8 11 14 19
5 9 11 14 19
1 6 12 14 19
1 7 12 14 19
1 8 12 14 19
1 9 12 14 19
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 9 13 14 19
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 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
173

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter second Parameter Third Parameter Fourth
Fifth Parameter
No. No. No. Parameter No. No.
1 7 10 15 19
1 8 10 15 19
1 9 10 15 19
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
6 10 15 19
5 7 10 15 19
5 8 10 15 19
5 9 10 15 19
1 6 11 15 19
1 7 11 15 19
1 8 11 15 19
1 9 11 15 19
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 15 19
5 9 11 15 19
1 6 12 15 19
1 7 12 15 19
1 8 12 15 19
1 9 12 15 19
2 6 12 15 19
2 7 12 15 19
2 8 12 15 19
174

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter second Parameter Third 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
4 7 12 15 19
4 8 12 15 19
4 9 12 15 19
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 7 13 15 19
1 8 13 15 19
1 9 13 15 19
2 6 13 15 19
2 7 13 15 19
2 8 13 15 19
2 9 13 15 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 7 10 16 19
1 8 10 16 19
1 9 10 16 19
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 19
175

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
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
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 7 11 16 19
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 16 19
3 6 11 16 19
3 7 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 8 12 16 19
1 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
176

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter second Parameter Third Parameter Fourth
Fifth Parameter
No. No. No. Parameter No. No.
9 12 16 19
1 6 13 16 19
1 7 13 16 19
1 8 13 16 19
1 9 13 16 19
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
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 19
1 7 10 17 19
1 8 10 17 19
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 6 11 17 19
1 7 11 17 19
1 8 11 17 19
1 9 11 17 19
2 6 11 17 19
177

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter second Parameter Third Parameter Fourth
Fifth Parameter
No. No. No. Parameter No. No.
2 7 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 19
4 7 11 17 19
4 8 11 17 19
4 9 11 17 19
6 11 17 19
5 7 11 17 19
5 8 11 17 19
5 9 11 17 19
1 6 12 17 19
1 7 12 17 19
1 8 12 17 19
1 9 12 17 19
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
1 6 13 17 19
1 7 13 17 19
1 8 13 17 19
1 9 13 17 19
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
178

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter second Parameter Third Parameter Fourth
Fifth Parameter
No. No. No. Parameter No. No.
3 9 13 17 19
4 6 13 17 19
4 7 13 17 19
4 8 13 17 19
4 9 13 17 19
6 13 17 19
5 7 13 17 19
5 8 13 17 19
5 9 13 17 19
1 6 10 14 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 6 11 14 20
1 7 11 14 20
1 8 11 14 20
1 9 11 14 20
2 6 11 14 20
2 7 11 14 20
2 8 11 14 20
2 9 11 14 20
3 6 11 14 20
3 7 11 14 20
3 8 11 14 20
3 9 11 14 20
4 6 11 14 20
4 7 11 14 20
4 8 11 14 20
4 9 11 14 20
5 6 11 14 20
179

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter second Parameter Third Parameter Fourth
Fifth Parameter
No. No. No. Parameter No. No.
7 11 14 20
5 8 11 14 20
5 9 11 14 20
1 6 12 14 20
1 7 12 14 20
1 8 12 14 20
1 9 12 14 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 6 13 14 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 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 14 20
1 6 10 15 20
1 7 10 15 20
1 8 10 15 20
180

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter second Parameter Third Parameter Fourth
Fifth Parameter
No. No. No. Parameter No. No.
1 9 10 15 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
6 10 15 20
5 7 10 15 20
5 8 10 15 20
5 9 10 15 20
1 6 11 15 20
1 7 11 15 20
1 8 11 15 20
1 9 11 15 20
2 6 11 15 20
2 7 11 15 20
2 8 11 15 20
2 9 11 15 20
3 6 11 15 20
3 7 11 15 20
3 8 11 15 20
3 9 11 15 20
4 6 11 15 20
4 7 11 15 20
4 8 11 15 20
4 9 11 15 20
5 6 11 15 20
5 7 11 15 20
5 8 11 15 20
5 9 11 15 20
1 6 12 15 20
1 7 12 15 20
1 8 12 15 20
1 9 12 15 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
181

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter second Parameter Third 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
6 12 15 20
5 7 12 15 20
5 8 12 15 20
5 9 12 15 20
1 6 13 15 20
1 7 13 15 20
1 8 13 15 20
1 9 13 15 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 6 10 16 20
1 7 10 16 20
1 8 10 16 20
1 9 10 16 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
182

CA 03083783 2020-05-27
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First Parameter second Parameter Third Parameter Fourth
Fifth Parameter
No. No. No. Parameter No. No.
4 9 10 16 20
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 7 11 16 20
1 8 11 16 20
1 9 11 16 20
2 6 11 16 20
2 7 11 16 20
2 8 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 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 9 12 16 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
183

CA 03083783 2020-05-27
WO 2019/113375 PCT/US2018/064335
First Parameter second Parameter Third Parameter Fourth
Fifth Parameter
No. No. No. Parameter No. No.
1 7 13 16 20
1 8 13 16 20
1 9 13 16 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
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 7 10 17 20
1 8 10 17 20
1 9 10 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 9 11 17 20
2 6 11 17 20
2 7 11 17 20
2 8 11 17 20
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First Parameter second Parameter Third Parameter Fourth
Fifth Parameter
No. No. No. Parameter No. No.
2 9 11 17 20
3 6 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 17 20
4 9 11 17 20
6 11 17 20
5 7 11 17 20
5 8 11 17 20
5 9 11 17 20
1 6 12 17 20
1 7 12 17 20
1 8 12 17 20
1 9 12 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 7 13 17 20
1 8 13 17 20
1 9 13 17 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 17 20
3 8 13 17 20
3 9 13 17 20
4 6 13 17 20
185

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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
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 7 10 14 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 8 11 14 21
1 9 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 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 14 21
5 8 11 14 21
186

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First Parameter second Parameter Third Parameter Fourth
Fifth Parameter
No. No. No. Parameter No. No.
9 11 14 21
1 6 12 14 21
1 7 12 14 21
1 8 12 14 21
1 9 12 14 21
2 6 12 14 21
2 7 12 14 21
2 8 12 14 21
2 9 12 14 21
3 6 12 14 21
3 7 12 14 21
3 8 12 14 21
3 9 12 14 21
4 6 12 14 21
4 7 12 14 21
4 8 12 14 21
4 9 12 14 21
5 6 12 14 21
5 7 12 14 21
5 8 12 14 21
5 9 12 14 21
1 6 13 14 21
1 7 13 14 21
1 8 13 14 21
1 9 13 14 21
2 6 13 14 21
2 7 13 14 21
2 8 13 14 21
2 9 13 14 21
3 6 13 14 21
3 7 13 14 21
3 8 13 14 21
3 9 13 14 21
4 6 13 14 21
4 7 13 14 21
4 8 13 14 21
4 9 13 14 21
5 6 13 14 21
5 7 13 14 21
5 8 13 14 21
5 9 13 14 21
1 6 10 15 21
1 7 10 15 21
1 8 10 15 21
1 9 10 15 21
2 6 10 15 21
187

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First Parameter second Parameter Third Parameter Fourth
Fifth Parameter
No. No. No. Parameter No. No.
2 7 10 15 21
2 8 10 15 21
2 9 10 15 21
3 6 10 15 21
3 7 10 15 21
3 8 10 15 21
3 9 10 15 21
4 6 10 15 21
4 7 10 15 21
4 8 10 15 21
4 9 10 15 21
6 10 15 21
5 7 10 15 21
5 8 10 15 21
5 9 10 15 21
1 6 11 15 21
1 7 11 15 21
1 8 11 15 21
1 9 11 15 21
2 6 11 15 21
2 7 11 15 21
2 8 11 15 21
2 9 11 15 21
3 6 11 15 21
3 7 11 15 21
3 8 11 15 21
3 9 11 15 21
4 6 11 15 21
4 7 11 15 21
4 8 11 15 21
4 9 11 15 21
5 6 11 15 21
5 7 11 15 21
5 8 11 15 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
188

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First Parameter second Parameter Third Parameter Fourth
Fifth Parameter
No. No. No. Parameter No. No.
3 9 12 15 21
4 6 12 15 21
4 7 12 15 21
4 8 12 15 21
4 9 12 15 21
6 12 15 21
5 7 12 15 21
5 8 12 15 21
5 9 12 15 21
1 6 13 15 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
2 8 13 15 21
2 9 13 15 21
3 6 13 15 21
3 7 13 15 21
3 8 13 15 21
3 9 13 15 21
4 6 13 15 21
4 7 13 15 21
4 8 13 15 21
4 9 13 15 21
5 6 13 15 21
5 7 13 15 21
5 8 13 15 21
5 9 13 15 21
1 6 10 16 21
1 7 10 16 21
1 8 10 16 21
1 9 10 16 21
2 6 10 16 21
2 7 10 16 21
2 8 10 16 21
2 9 10 16 21
3 6 10 16 21
3 7 10 16 21
3 8 10 16 21
3 9 10 16 21
4 6 10 16 21
4 7 10 16 21
4 8 10 16 21
4 9 10 16 21
5 6 10 16 21
189

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First Parameter second Parameter Third Parameter Fourth
Fifth Parameter
No. No. No. Parameter No. No.
7 10 16 21
5 8 10 16 21
5 9 10 16 21
1 6 11 16 21
1 7 11 16 21
1 8 11 16 21
1 9 11 16 21
2 6 11 16 21
2 7 11 16 21
2 8 11 16 21
2 9 11 16 21
3 6 11 16 21
3 7 11 16 21
3 8 11 16 21
3 9 11 16 21
4 6 11 16 21
4 7 11 16 21
4 8 11 16 21
4 9 11 16 21
5 6 11 16 21
5 7 11 16 21
5 8 11 16 21
5 9 11 16 21
1 6 12 16 21
1 7 12 16 21
1 8 12 16 21
1 9 12 16 21
2 6 12 16 21
2 7 12 16 21
2 8 12 16 21
2 9 12 16 21
3 6 12 16 21
3 7 12 16 21
3 8 12 16 21
3 9 12 16 21
4 6 12 16 21
4 7 12 16 21
4 8 12 16 21
4 9 12 16 21
5 6 12 16 21
5 7 12 16 21
5 8 12 16 21
5 9 12 16 21
1 6 13 16 21
1 7 13 16 21
1 8 13 16 21
190

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First Parameter second Parameter Third Parameter Fourth
Fifth Parameter
No. No. No. Parameter No. No.
1 9 13 16 21
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
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 10 17 21
1 8 10 17 21
1 9 10 17 21
2 6 10 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
3 8 10 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 6 11 17 21
1 7 11 17 21
1 8 11 17 21
1 9 11 17 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
191

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First Parameter second Parameter Third Parameter Fourth
Fifth Parameter
No. No. No. Parameter No. No.
3 7 11 17 21
3 8 11 17 21
3 9 11 17 21
4 6 11 17 21
4 7 11 17 21
4 8 11 17 21
4 9 11 17 21
6 11 17 21
5 7 11 17 21
5 8 11 17 21
5 9 11 17 21
1 6 12 17 21
1 7 12 17 21
1 8 12 17 21
1 9 12 17 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 6 13 17 21
1 7 13 17 21
1 8 13 17 21
1 9 13 17 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
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First Parameter second Parameter Third Parameter Fourth
Fifth Parameter
No. No. No. Parameter No. No.
4 9 13 17 21
6 13 17 21
5 7 13 17 21
5 8 13 17 21
5 9 13 17 21
CXCR2 Agonists
5 Gro-,8, Gro-,8 T, and variants thereof
[0190] Exemplary CXCR2 agonists that may be used in conjunction with the
compositions
and methods described herein are Gro-(3 and variants thereof. Gro-(3 (also
referred to as
growth-regulated protein (3, chemokine (C-X-C motif) ligand 2 (CXCL2), and
macrophage
inflammatory protein 2-a (MIP2-a)) is a cytokine capable of mobilizing
hematopoietic 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-(3, exemplary CXCR2 agonists that may be used in
conjunction
with the compositions and methods described herein are truncated forms of Gro-
(3, such as
those that feature a deletion at the N-terminus of Gro-(3 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-(3 T, which is characterized by a
deletion of the first
four amino acids from the N-terminus of Gro-(3. Gro-(3 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-(3 by multiple orders of
magnitude. Gro-(3
and Gro-(3 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-f3 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-I3N69D, retains the hematopoietic stem and
progenitor cell-
mobilizing functionality of Gro-f3, 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-f3 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-f3 T N65D,
not only retains hematopoietic stem and progenitor cell-mobilizing capacity,
but exhibits a
potency that is substantially greater than that of Gro-f3 T. Gro-I3N69D and
Gro-f3 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-f3, Gro-f3 T, Gro-I3N69D, and Gro-f3 T
N65D are set
forth in TABLE 7, below.
TABLE 7. Amino acid sequences of Gro-f3 and select variants thereof
SEQ ID Description Amino Acid Sequence
NO.
APLATELRCQCLQTLQGIHLKN
1 G IQSVKVKSPGPHCAQTEVIATL
ro-f3
KNGQKACLNPASPMVKKIIEK
MLKNGKSN
TELRCQCLQTLQGIHLKNIQSV
2 G KVKSPGPHCAQTEVIATLKNGQ
ro-f3-T
KACLNPASPMVKKIIEKMLKNG
KSN
APLATELRCQCLQTLQGIHLKN
3 G ro-f3N69D IQSVKVKSPGPHCAQTEVIATL
KNGQKACLNPASPMVKKIIEK
MLKDGKSN
TELRCQCLQTLQGIHLKNIQSV
4 G KVKSPGPHCAQTEVIATLKNGQ
ro-f3-T N65D
KACLNPASPMVKKIIEKMLKDG
KSN
[0194] Additional CXCR2 agonists that may be used in conjunction with the
compositions
and methods described herein include other variants of Gro-f3, such as
peptides that have one
or more amino acid substitutions, insertions, and/or deletions relative to Gro-
P. 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-f3 T, such as peptides that
have one or more
amino acid substitutions, insertions, and/or deletions relative to Gro-f3 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-I3N69D, such as peptides that
have one or
more amino acid substitutions, insertions, and/or deletions relative to Gro-
I3N69D. 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-f3 T N65D, such as
peptides that have one
or more amino acid substitutions, insertions, and/or deletions relative to Gro-
f3 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-f3 or a variant or truncation thereof, such as
Gro-f3 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-f3 or a variant or truncation thereof, such as Gro-f3 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 a tandem di-scFv. In some
embodiments,
the antibody has an isotype selected from the group consisting of IgG, IgA,
IgM, IgD, and
IgE.
Synthetic CXCR2 Agonists
[0200] The peptidic CXCR2 agonists described herein, such as Gro-f3, Gro-f3 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;
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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 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 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 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 (NMNI). 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-f3 and Gro-f3 T in
aqueous
solution, a particular C-terminal asparagine residue (Asn69 within Gro-f3 and
Asn65 within
Gro-f3 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-f3 and Gro-f3 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-f3, Gro-f3 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-
(3, Gro-f3 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%,
970, 97.50
,
98%, 98.5%, 990, 99.50, 99.6%, 99.70, 99.8%, 99.90, 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-
f3, Gro-f3 T,
and variants thereof may have, for instance, a purity of from about 950 to
about 99.99%,
such as a purity of from about 9500 to about 99.990, about 96 A to about
99.990, about 970
to about 99.990, about 98 A to about 99.990, about 990 to about 99.990, about
99.90 to
about 99.990, about 950 to about 99.50, about 96 A to about 99.50, about 950
to about
990, or about 970 to about 990 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 ¨ 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) an amine represented by formula (IA)
A
N
(IA)
wherein A includes a monocyclic or bicyclic fused ring system including at
least one nitrogen atom and B is H or a sub stituent of from 1 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)
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N 1
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)õ ¨ X (IC)
wherein each R is independently H or Ci-C6 alkyl, n is 1 or 2, and X is an
aryl
or heteroaryl group or a mercaptan;
wherein the linker is a bond, optionally substituted alkylene (e.g.,
optionally substituted C1-
C6 alkylene), optionally substituted heteroalkylene (e.g., optionally
substituted Ci-C6
heteroalkylene), optionally substituted alkenylene (e.g., optionally
substituted C2-C6
alkenylene), optionally substituted heteroalkenylene (e.g., optionally
substituted C2-C6
heteroalkenylene), optionally substituted alkynylene (e.g., optionally
substituted C2-C6
alkynylene), optionally substituted heteroalkynylene (e.g., optionally
substituted C2-C6
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
1,4,8,11-tetraazocyclotetradecane.
[0207] In some embodiments, the linker is represented by formula (ID)
D
(ID)
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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
Cl-C6 alkylene), optionally substituted heteroalkylene (e.g., optionally
substituted Ci-C6
heteroalkylene), optionally substituted alkenylene (e.g., optionally
substituted C2-C6
alkenylene), optionally substituted heteroalkenylene (e.g., optionally
substituted C2-c6
heteroalkenylene), optionally substituted alkynylene (e.g., optionally
substituted C2-c6
alkynylene), or optionally substituted heteroalkynylene (e.g., optionally
substituted C2-C6
heteroalkynylene).
[0208] As an example, the linker may be represented by formula (IE)
,,,(x
Y A (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 substituted heteroalkylene (e.g., optionally
substituted C1-C6
heteroalkylene), optionally substituted C2-C6 alkenylene (e.g., optionally
substituted c2-C6
alkenylene), optionally substituted heteroalkenylene (e.g., optionally
substituted C2-c6
heteroalkenylene), optionally substituted alkynylene (e.g., optionally
substituted C2-c6
alkynylene), or optionally substituted heteroalkynylene (e.g., optionally
substituted C2-C6
heteroalkynylene). In some embodiments, X and Y are each independently
optionally
substituted C1-C6 alkylene. In some embodiments, X and Y are identical
substituents. In
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).
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[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.
FIN-
NH
I 1
J
õ
1,õ1,1H
(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-tetra-azacyclotetradecane; 1,1141,4-phenylene-bis-(methylene)]-bis-
1,4,8,11-
tetraazacyclotetradecane; bis-zinc or bis-copper complex of 1,1'-[1,4-
phenylene-bis-
(methyl ene)] -bi s-1,4,8,11-tetraazacyclotetradecane; 1,1'- [3,3 '-
biphenylene-bis-(methylene)]-
bis-1,4,8,11-tetraazacyclotetradecane; 11,11'-[1,4-phenylene-bis-(methylene)]-
bis-1,4,7,11-
tetraazacyclotetradecane; 1,11'-[1,4-phenylene-bis-(methylene)]-1,4,8,11-
tetraazacyclotetradecane-1, 4,7,11-tetraazacyclotetradecane; 1,1142,6-pyridine-
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 -thi ophene-bi s-(methylene)]-bi
s-1,4,8,11-
tetraazacyclotetradecane; 1,1'-[4,41-(2,2'-bipyridine)-bis-(methylene)]-bis-
1,4,8,11-
tetraazacyclotetradecane; 1,1 '-[2,9-(1, 10-phenanthroline)-bi s-(methylene)]-
bi s-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-
tetraazacyclotetradecane; 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; 1,1 '- [1,4-naphthyl ene-bi s-(methylene)]bi s-
1,4,8,11-
tetraazacyclotetradecane; 1,1'-[1,3-phenylenebis-(methylene)]bis-1,5,9-
triazacyclododecane;
1,1141,4-phenylene-bis-(methylene)]-1,5,9-triazacyclododecane; 1,1142,5-
dimethy1-1,4-
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-
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phenylenebis-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane; and 1,11-[6-
pheny1-2,4-
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-
tetraazacycl otetradecane; 1,4,7-triazacyclotetradecane; and 4,7,10-
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-triazacyclotetradecany1)-1,4-
phenylenebis(methylene)]-
2-(aminomethyl)pyridine; N-[4-(11,11-difluoro-1,4,7-triazacyclotetradecany1)-
1,4-
phenylenebis(methylene)]-2-(aminomethyl)pyridine; N44-(1,4,7-
triazacyclotetradecan-2-
ony1)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[12-(5-oxa-1,9-
diazacyclotetradecany1)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine;
N-[4-(11-
oxa-1,4,7-tri azacycl otetradecany1)-1,4-phenyl enebi s(methyl ene)] -2-
(aminomethyl)pyri dine;
N-[4-(11-thi a-1,4,7-tri azacycl otetradecany1)-1,4-phenyl enebi s(methyl
ene)] -2-
(aminomethyl)pyri dine; N-[4-(11-sulfoxo-1,4,7-triazacyclotetradecany1)-1,4-
phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[4-(11-sulfono-1,4,7-
triazacyclotetradecany1)-1,4-phenylenebis(methyl ene)] -2-
(aminomethyl)pyridine; and N- [4-
(3 -carb oxo-1,4,7-tri azacyclotetradecany1)-1,4-phenyl enebi s(methyl ene)] -
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-tetraazacycl otetra-decany1-1,4-phenyl enebi s-(methyl ene)] -
2-
(aminomethyl)pyri dine; N-[1,4,8,11-tetraazacyclotetra-decany1-1,4-
phenylenebis(methylene)]-N-methyl-2-(aminomethyl)pyridine; N-[1,4,8,11-
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tetraazacyclotetra-decany1-1,4-phenylenebis(methylene)]-4-
(aminomethyl)pyridine; N-
[1,4,8,11-tetraazacyclotetra-decany1-1,4-phenylenebis(methylene)]-3-
(aminomethyl)pyridine;
N-[1,4,8,11-tetraazacyclotetra-decany1-1,4-phenylenebis(methylene)]-(2-
aminomethy1-5-
methyl)pyrazine; N-[1,4,8,11-tetraazacyclotetra-decany1-1,4-
phenylenebis(methylene)]-2-
(aminoethyl) pyridine; N-[1,4,8,11-tetraazacyclotetra-decany1-1,4-
phenylenebis(methylene)]-
2-(aminomethyl)thiophene; N-[1,4,8,11-tetraazacyclotetra-decany1-1,4-
phenylenebis(methylene)]-2-(aminomethyl)mercaptan; N-[1,4,8,11-
tetraazacyclotetra-
decany1-1,4-phenylenebis(methylene)]-2-amino benzylamine; N-[1,4,8,11-
tetraazacyclotetra-
decany1-1,4-phenylenebis(methylene)]-4-amino benzylamine; N-[1,4,8,11-
tetraazacyclotetra-
.. decany1-1,4-phenylenebis(methylene)]-4-(aminoethyl)imidazole; N-[1,4,8,11-
tetraazacyclotetra-decany1-1,4-phenylenebis(methylene)]-benzylamine; N-[4-
(1,4,7-
triazacyclotetra-decany1)-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-
phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[7-(4,7,10-
triazabicyclo[13.3.1]heptadeca-1(17),13,15-trieny1)-1,4-
phenylenebis(methylene)]-2-
(aminomethyl)pyridine; N41-(1,4,7-triazacyclotetra-decany1)-1,4-
phenylenebis(methylene)]-
2-(aminomethyppyridine; 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-
triazabicyclo[13.3.1]heptadeca-1(17),13,15-trieny1]-1,4-
phenylenebis(methylene)]-2-
(aminomethyl)pyridine; N-[1,4,8,11-tetraazacyclotetradecany1-1,4-
phenylenebis(methylene)]-purine; 1-[1,4,8,11-tetraazacyclotetradecany1-1,4-
phenylenebix(methylene)]-4-phenylpiperazine; N-[4-(1,7-diazacyclotetradecany1)-
1,4-
phenylenebis(methylene)]-2-(aminomethyl)pyridine; and N-[7-(4,10-
diazabicyclo[13 .3 .1]heptadeca-1(17),13,15-trieny1)-1,4-phenylenebi s(methyl
ene)]-2-
.. (aminomethyl)pyridine.
[0215] In some embodiments, the CXCR4 antagonist is a compound selected from
the group
consisting of: 1-[2,6-dimethoxypyrid-4-yl(methylene)]-1,4,8,11-
tetraazacyclotetradecane; 1-
[2-chl oropyri d-4-yl(methyl ene)]-1,4,8,11-tetraazacyclotetradecane; 1-[2,6-
dimethylpyrid-4-
yl(methylene)]-1,4,8,11-tetraazacyclotetradecane; 142-methylpyrid-4-
yl(methylene)]-
1,4,8,11-tetraazacyclotetradecane; 1- [2,6-dichl oropyrid-4-yl(methyl ene)] -
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-
tetraazabicyclo[13 .3 .1]heptadeca-1(17),13,15-triene.
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[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: 7,7'41,4-phenylene-bis(methylene)This-
3,7, 11,17-
tetraazabicyclo[13.3.1]heptadeca-1(17),13,15-triene; 7,7'-[1,4-phenylene-
bis(methylene)]bis[15-chloro-3,7,11,17-tetraazabicyclo [13.3.1]heptadeca-1
(17),13,15-
triene]; 7,7141,4-phenylene-bis(methylene)This[15-methoxy-3,7,11,17-
tetraazabicyclo[13.3.1]heptadeca-1(17),13,15-triene]; 7,7'41,4-phenylene-
bis(methylene)This-3,7,11,17-tetraazabicyclo[13.3.1]-heptadeca-13,16-triene-15-
one; 7,7'-
[1,4-phenylene-bis(methylene)]bis-4,7,10,17-tetraazabicyclo[13.3.1]-heptadeca-
1(17),13,15-
triene; 8,8141,4-phenylene-bis(methylene)This-4,8,12,19-
tetraazabicyclo[15.3.1]nonadeca-
1(19),15,17-triene; 6,6141,4-phenylene-bis(methylene)This-3,6,9,15-
tetraazabicyclo[11.3.1]pentadeca-1 (15),11,13-triene; 6,6'-[1,3-phenylene-
bis(methylene)]bis-
3,6,9,15-tetraazabicyclo[11.3.1]pentadeca-1 (15),11,13-triene; and 17,17'-[1,4-
phenylene-
bis(methylene)]bis-3,6, 14,17,23,24-hexaazatricyclo[17.3.1.18'12]tetracosa-
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-
tetraaza cyclohexadecane); 3,3'-(bis-1,5,8,11,14-pentaazacyclohexadecane);
methylene (or
polymethylene) di-l-N-1,4,8,11-tetraaza cyclotetradecane; 3,3'-bis-1,5,9,13-
tetraazacyclohexadecane; 3,3 '-bi s-1,5,8, I 1,14-pentaazacyclohexadecane; 5,5
'-bi s-1,4,8, I 1-
tetraazacyclotetradecane; 2,5 '-bi s-1,4,8,11-tetraazacycl otetradecane; 2,6 '-
b i s-1,4,8,11-
tetraazacyclotetradecane; 11,11 '-(1,2-ethanediy1)b i s-1,4,8,11-tetraazacycl
otetradecane;
11,11 '-(1,2-propanediy1)bi s-1,4,8,11-tetraazacycl otetradecane; 11,11 '-(1,2-
butanediy1)bi s-
1,4,8,11-tetraazacycl otetradecane; 11,11 '-(1,2-pentanediy1)bi s-1,4,8,11-
tetraazacycl otetradecane; and 11,11'-(1,2-hexanediy1)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-
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tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-
(6,7-dihydro-
5H-cyclopenta[b]pyridin-7-y1)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-
N'-
(1,2,3,4-tetrahydro-l-naphthal eny1)-i,4-benzenedimethanamine; N-(2-pyri
dinylmethyl)-N'-
(1-naphthaleny1)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(8-
quinoliny1)-1,4-
benzenedimethanamine; N-(2-pyridinylmethyl)-N'42-[(2-pyridinylmethyl)amino]
ethyl] -N'-
(1-methyl -1,2,3,4-tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine; N-(2-
pyridinylmethyl)-N'42-[(1H-imidazol -2-ylmethyl)amino] ethyl] -N'-(1-methy1-
1,2,3,4-
tetrahydro-8-quinoliny1)-i,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-
(1,2,3,4-
tetrahydro-8-quinoliny1)-i,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-[2-
[(1H-
imidazol-2-ylmethyl)amino]ethy1]-N'-(1,2,3,4-tetrahydro-l-naphthaleny1)-1,4-
benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(2-pheny1-5,6,7,8-tetrahydro-8-
quinoliny1)-1,4-benzenedimethanamine; N,N1-bis(2-pyridinylmethyl)-N'-(2-pheny1-
5,6,7,8-
tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine;
[0219] N-(2-pyri dinylmethyl)-N'-(5,6,7,8-tetrahydro-5-quinoliny1)-1,4-
benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(1H-imidazol-2-ylmethyl)-N'-
(5,6,7,8-
tetrahydro-5-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-
(1H-
imidazol-2-ylmethyl)-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-
benzenedimethanamine; N-(2-
pyri dinylmethyl)-N'-[(2-amino-3 -phenyl)propyl] -N'-(5,6,7,8-tetrahydro-8-
quinoliny1)-1,4-
benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(1H-imidazol-4-ylmethyl)-N'-
(5,6,7,8-
tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(2-
quinolinylmethyl)-N'-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-
benzenedimethanamine; N-(2-
pyri dinylmethyl)-N'-(2-(2-naphthoyl)aminoethyl)-N'-(5,6,7,8-tetrahydro-8-
quinoliny1)-1,4-
benzenedimethanamine; N-(2-pyridinylmethyl)-N'-[(S)-(2-acetyl amino-3 -
phenyl)propy1]-N'-
(5,6,7,8-tetrahydro-8-quinoliny1)-i,4-benzenedimethanamine; N-(2-pyri di
nylmethyl)-N'-[(S)-
(2-acetyl amino-3 -phenyl)propyl] -N'-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-
benzenedimethanamine; N-(2-pyridinylmethyl)-N'4342-
naphthalenylmethypamino)propyl]-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-
benzenedimethanamine; N-(2-pyridinylmethyl)-N'42-(S)-pyrollidinylmethy1]-N'-
(5,6,7,8-
tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'42-
(R)-
pyrolli dinylmethyl] -N'-(5,6,7,8-tetrahydro-8-quinoliny1)-i,4-
benzenedimethanamine; N-(2-
pyri dinylmethyl)-N'-[3 -pyrazolylmethyl] -N'-(5,6,7,8-tetrahydro-8-
quinoliny1)-1,4-
benzenedimethanamine; N-(2-pyridinylmethyl)-N'-[2-pyrrolylmethy1]-N'-(5,6,7,8-
tetrahydro-
8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyri dinylmethyl)-N'42-thi
opheneylmethyl] -
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N'-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine; N-(2-
pyridinylmethyl)-N'-
[2-thiazolylmethy1]-N'-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-
benzenedimethanamine; N-(2-
pyridinylmethyl)-N'42-furanylmethy1]-N'-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-
benzenedimethanamine; N-(2-pyridinylmethyl)-N'42-[(phenylmethyl)amino]ethyl]-
N'-
(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine; N-(2-
pyridinylmethyl)-N'-(2-
aminoethyl)-N'-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine; N-
(2-
pyri dinylm ethyl)-N'-3 -pyrrolidinyl-N'-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-
benzenedimethanamine; N-(2-pyridinylmethyl)-N'-4-piperidinyl-N'-(5,6,7,8-
tetrahydro-8-
quinoliny1)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'42-
[(phenyl)amino]ethyl]-
N'-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine; N-(2-
pyridinylmethyl)-N'-
(7-methoxy-1,2,3,4-tetrahydro-2-naphthaleny1)-1,4-benzenedimethanamine; N-(2-
pyridinylmethyl)-N'-(6-methoxy-1,2,3,4-tetrahydro-2-naphthaleny1)-1,4-
benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(1-methy1-1,2,3,4-tetrahydro-2-
naphthaleny1)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(7-methoxy-
3,4-
dihydronaphthaleny1)-1-(aminomethyl)-4-benzamide; N-(2-pyridinylmethyl)-N'-(6-
methoxy-
3,4-dihydronaphthal eny1)-1-(aminomethyl)-4-b enzami de; N-(2-pyridinylmethyl)-
N'-(1H-
imidazol-2-ylmethyl)-N'-(7-methoxy-1,2,3,4-tetrahydro-2-naphthalenyl)-1,4-
benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(8-hydroxy-1,2,3,4-tetrahydro-2-
naphthaleny1)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(1H-imidazol-
2-
ylmethyl)-N'-(8-hydroxy-1,2,3,4-tetrahydro-2-naphthaleny1)-1,4-
benzenedimethanamine; N-
(2-pyridinylmethyl)-N'-(8-Fluoro-1,2,3,4-tetrahydro-2-naphthaleny1)-1,4-
benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(1H-imidazol-2-ylmethyl)-N'-(8-
Fluoro-
1,2,3,4-tetrahydro-2-naphthalenyl)-1,4-benzenedimethanamine; N-(2-
pyridinylmethyl)-N'-
(5,6,7,8-tetrahydro-7-quinoliny1)-1,4-benzenedimethanamine; N-(2-
pyridinylmethyl)-N'-(1H-
imidazol-2-ylmethyl)-N'-(5,6,7,8-tetrahydro-7-quinolinyl)-1,4-
benzenedimethanamine; N-(2-
pyridinylmethyl)-N'42-[(2-naphthalenylmethyl)amino]ethy1]-N'-(5,6,7,8-
tetrahydro-8-
quinoliny1)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'42-
(isobutylamino)ethy1]-
N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-
pyridinylmethyl)-N'-
[2-[(2-pyridinylmethyl)amino]ethy1]-N'-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-
benzenedimethanamine; N-(2-pyridinylmethyl)-N'42-[(2-
furanylmethyl)amino]ethy1]-N'-
(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine; N-(2-pyri di
nylmethyl)-N'-(2-
guanidinoethyl)-N'-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine;
N-(2-
pyridinylmethyl)-N'42-[bis-[(2-methoxy)phenylmethyl]amino]ethy1]-N'-(5,6,7,8-
tetrahydro-
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8-quinoliny1)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-[2-[(1H-
imidazol-4-
ylmethyl)amino]ethyl]-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-
benzenedimethanamine; N-
(2-pyridinylmethyl)-N'42-[(1H-imidazol-2-ylmethyl)amino]ethy1]-N'-(5,6,7,8-
tetrahydro-8-
quinoliny1)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'42-
(phenylureido)ethy1]-
N'-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine; N-(2-
pyridinylmethyl)-N'-
[[N"-(n-butyl)carboxamido]methy1]-N'-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-
benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(carboxamidomethyl)-N'-(5,6,7,8-
tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-
[(N"-
phenyl)carboxamidomethy1]-N'-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-
benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(carboxymethyl)-N'-(5,6,7,8-
tetrahydro-8-
quinoliny1)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(phenylmethyl)-
N'-
(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine; N-(2-
pyridinylmethyl)-N'-(1H-
benzimidazol-2-ylmethyl)-N'-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-
benzenedimethanamine;
N-(2-pyridinylmethyl)-N'-(5,6-dimethy1-1H-benzimidazol-2-ylmethyl)-N'-(5,6,7,8-
tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine (hydrobromide salt); N-(2-
pyridinylmethyl)-N'-(5-nitro-1H-benzimidazol-2-ylmethyl)-N'-(5,6,7,8-
tetrahydro-8-
quinoliny1)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-[(1H)-5-
azabenzimidazol-
2-ylmethyl]-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-
(2-
pyridinylmethyl)-N-(4-pheny1-1H-imidazol-2-ylmethyl)-N'-(5,6,7,8-tetrahydro-8-
quinolinyl)-
1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'42-(2-pyridinyl)ethy1FN'-
(5,6,7,8-
tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(2-
benzoxazoly1)-N'-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine; N-
(2-
pyridinylmethyl)-N'-(trans-2-aminocyclohexyl)-N'-(5,6,7,8-tetrahydro-8-
quinoliny1)-1,4-
benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(2-phenylethyl)-N'-(5,6,7,8-
tetrahydro-8-
quinoliny1)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(3-
phenylpropy1)-N'-
(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-
pyridinylmethyl)-N'-
(trans-2-aminocyclopenty1)-N'-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-
benzenedimethanamine;
N-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methy1]-N-(5,6,7,8-tetrahydro-8-
quinoliny1)-glycinamide; N4[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methy1]-
N-
(5,6,7,8-tetrahydro-8-quinoliny1)-(L)-alaninamide; N4[4-[[(2-
pyridinylmethyl)amino]methyl]phenyl]methyl]-N-(5,6,7,8-tetrahydro-8-
quinolinyl)-(L)-
aspartamide; N4[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-N-(5,6,7,8-
tetrahydro-8-quinoliny1)-pyrazinamide; N-[[4-[[(2-
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pyridinylmethypamino]methyl]phenyl]methy1]-N-(5,6,7,8-tetrahydro-8-quinolinyl)-
(L)-
prolinamide; N-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-N-(5,6,7,8-
tetrahydro-8-quinoliny1)-(L)-lysinamide; N-[[4-[[(2-
pyridinylmethypamino]methyl]phenyl]methy1]-N-(5,6,7,8-tetrahydro-8-quinolinyl)-
benzamide; N-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-N-(5,6,7,8-
tetrahydro-
8-quinoliny1)-picolinamide; N'-Benzyl-N-[[4-[[(2-
pyridinylmethyl)amino]methyl]phenyl]methyl]-N-(5,6,7,8-tetrahydro-8-
quinoliny1)-urea; N'-
phenyl-N-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methy1]-N-(5,6,7,8-
tetrahydro-8-
quinoliny1)-urea; N-(6,7,8,9-tetrahydro-5H-cyclohepta[bacteriapyridin-9-y1)-4-
[[(2-
pyridinylmethyl)amino]methyl]benzamide; N-(5,6,7,8-tetrahydro-8-quinol iny1)-4-
[[(2-
pyridinylmethyl)amino]methyl]benzamide; N,N'-bis(2-pyridinylmethyl)-N'-
(5,6,7,8-
tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine; N,N'-bis(2-pyridinylmethyl)-
N'-
(6,7,8,9-tetrahydro-5H-cyclohepta[bacteriapyridin-9-y1)-1,4-
benzenedimethanamine; N,N'-
bis(2-pyridinylmethyl)-N'-(6,7-dihydro-5H-cyclopenta[bacteriapyridin-7-y1)-1,4-
benzenedimethanamine; N,N1-bis(2-pyridinylmethyl)-N'-(1,2,3,4-tetrahydro-1-
naphthaleny1)-
1,4-benzenedimethanamine; N,N'-bis(2-pyridinylmethyl)-N'-[(5,6,7,8-tetrahydro-
8-
quinolinyl)methy1]-1,4-benzenedimethanamine; N,N1-bis(2-pyridinylmethyl)-
NI(6,7-
dihydro-5H-cyclopenta[bacteriapyridin-7-yl)methyl]-1,4-benzenedimethanamine; N-
(2-
pyridinylmethyl)-N-(2-methoxyethyl)-N'-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-
benzenedimethanamine; N-(2-pyridinylmethyl)-N42-(4-methoxyphenyl)ethyl]-N'-
(5,6,7,8-
tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine; N,N'-bis(2-pyridinylmethyl)-
1,4-
(5,6,7,8-tetrahydro-8-quinolinyl)benzenedimethanamine; N-[(2,3-
dimethoxyphenyl)methy1]-
N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-
benzenedimethanamine;
N,N1-bis(2-pyridinylmethyl)-N41-(N"-phenyl-N"-methylureido)-4-piperidinyl]-1,3-
benzenedimethanamine; N,N1-bis(2-pyridinylmethyl)-N4N"-p-
toluenesulfonylphenylalany1)-
4-piperidinyl]-1,3-benzenedimethanamine; N,N'-bis(2-pyridinylmethyl)-N-[1-[3-
(2-
chloropheny1)-5-methyl-isoxazol-4-oyl]-4-piperidinyl]-1,3-
benzenedimethanamine; N-[(2-
hydroxyphenyl)methy1]-N'-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-
cyclohepta[bacteriapyridin-9-y1)-1,4-benzenedimethanamine; N-[(4-
cyanophenyl)methy1]-N'-
(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[bacteriapyridin-9-y1)-
1,4-
benzenedimethanamine; N-[(4-cyanophenyl)methy1]-N'-(2-pyridinylmethyl)-N-
(5,6,7,8-
tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[(4-
acetamidophenyl)methy1]-N'-(2-
pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine;
N-[(4-
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phenoxyphenyl)methy1]-N'-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-
cyclohepta[bacteriapyridin-9-y1)-1,4-benzenedimethanamine; N-[(1-methy1-2-
carboxamido)ethy1]-N,N1-bis(2-pyridinylmethyl)-1,3-benzenedimethanamine; N-[(4-
benzyloxyphenyl)methy1]-N'-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-
cyclohepta[bacteriapyridin-9-y1)-1,4-benzenedimethanamine; N-[(thiophene-2-
yl)methy1]-N'-
(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[bacteriapyridin-9-y1)-
1,4-
benzenedimethanamine; N41-(benzy1)-3-pyrrolidinyl]-N,N1-bis(2-pyridinylmethyl)-
1,3-
benzenedimethanamine; N-[[1-methy1-3-(pyrazol-3-y1)]propyl]-N,N1-bis(2-
pyridinylmethyl)-
1,3-benzenedimethanamine; N41-(phenyl)ethy1]-N,N1-bis(2-pyridinylmethyl)-1,3-
benzenedimethanamine; N-[(3,4-methylenedioxyphenyl)methy1]-N'-(2-
pyridinylmethyl)-N-
(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-y1)-1,4-benzenedimethanamine; N-
[1-benzy1-
3-carboxymethy1-4-piperidinyl]-N,N1-bis(2-pyridinylmethyl)-1,3-
benzenedimethanamine; N-
[(3,4-methylenedioxyphenyl)methy1]-N'-(2-pyridinylmethyl)-N-(5,6,7,8-
tetrahydro-8-
quinoliny1)-1,4-benzenedimethanamine; N-(3-pyridinylmethyl)-N'-(2-
pyridinylmethyl)-N-
(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-y1)-1,4-benzenedimethanamine; N-
[[1-
methy1-2-(2-tolyl)carboxamido]ethyl]-N,N1-bis(2-pyridinylmethyl)-1,3-
benzenedimethanamine; N-[(1,5-dimethy1-2-pheny1-3-pyrazolinone-4-yl)methyl]-N'-
(2-
pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine;
N-[(4-
propoxyphenyl)methy1]-N'-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-
cyclohepta[b]pyridin-9-y1)-1,4-benzenedimethanamine; N-(1-pheny1-3,5-
dimethylpyrazolin-
4-ylmethyl)-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-
benzenedimethanamine; N4H-imidazol-4-ylmethy1]-N,N1-bis(2-pyridinylmethyl)-1,3-
benzenedimethanamine; N-[(3-methoxy-4,5-methylenedioxyphenyl)methy1]-N'-(2-
pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-y1)-1,4-
benzenedimethanamine; N-[(3-cyanophenyl)methy1]-N'-(2-pyridinylmethyl)-N-
(6,7,8,9-
tetrahydro-5H-cyclohepta[b]pyridin-9-y1)-1,4-benzenedimethanamine; N-[(3-
cyanophenyl)methy1]-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-
1,4-
benzenedimethanamine; N-(5-ethylthiophene-2-ylmethyl)-N'-(2-pyridinylmethyl)-N-
(6,7,8,9-
tetrahydro-5H-cyclohepta[b]pyridin-9-y1)-1,4-benzenedimethanamine; N-(5-
ethylthiophene-
2-ylmethyl)-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-
benzenedimethanamine; N-[(2,6-difluorophenyl)methy1]-N'-(2-pyridinylmethyl)-N-
(6,7,8,9-
tetrahydro-5H-cyclohepta[b]pyridin-9-y1)-1,4-b enzenedimethanamine; N-[(2,6-
difluorophenyl)methy1]-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-
quinolinyl)-1,4-
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benzenedimethanamine; N-[(2-difluoromethoxyphenyl)methyl]-N'-(2-
pyridinylmethyl)-N-
(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-y1)-1,4-benzenedimethanamine; N-
(2-
difluoromethoxyphenylmethyl)-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-
quinoliny1)-
1,4-benzenedimethanamine; N-(1,4-b enzodioxan-6-ylmethyl)-N'-(2-
pyridinylmethyl)-N-
(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-y1)-1,4-benzenedimethanamine;
N,N'-bis(2-
pyridinylmethyl)-N-[1-(N"-phenyl-N"-methylureido)-4-piperidiny1]-1,4-
benzenedimethanamine; N,N1-bis(2-pyridinylmethyl)-N4N"-p-
toluenesulfonylphenylalanyl)-
4-piperidinyl]-1,4-benzenedimethanamine; N-[1-(3-pyridinecarboxamido)-4-
piperidiny1]-
N,N1-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-[1-
(cyclopropylcarboxamido)-4-
.. piperidiny1]-N,N1-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N41-(1-
phenylcyclopropylcarboxamido)-4-piperidiny1]-N,N1-bis(2-pyridinylmethyl)-1,4-
benzenedimethanamine; N-(1,4-benzodioxan-6-ylmethyl)-N'-(2-pyridinylmethyl)-N-
(5,6,7,8-
tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine; N-[1-[3-(2-chloropheny1)-5-
methyl-
isoxazol-4-carboxamido]-4-piperidiny1]-N,N1-bis(2-pyridinylmethyl)-1,4-
benzenedimethanamine; N-[1-(2-thiomethylpyridine-3-carboxamido)-4-piperidiny1]-
N,N1-
bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-[(2,4-
difluorophenyl)methy1]-N'-(2-
pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine;
N-(1-
methylpyrrol-2-ylmethyl)-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-
quinoliny1)-1,4-
benzenedimethanamine; N-[(2-hydroxyphenyl)methy1]-N'-(2-pyridinylmethyl)-N-
(5,6,7,8-
tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine; N-[(3-methoxy-4,5-
methylenedioxyphenyl)methy1]-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-
quinolinyl)-
1,4-benzenedimethanamine; N-(3-pyridinylmethyl)-N'-(2-pyridinylmethyl)-N-
(5,6,7,8-
tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine; N-[2-(N"-morpholinomethyl)-
1-
cyclopenty1]-N,N1-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-[(1-
methy1-3-
piperidinyl)propy1]-N,N1-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-(1-
methylbenzimidazol-2-ylmethyl)-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-
quinoliny1)-1,4-benzenedimethanamine; N41-(benzy1)-3-pyrrol idiny1]-N,N1-bis(2-
pyridinylmethyl)-1,4-benzenedimethanamine; N-[[(1-pheny1-3-(N"-
morpholino)]propy1]-
N,N1-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N41-(iso-propy1)-4-
piperidinyl]-
N,N'-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N41-(ethoxycarbony1)-4-
piperidiny1]-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-
benzenedimethanamine; N-[(1-methy1-3-pyrazolyl)propyl]-N'-(2-pyridinylmethyl)-
N-
(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine; N-[1-methy1-2-
(N",N"-
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diethylcarboxamido)ethy1]-N,N1-bis(2-pyridinylmethyl)-1,4-
benzenedimethanamine; N-[(1-
methy1-2-phenylsulfonyl)ethyl]-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-
quinoliny1)-
1,4-benzenedimethanamine; N-[(2-chloro-4,5-methylenedioxyphenyl)methy1]-N'-(2-
pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine;
N-[1-
methy1-2-[N"-(4-chlorophenyl)carboxamido]ethyl]-N'-(2-pyridinylmethyl)-N-
(5,6,7,8-
tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(1-acetoxyindo1-3-
ylmethyl)-N'-(2-
pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-y1)-1,4-
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-(3-
quinolylmethyl)-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-
benzenedimethanamine; N-[(8-hydroxy)-2-quinolylmethy1]-N'-(2-pyridinylmethyl)-
N-
(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-y1)-1,4-benzenedimethanamine; N-
(2-
quinolylmethyl)-N'-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-
cyclohepta[b]pyridin-9-
y1)-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;
N41H-
imidazol-2-ylmethyl]-N,N1-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-
(3-
quinolylmethyl)-N'-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-
cyclohepta[b]pyridin-9-
y1)-1,4-benzenedimethanamine; N-(2-thiazolylmethyl)-N'-(2-pyridinylmethyl)-N-
(6,7,8,9-
tetrahydro-5H-cyclohepta[b]pyridin-9-y1)-1,4-benzenedimethanamine; N-(4-
pyridinylmethyl)-N'-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-
cyclohepta[b]pyridin-9-
y1)-1,4-benzenedimethanamine; N-[(5-benzyloxy)benzo[b]pyrrol-3-ylmethyl]-N,N1-
bis(2-
pyridinylmethyl)-1,4-benzenedimethanamine; N-(1-methylpyrazol-2-ylmethyl)-N'-
(2-
pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-y1)-1,4-
benzenedimethanamine; N-[(4-methyl)-1H-imidazol-5-ylmethyl]-N,N1-bis(2-
pyridinylmethyl)-1,4-benzenedimethanamine; N-[[(4-dimethylamino)-1-
napthalenyl]methy1]-
N,N1-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-[1,5-dimethy1-2-pheny1-
3-
pyrazolinone-4-ylmethyl]-N,N1-bis(2-pyridinylmethyl)-1,4-benzenedimethanamine;
N-[1-[(1-
acety1-2-(R)-proliny1]-4-piperidinyl]-N42-(2-pyridinyl)ethyl]-N'-(2-
pyridinylmethyl)-1,3-
benzenedimethanamine; N- [142-acetamidobenzoy1-4-piperidiny1]-4-piperidiny1]-
N42-(2-
pyridinyl)ethy1]-N'-(2-pyridinylmethyl)-1,3-benzenedimethanamine; N-[(2-cyano-
2-
phenyl)ethy1]-N'-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-
cyclohepta[b]pyridin-9-y1)-
1,4-benzenedimethanamine; N-[(N"-acetyltryptophany1)-4-piperidinyl]-N42-(2-
pyridinyl)ethyl]-N'-(2-pyridinylmethyl)-1,3-benzenedimethanamine; N-RN"-
benzoylvaliny1)-
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4-piperidiny1]-N42-(2-pyridinyl)ethyl]-N'-(2-pyridinylmethyl)-1,3-
benzenedimethanamine;
N-[(4-dimethylaminophenyl)methyl]-N'-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-
5H-
cyclohepta[b]pyridin-9-y1)-1,4-benzenedimethanamine; N-(4-pyridinylmethyl)-N'-
(2-
pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine;
N-(1-
methylbenzimadazol-2-ylmethyl)-N'-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-
cyclohepta[b]pyridin-9-y1)-1,4-benzenedimethanamine; N41-buty1-4-piperidinyl]-
N42-(2-
pyridinyl)ethyl]-N'-(2-pyridinylmethyl)-1,3-benzenedimethanamine; N-[1-benzoy1-
4-
piperidiny1]-N42-(2-pyridinyl)ethyl]-N'-(2-pyridinylmethyl)-1,3-
benzenedimethanamine; N-
[1-(benzy1)-3-pyrrolidiny1]-N-[2-(2-pyridinyl)ethyl]-N'-(2-pyridinylmethyl)-
1,3-
benzenedimethanamine; N-[(1-methyl)benzo[b]pyrrol-3-ylmethy1]-N-[2-(2-
pyridinyl)ethyl]-
N'-(2-pyridinylmethyl)-1,3-benzenedimethanamine; N41H-imidazol-4-ylmethyl]-N-
[2-(2-
pyridinyl)ethyl]-N'-(2-pyridinylmethyl)-1,3-benzenedimethanamine; N-[1-
(benzy1)-4-
piperidiny1]-N42-(2-pyridinyl)ethyl]-N'-(2-pyridinylmethyl)-1,4-
benzenedimethanamine; N-
[1-methylb enzimidazol-2-ylmethy1]-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-pyridinylmethyl)-1,4-benzenedimethanamine; N-[(6-methylpyridin-2-
yl)methyl]-N'-(2-
pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinoliny1)-1,4-benzenedimethanamine;
N-(3-
methy1-1H-pyraz ol-5-ylm ethyl)-N'-(2-pyri dinylm ethyl)-N-(5,6, 7, 8-
tetrahydro-8-quinoliny1)-
1,3-benzenedimethanamine; N-[(2-methoxyphenyl)methy1]-N'-(2-pyridinylmethyl)-N-
(5,6,7,8-tetrahydro-8-quinoliny1)-1,3-benzenedimethanamine; N-[(2-
ethoxyphenyl)methy1]-
N'-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-y1)-1,3-
benzenedimethanamine; N-(benzyloxyethyl)-N'-(2-pyridinylmethyl)-N-(5,6,7,8-
tetrahydro-8-
quinoliny1)-1,3-benzenedimethanamine; N-[(2-ethoxy-l-naphthalenyl)methy1]-N'-
(2-
pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinoliny1)-1,3-benzenedimethanamine;
N-[(6-
.. methylpyridin-2-yl)methy1]-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-
quinoliny1)-1,3-
benzenedimethanamine; 1-[[4-[[(2-
pyridinylmethyl)amino]methyl]phenyl]methyl]guanidine;
N-(2-pyri di nyl m ethyl)-N-(8-m ethy1-8-az ab i cycl o [3 .2 .1] octan-3 -y1)-
1,4-
benzenedimethanamine; 1-[[4-[[(2-
pyridinylmethyl)amino]methyl]phenyl]methyl]homopiperazine; 1-[[3-[[(2-
pyridinylmethyl)amino]methyl]phenyl]methyl]homopiperazine; trans and cis-14[4-
[[(2-
pyridinylmethypamino]methyl]phenyl]methyl]-3,5-piperidinediamine; N,N1-[1,4-
Phenylenebis(methylene)]bis-4-(2-pyrimidyl)piperazine; 14[4-[[(2-
pyridinylmethypamino]methyl]phenyl]methyl]-1-(2-pyridinyl)methylamine; 2-(2-
pyridiny1)-
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5-[[(2-pyridinylmethyl)amino]methy1]-1,2,3,4-tetrahydroisoquinoline; 14[4-[[(2-
pyridinylmethypamino]methyl]phenyl]methyl]-3,4-diaminopyrrolidine; 14[4-[[(2-
pyridinylmethypamino]methyl]phenyl]methyl]-3,4-diacetylaminopyrrolidine; 84[4-
[[(2-
pyridinylmethypamino]methyl]phenyl]methyl]-2,5,8-triaza-3-oxabicyclo
[4.3.0]nonane; and
84[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methy1]-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
N'NR1 N
\/¨R3
R4 (III)
in which:
L is selected from ¨NR5a(CH2)2-3, ¨NR5a(CH2)2NR5b¨, ¨NR5a(CH2)2S¨, ¨
NR5aCH2CH(OH)¨ and ¨NR5aCH(CH3)CH2¨; wherein R5a and R5b are independently
selected from hydrogen and C1-4 alkyl;
R1 is selected from thiophenyl, 1H-benzoimidazolyl, isoquinolinyl, 1H-
imidazopyridinyl,
benzothiophenyl, pyrimidinyl, pyridinyl, pyrazinyl, pyridazinyl, and
thiazolyl; In some
embodiments, wherein the thiophenyl, 1H-benzoimidazolyl, isoquinolinyl, 1H-
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imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyridinyl, pyrazinyl,
pyridazinyl, or
thiazolyl of R1 can be optionally substituted by 1 to 3 radicals independently
selected from
cyano, hydroxy, Ci.4 alkyl, Ci.4 alkoxy, halo, halo-substituted-C14 alkyl,
halo-substituted-C1.
4a1koxy, amino, -C(0)R8a, -S(0)0.2R8a, -C(0)0R8a and -C(0)NR8aR8b; wherein Rga
and
Rgb are independently selected from hydrogen and Ci.4alkyl;
R2 is selected from -S(0)2NR6aR6b, -NR6aC(0)R6b-, -NR6aC(0)NR6bR6c, phenyl, 1H-
pyrrolopyridin-3-yl, 1H-pyrrolopyridin-5-yl, 1H-indoly1 thiophenyl, pyridinyl,
1H-1,2,4-
triazolyl, 2-oxoimidazolidinyl, 1H-pyrazolyl, 2-oxo-2,3-dihydro-1H-
benzoimidazoly1 and
1H-indazoly1; wherein R6a, R6b and R6 are independently selected from hydrogen
and C1.
4a1ky1; 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-benzoimidazoly1 or 1H-indazoly1 of R2 is optionally substituted
with 1 to 3
radicals independently selected from hydroxy, halo, methyl, methoxy, amino, -
0(CH2)2NR7aR7b, -S(0)2NR7aR7b, -0S(0)2NR7aRm and -NR7aS(0)2R7b; wherein It7a
and
R7b are independently selected from hydrogen and C1-4 alkyl;
R3 is selected from hydrogen, C1_4 alkyl and biphenyl; and
R4 is selected from Ci_io alkyl, prop-1-en-2-yl, cyclohexyl, cyclopropyl, 2-(2-
oxopyrrolidin-l-
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-
(1-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-y1)-1H-1,2,3-triazol-4-yl)ethyl
wherein said alkyl,
cyclopropyl, cyclohexyl, 2-(2-oxopyrrolidin-l-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-pentylphenyl)(phenyl)methyl or 1-(1-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-
y1)-1H-
1,2,3-triazol-4-yl)ethyl can be optionally substituted with 1 to 3 radicals
independently
selected from hydroxy, Ci_4alkyl and halo-substituted-C1.4alkyl; or a 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.
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OH
HN
NN
(1)
Methods for the Recombinant Expression of Peptides and Proteins
[0224] Peptides and proteins described herein (e.g., CXCR2 agonists, such as
Gro-f3, Gro-f3
T, Gro-f3 N69D, Gro-f3 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-f3, Gro-f3 T, Gro-f3 N69D, Gro-f3 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, the disclosure of which is incorporated herein by reference. A
similar technique,
NucleofectionTm, utilizes an applied electric field in order to stimulate the
uptake of
exogenous polynucleotides into the nucleus of a eukaryotic cell.
NucleofectionTm and
protocols useful for performing this technique are described in detail, e.g.,
in Distler et at.
(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 at.
(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 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, 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 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-f3, Gro-f3 T, Gro-I3N69D, Gro-f3 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 at., 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 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 (MD),
lymphocytic colitis, acute disseminated encephalomyelitis (ADEM), Addison's
disease,
alopecia universalis, ankylosing spondylitis, antiphospholipid antibody
syndrome (AP S),
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
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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' s syndrome, rheumatic fever, sarcoidosis,
scleroderma,
Sjogren's syndrome, stiff person syndrome, Takayasu's arteritis, temporal
arteritis (also
known as "giant cell arteritis"), ulcerative colitis, collagenous colitis,
uveitis, vasculitis,
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.
CD34thm cells
[0244] When the donor and patient are distinct, methods of treatment using
hematopoietic
stem cells comprising CD34d1m cells are particularly useful, in part because
CD34d1m cells are
capable of suppressing alloreactive T lymphocyte proliferation when
administered to a
recipient, thereby reducing the risk of graft vs. host disease (GVHD).
(D'Aveni et al. (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 CD34clim cells from the bone marrow of a donor into peripheral
blood. The
CD34d1m cells are present in a higher amount in the peripheral blood as
compared to
peripheral blood from an unmobilized mammal. In certain embodiments, the
CD34d1m 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-f3, Gro-f3 T, and variants
thereof at a dose
of from about 50 [tg/kg to about 1,000 [tg/kg and (ii) a CXCR4 antagonist.
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[0246] In addition, CD34d1th 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 at.
"CD34+ monocytes mobilized by G-CSF in donor PB and clinical outcomes after
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
CD34d1th 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-f3, Gro-f3 T, and variants thereof
at a dose of from
about 501.tg/kg to about 1,00011g/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% at least 10%, at least 15%, at least 20% or at least 20% or more CD34c1m
cells 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% CD34thin cells as compared to
peripheral 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
CD34d1m cells as
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
17-fold more CD34d1m cells as compared to peripheral blood from an 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 CD34d1m
cells than 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 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% CD34d1m cells than if
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,
at least 15-fold, at least 20-fold, at least 30-fold, at least 50-fold more
CD34d1m cells than 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 CD34d1m cells than if the hematopoietic stem cells were
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
ARCUSTm meganucleases that can be rationally designed so as to site-
specifically 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 1.6 hours, about 40 minutes to about 1.5 hours (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, 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 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, 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-f3, Gro-f3 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+ CD45RAT cells (hematopoietic stem cells), and reduce the
mobilization of
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.)
and in 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
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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 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-I3 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-f3 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+
CD48+). Grafts containing cells mobilized by Gro-0 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 (FIG. 1B).
[0270] In this example, data are presented to demonstrate that combination
treatment with
Gro-f3 T and AMD3100 results in significantly enhanced mobilization of CD34+
cells and
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-f3 T and plerixafor (also referred to as AMD3100) as described below.
Methods
[0272] Male rhesus macaques were treated with AMD3100 alone or in combination
with
Gro-0 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-0 T
when
administered intravenously to Rhesus monkeys. FIG. 2B shows the
pharmacokinetic profile
of various dosages of Gro-0 T when administered subcutaneously to Rhesus
monkeys. In all
experiments, Gro-0 T was administered to subjects concurrently with
plerixafor.
[0274] As shown in FIGS. 3-6, Gro-f3 T and AMD3100 mobilizes white blood cells
into the
peripheral blood. Animals were mobilized with AMD3100 alone or in combination
with
Gro-0 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 !IL of peripheral blood were
determined. Data
shown in FIGS. 2-5 are expressed as mean SEM and represent 5 animals 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-f3 T and AMD3100 induces
robust
mobilization of CD34+ cells into the peripheral blood. The data shown in FIGS.
7 and
8inc1ude the percentage of whole blood accounted for by CD34+ hematopoietic
stem and
progenitor cells for each treatment group. Absolute numbers and fold change in
CD34+ cells
per [tL of peripheral blood were quantified with a single platform
quantitative method. Data
shown in FIGS. 7 and 8 are expressed as mean SEM and represent 5 animals per
group.
Statistical significance was determined based on 2-way ANOVA with post-hoc
Dunnett's
multiple comparisons test (* p <0.05, ** p <0.01, **** p <0.0001).
[0276] Additionally, as shown in FIGS. 9 and 10, CD34+ cells mobilized in
response to Gro-
.. 0 T + AMD3100 are enriched for primitive CD34+ CD90+ CD45RA- stem and
progenitor
cells. The data shown in FIGS. 9 and 10 include the percentage of whole blood
accounted
for by CD34+ CD90+ CD45RAT hematopoietic stem and progenitor cells for each
treatment
group. Absolute numbers and fold change in CD34+ CD90+ CD45RAT cells per [tL
of
peripheral blood are shown. Data shown in FIGS. 9 and 10 are expressed as mean
SEM
and represent 5 animals per group. Statistical significance was determined
based on 2-way
ANOVA with post-hoc Dunnett's multiple comparisons test (* p <0.05. **p <0.01,
*** p <
0.001, **** p < 0.0001).
[0277] Further, as shown in FIG. 11, Gro-f3 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 represent 3-5 animals per group. Statistical
significance
was determined based on 2-way ANOVA with post-hoc Dunnett's multiple
comparisons test
(* p < 0.05). The ratio of MMP-9 to TIMP-1 is additionally elevated following
treatment
with Gro-f3 T and AMD3100 (FIGS. 12-14).
[0278] Additional data summarizing the mobilization of CD34+ cells (e.g.,
CD34+ 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)."
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TABLE 8. Mobilization response observed in Rhesus monkeys upon administration
of
plerixafor (1 mg/kg, subcutaneously)
Ratio of Ratio of
Quantity of quantity of
fold
Ratio of CD34+ Fold increase
Ratio of fold increase of
cells observed 4
quantity of CD90+ vs. baseline
increase of CD34
hours following
+
Cell type CD34 cells CD45RA- following sc CD34+ cells to
CD90+
sc
to other cell cells to administration
other cell CD45RA-
administration
of AMD3100: populations other cell of AMD3100:
populations cells to
population
other cell
s
populations
0.352
41054 (23012 -
1.4 (0.6 -
CD34+ -- (0.259 - 9.1 (7.8 -
16.5) --
58496) 2.6)
0.551)
CD34+
14820 (6123 - 2.838 (1.816 - 14.9 (5.3 -
CD90+ -- 0.7 (0.4 - 1.7)
--
17893) 3.864) 23.3)
CD54RA-
0.0009 0.0003
3.90E+07 (3.16 -
WBCs (0.0006 - (0.0002 - 2.3 (1.6 -4.6)
2.3 (1.6 -4.6) 3.7 (1.0 -
4.97E+07)7.9)
0.0012) 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 -
Lymphocytes
1.62E+07) (0.0020 - (0.0005 - 2.5 (1.3 - 3.1) 4.0 (2.9 -
8.2) 5.7 (2.4 -
14.0)
0.0043) 0.0013)
0.0111 0.0039
3.10E+06 (1.55 -
1.6 (0.7 -
Monocytes (0.0047 - (0.0020 - 7.9 (5.4 -
14.1) 1.2 (0.6 -2.0)
4.93E+06)3.5)
0.0377) 0.0115)
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TABLE 9. Mobilization response observed in Rhesus monkeys upon administration
of Gro-
f3 T (450 pg/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. baseline
Ratio of fold of CD34+
4 hours quantity of CD34+ CD90+
following iv increase of
CD90+
Cell type following iv CD34+ cells CD45RA- administratio CD34+
cells
CD45RA-
administration to other cell cells
to other n of Gro-beta to other cell
cells to
of Gro-beta T populations cell T at 450
populations
other cell
at 450 g/kg: populations iug/kg:
populatio
ns
CD34 47194 (38004 - -- 0.676 (0.393 - 21.7 (11.2 - --
1.2 (1.1 -
+
103119) 0.745) 27.2)
4.8)
CD34+ CD90+ 25701 (16770 - 1.479 (1.341 - __ 30.7 (18.3 -
0.8 (0.2 -
__
CD54RA- 76870) 2.545) 73.9) 0.9)
0.0014 0.0009
3.84E+07 (2.72 5.1 (3.4 -
6.0 (5.5 -
WBCs (0.0008 - (0.0003 - 3.8 (2.7 - 5.4)
-
5.27E+07) 6.9) 26.9)
0.0021) 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 -5.2)
-
2.44E+07)8.1) 22.0)
0.0058) 0.0043)
0.0031 0.0021
1.76E+07 (0.8 - 5.7 (4.8 -
9.3 (5.6 -
(0.0021 - (0.0008 - 3.2 (2.0 - 5.4)
Lymphocytes
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 _ 599E+06) (0.0071 - (0.0028 -
.99E+06) 21.1) 2.3) 8.5)
0.0174) 0.0130)
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TABLE 10. Mobilization response observed in Rhesus monkeys upon administration
of
Gro-f3 T (450 ug/kg, subcutaneously and plerixafor (1 mg/kg, subcutaneously)
Ratio of
Quantity of Ratio of fold
cells observed Ratio of quantity of Fold
increase Ratio of
CD34+ vs. baseline fold
increase of
6 hours quantity of
CD34+ CD90+
CD90+ following sc increase
of
Cell type following sc CD34+ cells
CD45RA-
CD45RA- administration CD34+ cells
administration to other cell
cells to other
of Gro-beta T populations cells to of Gro-
beta T to other cell
cell
other cell at 450 g/kg:
populations
at 450 g/kg: populations
populations
41178 (19413 - 0.359 (0.318
CD34+ -- 6.3 (4.8- 13.2)
-- 1.0 (1.0 - 1.3)
72140) -0.441)
CD34+ CD90+ 14782 (6177 - 2.786 (2.266 1.0 (0.8 -
CD54RA- 31841) - 3.143) -- 6.6 (6.1 - 13.6)
1.0) --
0.0007 0.0003
6.31E+07 (5.83 1.3 (0.9 -
WBCs (0.0003 - (0.0001 - 5.4 (4.8 - 5.8)
1.4 (1.1 -2.3)
- 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 - 6.1)
1.4 (1.0 - 2.4)
- 4.98E+07)
2.4)
0.0018) 0.0008)
0.0022 0.0008
1.85E+07 (1.57
1.4 (1.2 -
Lymphocytes _
1.97E+07) (0.0012 - (0.0004 - 4.5 (3.9 - 5.7)
1.6(1.5 -2.4)
0.0037) 0.0016) 2.3)
0.0185 0.0066
2.23E+06 (2.05 0.7 (0.6 -
Monocytes _ 2.60E+06) (0.0075 - (0.0024 - 9.7
(7.4 - 17.1) -- 0.8 (0.7 - 0.8)
0.0352) 0.0155) 0.8)
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TABLE 11. Mobilization response observed in Rhesus monkeys upon administration
of
Gro-f3 T (1.2 mg/kg, subcutaneously) and plerixafor (1 mg/kg, subcutaneously)
Ratio of
Ratio of fold
Quantity of
cells observed Ratio of quantity of Fold increase
Ratio of fold
increase of
CD34+ vs. baseline
CD34+
4 hours quantity of increase of
CD90+ following sc
CD90+
Cell type following sc CD34+ cells
CD34+ cells
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+
1.3 (0.8 - 1.9)
71704) -0.548) 15.0)
CD34+
17471 (7645 - 2.232 (1.826 13.1 (10.3 -
CD90+ 0.8 (0.5 - 1.3)
25964) - 3.594) 23.9)
CD54RA-
0.0008 0.0003
4.94E+07 (4.07
WBCs (0.0005 - (0.0001 - 5.7 (4.5 -9.7)
1.8 (1.4 -2.7) 2.5 (1.1 -4.2)
- 6.66E+07)
0.0011) 0.0005)
0.0013 0.0006
3.23E+07 (2.31
Neutrophils (0.0008 - (0.0002 - 9.7 (5.5 - 15.1)
1.2 (0.7 - 1.9) 1.6 (0.7 -2.6)
- 3.52E+07)
0.0023) 0.0008)
0.0024 0.0010
1.36E+07 (0.66
4.9 (2.0 -
Lymphocytes (0.0019 - (0.0005 - 3.3 (1.7 -5.2)
3.6 (2.4 - 8.9)
- 2.91E+07)12.0)
0.0068) 0.0037)
0.0130 0.0052
4.08E+06 (1.16 13.7 (7.2 -
Monocytes - 5.37E+06) (0.0068 - (0.0029 -
23.2) 0.8 (0.5 -2.1) 1.1 (0.6 -2.8)
0.0237) 0.0078)
Conclusions
[0279] A
single treatment of Gro-f3 T, in combination with AMD3100, induces robust
mobilization of stem and progenitor cells within four hours of administration
in nonhuman
primates. Additionally, Gro-f3 T, in combination with AMD3100, results in 2-3
fold more
CD34+ CD90+ CD45RAT stem and progenitor cells relative to AMD3100 alone,
suggesting a
significant graft quality improvement.
[0280]
Further, as evidenced by these data, Gro-f3 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.
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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,
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).
[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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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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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 1 x 103 to 1 x 109 hematopoietic stem cells/kg (e.g., about 1 x 105 CD34+
cells/kg to
about 1 x 107 CD34+ cells/kg, about 2 x 105 CD34+ cells/kg to about 9 x 106
CD34+ cells/kg,
about 3 x 105 CD34+ cells/kg to about 8 x 106 CD34+ cells/kg, about 4 x 105
CD34+ cells/kg
to about 7 x 106 CD34+ cells/kg, depending on various factors, such 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
concentration of hematopoietic stem cells or cells of the hematopoietic
lineage (such as
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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.
Example 4: Mobilization of CD34thm cells using a CXCR2 agonist and/or a CXCR4
antagonist
[0290] In this example, data are presented to demonstrate that combination
treatment with
Gro-f3 T and AMD3100 (plerixafor) results in significantly enhanced
mobilization of
CD34d1m cells compared to that achieved with AMD3100 alone in nonhuman
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 45011g/kg Gro-f3 T
and
subcutaneous administration of 1 mg/kg AMD3100 surprisingly leads to the
mobilization of a
population of cells that are CD34d1m CD11b+. Subpopulations within this
population are
CD14+ CD16+, CD14- CD16-, and combinations thereof FIG. 16 shows that
mobilization of
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the CD34d1m cells is significantly enhanced upon administration of the
combination of Gro-0
T and AMD3100 as compared to AMD3100 alone.
Example 5: Gro-I3 T in combination with AMD3100 mobilizes immunosuppressive
monocytes that inhibit graft vs. host disease
[0293] This example demonstrates that combination treatment with Gro-f3 T and
AMD3100
(plerixafor) results in significantly enhanced mobilization of CD34d1m cells
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
having a lower concentration of CD34d1m cells.
[0294] Rhesus macaques were treated with Gro-f3 T, AMD3100, Gro-f3 T and
AMD3100, or
G-CSF alone. A sample of peripheral blood was tested using a flow cytometry.
Animals
mobilized with Gro-f3 T and AMD3100 showed a more than fifteen-fold increase
over
baseline in the number of CD34d1m cells at 4 hours post-treatment (p<0.0001,
n=13, FIG. 17).
By comparison, animals mobilized with G-CSF alone showed only about a nine-
fold increase
over baseline in the number of CD34d1m cells at 4 hours after the last dose of
G-CSF
(p<0.0001, n=3, FIG. 17).
[0295] Further, the composition of unmobilized cells and grafts mobilized by G-
CSF, Gro-0
T and AMD3100 and AMD3100 alone is provided in FIG 18. As shown, grafts
mobilized
using Gro-f3 T and AMD3100 show a 3 fold increase in CD34d1m cells and a 3
fold increase in
T-cells as compared to grafts mobilized using G-CSF.
[0296] To determine if CD34d1m cells had immunosuppressive properties, they
were sorted
from the peripheral blood of Rhesus macaques treated with Gro-f3 T and
AMD3100. The
CD34d1m cells were co-cultured with carboxyfluorescein succinimidyl ester
(CFSE)-labeled
autologous T cells stimulated with anti-CD2/CD3/CD28-coated beads in vitro. As
shown in
FIG. 19, Gro-f3 T and AMD3100 mobilized CD34d1m cells suppressed T-cell
proliferation 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-f3 T and AMD3100 mobilized peripheral blood (6 x 106 PBMCs)
containing a
high percentage of CD34d1m cells were injected into sublethally irradiated NSG
mice. This
was compared to unmobilized primate PBMCs (6 x 106 PBMCs) containing a
relatively low
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numbers of CD34d1m cells and G-CSF mobilized primate PBMCs (6 x 106 PBMCs). 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-0 T and AMD3100-mobilized PBMCs. At day
60
post-transplant, 15/16 mice transplanted with Gro-f3 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-f3 T and AMD3100 mobilized PBMCs
("Gro-f3
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-f3 T and
AMD3100-
mobilized PBMCs.
[0298] To confirm that the CD34d1m cells in the Gro-f3 T and AMD3100-mobilized
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
PBMCs from
Gro-f3 T and AMD3100-mobilized rhesus monkeys, where CD34d1m cells were first
depleted
by fluorescence activated cell sorting and T cell numbers and survival were
compared to
cohorts transplanted with Gro-0 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-f3 T and AMD3100-mobilized PBMCs ("Gro-f3 T +
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-f3 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-f3 T and AMD3100-mobilized PBMCs ("Gro-f3 T +
plerixafor")
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(p<0.05) and only 4/10 mice transplanted with Gro-f3 T and AMD3100 mobilized
PBMCs
that were depleted of CD34dim cells ("Gro-f3 T + plerixafor CD34thm-depleted")
(no
significant difference versus Unmobilized).
[0299] Accordingly, these data demonstrate that co-administration of Gro-f3 T
and AMD3100
results not only in rapid and efficacious mobilization of highly enriched HSCs
but that these
HSCs are enriched in a CD34d1m cell population with potent immunosuppressive
activity
compared to AMD3100 alone or the current standard of care, G-CSF, and that
transplant of
this CD34d1m cell population can result in a reduced incidence of or risk for
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.
245